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[ [ "Electrophoretic analysis method", "By capillary electrophoresis, an effective mobility to play an important role for identifying an unknown sample is quickly determined highly precisely.", "A standard substance is added to a sample containing an unknown substance (unknown sample)(S101).", "This unknown sample to which the standard substance is added is introduced into a capillary, and capillary electrophoresis is executed (s103).", "Next, by executing this capillary electrophoresis, the various parameters of the standard substance and the unknown sample are determined (S107).", "Next, on the basis of the various parameters and a theoretical equation, the effective mobility of the unknown sample is calculated.", "On the basis of the calculated effective mobility of the unknown sample, the substance of the unknown sample is identified (S109)." ], [ "1.An electrophoretic analysis method including the steps of: adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis and a migration time of a substance having a mobility of 0 or a migration time of a system peak; and determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample and the migration time of the substance having a mobility of 0 or the migration time of a system peak as measured in the measuring step, and a known effective mobility of the standard substance at a standard temperature.", "2.The electrophoretic analysis method according to claim 1, wherein the step of determining an effective mobility of the unknown sample is to determine an effective mobility m0 of the unknown sample on the basis of a migration time tS of the standard substance, a migration time t of the unknown sample, a migration time teof of the substance having a mobility of 0 or of the system peak, and an effective mobility m0, S of the standard substance at the standard temperature according to the following equation: m0={(teof/t−1)/(teof/ts−1)}·mo, s 3.The electrophoretic analysis method according to claim 1, wherein the step of determining an effective mobility of the unknown sample is to determine the effective mobility of the unknown sample on the basis of the migration times of the two or more standard substances, the migration time of the unknown sample, the migration time of the substance having a mobility of 0 or of the system peak, and the effective mobilities of the standard substances at the standard temperature.", "4.The electrophoretic analysis method according to claim 3, wherein the step of determining an effective mobility of the unknown sample is to determine an effective mobility m0 of the unknown sample on the basis of migration times tA and tB of arbitrary two substances of the two or more standard substances, a migration time t of the unknown sample, a migration time teof of the substance having a mobility of 0 or of the system peak, and effective mobilities m0, A and m0, B of the standard substances at the standard temperature according to the following equations: m0={(tA−τ)(teof−t)/(t−τ)(teof−tA)}·m0, A τ={m0, AtA(teof−tB)−m0, BtB(teof−tA)}/{m0, A(teof−tB)−m0, B(teof−tA)} 5.The electrophoretic analysis method according claim 1, further including a step of identifying the substance of the unknown sample from the effective mobility of the unknown sample.", "6.An electrophoretic analysis method including the steps of: adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis; and determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample as measured in the measuring step, and a known effective mobility of the standard substance at a standard temperature.", "7.The electrophoretic analysis method according to claim 6, wherein the step of determining an effective mobility of the unknown sample is to determine an effective mobility m0 of the unknown sample on the basis of migration times tA, tB and tC of arbitrary three substances of the three or more standard substances, a migration time t of the unknown sample, and effective mobilities m0, A and m0, B and m0, C of the standard substances at the standard temperature according to the following equations: m0={(ti−t)(tj−τ)(m0, j−m0, i)}/{(t−τ)(ti−tj)}+m0, i wherein i and j are any one of the standard substances A, B and C, and i is not equal to j, τ={m0, AtA(tC−tB)+m0, BtB(tA−tC)+m0, CtC(tB−tA)}/{m0, A(tC−tB)+m0, B(tA−tC)+m0, C(tB−tA)} 8.The electrophoretic analysis method according to claim 2 further including a step of identifying the substance of the unknown sample from the effective mobility of the unknown sample.", "9.The electrophoretic analysis method according to claim 3 further including a step of identifying the substance of the unknown sample from the effective mobility of the unknown sample.", "10.The electrophoretic analysis method according to claim 4 further including a step of identifying the substance of the unknown sample from the effective mobility of the unknown sample." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In recent years, in the case where polymers, colloid particles, etc.", "contained in various solutions are identified, an analysis method utilizing a phenomenon in which the polymers or colloid particles move (migrate) corresponding to potential differences, so-called electrophoresis, become widespread.", "The migration velocity of the polymers or colloid particles in the solution that is influenced by not only the kind and concentration of an electrolyte in the solution but also the shape and size of the particles of the polymers or colloid particles themselves.", "For this reason, capillary zone electrophoresis (CZE) that is one of the analysis methods utilizing this electrophoresis is being watched as, for example, an important analysis method for analyzing the length of DNA fragments contained in proteins, etc.", "This capillary zone electrophoretic analysis method is required to have high reproducibility regarding a peak position to be used directly as a quantitative index of an unknown substance (unknown sample) in a solution.", "In the case where the analysis results of this capillary zone electrophoresis is expressed by a pherogram based on coordinate axes in which an abscissa represents the migration time by an unknown sample in a solution, and an ordinate represents the peak position, in order to obtain a peak position with good reproducibility, it is a very important factor to control each of a migration voltage (current), a temperature of a separation chamber, and electroosmotic flow.", "It is easily possible to control the migration voltage (current) and the temperature of the separation chamber by making the analysis conditions (such as an inner diameter of a capillary and a supporting electrolyte) identical.", "On the other hand, a temperature increase of a separation tube by a Joule's heat is inevitable and the control of the electroosmotic flow becomes difficult.", "For these reasons, the reproducibility of the electrophoresis of a usual capillary zone electrophoretic pherogram having a time axis is not high as compared with that in high-performance liquid chromatography (HPLC).", "As factors of disturbing the reproducibility of the migration time of an unknown sample in a solution are enumerated a change of electroosmotic flow, a relaxation effect of potential gradient (RPG), and a temperature increase by a Joule's heat generated with the application of a high voltage.", "Since these occur due to complex factors, it is difficult to control them.", "In the case where the measurement is carried out by using different equipments, it is assumed that even if the sample or supporting electrolyte is identical, it is difficult to obtain the same migration time.", "Accordingly, in order to correctly compare the measurement results in the case of using different equipments, it is necessary to standardize the electrophoresis data.", "On the other hand, Lee, et al.", "proposed a migration index (MI) using a value obtained by dividing a quantity of electricity, i.e., an integral value of current, by a cross-sectional area of a capillary and a whole length of the capillary and an adjusted migration index (AMI) obtained by correcting MI of the osmotic flow (the units of MI and AMI in the description being “μC/m 3 ”).", "It was considered that if the supporting electrolyte is identical, AMI could become a considerably good quantitative index.", "However, since a conductivity of the supporting electrolyte is included as one of the parameters of AMI, even a slight difference in the composition (for example, the case where even when a pH is substantially the same, counter ions are different) produces different measurement results." ], [ "<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>In order to prevent the matters as described above, it may be required to propose an electrophoretic analysis method using an effective mobility relying upon only conditions of a sample ion and a supporting electrolyte as a quantitative index.", "However, in the conventional conversion from the migration time to the effective mobility, in many cases, the change of the mobility by a Joule's heat and the relaxation effect of potential gradient were not taken into consideration.", "Here, the technology related to the invention will be described.", "As the method of correcting the change of the mobility by a Joule's heat and the relaxation effect of potential gradient, the present inventors previously filed an application for patent with respect to a method in which a standard substance is added to an unknown sample, capillary zone electrophoresis is executed, a migration time of the standard substance is determined, and assuming that an osmotic flow velocity linearly changes with respect to the time, the migration time of the standard substance is converted into an effective mobility (Japanese Patent Application No.", "11-251604).", "This method does not correct temperature dependency of the effective mobility of the sample (temperature dependency of a sample ion itself and temperature dependency to a supporting electrolyte) and so on in relation with a strict physical phenomenon but uses a hypothetic osmotic flow velocity on the assumption that the osmotic flow velocity linearly changes with respect to the time.", "Accordingly, it is assumed that all of phenomena are included in coefficients in empirical equations representing this linear change, whereby physical meanings become vague.", "Thus, the present inventors grasped three factors that lower the reproducibility (i.e., temperature dependency of the sample ion itself influencing the migration velocity, temperature dependency to the supporting electrolyte, and a delay of the migration time based on the relaxation effect of potential gradient) as original physical phenomena and groped for a method of theoretically eliminating these factors.", "Under these circumstances, the invention is aimed to quickly determine with high precision an effective mobility to play an important role for identifying an unknown sample by capillary zone electrophoresis.", "Further, the invention is aimed to eliminate a scattering of the effective mobility by operational conditions of an analysis equipment and to enhance reliability of data as a standardized index of every unknown sample.", "Moreover, the invention is aimed to use the same index in different analysis equipments.", "According to the first dissolution means of the invention, there is provided an electrophoretic analysis method including: a step of adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis and a migration time of a substance having a mobility of 0 or a migration time of a system peak, and a step of determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample and the migration time of the substance having a mobility of 0 or the migration time of a system peak as measured in the foregoing measuring step, and a known effective mobility of the standard substance at a standard temperature.", "According the second dissolution means of the invention, there is provided an electrophoretic analysis method including: a step of adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis, and a step of determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample as measured in the foregoing measuring step, and a known effective mobility of the standard substance at a standard temperature.", "A more detailed explanation of the invention is provided in the following description and appended claims take in conjunction with the accompanying drawings." ], [ "CROSS REFERENCES TO RELATED APPLICATIONS This application is based upon priority International Application PCT/JP01/05213 filed Jun.", "19, 2001, International Publication No.", "WO 01/98768 A1 published Dec. 27, 2001, which is based upon Japanese Application 2000-185276 filed Jun.", "20, 2000.FIELD OF THE INVENTION The present invention relates to an electrophoretic analysis method, and particularly to an electrophoretic analysis method in which by capillary zone electrophoresis, a standard substance is added to an unknown sample to convert respective migration times, from which an effective mobility of the unknown sample is determined, and the unknown sample is quickly identified with high precision.", "BACKGROUND OF THE INVENTION In recent years, in the case where polymers, colloid particles, etc.", "contained in various solutions are identified, an analysis method utilizing a phenomenon in which the polymers or colloid particles move (migrate) corresponding to potential differences, so-called electrophoresis, become widespread.", "The migration velocity of the polymers or colloid particles in the solution that is influenced by not only the kind and concentration of an electrolyte in the solution but also the shape and size of the particles of the polymers or colloid particles themselves.", "For this reason, capillary zone electrophoresis (CZE) that is one of the analysis methods utilizing this electrophoresis is being watched as, for example, an important analysis method for analyzing the length of DNA fragments contained in proteins, etc.", "This capillary zone electrophoretic analysis method is required to have high reproducibility regarding a peak position to be used directly as a quantitative index of an unknown substance (unknown sample) in a solution.", "In the case where the analysis results of this capillary zone electrophoresis is expressed by a pherogram based on coordinate axes in which an abscissa represents the migration time by an unknown sample in a solution, and an ordinate represents the peak position, in order to obtain a peak position with good reproducibility, it is a very important factor to control each of a migration voltage (current), a temperature of a separation chamber, and electroosmotic flow.", "It is easily possible to control the migration voltage (current) and the temperature of the separation chamber by making the analysis conditions (such as an inner diameter of a capillary and a supporting electrolyte) identical.", "On the other hand, a temperature increase of a separation tube by a Joule's heat is inevitable and the control of the electroosmotic flow becomes difficult.", "For these reasons, the reproducibility of the electrophoresis of a usual capillary zone electrophoretic pherogram having a time axis is not high as compared with that in high-performance liquid chromatography (HPLC).", "As factors of disturbing the reproducibility of the migration time of an unknown sample in a solution are enumerated a change of electroosmotic flow, a relaxation effect of potential gradient (RPG), and a temperature increase by a Joule's heat generated with the application of a high voltage.", "Since these occur due to complex factors, it is difficult to control them.", "In the case where the measurement is carried out by using different equipments, it is assumed that even if the sample or supporting electrolyte is identical, it is difficult to obtain the same migration time.", "Accordingly, in order to correctly compare the measurement results in the case of using different equipments, it is necessary to standardize the electrophoresis data.", "On the other hand, Lee, et al.", "proposed a migration index (MI) using a value obtained by dividing a quantity of electricity, i.e., an integral value of current, by a cross-sectional area of a capillary and a whole length of the capillary and an adjusted migration index (AMI) obtained by correcting MI of the osmotic flow (the units of MI and AMI in the description being “μC/m3”).", "It was considered that if the supporting electrolyte is identical, AMI could become a considerably good quantitative index.", "However, since a conductivity of the supporting electrolyte is included as one of the parameters of AMI, even a slight difference in the composition (for example, the case where even when a pH is substantially the same, counter ions are different) produces different measurement results.", "BRIEF SUMMARY OF THE INVENTION In order to prevent the matters as described above, it may be required to propose an electrophoretic analysis method using an effective mobility relying upon only conditions of a sample ion and a supporting electrolyte as a quantitative index.", "However, in the conventional conversion from the migration time to the effective mobility, in many cases, the change of the mobility by a Joule's heat and the relaxation effect of potential gradient were not taken into consideration.", "Here, the technology related to the invention will be described.", "As the method of correcting the change of the mobility by a Joule's heat and the relaxation effect of potential gradient, the present inventors previously filed an application for patent with respect to a method in which a standard substance is added to an unknown sample, capillary zone electrophoresis is executed, a migration time of the standard substance is determined, and assuming that an osmotic flow velocity linearly changes with respect to the time, the migration time of the standard substance is converted into an effective mobility (Japanese Patent Application No.", "11-251604).", "This method does not correct temperature dependency of the effective mobility of the sample (temperature dependency of a sample ion itself and temperature dependency to a supporting electrolyte) and so on in relation with a strict physical phenomenon but uses a hypothetic osmotic flow velocity on the assumption that the osmotic flow velocity linearly changes with respect to the time.", "Accordingly, it is assumed that all of phenomena are included in coefficients in empirical equations representing this linear change, whereby physical meanings become vague.", "Thus, the present inventors grasped three factors that lower the reproducibility (i.e., temperature dependency of the sample ion itself influencing the migration velocity, temperature dependency to the supporting electrolyte, and a delay of the migration time based on the relaxation effect of potential gradient) as original physical phenomena and groped for a method of theoretically eliminating these factors.", "Under these circumstances, the invention is aimed to quickly determine with high precision an effective mobility to play an important role for identifying an unknown sample by capillary zone electrophoresis.", "Further, the invention is aimed to eliminate a scattering of the effective mobility by operational conditions of an analysis equipment and to enhance reliability of data as a standardized index of every unknown sample.", "Moreover, the invention is aimed to use the same index in different analysis equipments.", "According to the first dissolution means of the invention, there is provided an electrophoretic analysis method including: a step of adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis and a migration time of a substance having a mobility of 0 or a migration time of a system peak, and a step of determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample and the migration time of the substance having a mobility of 0 or the migration time of a system peak as measured in the foregoing measuring step, and a known effective mobility of the standard substance at a standard temperature.", "According the second dissolution means of the invention, there is provided an electrophoretic analysis method including: a step of adding a standard substance to a sample containing an unknown sample and measuring migration times of the standard substance and the unknown sample by electrophoresis, and a step of determining an effective mobility of the unknown sample on the basis of the migration times of the standard substance and the unknown sample as measured in the foregoing measuring step, and a known effective mobility of the standard substance at a standard temperature.", "A more detailed explanation of the invention is provided in the following description and appended claims take in conjunction with the accompanying drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic configurative diagram of a general capillary zone electrophoresis equipment.", "FIG.", "2 is a flow chart of the electrophoretic analysis method according to the invention.", "FIG.", "3 shows pherograms obtained from an experiment not applying the migration time-effective mobility conversion according to the invention.", "FIG.", "4 shows pherograms at calculation of the mobilities using equation (8).", "FIG.", "5 shows pherograms at calculation of the mobilities using equations (14) and (16).", "FIG.", "6 shows pherograms in which the abscissa is MI.", "FIG.", "7 shows pherograms in which the abscissa is AMI.", "FIG.", "8 shows pherograms according to the conventional analysis method, in which the abscissa is the effective mobility.", "FIG.", "9 shows pherograms according to the analysis method as previously applied for patent by the present inventors, in which the abscissa is the effective mobility.", "DETAILED DESCRIPTION OF THE INVENTION A detailed description of the preferred embodiments, examples and best modes for practicing the invention are described therein.", "1-1.Capillary Zone Electrophoresis: As the analysis method using electrophoresis are known U-shaped tube electrophoresis, agarose electrophoresis, capillary zone electrophoresis, etc.", "In the invention, since an effective mobility to play an important role for identifying an unknown sample is determined by using the capillary zone electrophoresis, the outline of the capillary zone electrophoresis will be first explained.", "The capillary zone electrophoresis is means for separation and analysis by a difference in the mobility of ions regardless of water or organic solvents and is an analysis method having a markedly high separation capability as compared with liquid chromatography, which is very watched in recent years.", "This capillary zone electrophoresis can be utilized so far as a material is soluble in some solvents and can conduct the electricity.", "Accordingly, the capillary zone electrophoresis can analyze substantially “all” materials so far as conditions for the separation (such as solvents, pH, and additives) are put in good order.", "However, since this capillary zone electrophoresis is hardly applied to neutral substances or giant molecules among of which no difference in the mobility is found as they are, in many cases, other electrophoresis modes are employed for the separation and analysis.", "As other electrophoretic methods, for example, in the case of neutral molecules, a method called micellar electrokinetic chromatography (MEKC) is employed, and in he case of giant molecules, a capillary gel electrophoresis (CGE) to add a gel to an electrolyte is employed.", "Especially, the capillary gel electrophoresis is one of key technologies of the human genome plan, and the base sequence is determined by using this means.", "Incidentally, there may be the case where the invention is applicable to these electrophoretic methods, too.", "Also, the capillary zone electrophoresis is watched to be useful for proteome (identification of functions and structures of proteins) that is considered to be a next stage of biochemical research of the genome project.", "In particular, it is reported that if a minute mobility can be determined, the capillary zone electrophoresis will become a very effective analysis method in the research of proteins by combining with a mass analysis equipment.", "Additionally, since an amount of the waste liquids is greatly low (several milliliters at maximum), it can be said that the capillary zone electrophoresis is an analysis method taking into consideration influences against the environment.", "FIG.", "1 is a schematic configurative diagram of a general capillary zone electrophoresis equipment.", "However, a subject of the analysis is cations within a sample, and an indirect absorption method as described later was applied as the analysis of these cations.", "This capillary zone electrophoresis equipment 1 is provided with, for example, a high voltage power supply section 2, vials 3 and 4, a capillary 5, a slit 6, a light source (such as UV lamps) 7, a diffraction grating 8, and a multi-channel photodiode 9.The high voltage power supply section 2 is a high voltage power supply having, for example, an output of from 0 to 30 kV and is connected to each of the vials 3 and 4 via a wiring.", "The vials 3 and 4 are placed on a turntable or translational table (not shown).", "Into the vials 3 and 4 are injected proper supporting electrolytes having UV absorption.", "Also, a sample having no UV absorption for the analysis is introduced into the vial 3.The capillary 5 is a tube having proper inner diameter and length, one end of which is inserted into the vial 3, with the other end being inserted into the vial 4.The capillary 5 is equipped with the slit 6 having a gap for controlling a width of ultraviolet rays to be irradiated from the light source 7.The light having passed through the slit 6 is decomposed into a proper spectrum by the diffraction grating 8.The obtained spectrum is digitalized as an absorbance of UV by the multi-channel photodiode 9.Incidentally, the high voltage power supply section 2, the vials 3 and 4, and the multi-channel diode 9 are controlled by a computer (not shown).", "The operational outline of the capillary zone electrophoresis equipment 1 will be explained.", "First of all, vial 3 filled by a supporting electrolyte to be injected into the capillary and the empty vial 4 are set.", "The vial 4 is sealed, and the supporting electrolyte injected into the vial 3 is sucked.", "The sucked supporting electrolyte is filled within the capillary 5.Next, a sample is injected in the vicinity of one end of the capillary 5 by, for example, hydrodynamic injection.", "Concretely, the supporting electrolyte (even water is employable) is introduced into the vial 4, and the sample is introduced into the vial 3.At this time, the liquid level of the vial 3 is set to be higher than the liquid level of the vial 4.The sample is injected in the vicinity of the capillary 5 from the vial 3 due to a principle of siphon by a difference in the liquid level between the vials 3 and 4.Next, the supporting electrolyte is injected, and the vials 3 and 4 having the same liquid level are set.", "Then, a high voltage is applied to the both ends of the capillary 5 from the high voltage supply section 2.The sample migrates within the capillary 5 by an effective length (a distance from the vicinity of one end of the capillary 5 into which the sample has been introduced to the slit 6 along the capillary 5).", "The migration direction of cations within the sample goes from the vial 3 towards the vial 4 as shown by an arrow in the drawing.", "Meanwhile, the respective cations are separated due to a difference in the migration velocity (effective mobility) of the respective cations.", "The sample separated due to the difference in the migration velocity (effective mobility) is detected as a spectral data every fixed period of time by the above-described diffraction grating 8.The spectral data is digitalized as an absorbance of UV by the above-described multi-channel photodiode 9.Incidentally, while the cations within the sample were analyzed herein, in the case where anions are analyzed, it is only required to make the sign of the high voltage supply section 2 reverse.", "Further, during the injection of the sample in the vicinity of the capillary 5, the hydrodynamic injection was employed, but examples of other methods include suction, pressurization, and electro-pressurization.", "According to the suction, the vial 3 is charged with the sample, and the vial 4 is made empty.", "Next, the vial 4 is sealed, and the sample is injected into the capillary 5 by suction.", "According to the pressurization, the vial 3 is charged with the sample, and the vial 4 is made empty.", "Next, the vial 3 is sealed, and the sample is injected into the capillary 5 by pressurization.", "According to the electro-pressurization, the vial 3 is charged with the sample, and the vial 4 is charged with the electrolyte (even water is employable).", "Next, the sample is injected into the capillary 5 by applying a high voltage.", "1-2.Calculation of Effective Mobility by Migration Time-Effective Mobility Conversion: An electrophoresis phenomenon in the capillary zone electrophoresis and an outline of calculation of an effective mobility will be explained.", "According to the conventional migration time-effective mobility conversion, an effective mobility m is determined according to the following manner.", "m=νion/E=(1/t−νeof)/(V/L) (1) In the equation, V represents an applied voltage; E represents a potential gradient; I represents an effective length; L represents a capillary whole length; νion represents a migration velocity of an unknown sample; and t represents a period of time (migration time) until the unknown sample is detected by a detector.", "Further, νeof (electroosmotic flow velocity) is obtained using a migration time teof of a substance having a mobility of 0 or of a system peak according to the following equation.", "νeof=1/teof (2) That is, the equation (1) will become as follows.", "m=(1/t−1/teof)/(V/L) (1)′ The system peak as referred to herein means a peak as if a substance were present, appearing in the case where when an indirect absorption method is used, a fluid within the capillary is flown by the electroosmotic flow, and a portion in which the sample is introduced reaches the detector.", "Accordingly, the migration time teof of the system peak means a period of time until the system peak appears.", "Further, the indirect absorption method as referred to herein is a detection method utilizing a phenomenon in which, for example, in the case where the sample does not have absorption of UV (ultraviolet rays), when a substance having UV absorption is used as the electrolyte, the substance of the electrolyte is reduced only in a portion of the sample, whereby the UV absorbance is lowered.", "Further, the electroosmotic flow as referred to herein means a phenomenon in which, for example, when a silica capillary is used, a silanol group present in an inner wall of the capillary dissociates, whereby the inner wall is negatively charged, and the solution within the capillary is apparently positively charged, and thus, when an electric field is applied, the whole of the liquid flows into the negative electrode side.", "In most cases, even when other substances are used for the capillary, this electroosmotic flow is generated (however, the direction of the flow may possibly change depending upon the substance or the composition of the electrolyte).", "Moreover, the migration time teof at a mobility of 0 means a period of time from the application of an electric field to the detection of a substance because by applying the electric field, the whole of the liquid within the capillary is pushed and flown by the electroosmotic flow as described above, and hence, even the substance having no mobility (namely, the mobility is 0) (such as neutral substances) is detected by the detector.", "Here, the change of the mobility with respect to the temperature will be investigated.", "It is considered that the temperature dependency of the effective mobility m of the sample may be split into a term f(T) of the sample ion itself and a term g(T)=∈/η (∈: dielectric constant, η: viscosity coefficient) of the temperature dependency to the supporting electrolyte as expressed in the following equation.", "m=f(T)·g(T) (3) The Taylor's expansion of this equation around a standard temperature T0 is as follows.", "m=(f0+f1ΔT+f2ΔT2+ .", ".", ".", ")(g0+g1ΔT+g2ΔT2+ .", ".", ". )", "(4) In the equation, ΔT=T−T0, f0=f(T0), and g0=g(T0).", "Here, since the change of ion size by the temperature change is very small as compared with the viscosity of a solvent and the temperature dependency of the dielectric constant, when the temperature dependency of influences against the ion itself is neglected, the effective mobility m is expressed as follows.", "m = f 0 ( g 0 + g 1 ⁢ Δ ⁢ ⁢ T + g 2 ⁢ Δ ⁢ ⁢ T 2 + … ⁢ ) = f 0 ⁢ g 0 ( 1 + g 1 / g 0 · Δ ⁢ ⁢ T + g 2 / g 0 · Δ ⁢ ⁢ T 2 + … ⁢ ) ( 5 ) Here, where ΔT is small, the secondary or sequent members can be neglected.", "Further, when the replacement of f0g0=m0 and g1/g0=α is made, the mobility is expressed below independent upon the substance.", "m=(1+αΔT)·m0 (6) In the equation, m0 represents an effective mobility of the sample at the standard temperature, and α represents a temperature coefficient independent upon the sample ion.", "In general, the value of the temperature coefficient α at 25° C. is about 0.02 (e.g., K: 0.0191, Li: 0.0228).", "1-3.Conversion Taking Into Consideration Only Temperature Coefficient: Next, the conversion taking into consideration only the temperature coefficient will be explained.", "An effective mobility m0, S of a known standard substance (effective mobility mS) at the standard temperature T0 is determined below using the equation (1)′.", "m0, s=(1/ts−1/teof)/{(1+αΔT)E} Additionally, from the equation (6), the relation between mS and m0, S is as follows.", "1+αΔT=ms/m0, s (7) Further, an effective mobility m0 of a sample substance (effective mobility m) at the standard temperature T0 is determined below using the equation (1)′.", "m0=(1/t−1/teof)/{(1+αΔT)E} Additionally, from the equation (6), the relation between m and m0 is as follows.", "1+αΔT=m/m0 From these equations, the effective mobility m0 of the sample substance (ion) at the temperature T0 is obtained in the following equation.", "m0=m/(1+αΔT)=(1/t−1/teof)/{(1+αΔT)E}={(teof/t−1)/(teof/ts−1)}·mo, s (8) According to the equation (8), the parameters depending upon the equipment, such as E, V, I, and L are eliminated, so that it becomes possible to standardize the electrophoresis data independent upon the conditions of the equipment.", "Accordingly, in this embodiment of the invention, in the capillary zone electrophoresis in which a standard substance is added to an unknown sample, a mobility is measured from the respective migration times, and the substance is identified from the mobility value based on the foregoing theory, first of all, as a correction method of the case where the effective mobility is influenced by only the temperature, the effective mobility m0 of the unknown substance is determined from the migration time teof of the substance having a mobility of 0 or of the system peak, the migration time tS of the standard substance, and the effective mobility m0, S of the standard substance at the standard temperature T0 according to the equation (8).", "Incidentally, the parameter m0, S included in the equation (8) is determined from articles or existing data.", "Further, teof is measured from, for example, unevenness in the concentration of the electrolyte within the capillary.", "In addition, with respect to teof, t and tS, it may be estimated what detection peak is corresponding to what substance is from existing data and experimental values, or estimations.", "Accordingly, the respective migration times can be determined from pherograms obtained from the experiments as described later.", "1-4.Conversion Taking Into Consideration Temperature and Time Correction: Next, the conversion taking into consideration not only the temperature but also the time correction will be explained.", "In general, it is considered that all migration times of ions are delayed by a fixed period of time by the relaxation effect of potential gradient.", "This relaxation effect of potential gradient is a phenomenon found by the present inventors through simulation (see J. Chromatogr.", "A, 898 (1999), 19-29).", "Concretely, in the case where a high voltage is applied to the both ends of the capillary using a constant voltage power supply, the current flowing within the capillary does not become constant immediately due to the time change of potential gradient in a portion into which the sample has been introduced, but becomes constant after a while.", "For this reason, a mean potential gradient (voltage/whole length of capillary) to be used for the determination of the mobility using the conventional electrophoresis is not obtained immediately.", "Accordingly, the relaxation effect of potential gradient refers to a phenomenon in which the mean potential gradient is delayed in detection by a fixed period of time as compared with the migration time as expected by the conventional calculation method (the delayed time being expressed by “τ” in the description).", "Here, assuming that the electroosmotic flow is constant during the measurement, when the relaxation effect of potential gradient is taken into consideration, the effective mobility m of the sample ion is expressed as follows.", "m=1/{(t−τ)E}−meof (9) Here, meof is the mobility of osmotic flow, νeof/E.", "That is, this mobility represents one at the temperature increased by a Joule's heat within the capillary.", "When this equation is substituted for the foregoing equation (6), the following equation from which the effective mobility m0 at the standard temperature T0 can be determined is obtained.", "m0={1/(t−τ)E−meof}/(1+αΔT) (10) However, the parameters τ, meof, and (1+αΔT) are unknown.", "Thus, these parameters are determined from the standard samples (standard substances) A and B whose mobilities are known, and the migration time teof of the substance having a mobility of 0 or of the system peak.", "The mobilities of the standard samples A and B and the system peak are expressed by the following equations.", "m0, A=[1/{(t A−τ)E}−meof]/(1+αΔT) (11) m0, B=[1/{(t B−τ)E}−meof]/(1+αΔT) (12) meof=1/{(teof−τ)E} (13) In the equations, tA and tB represent migration times of the standard substances A and B, respectively; and m0, A and m0, B represent effective mobilities of the standard substances A and B at the standard temperature T0, respectively.", "The following equations are obtained from the equations (11) to (13).", "τ={m0, AtA(teof−tB)−m0, BtB(teof−tA)}/{m0, A(teof−tB)−m0, B(teof−tA)} (14) Further, the following equation is obtained by modifying the equation (11).", "1+αΔT=[1/{(tA−τ)E}−meof]/m0, A (15) Moreover, when the equations (13) and (15) are substituted for the equation (10), the following equation is obtained.", "m0={(tA−τ)(teof−t)/(t−τ)(teof−tA)}·m0, A (16) According to the equation (16), the parameters depending upon the equipment, such as E, V, I, and L, are eliminated, so that it becomes possible to standardize the electrophoresis data independent upon the conditions of the equipment.", "Accordingly, in this embodiment of the invention, as a correction method of the case where the effective mobility m0 is influenced by not only the temperature but also the delay in migration time by the relaxation effect of potential gradient, the effective mobility m0 of the unknown substance is determined from the migration time teof of the substance having a mobility of 0 or of the system peak, the migration times tA and tB of the two or more standard substances, and the effective mobilities m0, A and m0, B of the standard substances at the standard temperature T0 according to the equations (14) and (16).", "Incidentally, the parameter m0, A and m0, B included in the equations (14) and (16) are determined from articles or existing data.", "Further, with respect to teof, t, tA, and tB, the relations with the substances can be grasped from existing data and experimental values, or estimations, as described above.", "Accordingly, the respective migration times can be determined from pherograms obtained from the experiments as described later.", "In addition, the conversion taking into consideration the case where the substance having a mobility of 0 cannot be added, or the system peak cannot be detected (that is, teof cannot be identified), will be explained.", "First of all, substances A, B and C whose mobilities are known are added a sample, and their migration times tA, tB and tc are measured, thereby determining necessary parameters.", "The mobilities of the standard substances A, B and C are expressed as follows.", "m0, A=[1/{(tA−τ)E}−meof]/(1+αΔT) (11) m0, B=[1/{(tB−τ)E}−meof]/(1+αΔT) (12) m0, C=[1/{(tC−τ)E}−meof]/(1+αΔT) (17) The following equations are obtained from the equations (11), (12) and (17).", "τ={m0, AtA(tC−tB)+m0, BtB(tA−tC)+m0, CtC(tB−tA)}/{m0, A(tC−tB)+m0, B(tAtC)+m0, C(tB−tA)} (18) 1+αΔT={1(ti−tj)}/{E(ti−τ)(tj−τ)(m0, i−m0, j)} (19) In the equations, i and j are any one of A, B and C, and i is not equal to j. meof=1/{(ti−τ)E}−m0, i(1+αΔT) (20) In the equation, i is any one of A, B and C. When the thus obtained τ, (1+αΔT), and meof are substituted for the equation (10), the following equation is obtained.", "m0={(ti−t)(tj−τ)(m0, j−m0, i)}/{(t−τ)(ti−tj)}+m0, i (21) In the equation, i and j are any one of A, B and C, and i is not equal to j.", "Here, when m0, C=0, tC=teof, i=C, and j=A, the following equations (14) to (16) are obtained.", "The leading steps will be described below.", "The following equations are obtained from the equations (11), (12) and (17).", "meof=1/{(tA−τ)}−m0, A(1+αΔT) (11)′ meof=1/{(tB−τ)}−m0, B(1+αΔT) (12)′ meof=1/{(tC−τ)}−m0, C(1+αΔT) (17)′ Further, the equation (12)′ and the equation (17)′ are subtracted from the equation (11)′, followed by putting in order to obtain the following equations.", "(tA−τ)(tB−τ)={1(tB−tA)}/{E(1+αΔT)(m0, A−m0, B)} (a) (tA−τ)(tC−τ)={1(tC−tA)}/{E(1+αΔT)(m0, A−m0, C)} (b) Moreover, (a)/(b) is expressed as follows.", "(tB−τ)/(tc−τ)={(tB−tA)(m0, A−m0, C)}/{(tC−tA)(m0, A−m0, B)}=K (c) Thus, the following equation is obtained.", "τ=(tB−KtC)/(1−K) (c)′ Here, the numerator and the denominator of τ are put in order as follows.", "Numerator of τ= tB−{(tB−tA)(m0, A−m0, C)tC}/{(tC−tA)(m0, A−m0, B)}={m0, AtA(tC−tB)+m0, BtB(tA−tC)+m0, CtC(tB−tA) }/{(tC−tA)(m0, A−m0, B)} Denominator of τ =1−{(tB−tA)(m0, A−m0, C)}/{(tC−tA)(m0, A−m0, B)}={m0, A(tC−tB)+m0, B(tA−tC)+m0, C(tB−tA)}/{(tC−tA)(m0, A−m0, B)} Further, the following equation is obtained.", "τ={m0, AtA(tC−tB)+m0, BtB(tA−tC)+m0, CtC(tB−tA)}/{m0, A(tC−tB)+m0, B(tA−tC)+m0, C(tB−tA)} (18) Moreover, the following equation is obtained from the equation (a) or (b).", "1+αΔT={1(ti−tj)}/{E(ti−τ)(tj−τ)(m0, i−m0, j)} (19) Still further, the following equation is obtained by using any one of the equations (11)′, (12)′ and (17)′.", "meof=1/{(ti−τ)}−m0, i(1+αΔT) (20) Thus, even in the case where the migration time teof of the substance having a mobility of 0 or of the system peak cannot be identified, the effective mobility m0 of the unknown substance can be determined from the migration times tA, tB and tC of the three or more standard substances and the effective mobilities m0, A, m0, B and m0, C of the standard substances at the standard temperature T0 according to the equations (18) and (21).", "However, when m0, C=0, tC=teof, i=C, and j=A, the equation (14) is equal to the equation (18), and the equation (16) is equal to the equation (21), respectively.", "Accordingly, in the case where the migration time teof of the substance having a mobility of 0 or of the system peak cannot be identified, the experiment is omitted.", "Incidentally, the parameter m0, A, m0, B and m0, C included in the equations (18) and (21) are determined from articles or existing data.", "Further, with respect to tA, tB, tC, and t, the relations with the substances can be grasped from existing data and experimental values, or estimations, as described above.", "Accordingly, the respective migration times can be determined from pherograms obtained from the experiments.", "1-5.Processing Procedures of Electrophoretic Analysis Method According to the Invention: FIG.", "2 is a flow chart of the electrophoretic analysis method according to the invention.", "Incidentally, for the sake of convenience of the explanation, the flow chart is briefly explained here, and specific experimental results will be described later.", "First of all, the standard substance is added to the sample containing the unknown substance (unknown sample) (S101).", "The unknown sample having the standard substance added thereto is introduced into the capillary, and the above-described capillary zone electrophoresis is executed (S103).", "Next, by executing the capillary zone electrophoresis, as explained above in the section 1-3, during determining the effective mobility, in the case where only influences by the temperature are taken into consideration, the migration time teof of the substance having a mobility of 0 or of the system peak, the migration time tS of the standard substance, and the migration time t of the unknown sample are obtained, and further, the known effective mobility m0, S of the standard substance at the standard temperature T0 is obtained (S105).", "The effective mobility m0 of the unknown sample is calculated on the basis of these parameter values and the equation (8) (S107).", "Incidentally, the substance of the unknown sample may be identified on the basis of the calculated effective mobility m0 of the unknown sample (S109).", "Also, as explained above in the section 1-4, during determining the effective mobility, in the case where influences by not only the temperature but also the delay in migration time by the relaxation effect of potential gradient are taken into consideration, the migration time teof of the substance having a mobility of 0 or of the system peak, the migration times tA and tB of the two or more standard substances, and the migration time t of the unknown sample are obtained, and further, the known effective mobilities m0, A and m0, B of the standard substances at the standard temperature T0 are obtained (S105).", "The effective mobility m0 of the unknown sample is calculated on the basis of these parameter values and the equations (14) and (16) (S107).", "Incidentally, the substance of the unknown sample may be identified on the basis of the calculated effective mobility m0 of the unknown sample (S109).", "2.Experiments and Comparison: The following experiments illustrate some examples of the electrophoretic analysis method.", "These examples shall not be regarded as restricting the scope of the invention, as they are only examples using the inventive method.", "2-1.Experimental Method: Here, a capillary zone electrophoresis equipment to be used for verifying the effectiveness of the invented method is equipped with a capillary having an inner diameter (I.D.)", "of 75 μm or 50 μm and a whole length of 40 cm (effective length: 27.5 cm).", "The capillary is set up at a temperature of 25° C., and a voltage of 15 kV or 30 kV is applied thereto.", "Further, in order to detect a wavelength (in order to put the measurement results on coordinate axes), an indirect absorption method to measure the absorption of 3,5-lutidine of 265 nm was employed.", "As the sample were used a 30 mM equimolar mixed sample and a 0.3 mM equimolar mixed sample, each consisting of KCl, LiCl, TRIS (trihydroxymethyl aminomethane), ∈-aminocaproic acid, and sodium octylsulfonate.", "The 30 mM and 0.3 mM equimolar samples were each injected by the above-described hydrodynamic injection methods (2 cm, 15 s) and (2 cm, 75 s), respectively.", "As the supporting electrolyte were used a solution of 20 mM 3,5-lutidine whose pH had been adjusted at 6.0 by the addition of HCl (supporting electrolyte A: SE20 mM) and a solution of 40 mM 3,5-lutidine whose pH had been adjusted at 6.0 by the addition of acetic acid (supporting electrolyte B: SE40 mM).", "2-2.Experiment in the State Not Applying the Migration Time-Effective Mobility Conversion According to the Invention: FIG.", "3 shows pherograms obtained from an experiment not applying the migration time-effective mobility conversion according to the invention.", "The pherograms were obtained by the measurement under the following conditions in order from the above.", "(A) sample 30 mM, SE20 mM (supporting electrolyte A), I.D.", "75 μm, V=30 kV (B) sample 30 mM, SE40 mM (supporting electrolyte B), I.D.", "75 μm, V=30 kV (C) sample 30 mM, SE40 mM (supporting electrolyte B), I.D.", "50 μm, V=30 kV (D) sample 30 mM, SE40 mM (supporting electrolyte B), I.D.", "50 μm, V=15 kV (E) sample 30 mM, SE40 mM (supporting electrolyte B), I.D.", "75 μm, V=15 kV (F) sample 0.3 mM, SE40 mM (supporting electrolyte B), I.D.", "75 μm, V=15 kV Further, in (C), a new capillary was used without processing the inner wall, and in other experiments, the capillaries that had been used several times were used.", "Table 1 shows the measurement results (unit: second) of the migration time.", "TABLE 1 K Na Li Tris EOF (A) 22.36 27.65 31.52 38.91 58.4 (B) 17.84 21.28 23.88 27.24 41.44 (C) 35.32 48.42 59.93 82.1 233.3 (D) 72.29 94.99 112.76 147.3 260.8 (E) 57.72 74.15 86.22 102.64 176.1 (F) 61.58 77.52 89.59 107.46 185.71 Accordingly, the peaks are assigned to K+, Na+, Li+, TRIS, ∈-aminocaproic acid, system peak, and octylsulfonic acid in order from the left.", "It is understood that even in the same sample and electrolyte, various pherograms are obtained by changing the experimental conditions.", "Accordingly, when different equipments are used as described above, only the pherograms having a different migration time are obtained.", "For this reason, in order to measure a number of unknown samples at the same time, the standardization method in which the same substance exhibits the same quantitative index as described above is effective.", "2-3.Experiment of the Migration Time-Effective Mobility Conversion Using the Equation (8) According to the Invention: FIG.", "4 shows pherograms at calculation (execution of S101 to S107) of the mobilities using the equation (8).", "The peaks are assigned to K+, Na+, Li+, and TRIS in order from the left.", "Further, in the drawing, the solid line shows the previously reported value, 34.9 (m0, S in the equation (8)) of Li used as the standard sample at 25° C. The dotted lines show the previously reported values, 69.9, 46.3 and 24.4 of K, Na and TRIS, respectively at 25° C. in order from the left.", "By this experiment, the scattering of the data was greatly improved, and averages (relative standard deviations) of the mobilities of Na, Li and TRIS were 66.6 (3.60%), 45.7 (1.16%) and 22.6 (8.07%), respectively.", "Further, these averages (relative standard deviations) were deviated from the previously reported values by 4.74%, 1.37% and 7.49%, respectively.", "Especially, in the sample (D) where the electric power was the smallest, the inner diameter was small, and the applied voltage was low, the mobilities of K and Na were deviated from the previously reported values only by 0.93% and 0.08%, respectively.", "However, other data were estimated such that the mobility of K was smaller than the previously reported value.", "That is, it is considered that since the delay time τ by the relaxation effect of potential gradient was not taken into consideration, an error of K having a small migration time became relatively slightly large.", "For example, in Na having a small mobility, the influence became small.", "2-4.Experiment of the Migration Time-Effective Mobility Conversion Using the Equation (16) According to the Invention: FIG.", "5 shows pherograms at calculation (execution of S101 to S107) of the mobilities using the equations (14) and (16).", "The peaks are assigned to K+, Na+, Li+, and TRIS in order from the left.", "Further, in the drawing, the solid lines show the previously reported values, 69.9 (m0, A in the equations (14) and (16)) of K and 34.9 (m0, B in the equations (14) and (16)) of Li, respectively used as the standard samples at 25° C. The dotted lines in the drawing show the previously reported values, 46.3 and 24.4 of Na and TRIS at 25° C. in order from the left.", "By this experiment, the scattering of the data was greatly improved, and averages (relative standard deviations) of the mobilities of Na and TRIS were 46.4 (0.30%) and 22.2 (7.68%), respectively.", "Further, these were deviated from the previously reported values by 0.29% and 9.17%, respectively.", "Incidentally, the large deviation of TRIS was caused by the occurrence of tailing.", "On the other hand, it was exhibited that it is possible to depress the deviation of the mobility of Na to 0.3% or less.", "Further, it is possible to convert the mobility into an effective mobility at the reference temperature.", "Thus, it is possible to compare it with an effective mobility determined by other methods such as the conductivity method, thereby enabling to improve the flexibility of data.", "3.Comparison with the Conventional Method: The following experiments illustrate some examples of the electrophoretic analysis method.", "These examples shall not be regarding as restricting the scope of the invention as they are only examples of using the inventive method.", "3-1.Experiment Using Migration Index (MI): FIG.", "6 shows pherograms in which the abscissa is MI.", "In the case where MI using a value obtained by dividing a quantity of electricity, i.e., an integral value of current, by a cross-sectional area of a capillary and a whole length of the capillary, as proposed by Lee, et al., is used, (B), (D), (E) and (F) in which the peaks appeared at quite different migration times in the pherograms shown in FIG.", "3 exhibited relatively good consistency (the peaks are assigned to K+, Na+, Li+ and TRIS in order from the left as in FIG.", "3) by putting them in order by MI.", "However, in (A) in which the supporting electrolyte is different and in (C) in which the velocity of the osmotic flow is largely different, the peaks appeared at the largely different MI values.", "That is, it is shown that though the pherogram using MI can eliminate the influences of the temperature, it cannot thoroughly eliminate the nature of the electrolyte and the influences of the osmotic flow by MI.", "3-2.Experiment Using Adjusted Migration Index (AMI): FIG.", "7 shows pherograms in which the abscissa is AMI.", "In the case where AMI in which the influences of the osmotic flow is further taken into consideration is used in place of MI, (B) to (F) using the same supporting electrolyte exhibit relatively good consistency.", "In the drawing, the dotted lines show average values of the peak positions of the respective substances of (B) to (F).", "The AMI values will be examined in detail while using the dotted lines as indexes.", "The relative standard deviations (RSD) are deviated by 4.83% (K), 3.77% (Na), 4.70% (Li), and 7.08% (Tris), respectively.", "Further, nevertheless the mobility was substantially the same, (A) exhibited a quite different pherogram.", "This difference is caused by the matter that the conductivity of the supporting electrolyte is different.", "That is, it is shown that though the pherogram using AMI can eliminate the influences of the temperature and osmotic flow, it cannot thoroughly eliminate the nature of the electrolyte.", "3-3.Experiment by the Conventional Analysis Method in which the Abscissa is the Effective Mobility: FIG.", "8 shows pherograms according to the conventional analysis method, in which the abscissa is the effective mobility.", "In this conventional analysis method, a velocity is determined from the migration time; from the determined velocity, a difference from the velocity of the osmotic flow is determined; this difference is divided by an applied voltage; and the obtained value is further multiplied by a whole length of the capillary, to calculate an effective mobility.", "In the pherograms in which the abscissa is the effective mobility, the peaks are assigned to K+, Na+, Li+ and TRIS in order from the left.", "In the drawing, the dotted lines show the previously reported values, 69.9, 46.3, 34.9 and 24.4 of K, Na, Li and Tris, respectively at 25° C. in order from the left.", "The mobilities of the respective peaks were greatly scattered, and averages (relative standard deviations) of the mobilities of K, Na, Li and Tris were 90.7 (17.5%), 62.5 (19.9%), 47.8 (21.0%) and 31.1 (26.0%), respectively.", "According to the consideration in more detail, it is estimated that the effective mobilities of (A) to (C) in which the applied voltage is 30 kV are larger than those of (D) to (F) in which the applied voltage is 15 kV.", "This is because the electric power generated within the system was large, and the internal temperature increased, whereby the affective mobility became large.", "In particular, in (B) in which the inner diameter is large, and the electrolyte having a high concentration is used, such tendency was remarkably observed.", "In addition, the mobilities of all substances were estimated to be larger than the previously reported values.", "Thus, in order to obtain data with reproducibility using the capillary zone electrophoresis to apply a high voltage, the data processing method for correcting the increase of the internal temperature is effective as in this embodiment of the invention as described above.", "3-4.Experiment According to the Analysis Method as Previously Applied For Patent by the Present Inventors, in which the Abscissa is the Effective Mobility: FIG.", "9 shows pherograms according to the analysis method as previously applied for patent by the present inventors, in which the abscissa is the effective mobility (Japanese Patent Application No.", "11-251604).", "In the pherograms in which the abscissa is the effective mobility as calculated by this analysis method, the peaks are assigned to K+, Na+, Li+ and TRIS in order from the left.", "In the drawing, the solid lines show the previously reported values, 69.9 of K and 34.9 of Li, respectively used as the standard samples at 25° C. The dotted lines in the drawing show the previously reported values, 46.3 and 24.4 of Na and Tris at 25° C. in order from the left.", "By this experiment, the scattering of the data was greatly improved, and averages (relative standard deviations) of the mobilities of Na and Tris were 46.9 (1.05%) and 21.8 (8.11%), respectively.", "Further, these were deviated from the previously reported values by 0.64% and 10.7%, respectively.", "Incidentally, with respect to the migration time of TRIS, as in the article of Mikkers, et al.", "reporting that its evaluation is difficult because of the occurrence of tailing, and the mobility of Tris was largely deviated from the previously reported value.", "However, Na showed substantial consistency with the previously reported value.", "Thus, this method is a very useful method.", "However, it is assumed that physical meanings become vague, as described above." ] ]

[ [ "Structure and Method For Bolting Neutron Reflector", "A neutron reflector bolt fastening structure is disclosed in which even upon relaxation in the fastening forces thereof being generated in tie rods for divided stage portions as a result of neutron irradiation, it is possible to press the neutron reflector firmly against a core vessel.", "The neutron reflector bolt fastening structure includes: a neutron reflector which includes of a plurality of divided stage portions and situated in a core vessel in a reactor vessel; a plurality of tie rods for fixing the neutron reflector to the core vessel; and a plurality of bolts for exclusively fixing the lowermost stage portion of the plurality of stage portions of the neutron reflector to the core vessel." ], [ "1.A fastening assembly for a neutron reflector, comprising: a neutron reflector comprising a plurality of divided stage portions situated in a core vessel in a reactor vessel and including a lower core plate; a plurality of tie rods configured for fixing the neutron reflector to said core vessel; and a plurality of vertical bolts configured for fixing a lowermost stage portion of said plurality of stage portions and said lower core plate of said neutron reflector to said core vessel, said lowermost stage being fixed to said core vessel exclusively by said plurality of vertical bolts, wherein said core vessel comprises a horizontal flange portion and wherein said lowermost stage portion comprises horizontal flange portions having vertical through-holes formed therein for positioning of said plurality of vertical bolts therein, said flange portions being fixed to said core vessel and said vertical bolts exclusively securing said lowermost stage portion and said lower core plate to said horizontal flange portion of said core vessel.", "2.", "(canceled) 3.A neutron reflector bolt fastening structure according to claim 1, wherein said flange portions are arranged at opposing positions in an outer periphery of said lowermost stage portion of said neutron reflector.", "4.A neutron reflector bolt fastening structure according to claim 1, wherein said flange portions are arranged at equal intervals in an outer periphery of said lowermost stage portion of said neutron reflector.", "5.", "(canceled) 6.A neutron reflector bolt fastening method comprising: fixing a neutron reflector composed of a plurality of divided stage portions which are interconnected by a plurality of tie rods and which are situated in a core vessel in a reactor vessel; fixing the lowermost stage portion of said plurality of stage portions of said neutron reflector to said core vessel exclusively by a plurality vertically oriented of bolts; equipping said core vessel with a horizontal flange and equipping a lowermost stage portion of said plurality of stage portions with a plurality of through-holes; and securing said lowermost stage portion and said lower core plate to said horizontal flange portion of said core vessel exclusively by said plurality of bolts." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>FIG.", "3 is a schematic longitudinal sectional view showing the general construction of an ordinary nuclear reactor.", "In the drawing, a reactor vessel 10 is equipped with a core vessel 12 in which a fuel assembly 14 is supported.", "The fuel assembly 14 in the core vessel 12 is surrounded by an upper core plate 16 on the upper side, by a lower core plate 18 on the lower side, and by a neutron reflector 20 at the periphery.", "Next, the assembly structure of this neutron reflector will be described with reference to FIG.", "4 .", "The neutron reflector 20 is formed by vertically stacking together eight substantially annular stage portions, which are fastened by eight tie rods 22 that are circumferentially arranged.", "The positioning when assembling each stage portion is effected by a positioning pin 23 .", "The lowermost stage portion 20 A of the neutron reflector 20 has at its periphery four flange portions 201 (only two of which are shown in the drawing); similarly, the top stage portion 20 C has four flange portions 202 at its periphery.", "As shown in FIG.", "5 , the tie rods 22 extend through the neutron reflector 20 from the top stage portion 20 C to the lowermost stage portion 20 A.", "The lowermost end portions of the tie rods 22 are passed through the lower core plate 18 and screwed into a flange portion 13 formed in the core vessel 12 .", "To the upper end portion of each tie rod 22 , there is mounted a nut 24 for pressing down the upper surface of the top stage portion 20 C of the neutron reflector.", "By turning these nuts 24 , the neutron reflector 20 is tightened between them and the lower core plate 18 , and is secured to the flange portion 13 of the core vessel 12 through the intermediation of the lower core plate 18 .", "The neutron reflector 20 has a large number of flow holes for cooling, through which cooling water flows.", "FIG.", "6 shows a structure of inlet portions of the flow holes 204 in the lowermost stage portion 20 A of the neutron reflector 20 .", "In the drawing, plugs 181 are provided in the lower core plate 18 mounted to the flange portion 13 of the core vessel 12 .", "Cooling water flowing in through these plugs 181 passes orifices 203 provided in the lowermost stage portion 20 A of the neutron reflector 20 and flows upwards through the flow holes 204 .", "These flow holes extend through the 8-stage neutron reflector 20 from the lowermost stage portion 20 A to the top stage portion 20 C, so that the cooling water flowing in through the orifices 203 of the lowermost stage portion 20 A rises through the flow holes 204 to flow out through the flow holes of the top stage portion 20 C of the neutron reflector 20 .", "Where the coolant water passes through the orifices 203 of the lowermost stage portion 20 A, pressure loss is generated, and, due to this pressure loss, a great lifting force is applied to the entire assembly structure of the neutron reflector 20 .", "Most of this lifting force is generated when the cooling water passes through the orifices 203 of the lowermost stage portion 20 A, and the force applied to the remaining seven stage portions, that is, from the second stage portion 20 B to the top stage portion 20 C is relatively small.", "In view of this lifting force, the eight tie rods 22 are fastened to thereby press the neutron reflector 20 against the lower core plate 18 .", "However, in a conventional structure, in which the neutron reflector 20 is pressed against the lower core plate 18 by the eight tie rods 22 , when relaxation or loosening is generated in the tie rods 22 as a result of neutron irradiation, there is a possibility of the fastening force for pressing down the neutron reflector 20 against the lifting force falling short of the required level." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to a main aspect of the present invention, a neutron reflector bolt fastening structure is characterized by including: a neutron reflector composed of a plurality of divided stage portions and situated in a core vessel in a reactor vessel; a plurality of tie rods for fixing the neutron reflector to the core vessel; and a plurality of bolts for solely fixing the lowermost stage portion of the plurality of stage portions of the neutron reflector to the core vessel.", "According to another aspect of the present invention, a neutron reflector bolt fastening method is characterized by including: fixing a neutron reflector composed of a plurality of divided stage portions and situated in a core vessel in a reactor vessel to the core vessel by means of a plurality of tie rods; and fixing the lowermost stage portion of the plurality of stage portions of the neutron reflector solely to the core vessel by means of a plurality of bolts.", "In accordance with the present invention, the lowermost stage portion, to which most of the lifting force on the entire neutron reflector is applied, is exclusively secured to the core vessel by means of bolts other than the tie rods, whereby the initial fastening force for the lowermost stage portion of the neutron reflector becomes very large.", "Thus, even if relaxation is generated in the tie rods as a result of neutron irradiation, it is possible to press the neutron reflector firmly against the core vessel." ], [ "FIELD OF THE INVENTION The present invention relates to an in-pile structure in a reactor vessel of a nuclear power plant and, in particular, to a neutron reflector bolt fastening structure to be used when securing in position a neutron reflector within a core vessel.", "The present invention also relates to a fastening method for the fastening structure.", "BACKGROUND OF THE INVENTION FIG.", "3 is a schematic longitudinal sectional view showing the general construction of an ordinary nuclear reactor.", "In the drawing, a reactor vessel 10 is equipped with a core vessel 12 in which a fuel assembly 14 is supported.", "The fuel assembly 14 in the core vessel 12 is surrounded by an upper core plate 16 on the upper side, by a lower core plate 18 on the lower side, and by a neutron reflector 20 at the periphery.", "Next, the assembly structure of this neutron reflector will be described with reference to FIG.", "4.The neutron reflector 20 is formed by vertically stacking together eight substantially annular stage portions, which are fastened by eight tie rods 22 that are circumferentially arranged.", "The positioning when assembling each stage portion is effected by a positioning pin 23.The lowermost stage portion 20A of the neutron reflector 20 has at its periphery four flange portions 201 (only two of which are shown in the drawing); similarly, the top stage portion 20C has four flange portions 202 at its periphery.", "As shown in FIG.", "5, the tie rods 22 extend through the neutron reflector 20 from the top stage portion 20C to the lowermost stage portion 20A.", "The lowermost end portions of the tie rods 22 are passed through the lower core plate 18 and screwed into a flange portion 13 formed in the core vessel 12.To the upper end portion of each tie rod 22, there is mounted a nut 24 for pressing down the upper surface of the top stage portion 20C of the neutron reflector.", "By turning these nuts 24, the neutron reflector 20 is tightened between them and the lower core plate 18, and is secured to the flange portion 13 of the core vessel 12 through the intermediation of the lower core plate 18.The neutron reflector 20 has a large number of flow holes for cooling, through which cooling water flows.", "FIG.", "6 shows a structure of inlet portions of the flow holes 204 in the lowermost stage portion 20A of the neutron reflector 20.In the drawing, plugs 181 are provided in the lower core plate 18 mounted to the flange portion 13 of the core vessel 12.Cooling water flowing in through these plugs 181 passes orifices 203 provided in the lowermost stage portion 20A of the neutron reflector 20 and flows upwards through the flow holes 204.These flow holes extend through the 8-stage neutron reflector 20 from the lowermost stage portion 20A to the top stage portion 20C, so that the cooling water flowing in through the orifices 203 of the lowermost stage portion 20A rises through the flow holes 204 to flow out through the flow holes of the top stage portion 20C of the neutron reflector 20.Where the coolant water passes through the orifices 203 of the lowermost stage portion 20A, pressure loss is generated, and, due to this pressure loss, a great lifting force is applied to the entire assembly structure of the neutron reflector 20.Most of this lifting force is generated when the cooling water passes through the orifices 203 of the lowermost stage portion 20A, and the force applied to the remaining seven stage portions, that is, from the second stage portion 20B to the top stage portion 20C is relatively small.", "In view of this lifting force, the eight tie rods 22 are fastened to thereby press the neutron reflector 20 against the lower core plate 18.However, in a conventional structure, in which the neutron reflector 20 is pressed against the lower core plate 18 by the eight tie rods 22, when relaxation or loosening is generated in the tie rods 22 as a result of neutron irradiation, there is a possibility of the fastening force for pressing down the neutron reflector 20 against the lifting force falling short of the required level.", "OBJECT OF THE INVENTION Thus, it is a principal object of the present invention to provide a neutron reflector bolt fastening structure capable of firmly pressing the neutron reflector against the flange portion of the core vessel through the intermediation of the lower core plate even if relaxation is generated in the tie rods as a result of neutron irradiation, as well as a fastening method for the structure.", "SUMMARY OF THE INVENTION According to a main aspect of the present invention, a neutron reflector bolt fastening structure is characterized by including: a neutron reflector composed of a plurality of divided stage portions and situated in a core vessel in a reactor vessel; a plurality of tie rods for fixing the neutron reflector to the core vessel; and a plurality of bolts for solely fixing the lowermost stage portion of the plurality of stage portions of the neutron reflector to the core vessel.", "According to another aspect of the present invention, a neutron reflector bolt fastening method is characterized by including: fixing a neutron reflector composed of a plurality of divided stage portions and situated in a core vessel in a reactor vessel to the core vessel by means of a plurality of tie rods; and fixing the lowermost stage portion of the plurality of stage portions of the neutron reflector solely to the core vessel by means of a plurality of bolts.", "In accordance with the present invention, the lowermost stage portion, to which most of the lifting force on the entire neutron reflector is applied, is exclusively secured to the core vessel by means of bolts other than the tie rods, whereby the initial fastening force for the lowermost stage portion of the neutron reflector becomes very large.", "Thus, even if relaxation is generated in the tie rods as a result of neutron irradiation, it is possible to press the neutron reflector firmly against the core vessel.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic diagram showing a neutron reflector fastening structure according to the present invention; FIG.", "2 is an end view taken along the line A-A of FIG.", "1; FIG.", "3 is a schematic longitudinal sectional view showing a general construction of an ordinary nuclear reactor; FIG.", "4 is a perspective view showing an assembly construction of a neutron reflector; FIG.", "5 is a schematic diagram showing a conventional fastening structure for a neutron reflector; and FIG.", "6 Is a schematic diagram showing a structure of flow hole inlet portions in a lowermost stage portion of a neutron reflector.", "DESCRIPTION OF THE PREFERRED EMBODIMENT Next, a preferred embodiment of the present invention will be described with reference to the accompanying drawings, in which the components which are the same as or equivalent to those of the conventional structure are indicated by the same reference numerals.", "FIG.", "1 is a schematic diagram showing a neutron reflector bolt fastening structure according to the present invention, and FIG.", "2 is an end view taken along the line A-A of FIG.", "1.In these drawings, the lowermost stage portion 20A of the neutron reflector 20 has flange portions 201, which has through-holes 205 for bolts 1.Further, the lower core plate 18 situated under the neutron reflector 20 also has through-holes 182 for the bolts 1.The flange portion 13 of the core vessel 12 supporting the neutron reflector 20 and the lower core plate 18 has screw holes 131 into which bolts 1 are to be fitted.", "By using the bolts 1, the lowermost stage portion 20A of the neutron reflector 20 is fastened to the flange portion 13 of the core vessel 12 through the intermediation of the lower core plate 18.In this embodiment, there are provided eight holes 205, 182, and 131, and eight bolts 1 for each flange portion 201 formed on the lowermost stage portion 20A.", "Namely, four flange portions 201 are provided (See FIG.", "4), so that the lowermost stage portion 20A of the neutron reflector 20 is fastened to the flange portion 13 by a total of 32 bolts.", "When fastening the neutron reflector by utilizing this neutron reflector bolt fastening structure, the entire 8-stage neutron reflector 20 is fastened to the core vessel 12 by the conventional eight tie rods 22 and, at the same time, the lowermost stage portion 20A of the neutron reflector 20 is solely fastened to the flange portion 13 of the core vessel 12 by means of a large number of bolts 1 according to the present invention.", "In this way, the lowermost stage portion 20A of the neutron reflector 20, to which most of the lifting force is applied, is solely fixed to the core vessel 12 by means of a plurality of bolts separate from and independent of the tie rods 22, whereby it Is possible to secure in position the lowermost stage portion 20A of the neutron reflector 20 with a much larger initial fastening force as compared with the force obtained from the eight tie rods 22.Thus, even if relaxation is generated in the tie rods 22 as a result of neutron irradiation, and the fastening force is reduced, it is possible to maintain a sufficient fastening force to cope with the lifting force since the initial fastening force of the large number of bolts 1 is sufficiently large.", "As a result, it is possible to keep the neutron reflector 20 in a state in which it is pressed against and fixed to the flange portion 13 of the core vessel 12.Regarding the remaining seven stage portions of she neutron reflector 20, the lifting force applied thereto is relatively small as compared with that applied to the lowermost stage portion 20A, so that the fastening with the conventional eight tie rods 22 suffices.", "Even if relaxation is generated in the tie rods and their axial force is weakened, it is possible to maintain the requisite fastening force.", "In the present invention, the bolts 1 are fastened where the neutron irradiation amount is small, so that it is possible to prevent a deterioration in fastening force due to relaxation, thus making it possible to reliably maintain the requisite fastening force.", "While in the above-described embodiment all the four flange portions 201 of the neutron reflector 20 are fastened to the flange portion 13 of the core vessel 12 through the intermediation of the lower core plate 18 by means of the bolts 1, it is also possible to fasten only two opposing flange portions 201.Further, while in the above-mentioned embodiment eight bolts 1 are used for each flange portion 201, the number of bolts for each flange portion may be more or less than eight as long as they provide a predetermined initial fastening force." ] ]

[ [ "Single molecule sequencing method", "The invention relates to a method for single molecule sequencing of nucleic acids and to a device suitable for carrying out said method." ], [ "1-36.", "(canceled) 37.A method for sequencing nucleic acids, comprising: (a) providing a support particle on which a nucleic acid molecule has been immobilized, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (b) introducing said support particle into a sequencing device comprising a micro channel, (c) arresting said support particle in said sequencing device, (d) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, (e) passing the removed nucleotide building blocks through a microchannel by means of a hydrodynamic flow and (f) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks.", "38.The method as claimed in claim 37, wherein said support particle is made of a material selected form the group consisting of plastic, glass, quartz, metal, semimetal, metal oxides and a composite material.", "39.The method as claimed in claim 37, wherein the diameter of the support particle is from 0.5 to 10 μm.", "40.The method as claimed in claim 37, wherein the nucleic acid molecule is immobilized on the support particle via it 5′-terminus by means of bioaffinity interactions.", "41.The method as claimed in claim 40, wherein a 5′-biotinylated nucleic acid molecule is immobilized to an avidine- or streptavidine-coated support particle.", "42.The method as claimed in claim 37, wherein the nucleic acid molecule is immobilized in single-stranded form on the support particle.", "43.The method as claimed in claim 37, wherein the nucleic acid molecule molecule is immobilized in double-stranded form on the support particle, it being possible for labeled nucleotide building blocks to be removed by cleavage only from one single strand.", "44.The method as claimed in claim 37, wherein essentially all nucleotide building blocks of at least two base types carry a fluorescent label.", "45.The method as claimed in claim 37, wherein the support particle is arrested using a capturing laser.", "46.The method as claimed in claim 37, wherein the support particles are arrested in a microchannel.", "47.The method as claimed in claim 37, wherein individual nucleotide building blocks are removed by cleavage by an exonuclease.", "48.The method as claimed in claim 47, wherein T7 DNA polymerase, E. coli exonuclease I or E. coli exonuclease III is used.", "49.The method as claimed in claim 37, wherein the removed nucleotide building blocks are passed through a microchannel having a diameter of from 1 to 100 μm.", "50.The method as claimed in claim 37, wherein the removed nucleotide building blocks are passed through a microchannel with a velocity of from 1 to 50 mm/s.", "51.The method as claimed in claim 37, wherein the determination is carried out by means of confocal fluorescence measurement in a detection volume element.", "52.The method as claimed in claim 51, wherein the determination is carried out by means of confocal single molecule detection, such as, for example, fluorescence correlation spectroscopy.", "53.The method as claimed in claim 37, wherein the determination is carried out by means of a time-resolved decay measurement or time gating in a detection volume element.", "54.The method as claimed in claim 37, wherein nucleic acid molecules are determined in parallel in a plurality of microchannels.", "55.The method as claimed in claim 37, wherein a sequence with known base sequence is attached to the nucleic acid molecule to be sequenced.", "56.The method as claimed in claim 37, wherein the support particles are arrested essentially in the center of the microchannel and the removed nucleotide building blocks are directed in a laminar flow to a detection volume element which has been positioned in the center of the channel.", "57.The method as claimed in claim 56, wherein the detection volume element is kept as small as possible in order to only just detect all removed nucleotide building blocks.", "58.A device for sequencing an analyte in a sample fluid, which comprises: (a) an optically transparent microchannel, (b) means for introducing a support particle on which a nucleic acid molecule has been immobilized into said microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) means for arresting said support particle at a predetermined position in said microchannel, (d) means for generating a hydrodynamic flow in said microchannel, (e) means for progressively removing cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, and (f) means for sequentially detecting the removed nucleotide building blocks.", "59.A method for sequencing nucleic acids, which comprises the following steps: (a) providing a support particle on which a nucleic acid molecule ahs been immobilized, with essentially all nucleotide building blocks in at least one strand of said nucleic acid molecule carrying a fluorescent label and with 2 fluorescent labels with different properties being used for the 4 bases.", "(b) introducing said support particle into a sequencing device comprising a microchannel, (c) arresting said support particle in said sequencing device.", "(d) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, (e) passing the removed nucleotide building blocks through a microchanel and (f) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks.", "60.The method as claimed in claim 59, wherein the different spectroscopic properties are selected from emission wavelength or/and lifetime.", "61.The method as claimed in claim 59, wherein step (a) comprises providing at least 2 support particles on which nucleic acid molecules have been immobilized which have at least partially overlapping sequences and in which the 2 fluorescent labels, in each case, have been assigned to different bases or/and base combinations.", "62.The method as claimed in claim 61, wherein a first support particle in each case a first fluorescent label is assigned to 2 bases, B1 and B2, and a second fluorescent label is assigned to B3 and B4, on a second support particle in each case a first fluorescent labile is assigned to 2 bases, B1 and B3, and a second fluorescent label is assigned to 2 bases, B2 and B4, and, where appropriate, on a third support particle in each case a first fluorescent label is assigned to 2 bases, B1 and B4, and a second fluorescent label is assigned to 2 bases, B2 and B3.63.The method as claimed in claim 61, wherein on a first support particle in each case a first fluorescent label is assigned to a base B1 and a second fluorescent label is assigned to 3 bases, B2, B3 and B4, on a second support particle in each case a first fluorescent label is assigned to a base B2 and a second fluorescent label is assigned to 3 bases, B1, B3 and B4, on a third support particle in each case a first fluorescent label is assigned to a base B3 and a second fluorescent label is assigned to 3 bases, B1, B2 and B4, and on a fourth support particle in each case a first fluorescent label is assigned to a base B4 and a second fluorescent label is assigned to 3 bases, B1, B2 and B3.64.The method as claimed in claim 61, wherein a first support particle a first fluorescent label is assigned to 2 bases and a second fluorescent label is assigned to the other 2 bases, and on a second support particle a first fluorescent label is assigned to one base and a second fluorescent label is assigned to the other three bases.", "65.A method for sorting particles in a microchannel, which comprises the following steps: (a) passing particles through a detection element in said microchannel, said detection element being adjusted in such a way that a capturing laser is activated if a predetermined parameter is present on the particle and that said capturing laser is not activated if said predetermined parameter is not present on said particle, (b) arresting a particle on which said predetermined parameter is present by said capturing laser in a measuring element, (c) interrupting the sorting process and (d) measuring the arrested particle.", "66.A device for sequencing nucleic acids, comprising: (a) an optically transparent microchannel, (b) means for introducing a support particle on which a nucleic acid molecule has been immobilized into said microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) means for arresting said support particle at a first predetermined position in said microchannel, (d) means for transporting said support particle to a second predetermined position of said mircochannel, (e) means for generating a flow in said microchannel, (f) means for progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at the second position, and (g) means for sequentially detecting the removed nucleotide building blocks.", "67.A device for sequencing nucleic acids, comprising: (a) a support comprising a system of microchannels which are in fluid communication with one another, (b) an opening for introducing a support particle having a nucleic acid molecule immobilized thereon into a microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) an opening for feeding a nucleic acid-degrading enzyme into a microchannel, (d) a plurality of openings for discharging fluid from said support, (e) an opening for feeding buffer into a microchannel, (f) means for generating a liquid stream in said microchannels, (g) means for capturing the support particle at a first predetermined position, (h) means for transporting a captured support particle to a second predetermined position, (i) means for progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at the second predetermined position, and (j) means for sequentially detecting the removed nucleotide building blocks.", "68.The device as claimed in claim 67, wherein the diameter of the microchannels leading to the discharge openings is larger, preferably at least 1.5 times larger, than the diameter of the microchannels leading to the feeding openings and: 69.The device as claimed in claim 66, wherein the means (f) for generating a hydrodynamic flow are provided in the support.", "70.The device as claimed in claim 66, wherein means for applying an electric field between the second predetermined position and the first predetermined position are provided.", "71.A method for sequencing nucleic acids, characterized in that a device as claimed in claim 66, is used.", "72.A method for sequencing nucleic acids, comprising: (a) providing a support particle on which a nucleic acid molecule has been immobilized, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (b) introducing said support particle into an opening of a support comprising a system of microchannels which are in fluid communication with one another, (c) arresting said support particle at a first predetermined position within said support, (d) transporting said support particle to a second predetermined position within said support, (e) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at said second predetermined position, (f) passing the removed nucleotide building blocks through a microchannel, and (g) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks.", "73.The method as claimed in claim 38, wherein the diameter of the support particle is from 0.5 to 10 μm.", "74.The method as claimed in claim 60, wherein step (a) comprises providing at least 2 support particles on which nucleic acid molecules have been immobilized which have at least partially overlapping sequences and in which the 2 fluorescent labels, in each case, have been assigned to different bases or/and base combinations." ], [ "The invention relates to a method for single molecule sequencing of nucleic acids and to a device suitable for carrying out said method.", "In order to sequence the human genome which consists of approx., 3×109 bases or the genome of other organisms and to determine and compare individual sequence variants, sequencing methods must be provided which, firstly, are rapid and, secondly, can be used routinely and at low cost.", "Although great efforts have been made in order to accelerate common sequencing methods, for example the enzymic chain termination method according to Sanger et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "USA 74 (1977) 5463), in particular by means of automation (Adams et al., Automated DNA Sequencing and Analysis (1994), New York, Academic Press), currently a maximum of only 2,000 bases per day can be determined using a sequencer.", "In recent years, novel approaches to overcoming the limitations of conventional sequencing methods have been developed, inter alia sequencing by scanning tunneling microscopy (Lindsay and Phillip, Gen. Anal.", "Tech.", "Appl.", "8 (1991), 8-13), by highly parallel capillary electrophoresis (Huang et al., Anal.", "Chem.", "64 (1992), 2149-2154; Kambara and Takahashi, Nature 361 (1993), 565-566), by oligonucleotide hybridization (Drmanac et al., Genomics 4 (1989), 114-128; Khrapko et al., FEBS Let.", "256 (1989), 118-122; Maskos and Southern, Nucleic Acids Res.", "20 (1992), 1675-1678 and 1679-1684) and by matrix-assisted laser desorption/ionization mass spectrometry (Hillenkamp et al., Anal, Chem.", "63 (1991), 1193A-1203A).", "Another approach is single molecule sequencing (Dörre et al., Bioimaging 5 (1997), 139-152) which comprises sequencing nucleic acids by progressive enzymic degradation of fluorescently labeled single-stranded DNA molecules and detecting the sequentially released monomeric molecules in a microstructure channel in which said monomeric molecules are directed electroosrnotically by pumping.", "This procedure has the advantage that in each case only a single molecule of the target nucleic acid is sufficient for carrying out a sequence determination.", "However, the method described in Dörre et al.", "has a disadvantage in that the sequentially released monomeric molecules can interact with the walls of said microstructures, and this may cause considerable problems during analysis.", "It was, therefore, the object of the present invention to provide a method for single molecule sequencing of nucleic acids which represents an improvement compared to the prior art and which, in particular, makes it possible to improve detection by avoiding wall effects.", "This object is achieved by a method for sequencing nucleic acids, which comprises the following steps: (a) providing a support particle on which a nucleic acid molecule has been immobilized, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (b) introducing said support particle into a sequencing device comprising a microchannel, (c) arresting said support particle in said sequencing device, (d) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, (e) passing the removed nucleotide building blocks through a microchannel by means of a hydrodynamic flow and (f) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks.", "The method of the invention is a sequencing method which comprises studying a single nucleic acid molecule immobilized on a support.", "The size of the support particle used for said method is such as to enable said support particle to move in microchannels and to be arrested at a desired position within a sequencing device The particle size is preferably in the range from 0.5 to 10 μm and particularly preferably from 1 to 3 μm.", "Examples of suitable materials of support particles are plastics such as polystyrene, glass, quartz, metals or semimetals such as silicon, metal oxides such as silicon dioxide or composite materials which contain several of the abovementioned components.", "Particular preference is given to using optically transparent support particles, for example made from plastics, or particles having a plastic core and a silicon dioxide shell.", "The nucleic acid molecules are immobilized on the support particle preferably via their 5′ ends.", "The nucleic acid molecules may be bound to the support via covalent or noncovalent interactions.", "For example, binding of the polynucleotides to the support can be mediated by high-affinity interactions between the partners of a specific binding pair, for example biotin/streptavidin or avidin, hapten/anti-hapten antibody, sugar/lectin, etc.", "Thus it is possible to couple biotinylated nucleic acid molecules to streptavidin-coated supports.", "As an alternative, it is possible to bind the nucleic acid molecules to the support by means of adsorption.", "Thus, nucleic acid molecules modified by incorporation of alkanethiol groups can bind to metallic supports, for example supports made from gold.", "Still another alternative is covalent immobilization which comprises the possibility of mediating polynucleotide binding via reactive silane groups on a silica surface.", "The method of the invention uses support particles to which only one single nucleic acid molecule is bound.", "Support particles of this kind may be generated by contacting the nucleic acid molecules intended for sequencing with the support particles in a molar ratio of preferably 1:5 to 1:20, for example 1:10, under conditions under which the nucleic acid molecules are immobilized on the support.", "The resulting support particles are then sorted, for example on the basis of the fluorescent labeling groups present on the nucleic acid molecules, and removed from particles to which no nucleic acid molecule has bound.", "Said sorting and removal may be carried out, for example, according to the methods described in Holm et al.", "(Analytical Methods and Instrumentation, Special Issue μTAS 96, 85-87), Eigen and Rigler (Proc.", "Natl.", "Acad.", "Sci.", "USA 91 (1994), 5740-5747) or Rigler (J. Biotech.", "41 (1995), 177-186), which involve detection by means of a confocal microscope.", "The support-bound nucleic acid molecules, for example DNA molecules or RNA molecules, may be present in single-stranded form or double-stranded form.", "In the case of double-stranded molecules it must be ensured that labeled nucleotide building blocks can be removed by cleavage only from one single strand.", "In the nucleic acid strands to be sequenced, essentially all nucleotide building blocks, for example at least 90%, preferably at least 95%, of all nucleotide building blocks, of at least one base type carry a fluorescent labeling group.", "Preferably, essentially all nucleotide building blocks of at least two base types, for example two, three or four base types, carry a fluorescent label, each base type advantageously carrying a different fluorescent labeling group.", "Nucleic acids labeled in this way may be generated by enzymic primer extension on a nucleic acid template, using a suitable polymerase, for example a DNA polymerase such as, for example, a DNA polymerase from Thermococcus gorgonarius or from other thermostable organisms (Hopfner et al., PNAS USA 96 (1999), 3600-3605) or a mutated Taq polymerase (Patel and Loeb, PNAS USA 97 (2000), 5095-510) and using fluorescently labeled nucleotide building blocks.", "The labeled nucleic acid strands may also be prepared by amplification reactions, for example PCR.", "Thus, an asymmetric PCR produces amplification products in which only one single strand contains fluorescent labels.", "Such asymmetric amplification products may be sequenced in double-stranded form.", "Symmetric PCR produces nucleic acid fragments in which both strands are fluorescently labeled.", "These two fluorescently labeled strands may be separated and immobilized separately in single-stranded form on support particles so that it is possible to determine the sequence of one or both complementary strands separately.", "As an alternative, any of the two strands may be modified on the 3′ end in such a way, for example by incorporating a PNA link, that removing monomeric building blocks by cleavage is no longer possible.", "In this case, double-strand sequencing is possible.", "It is possible, where appropriate, to attach also a “sequence identifier”, i.e.", "a labeled nucleic acid of known sequence, to the nucleic acid strand to be studied, for example via enzymic reaction using ligase or/and terminal transferase, so that at the start of sequencing initially a known fluorescence pattern is obtained and only thereafter the fluorescence pattern corresponding to the unknown sequence to be studied is obtained.", "The nucleic acid template whose sequence is to be determined may be selected, for example, from DNA templates such as genomic DNA fragments, cDNA molecules, plasmids, etc., or else from RNA templates such as mRNA molecules.", "The fluorescent labeling groups may be selected from known fluorescent labeling groups used for labeling biopolymers, for example nucleic acids, such as, for example, fluorescein, rhodamine, phycoerythrin, Cy3, Cy5 or derivatives therefrom, etc.", "Step (b) comprises introducing a loaded support particle into a sequencing device containing a microchannel.", "The support particle can be arrested in a capillary or a microchannel with the aid of a capturing laser, for example an IR laser, according to method step Cc).", "Methods of this kind are described, for example, in Ashkin et al.", "(Nature 330 (1987), 24-31) and Chu (Science 253 (1991), 861-866).", "Preferably, the support particle is arrested using an automated process.", "For this purpose, the support particles are passed through the microchannel in a hydrodynamic flow, passing a detection element in the process.", "The detector in the detection window is adjusted so as to recognize a labeled sphere owing to the fluorescently labeled DNA located thereon and/or an additional fluorescently labeled probe and, as a result, to activate automatically the capturing laser in the measuring space.", "Support particles which have not been classified as positive by the detector can pass through.", "After capturing a support particle, the sorting process is stopped and the remaining support particles are removed by washing.", "This is followed by carrying out the sequencing reaction on the immobilized support particle.", "The sequencing reaction of the method of the invention comprises progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecules.", "An enzymic cleavage is preferably carried out using an exonuclease, and it is possible to use single-strand and double-strand exonucleases degrading either in the 5′→3′ direction or in the 3′→5′ direction, depending on the type of immobilization of the nucleic acid strands on the support.", "Exonucleases which are particularly preferably used are T7 DNA polymerase, E. coli exonuclease I and E. coli exonuclease III.", "The invention is based on passing the nucleotide building blocks released by the cleavage reaction through a microchannel by means of a hydrodynamic flow and determining them during their flow through said microchannel.", "The hydrodynamic flow makes it possible to increase the flow rate, and this in turn increases the probability of detection of a nucleotide building block.", "Furthermore, the hydrodynamic flow which is generated, for example, by suction action or by applying pressure can reduce the occurrence of wall effects, compared to electroosmotic pumping known in the prior art.", "The hydrodynamic flow through the microchannel preferably has a parabolic flow profile, i.e.", "the flow rate is highest in the center of the microchannel and then decreases with a parabolic function toward the edges down to a minimum rate.", "The flow rate through the microchannel, at maximum, is preferably in the range from 1 to 50 mm/s, particularly preferably in the range from 5 to 10 mm/s.", "The microchannel diameter is preferably in the range from 1 to 100 μm, particularly preferably from 10 to 50 μm.", "Preference is given to carrying out the measurement in a linear microchannel whose diameter is essentially constant.", "Fluorescently labeled nucleotide building blocks can be identified according to step (e) of the method of the invention by means of any method of measurement, for example using space- or/and time-resolved fluorescence spectroscopy, which is capable of recording fluorescence signals down to single-photon counting in a very small volume element, such as one given in a microchannel.", "It is possible, for example, to carry out detection by means of confocal single molecule detection, for example by fluorescence correlation spectroscopy, wherein a very small, preferably confocal, volume element, for example 0.1×10−15 to 20×10−12 l of the sample fluid flowing through the microchannel is subjected to excitation light from a laser, which causes the receptors contained in said measuring volume to emit fluorescence light, and the fluorescence light emitted from the measuring volume is measured by means of a photodetector, followed by correlating the time-dependent change in the emission measured and the relative flow rate of the molecules involved so that it is possible, at an appropriately high dilution, to identify individual molecules in said measuring volume.", "For details of carrying out the method and of the devices used for detection, reference is made to the disclosure of European patent 0 679 251.Confocal single molecule determination is also described in Rigler and Mets (Soc.Photo-Opt.Instrum.Eng.", "1921 (1993), 239 ff.)", "and Mets and Rigler (J. Fluoresc, 4 (1994) 259-264).", "Alternatively or additionally, detection may also be carried out by time-resolved decay measurement, so-called time gating, as described, for example, by Rigler et al., “Picosecond Single Photon Fluorescence Spectroscopy of Nucleic Acids”, in: “Ultrafast Phenomenenes”, D. H. Auston, Ed., Springer 1984.Here the fluorescence molecules are excited in a measuring volume, followed by, preferably after an interval of ≧100 ps, opening a detection interval on the photodetector.", "In this way, it is possible to keep background signals generated by Raman effects sufficiently low in order to enable essentially interference-free detection.", "In a particularly preferred embodiment, detection is carried out using a laser device which has a deflecting element or a phase-modulating element in the laser beam path, which, where appropriate in combination with one or more optical imaging elements, has been fitted for the purpose of generating from the laser beam a deflection pattern in the form of a linear or two-dimensional array of focal regions in the microchannel, the optical arrangement being fitted for the purpose, of projecting confocally each focal region for fluorescence detection by the photodetector arrangement.", "In a further preferred embodiment, the detection device is integrated into two walls facing one another and forming the microchannel, with one wall having an array of laser elements emitting into said microchannel as fluorescence excitation light source and the other one having an array of photodetector elements, in each case assigned to the opposite laser elements, as fluorescence light detectors.", "DE 100 23 423.2 discloses these two embodiments in detail.", "An increase in the probability of detection of nucleotide building blocks, substantial to the invention, and thus an improvement in sensitivity is achieved by the hydrodynamic flow profile in the microchannel of the sequencing device.", "The hydrodynamic flow in the microchannel can be adjusted and regulated via suitable electronic controlling equipment.", "In addition to the hydrodynamic flow in the microchannel, electrophoretic and electroosmotic methods for transporting reagents may also be employed in the sequencing device.", "The method of the invention also allows parallel sequencing of a plurality of support particle-bound nucleic acid molecules in in each case different microchannels which are preferably arranged in parallel.", "In a particularly preferred embodiment, the nucleic acid coupled to a support particle is essentially arrested in the center of the microchannel, the fluorescently labeled nucleotides removed by cleavage are directed in a laminar flow to a detection volume element downstream which is essentially positioned in the channel center which is the place with the highest flow rate.", "Preferably, the flow rate here is so high that the nucleotide arrives and is registered in the detector field, despite thermal broadening of the flow trajectories by Brownian diffusion.", "In this connection, the detector field is kept as small as possible so as to detect the nucleotide bases completely, while there is only a smallest possible fraction of background contamination (detector cross section to channel cross section ratio) in the detector.", "The invention further relates to a device for sequencing an analyte in a sample fluid, comprising: (a) an optically transparent microchannel, (b) means for introducing a support particle on which a nucleic acid molecule has been immobilized into said microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) means for arresting said support particle at a predetermined position in said microchannel, (d) means for generating a hydrodynamic flow in said microchannel, (e) means for progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, and (f) means for sequentially detecting the removed nucleotide building blocks.", "The device furthermore preferably comprises automatic manipulation devices, heating or cooling equipment such as Peltier elements, means for sorting support particles, reservoirs and, where appropriate, supply lines for sample fluid and reagents and also electronic apparatuses for analysis.", "The device is particularly suitable for carrying out the method of the invention.", "Still a further embodiment of the invention relates to a method for single molecule sequencing using only two fluorescent labels.", "Thus the invention relates to a method for sequencing nucleic acids, which comprises the following steps: (a) providing a support particle on which a nucleic acid molecule has been immobilized, with essentially all nucleotide building blocks in at least one strand of said nucleic acid molecule carrying a fluorescent label and with 2 fluorescent labels with different properties being used for the 4 bases, (b) introducing said support particle into a sequencing device comprising a microchannel, (c) arresting said support particle in said sequencing device, (d) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule, (e) passing the removed nucleotide building blocks through a microchannel and (f) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks.", "In a preferred embodiment, at least two support particles having nucleic acid molecules immobilized thereon which have at least partially overlapping sequences and in which the two fluorescent labels used for the two-color sequencing are in each case assigned to different bases or/and base combinations.", "In a first variant, it is possible to assign on a support particle in each case a first fluorescent label to two bases B1 and B2 and a second fluorescent label to two bases B3 and B4, on a second support particle to assign in each case a first fluorescent label to two bases B, and B3 and a second fluorescent label to two bases B2 and B4, and, where appropriate, on a third support particle to assign in each case a first fluorescent label to two bases B1 and B4 and a second fluorescent label to two bases B2 and B3.B1, B2, B3 and B4 represent the four different bases present in the nucleic acid to be sequenced, i.e.", "usually A, G, C and T. In a further variant, on a first support particle in each case a first fluorescent label is assigned to a base B1 and a second fluorescent label to three bases B2, B3 and B4, on a second support particle in each case a first fluorescent label is assigned to a base B2 and a second fluorescent label to three bases B1, B3 and B4, on a third support particle in each case a first fluorescent label is assigned to a base B3 and a second fluorescent label to three bases B1, B2 and B4, and on a fourth support particle in each case a first fluorescent label is assigned to a base B4 and a second fluorescent label to three bases B1, B2 and B3.In yet another variant, on a first support particle a first fluorescent label is assigned to two bases and a second fluorescent label to the other two bases, and on a second support particle a first fluorescent label is assigned to one base and a second fluorescent label to the other three bases.", "In this embodiment, it is possible, where appropriate, to use still further support particles having different 2/2 or/and 1/3 combinations.", "The base sequence of a DNA sequence may also be completely determined by using only 2 fluorescent labeling groups with different spectroscopic properties such as, for example, emission wavelength or/and lifetime of the excited state.", "For this purpose, for example, two nucleotide bases can be provided with a first labeling group and the other two nucleotide bases with a second labeling group.", "Parallelly, the nucleic acid to be sequenced is labelled, in a parallel reaction, in such a way that other base combinations are provided with in each case the same labeling group.", "An example of this embodiment is listed below: (1) AT green, CG red: ACGACATGCAATTGGGCAAAT TGCTGTACGTTAACCCGTTTA (2) AC green, TG red: ACGACATGCAATTGGGCAAAT CATCACGTACCGGTTTACCCG (3) AG green, TC red: ACGACATGCAATTGGGCAAAT GTAGTGCATGGCCAAATGGGC A sequence can be obtained if AT, AC or AG are present in one color and CG, GT or CT are present in a different color (see combinations 1, 2 and 3).", "In order to obtain the complete base sequence, it is sufficient to sequence the color combinations 1 and 2, 2 and 3 or 1 and 3.A combination of 1, 2 and 3 is not absolutely needed.", "However, such a combination of 3 mixtures is sometimes convenient in order to reduce the probability of errors.", "In the case of sequencing with two colors, there exist 22×22=16 possibilities for each color combination.", "As discussed above, it is possible to determine a complete sequence by using two dual base/fluorescent label combinations.", "However, for particular types of sequences, for example for the determination of mutations, a single dual combination may be sufficient.", "Furthermore, it is possible, where appropriate, to use only a single base of the first color and the other three bases in a different color.", "In this case, four mixtures must be sequenced in order to obtain the complete sequence.", "A combination of the dual color combinations (e.g.", "two green and two red bases) with a single color combination (e.g.", "one green base and 3 red bases) is also interesting for particular embodiments.", "The invention furthermore relates to a method for sorting particles in a microchannel, which comprises the following steps: (a) passing particles through a detection element in said microchannel, said detection element being adjusted in such a way that a capturing laser, for example an IR laser, is activated if a predetermined parameter, for example a fluorescent label, is present on the particle and that said capturing laser is not activated if said predetermined parameter is not present on said particle, (b) arresting a particle on which said predetermined parameter is present by said capturing laser in a measuring element, (c) interrupting the sorting process and (d) measuring the arrested particle.", "The detection or/and measuring element are preferably confocal volume elements, the detection element being arranged in the microchannel upstream with respect to the measuring element.", "Measuring the arrested particle may comprise, for example, a sequencing as described above.", "Furthermore, the invention is to be illustrated by the following figures in which: FIG.", "1 depicts a section of a device for carrying out the method of the invention.", "In a microchannel (2) a support particle (4) is arrested by means of a capturing laser (6).", "On the support particle (4) a nucleic acid molecule (8) is immobilized from which individual nucleotide building blocks (10) are sequentially removed by enzymic digest and are transported by a hydrodynamic flow through the microchannel to a detection element (12), preferably a confocal detection element, and are detected there.", "The fluid flowing through the microchannel contains the enzyme used for digesting the immobilized nucleic acid molecule, preferably an exonuclease.", "The flow rate through the microchannel is adjusted such that Brownian motion-caused broadening of the migration path of the nucleotide building blocks removed by cleavage is so low that said building blocks can be detected with sufficient statistical probability in the detection volume (12).", "FIG.", "2 depicts a larger section of the device of the invention, comprising the section (20a) of the microchannel (20), depicted in FIG.", "1, with the capturing laser and the confocal detection element (not shown here).", "The device furthermore contains an inlet (22) and an outlet (28) for liquids, for example solvent, between which the hydrodynamic flow in the microchannel (20) is generated by applying pressure or by suction action.", "Furthermore, the device contains an opening for supplying support particles (24) and an opening for supplying enzyme (26).", "Enzyme and support particles may, where appropriate, be introduced by electroosmotic flow, with a negative electrode being applied at (24) and (26) and a positive electrode being applied at (28).", "Hydrodynamic flow through the microchannel (20) can be carried out by electronically controlled pumps which may be located outside the microstructure but may also be integrated therein.", "The method is carried out by passing support particles through the opening (24) into the channel (20).", "A single support particle which is loaded with a nucleic acid molecule is arrested by the capturing laser.", "Other particles and contaminations are removed by washing.", "This is followed by adding enzyme through the opening (26) and carrying out the sequencing reaction.", "After sequencing has finished, the support particle is washed out of the microchannel.", "Thereafter, another sequencing cycle using a new microparticle can be carried out.", "This procedure may be automated using appropriate electronic controllers.", "Furthermore, the device may contain a plurality of microchannels for parallel sequencing of a plurality of support particles.", "FIG.", "3 depicts a preferred embodiment of an inventive device for single molecule sequencing.", "This device comprises a support with at least 6 openings for microfluidic channels.", "The opening (2) serves to supply the sample or the microparticles contained therein and, where appropriate, a buffer.", "The opening (4) serves to supply exonuclease.", "The opening (6) is provided for discharging used solution from the support.", "The openings (8) and (12) are likewise provided for discharging used solutions from the support.", "The opening (10) serves to supply buffer.", "Where appropriate, the device may contain still further openings for supplying or discharging liquids.", "The diameter of the microfluidic channels in the support is, in the case of the channels provided for supplying, preferably in the range from 40-80 μm, in particular approx.", "50 μm.", "The discharging channels may have a considerably larger diameter, for example of up to 500 μm.", "The diameter may become wider immediately after the point of intersection of the channels and is intended to improve the control and to increase stability within the system.", "The microparticle introduced into the device through the opening (2) is first captured in the region of position (20), for example by an IR laser, while the transport fluid is discharged from the support through opening (8).", "The captured microparticle is then transported within the support to position 22, for example by a liquid stream or/and by an IR laser, where it is again arrested, for example by an IR laser, and then subjected to enzymic degradation.", "For this purpose, enzyme is passed through the opening (4) to position (22) with the arrested microparticle and then transported out of the device again through the opening (12).", "The nucleotides released at position (22) by enzymic degradation of the nucleic acid on the microparticle are detected downstream at position (24).", "After finishing the measurement, buffer may be introduced into the device through the opening (10) and discharged again through the openings (8) or/and (12).", "In this way, the enzyme present in the device is washed out through opening (12), preventing, at the same time, a new microparticle introduced into the device, for example at position (20) from contacting the enzyme prematurely.", "Where appropriate, an electric field may be applied in the channels, for example via a positive electrode in the region of position (24) and a negative electrode in the region of opening (2).", "In this way it is possible to stretch the DNA to be sequenced which is immobilized on the microparticle and thus make it more accessible to enzymic treatment.", "Reservoirs (not shown) are provided for the liquids to be introduced into the device and for the liquids discharged from the device.", "Thus the invention further relates to a device for sequencing nucleic acids, comprising: (a) an optically transparent microchannel, (b) means for introducing a support particle on which a nucleic acid molecule has been immobilized into said microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) means for arresting said support particle at a first predetermined position in said microchannel, (d) means for transporting said support particle to a second predetermined position of said microchannel, (e) means for generating a flow in said microchannel, (f) means for progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at the second predetermined position, and (g) means for sequentially detecting the removed nucleotide building blocks.", "In a preferred embodiment, this device comprises; (a) a support comprising a system of microchannels which are in fluid communication with one another, (b) an opening (2) for introducing a support particle having a nucleic acid molecule immobilized thereon into a microchannel, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (c) an opening (4) for feeding a nucleic acid-degrading enzyme into a microchannel, (d) a plurality of openings (6, 8, 12) for discharging fluid from said support, (e) an opening (10) for feeding buffer into a microchannel, (f) means for generating a liquid stream in said microchannels, (g) means for capturing the support particle at a first predetermined position (22), (h) means for transporting a captured support particle to a second predetermined position (24), (i) means for progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at the second predetermined position (24), and (j) means for sequentially detecting the removed nucleotide building blocks.", "An essential feature of this device is the fact that the support has means for transporting the particle containing the nucleic acid to be sequenced from a first predetermined position, the capturing position, to a second predetermined position, the degradation position.", "In this way, sequential operation of the device, i.e.", "successive analysis of a plurality of particles is facilitated, in particular because a contact with the nucleic acid-degrading enzyme can be prevented more easily in the capturing position.", "In the device of the invention, microchannels in the direction of the discharge opening preferably have a diameter which is larger, preferably at least 1.5 times larger, than the diameter of microchannels in the direction of the supply openings.", "The flow within the device is preferably a hydrodynamic flow.", "Preference is furthermore given to providing means for supplying an electric field between the second predetermined position and the first predetermined position.", "Finally, the invention relates to a method for sequencing nucleic acids, using a device as described above.", "This method preferably comprises the following steps: (a) providing a support particle on which a nucleic acid molecule has been immobilized, with essentially all nucleotide building blocks of at least one base type in at least one strand of said nucleic acid molecule carrying a fluorescent label, (b) introducing said support particle into an opening (2) of a support comprising a system of microchannels which are in fluid communication with one another, (c) arresting said support particle at a first predetermined position (22) within said support, (d) transporting said support particle to a second predetermined position (24) within said support, (e) progressively removing by cleavage individual nucleotide building blocks from the immobilized nucleic acid molecule at said second predetermined position, (f) passing the removed nucleotide building blocks through a microchannel, and (g) determining the base sequence of said nucleic acid molecule based on the sequence of said removed nucleotide building blocks." ] ]

[ [ "Methods and materials relating to carcinoembryonic antigen-like (cea-like) polypeptides and polynucleotides", "The invention provides novel polynucleotides and polypeptides encoded by such polynucleoddes and mutants or variants thereof that correspond to a novel human secreted CEA-like polypeptide.", "These polynucleotides comprise nucleic acid sequences isolated from cDNA library from rectum (Hyseq clone identification numbers 15456780 (SEQ ID NO: 1)).", "Other aspects of the invention include vectors containing processes for producing novel human secreted CEA-like polypeptides, and antibodies specific for such polypeptides." ], [ "1.An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 2-3 and 5, the translated protein coding portion thereof, the mature protein coding portion thereof, the extracellular portion thereof, or the active domain thereof.", "2.An isolated polynucleotide encoding a polypeptide with biological activity, which polynucleotide hybridizes to the complement of a polynucleotide of claim 1 under stringent hybridization conditions.", "3.An isolated polynucleotide encoding a polypeptide with biological activity, said polynucleotide having greater than about 90% sequence identity with the polynucleotide of claim 1.4.The polynucleotide of claim 1 which is a DNA sequence.", "5.An isolated polynucleotide which comprises the complement of the polynucleotide of claim 1.6.A vector comprising the polynucleotide of claim 1.7.An expression vector comprising the polynucleotide of claim 1.8.A host cell genetically engineered to express the polynucleotide of claim 1.9.The host cell of claim 8 wherein the polynucleotide is in operative association with a regulatory sequence that controls expression of the polynucleotide in the host cell.", "10.An isolated polypeptide comprising an amino acid sequence which is at least 80% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 6-10, the translated protein coding portion thereof, the mature protein coding portion thereof, the extracellular portion thereof, or the active domain thereof.", "11.A composition comprising the polypeptide of claim 10 and a carrier.", "12.A polypeptide, having CEA-like activity, comprising at least ten consecutive amino acids from the polypeptide sequences selected from the group consisting of SEQ ID NO: 4 and 6-10.13.The polypeptide of claim 12, comprising at least five consecutive amino acids from the polypeptide sequences selected from the group consisting of SEQ ID NO.", "4 and 6-10.14.A polynucleotide encoding a polypeptide according to claim 12.15.A polynucleotide encoding a polypeptide according to claim 13.16.A polynucleotide encoding a polypeptide according to claim 10.17.An antibody specific for the polypeptide of claim 10.18.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.", "19.A method for detecting the polynucleotide of claim 1 in a sample, comprising: a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions; b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and c) detecting said product and thereby the polynucleotide of claim 1 in the sample.", "20.The method of claim 19, wherein the polynucleotide comprises an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.", "21.A method for detecting the polypeptide of claim 10 in a sample, comprising: a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.", "22.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10 under conditions and for a time sufficient to form a polypeptide/compound complex; and b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.", "23.A method for identifying a compound that binds to the polypeptide of claim 10, comprising: a) contacting the compound with the polypeptide of claim 10, in a cell, for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.", "24.A method of producing a CEA-like polypeptide, comprising, a) culturing the host cell of claim 8 under conditions sufficient to express the polypeptide in said cell; and b) isolating the polypeptide from the cell culture or cells of step (a).", "25.A kit comprising the polypeptide of claim 10.26.A nucleic acid array comprising the polynucleotide of claim 1 or a unique segment of the polynucleotide of claim 1 attached to a surface.", "27.The array of claim 26, wherein the array detects full-matches to the polynucleotide or a unique segment of the polynucleotide of claim 1.28.The array of claim 26, wherein the array detects mismatches to the polynucleotide or a unique segment of the polynucleotide of claim 1.29.A method of treatment of a subject in need of enhanced activity or expression of CEA-like polypeptide of claim 10 comprising administering to the subject a composition selected from the group consisting of: (a) a therapeutic amount of a agonist of said polypeptide; (b) a therapeutic amount of the polypeptide; and (c) a therapeutic amount of a polynucleotide encoding the polypeptide in a form and under conditions such that the polypeptide is produced, and a pharmaceutically acceptable carrier.", "30.A method of treatment of a subject having need to inhibit activity or expression of CEA-like polypeptide of claim 10 comprising administering to the subject a composition selected from the group consisting of: (a) a therapeutic amount of an antagonist to said polypeptide; (b) a therapeutic amount of a polynucleotide that inhibits the expression of the nucleotide sequence encoding said polypeptide; and (c) a therapeutic amount of a polypeptide that competes with the CEA-like polypeptide for its ligand and a pharmaceutically acceptable carrier." ], [ "<SOH> 2.BACKGROUND ART <EOH>Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.", "Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.", "It is to these polypeptides and the polynucleotides encoding them that the present invention is directed.", "In particular, this invention is directed to novel soluble CEA-like polypeptides and polynucleotides.", "Many tumors express genes whose products are required for either inducing or maintaining the malignant state (Abbas et al (2000) Cellular and Molecular Immunology, Saunders (Publishers) pp 386).", "These proteins may serve as markers for the tumor detection or even may provide therapeutic targets.", "Carcinoembryonic antigens (CEAs, e.g.", "CD66a-CD66d) were first described as antigens expressed on many carcinomas of colon, pancreas, stomach, and breast.", "However, it has become apparent with more sensitive detection techniques that these proteins are also expressed during inflammation and also, in minute quantities in normal tissues.", "Carcinoembryonic antigens are integral membrane glycoproteins belonging to immunoglobulin (Ig) superfamily of receptors.", "CEA cell adhesion molecule (CEA-CAM), also known as billiary glycoprotein (BGP) and CD66a, is a protein of about 85 kDa and, is highly glycosylated and exhibits at least two tissue specific, alternatively spliced, variants.", "Immunoglobulin superfamily members that serve as receptors can be classified into three groups according to their cytoplasmic domain characteristics.", "Transmembrane molecules with immunoreceptor tyrosine activation motifs (ITAMs) (YxxL where x is any amino acid) are usually activating receptors.", "Those possessing immunoreceptor tyrosine inhibition motifs (ITIMs) (I/L/VxxYxxL/V where x is any amino acid) are inhibitory in nature.", "There appears to be a third class of short transmembrane receptors like LIR-4, or alternately spliced soluble forms of FDF03 that have no known activating or inhibitory motifs.", "These molecules by their virtue of extracellular MHC binding domain could function as a “molecular sink” and could inhibit the functions of cognate transmembrane receptors.", "Several functions have been attributed to CEA.", "The first Ig domain of CD66a serves as an adhesive module to bind E-selectin and initiate the inflammatory cascade.", "The mouse CEAs have been shown to be the receptors for mouse hepatitis virus, whereas human CEA has been shown to be a receptor for bacterial proteins from Neisseria gonorrhoeae, Salmonella , and Escherichia coli .", "More recently, CEA has been shown to be a negative regulator, and therefore a tumor suppressor protein for colonic, prostate and breast carcinomas (Huber et al (1999) J. Biol.", "Chem.", "274, 335-344).", "CEA-CAM or CD66a has been implicated in transducing signals by its cytoplasmic domain.", "Several physiological events promote the phosphorylation of tyrosines in the cytoplasmic domain of CEA.", "It is also reported that stimulation of BGP1 in neutrophils leads to activation of Rac1, PAK, and Jun N-terminal kinase.", "Similarly, it is reported that the ITIM sequences in the BGP1 cytoplasmic domains interact with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells (Huber et al (1999) J. Biol.", "Chem.", "274, 335-344).", "CEA expression is greatly increased in colonic, pancreatic, gastric, and breast carcinomas resulting in a rise in serum levels.", "Furthermore, post-translational processing of CEA may be altered in tumor cells.", "Serum CEA is accordingly used to monitor the occurrence or recurrence of metastatic carcinoma after primary treatment.", "CEA functions as an intercellular adhesion molecule, promoting the binding of tumor cells to one another.", "Thus CEA may play a role in the way tumor cells interact with one another and with tissues in which they are growing.", "Thus, CEA appears to be involved in cell adhesion and subsequent signal transduction during normal fetal development and also during inflammation and carcinogenesis.", "Polynucleotides encoding CEA and polypeptides thereof could serve as potential therapeutics in the treatment of breast, prostate, colon and other cancers.", "CEA and compounds which bind to CEA could also be useful in treating disorders relating to inflammation and autoimmunity.", "Soluble CEA could also be used as immunosuppressant in organ transplant patients.", "Soluble CEA molecule could serve as a decoy receptor in above mentioned bacterial and viral infections." ], [ "<SOH> 3.SUMMARY OF THE INVENTION <EOH>This invention is based on the discovery of novel CEA-like polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.", "Specifically, the polynucleotides of the present invention are based on a CEA-like polynucleotide isolated from a cDNA library prepared from rectum (Hyseq clone identification numbers 15456780 (SEQ ID NO: 1).", "The compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.", "The compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in the SEQ ID NO: 1-3 or 5; or a fragment of SEQ ID NO: 1-3 or 5; a polynucleotide comprising the full length protein coding sequence of the SEQ ID NO: 1-3 or 5 (for example, SEQ ID NO: 4); and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of any of SEQ ID NO: 1-3 or 5.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-3 or 5; (b) a nucleotide sequence encoding any of SEQ ID NO: 4, 6-10; a polynucleotide which is an allelic variant of any polynucleotides recited above having at least 70% polynucleotide sequence identity to the polynucleotides; a polynucleotide which encodes a species homolog (e.g.", "orthologs) of any of the peptides recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide comprising SEQ ID NO: 4.A collection as used in this application can be a collection of only one polynucleotide.", "The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array.", "In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment.", "The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.", "The collection can also be provided in a computer-readable format.", "This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors.", "Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.", "Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.", "In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.", "Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.", "A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.", "The compositions of the present invention include polypeptides comprising, but not limited to, an isolated polypeptide selected from the group comprising the amino acid sequence of SEQ ID NO: 4, 6-10; or the corresponding full length or mature protein.", "Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: 1-3 or 5; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.", "Biologically or immunologically active variants of any of the protein sequences listed as SEQ ID NO: 4, 6-10 and substantial equivalents thereofthat retain biological or immunological activity are also contemplated.", "The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.", "host cells) of the invention.", "The invention also provides compositions comprising a polypeptide of the invention.", "Pharmaceutical compositions of the invention may comprise a polypeptide of the invention and an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.", "The invention also relates to methods for producing a polypeptide of the invention comprising culturing host cells comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein or peptide from the culture or from the host cells.", "Preferred embodiments include those in which the protein produced by such a process is a mature form of the protein.", "Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.", "These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like.", "For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.", "In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.", "The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.", "For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.", "Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.", "The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.", "Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier.", "In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as potential therapeutics in the treatment of breast, prostate, colon and other cancers.", "CEA-like polypeptide could also be useful in treating disorders relating to inflammation and autoimmunity.", "CEA-like polypeptide may also be used as immunosuppressant in organ transplant patients.", "Also, a soluble CEA-like molecule could serve as a decoy receptor in certain bacterial and viral infections.", "The methods of the invention also provides methods for the treatment of disorders as recited herein which comprise the administration of a therapeutically effective amount of a composition comprising a polynucleotide or polypeptide of the invention and a pharmaceutically acceptable carrier to a mammalian subject exhibiting symptoms or tendencies related to disorders as recited herein.", "In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering a composition comprising compounds and other substances that modulate the overall activity of the target gene products and a pharmaceutically acceptable carrier.", "Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.", "Specifically, methods are provided for preventing, treating or ameliorating a medical condition, including viral diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) CEA-like polypeptides of the invention.", "The mechanics of the particular condition or pathology will dictate whether the polypeptides of the invention or binding partners (or inhibitors) of these would be beneficial to the individual in need of treatment.", "According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of cellular function.", "A polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies.", "Conversely, protein or other active ingredients of the present invention may be included in formulations of a particular agent to minimize side effects of such an agent.", "The invention further provides methods for manufacturing medicaments useful in the above-described methods.", "The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample).", "Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.", "The invention provides a method for detecting a polypeptide of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting formation of the complex, so that if a complex is formed, the polypeptide is detected.", "The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.", "Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.", "The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.", "Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.", "Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.", "The invention provides a method for identifying a compound that binds to the polypeptide of the present invention comprising contacting the compound with the polypeptide under conditions and for a time sufficient to form a polypeptide/compound complex and detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.", "Also provided is a method for identifying a compound that binds to the polypeptide comprising contacting the compound with the polypeptide in a cell for a time sufficient to form a polypeptide/compound complex wherein the complex drives expression of a reporter gene sequence in the cell and detecting the complex by detecting reporter gene sequence expression so that if the polypeptide/compound complex is detected a compound that binds to the polypeptide is identified." ], [ "1.TECHNICAL FIELD The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.", "In particular, the invention relates to a novel carcinoembryonic antigen-like (CEA-like) polypeptide.", "2.BACKGROUND ART Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.", "Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.", "It is to these polypeptides and the polynucleotides encoding them that the present invention is directed.", "In particular, this invention is directed to novel soluble CEA-like polypeptides and polynucleotides.", "Many tumors express genes whose products are required for either inducing or maintaining the malignant state (Abbas et al (2000) Cellular and Molecular Immunology, Saunders (Publishers) pp 386).", "These proteins may serve as markers for the tumor detection or even may provide therapeutic targets.", "Carcinoembryonic antigens (CEAs, e.g.", "CD66a-CD66d) were first described as antigens expressed on many carcinomas of colon, pancreas, stomach, and breast.", "However, it has become apparent with more sensitive detection techniques that these proteins are also expressed during inflammation and also, in minute quantities in normal tissues.", "Carcinoembryonic antigens are integral membrane glycoproteins belonging to immunoglobulin (Ig) superfamily of receptors.", "CEA cell adhesion molecule (CEA-CAM), also known as billiary glycoprotein (BGP) and CD66a, is a protein of about 85 kDa and, is highly glycosylated and exhibits at least two tissue specific, alternatively spliced, variants.", "Immunoglobulin superfamily members that serve as receptors can be classified into three groups according to their cytoplasmic domain characteristics.", "Transmembrane molecules with immunoreceptor tyrosine activation motifs (ITAMs) (YxxL where x is any amino acid) are usually activating receptors.", "Those possessing immunoreceptor tyrosine inhibition motifs (ITIMs) (I/L/VxxYxxL/V where x is any amino acid) are inhibitory in nature.", "There appears to be a third class of short transmembrane receptors like LIR-4, or alternately spliced soluble forms of FDF03 that have no known activating or inhibitory motifs.", "These molecules by their virtue of extracellular MHC binding domain could function as a “molecular sink” and could inhibit the functions of cognate transmembrane receptors.", "Several functions have been attributed to CEA.", "The first Ig domain of CD66a serves as an adhesive module to bind E-selectin and initiate the inflammatory cascade.", "The mouse CEAs have been shown to be the receptors for mouse hepatitis virus, whereas human CEA has been shown to be a receptor for bacterial proteins from Neisseria gonorrhoeae, Salmonella, and Escherichia coli.", "More recently, CEA has been shown to be a negative regulator, and therefore a tumor suppressor protein for colonic, prostate and breast carcinomas (Huber et al (1999) J. Biol.", "Chem.", "274, 335-344).", "CEA-CAM or CD66a has been implicated in transducing signals by its cytoplasmic domain.", "Several physiological events promote the phosphorylation of tyrosines in the cytoplasmic domain of CEA.", "It is also reported that stimulation of BGP1 in neutrophils leads to activation of Rac1, PAK, and Jun N-terminal kinase.", "Similarly, it is reported that the ITIM sequences in the BGP1 cytoplasmic domains interact with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells (Huber et al (1999) J. Biol.", "Chem.", "274, 335-344).", "CEA expression is greatly increased in colonic, pancreatic, gastric, and breast carcinomas resulting in a rise in serum levels.", "Furthermore, post-translational processing of CEA may be altered in tumor cells.", "Serum CEA is accordingly used to monitor the occurrence or recurrence of metastatic carcinoma after primary treatment.", "CEA functions as an intercellular adhesion molecule, promoting the binding of tumor cells to one another.", "Thus CEA may play a role in the way tumor cells interact with one another and with tissues in which they are growing.", "Thus, CEA appears to be involved in cell adhesion and subsequent signal transduction during normal fetal development and also during inflammation and carcinogenesis.", "Polynucleotides encoding CEA and polypeptides thereof could serve as potential therapeutics in the treatment of breast, prostate, colon and other cancers.", "CEA and compounds which bind to CEA could also be useful in treating disorders relating to inflammation and autoimmunity.", "Soluble CEA could also be used as immunosuppressant in organ transplant patients.", "Soluble CEA molecule could serve as a decoy receptor in above mentioned bacterial and viral infections.", "3.SUMMARY OF THE INVENTION This invention is based on the discovery of novel CEA-like polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.", "Specifically, the polynucleotides of the present invention are based on a CEA-like polynucleotide isolated from a cDNA library prepared from rectum (Hyseq clone identification numbers 15456780 (SEQ ID NO: 1).", "The compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.", "The compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in the SEQ ID NO: 1-3 or 5; or a fragment of SEQ ID NO: 1-3 or 5; a polynucleotide comprising the full length protein coding sequence of the SEQ ID NO: 1-3 or 5 (for example, SEQ ID NO: 4); and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of any of SEQ ID NO: 1-3 or 5.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-3 or 5; (b) a nucleotide sequence encoding any of SEQ ID NO: 4, 6-10; a polynucleotide which is an allelic variant of any polynucleotides recited above having at least 70% polynucleotide sequence identity to the polynucleotides; a polynucleotide which encodes a species homolog (e.g.", "orthologs) of any of the peptides recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide comprising SEQ ID NO: 4.A collection as used in this application can be a collection of only one polynucleotide.", "The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array.", "In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment.", "The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment.", "The collection can also be provided in a computer-readable format.", "This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors.", "Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.", "Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.", "In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.", "Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.", "A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.", "The compositions of the present invention include polypeptides comprising, but not limited to, an isolated polypeptide selected from the group comprising the amino acid sequence of SEQ ID NO: 4, 6-10; or the corresponding full length or mature protein.", "Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: 1-3 or 5; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions.", "Biologically or immunologically active variants of any of the protein sequences listed as SEQ ID NO: 4, 6-10 and substantial equivalents thereofthat retain biological or immunological activity are also contemplated.", "The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g.", "host cells) of the invention.", "The invention also provides compositions comprising a polypeptide of the invention.", "Pharmaceutical compositions of the invention may comprise a polypeptide of the invention and an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.", "The invention also relates to methods for producing a polypeptide of the invention comprising culturing host cells comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein or peptide from the culture or from the host cells.", "Preferred embodiments include those in which the protein produced by such a process is a mature form of the protein.", "Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology.", "These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like.", "For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.", "In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.", "The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins.", "For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide.", "Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue.", "The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.", "Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier.", "In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, as potential therapeutics in the treatment of breast, prostate, colon and other cancers.", "CEA-like polypeptide could also be useful in treating disorders relating to inflammation and autoimmunity.", "CEA-like polypeptide may also be used as immunosuppressant in organ transplant patients.", "Also, a soluble CEA-like molecule could serve as a decoy receptor in certain bacterial and viral infections.", "The methods of the invention also provides methods for the treatment of disorders as recited herein which comprise the administration of a therapeutically effective amount of a composition comprising a polynucleotide or polypeptide of the invention and a pharmaceutically acceptable carrier to a mammalian subject exhibiting symptoms or tendencies related to disorders as recited herein.", "In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering a composition comprising compounds and other substances that modulate the overall activity of the target gene products and a pharmaceutically acceptable carrier.", "Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.", "Specifically, methods are provided for preventing, treating or ameliorating a medical condition, including viral diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) CEA-like polypeptides of the invention.", "The mechanics of the particular condition or pathology will dictate whether the polypeptides of the invention or binding partners (or inhibitors) of these would be beneficial to the individual in need of treatment.", "According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of cellular function.", "A polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies.", "Conversely, protein or other active ingredients of the present invention may be included in formulations of a particular agent to minimize side effects of such an agent.", "The invention further provides methods for manufacturing medicaments useful in the above-described methods.", "The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample).", "Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.", "The invention provides a method for detecting a polypeptide of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting formation of the complex, so that if a complex is formed, the polypeptide is detected.", "The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention.", "Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.", "The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention.", "Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein.", "Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.", "The invention provides a method for identifying a compound that binds to the polypeptide of the present invention comprising contacting the compound with the polypeptide under conditions and for a time sufficient to form a polypeptide/compound complex and detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.", "Also provided is a method for identifying a compound that binds to the polypeptide comprising contacting the compound with the polypeptide in a cell for a time sufficient to form a polypeptide/compound complex wherein the complex drives expression of a reporter gene sequence in the cell and detecting the complex by detecting reporter gene sequence expression so that if the polypeptide/compound complex is detected a compound that binds to the polypeptide is identified.", "4.BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1A shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that in this region the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.", "Gaps are presented as dashes.", "FIG.", "1B shows a second BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that in this region the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid.", "F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.", "Gaps are presented as dashes.", "FIG.", "2A shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that in this region the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine.", "H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.", "Gaps are presented as dashes.", "FIG.", "2B shows a second BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that in this region the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.", "Gaps are presented as dashes.", "5.DETAILED DESCRIPTION OF THE INVENTION The CEA-like polypeptide of SEQ ID NO: 4 is an approximately 425-amino acid secreted, transmembrane protein with a predicted molecular mass of approximately 47 kDa unglycosylated.", "Protein database searches with the BLASTP algorithm (Altschul S. F. et al., J. Mol.", "Evol.", "36:290-300 (1993) and Altschul S. F. et al., J. Mol.", "Biol.", "21:403-10 (1990), herein incorporated by reference) indicate that SEQ ID NO: 4 is homologous to human CEA protein.", "FIG.", "1A shows the BLASTP amino acid sequence alignment between a region of the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that in this region the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4.FIG.", "1B shows the BLASTP amino acid sequence alignment between another region of the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that in this region the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4.FIG.", "2A shows the BLASTP amino acid sequence alignment between a region of the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that in this region the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4.FIG.", "2B shows the BLASTP amino acid sequence alignment between another region of the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that in this region the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4.The sequences of the present invention are expected to have CEA-like activity.", "A predicted approximately twenty-residue signal peptide is encoded from approximately residue 1 through residue 20 of SEQ ID NO: 4 (SEQ ID NO: 8).", "The extracellular portion is useful on its own.", "This can be confirmed by expression in mammalian cells and sequencing of the cleaved product.", "The signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp.", "105-31 (1982), incorporated herein by reference).", "One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.", "Using eMATRIX software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.", "Biol., vol.", "6, pp.", "219-235 (1999), herein incorporated by reference), soluble CEA-like polypeptide is expected to have a CEA precursor amino-terminal domain at residues 363-407 of SEQ ID NO: 4 (SEQ ID NO: 6).", "Also, using eMATRIX software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.", "Biol., vol.", "6, pp.", "219-235 (1999), herein incorporated by reference), soluble CEA-like polypeptide is expected to have a CEA precursor amino-terminal domain at residues 68-112 of SEQ ID NO: 4 (SEQ ID NO: 7).", "The domains corresponding to SEQ ID NO: 6-7 are as follows wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine.", "I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine: Carcinoembryonic antigen precursor amino-terminal domain.", "SQLPSGTWIAGPAHTGREVGFPNCSLLVQKLNLTDTGRYTLKTVT designated as SEQ ID NO: 6) p-value of 8.920e-1 5, DM00372B (identification number correlating to signature); located at residues 363-407 of SEQ ID NO: 4, Carcinoembryonic antigen precursor amino-terminal domain.", "YIVSTGDETPGPAHTGREAVRPDGSLDIQGILPRHSGTYILQTFN designated as SEQ ID NO: 7) p-value of 3.329e-12, DM00372B (identification number correlating to signature); located at residues 68-112 of SEQ ID NO: 4.Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, Calif.) further show that the region of 32 through 120 residues of SEQ ID NO: 4 is structurally homologous to CD2 from human recombinant form expressed in Chinese Hamster (Cricetulus griseus) ovary, chain id=1hnf with a verify score of 0.07 using SeqFold and PB90 methods.", "The polypeptides and polynucleotides of the invention could serve as potential therapeutics in the treatment of breast, prostate, colon and other cancers.", "CEA could also be useful in treating disorders relating to inflammation and autoimmunity.", "CEA could also be as immunosuppressant in organ transplant patients.", "Also, a soluble CEA molecule could serve as decoy receptor in above mentioned bacterial and viral infections.", "5.1 Definitions It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.", "The term “active” refers to those forms of the polypeptide that retain the biologic and/or immunologic activities of any naturally occurring polypeptide.", "According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule.", "Likewise “biologically active” or “biological activity” refers to the capability of the natural, recombinant or synthetic CEA-like peptide, or any peptide thereof, to induce a specific biological response in appropriate animals or cells and to bind with specific antibodies.", "The term “CEA-like biological activity” refers to biological activity that is similar to the biological activity of a CEA peptide.", "The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.", "The terms “complementary” or “complementary” refer to the natural binding of polynucleotides by base pairing.", "For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.", "Complementary between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementary exists between the single stranded molecules.", "The degree of complementary between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.", "The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells.", "The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes.", "The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells.", "PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal.", "Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.", "The term “expression modulating fragment,” EMF, means a series of nucleotides that modulates the expression of an operably linked ORF or another EMF.", "As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF.", "EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements).", "One class of EMFs is nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.", "The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides.", "These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material.", "It is understood that where the polynucleotide is RNA the T (thymine) in the sequences set forth herein may be substituted with U (uracil).", "Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.", "The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeably and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides.", "The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides.", "Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides.", "Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules.", "A fragment or segment may uniquely identify each polynucleotide sequence of the present invention.", "Preferably the fragment comprises a sequence substantially similar to a portion of SEQ ID NO: 1-3 or 5.Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al.", "(Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250).", "They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art.", "Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.", "The nucleic acid sequences of the present invention also include the sequence information from any of the nucleic acid sequences of SEQ ID NO: 1-3 or 5.The sequence information can be a segment of SEQ ID NO: 1-3 or 5 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-3 or 5.One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300.In the human genome, there are three billion base pairs in one set of chromosomes.", "Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosome.", "Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5.When these segments are used in arrays for expression studies, fifteen-mer segments can be used.", "The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.", "Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer.", "The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25).", "The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five.", "The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.", "The term “open reading frame,” ORF, means a series of nucleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.", "The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences.", "For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence.", "While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g.", "repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.", "The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism.", "A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.", "The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules.", "A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids.", "The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids.", "Preferably the peptide is from about 5 to about 200 amino acids.", "To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.", "The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.", "The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or a processing sequence.", "The term “mature protein coding sequence” refers to a sequence which encodes a peptide or protein without any leader/signal sequence.", "The peptide may have the leader sequences removed during processing in the cell or the protein may have been produced synthetically or using a polynucleotide only encoding for the mature protein coding sequence.", "The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as omithine, which do not normally occur in human proteins.", "The term “variant” (or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e g., recombinant DNA techniques.", "Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.", "Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code.", "Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.", "Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.", "Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e.", "conservative amino acid replacements.", "“Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.", "For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine.", "and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.", "“Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids.", "The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.", "Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides.", "Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.", "For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.", "Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.", "For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.", "The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.", "In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).", "The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.", "In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion., or other components normally present in a solution of the same.", "The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.", "The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems.", "“Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems.", "As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation.", "Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.", "The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence.", "An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences.", "Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.", "Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue.", "This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.", "The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally.", "Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed.", "This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.", "Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.", "The cells can be prokaryotic or eukaryotic.", "The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell.", "“Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed.", "“Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum.", "“Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g.", "Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g.", "Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al.", "(1998) Annu.", "Rev.", "Immunol.", "16:27-55) Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell.", "Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.", "The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent.", "Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.).", "Other exemplary hybridization conditions are described herein in the examples.", "In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligonucleotides), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).", "As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences.", "Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less).", "Such a sequence is said to have 65% sequence identity to the listed sequence.", "In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity).", "Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.", "Substantially equivalent nucleotide sequence of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code.", "Preferably, the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, more preferably at least about 80% sequence identity, more preferably at least 85 % sequence identity, more preferably at least 90% sequence identity, more preferably at least about 95% sequence identity, more preferably at least 98% sequence identity, and most preferably at least 99% sequence identity.", "For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent.", "For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded.", "Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J.", "(1990) Methods Enzymol.", "183:626-645).", "Identity between sequences can also be deternined by other methods known in the art, e.g.", "by varying hybridization conditions.", "The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.", "The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration.", "The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed.", "The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.", "As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell.", "UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below.", "The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence.", "The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined.", "As described above, a UMF will increase the frequency of uptake of a linked marker sequence.", "Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.", "5.2 Nucleic Acids of the Invention The invention is based on the discovery of a novel secreted CEA-like polypeptide, the polynucleotides encoding the CEA-like polypeptide and the use of these compositions for the diagnosis, treatment or prevention of cancers and other immunological disorders.", "The isolated polynucleotides of the invention include, but are not limited to a polynucleotide comprising any of the nucleotide sequences of SEQ ID NO: 1-3 or 5; a fragment of SEQ ID NO: 1-3 or 5; a polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1-3 or 5 (for example SEQ ID NO: 4); and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-3 or 5.The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of the SEQ ID NO: 1-3 or 5; (b) a polynucleotide encoding any one of the polypeptides of SEQ ID NO: 4 or 6-10; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 4 or 6-10.Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.", "The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA.", "The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.", "The present invention also provides genes corresponding to the cDNA sequences disclosed herein.", "The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.", "Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.", "Further 5′ and 3′ sequence can be obtained using methods known in the art.", "For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of the SEQ ID NO: 1-3 or 5 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of the SEQ ID NO: 1-3 or 5 or a portion thereof as a probe.", "Alternatively,the polynucleotides of the SEQ ID NO: 1-3 or 5 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.", "The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene.", "The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.", "The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above.", "Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more typically at least about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%, 93%, 94%, and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence identity to a polynucleotide recited above.", "Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of the SEQ ID NO: 1-3 or 5, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides.", "Fragments of, e.g.", "15, 17, or 20 nucleotides or more that are selective for (i.e.", "specifically hybridize to any one of the polynucleotides of the invention) are contemplated.", "Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.", "The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof.", "Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-3 or 5, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NO: 1-3 or 5 with a sequence from another isolate of the same species.", "Furthermore.", "to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein.", "In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.", "The nearest neighbor result for the nucleic acids of the present invention, including SEQ ID NO: 1-3 or 5, can be obtained by searching a database using an algorithm or a program.", "Preferably, a BLAST which stands for Basic Local alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J Mol.", "Evol.", "36 290-300 (1993) and Altschul S. F. et al.", "J. Mol.", "Biol.", "21:403-410(1990)) Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention.", "Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.", "The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.", "The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids.", "These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide.", "There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation.", "Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature.", "These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions).", "Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.", "Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.", "Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.", "Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.", "Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.", "In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis.", "This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site being changed.", "In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983).", "A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res.", "10:6487-6500 (1982).", "PCR may also be used to create amino acid sequence variants of the novel nucleic acids.", "When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.", "PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer.", "The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.", "A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al.", "Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids.", "Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.", "Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.", "The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.", "The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA.", "Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.", "In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 4 or 6-10, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells.", "Also included are the cDNA inserts of any of the clones identified herein.", "A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al.", "(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).", "Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art.", "Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide.", "In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell.", "Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.", "A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.", "The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of the SEQ ID NO: 1-3 or 5 or a fragment thereof or any other polynucleotides of the invention.", "In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of the SEQ ID NO: 1-3 or 5 or a fragment thereof is inserted, in a forward or reverse orientation.", "In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF.", "Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention.", "The following vectors are provided by way of example.", "Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).", "Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).", "The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res.", "19, 4485-4490 (1991), in order to produce the protein recombinantly.", "Many suitable expression control sequences are known in the art.", "General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman.", "Methods in Enzymology 185, 537-566 (1990).", "As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.", "Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.", "Two appropriate vectors are pKK232-8 and pCM7.Particular named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, and trc.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.", "Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.", "Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.", "Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.", "Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.", "Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.", "The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.", "Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.", "As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).", "Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA).", "These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.", "Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.", "Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.", "Polynucleotides of the invention can also be used to induce immune responses.", "For example, as described in Fan et al., Nat.", "Biotech.", "17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA.", "The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.", "5.3 Antisense Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1-3, or 5, or fragments, analogs or derivatives thereof.", "An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.", "In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire coding strand, or to only a portion thereof.", "Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a protein of any of SEQ ID NO: 1-3, or 5 or antisense nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-3 or 5 are additionally provided.", "In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence of the invention.", "The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.", "In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence of the invention.", "The term “noncoding region” refers to 5′ and 3′ sequences that flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).", "Given the coding strand sequences encoding a nucleic acid disclosed herein (e.g., SEQ ID NO: 1-3, or 5, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.", "The antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of an mRNA.", "For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of an mRNA.", "An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.", "An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.", "For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.", "Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.", "Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).", "The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein according to the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.", "The hybridization can be by conventional nucleotide complementary to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.", "An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.", "Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.", "For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.", "The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.", "To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.", "In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule.", "An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al.", "(1987) Nucleic Acids Res 15: 6625-6641).", "The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al.", "(1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.", "(1987) FEBS Lett 215: 327-330).", "5.4 Ribozymes and PNA Moieties In still another embodiment, an antisense nucleic acid of the invention is a ribozyme.", "Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.", "Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of an mRNA.", "A ribozyme having specificity for a nucleic acid of the invention can be designed based upon the nucleotide sequence of a DNA disclosed herein (i.e., SEQ ID NO: 1-3, or 5).", "For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a SECX-encoding mRNA.", "See, e.g., Cech et al.", "U.S. Pat.", "No.", "4,987,071; and Cech et al.", "U.S. Pat.", "No.", "5,116,742.Alternatively, SECX mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.", "See, e.g., Bartel et al., (1993) Science 261:1411-1418.Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region (e.g., promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells.", "See generally, Helene.", "(1991) Anticancer Drug Des.", "6: 569-84; Helene.", "et al.", "(1992) Ann.", "N.Y. Acad.", "Sci.", "660:27-36; and Maher (1992) Bioassays 14: 807-15.In various embodiments, the nucleic acids of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.", "For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al.", "(1996) Bioorg Med Chem 4: 5-23).", "As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.", "The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.", "The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al.", "(1996) above; Perry-O'Keefe et al.", "(1996) PNAS 93: 14670-675.PNAs of the invention can be used in therapeutic and diagnostic applications.", "For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.", "PNAs of the invention can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup B.", "(1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al.", "(1996), above; Perry-O'Keefe (1996), above).", "In another embodiment, PNAs of the invention can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.", "For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.", "Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.", "PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).", "The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al.", "(1996) Nucl Acids Res 24: 3357-63.For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al.", "(1989) Nucl Acid Res 17: 5973-88).", "PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al.", "(1996) above).", "Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment.", "See, Petersen et al.", "(1975) Bioorg Med Chem Lett 5: 1119-11124.In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.", "Natl.", "Acad.", "Sci.", "84:648-652; PCT Publication No.", "W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.", "W089/10134).", "In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al., 1988, Bio Techniques 6:958-976) or intercalating agents.", "(See, e.g., Zon, 1988, Pharm.", "Res.", "5: 539-549).", "To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.", "5.5 Hosts The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention.", "For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods.", "The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.", "Knowledge of CEA-like DNA sequences allows for modification of cells to permit, or increase, expression of CEA-like polypeptide.", "Cells can be modified (e.g., by homologous recombination) to provide increased CEA-like polypeptide expression by replacing, in whole or in part, the naturally occurring CEA-like promoter with all or part of a heterologous promoter so that the cells CEA-like polypeptide is expressed at higher levels.", "The heterologous promoter is inserted in such a manner that it is operatively linked to CEA-like encoding sequences.", "See, for example, PCT International Publication No.", "WO94/12650, PCT International Publication No.", "WO92/20808, and PCT International Publication No.", "WO91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.", "If linked to the CEA-like coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the CEA-like coding sequences in the cells.", "The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.", "Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran-mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)).", "The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.", "Any host/vector system can be used to express one or more of the ORFs of the present invention.", "These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-I cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis.", "The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level.", "Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.", "Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.", "Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual.", "Second Edition, Cold Spring Harbor, New York (1989), the disclosure of which is hereby incorporated by reference.", "Various mammalian cell culture systems can also be employed to express recombinant protein.", "Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981).", "Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells.", "BHK, HL-60, U937, HaK or Jurkat cells.", "Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.", "DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.", "Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps.", "Protein refolding steps can be used, as necessary, in completing configuration of the mature protein.", "Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.", "Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.", "Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria.", "Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.", "Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins.", "If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.", "Such covalent attachments may be accomplished using known chemical or enzymatic methods.", "In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.", "As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.", "Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.", "Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.", "These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.", "The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.", "Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.", "Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.", "Here, the naturally occurring sequences are deleted and new sequences are added.", "In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.", "The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.", "Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.", "The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.", "No.", "5,272,071 to Chappel; U.S. Pat.", "No.", "5,578,461 to Sherwin et al.", "; International Application No.", "PCT/US92/09627 (WO93/09222) by Selden et al.", "; and International Application No.", "PCTIUS90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.", "5.6 Polypeptides of the Invention The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequence set forth as any one of SEQ ID NO: 4 or 6-10 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-3 or 5 or the corresponding full length or mature protein.", "Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in the SEQ ID NO: 1-3 or 5 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 4 or 6-10 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions.", "The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 4 or 6-10 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85 %, 86%, 87%, 88%, 89%, at least about 90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96%, 97%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity.", "Polypeptides encoded by allelic variants may have a similar.", "increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 4 or 6-10.Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention.", "Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer.", "Chem.", "Soc.", "114, 9245-9253 (1992), both of which are incorporated herein by reference.", "Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.", "The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins.", "The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences.", "The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell.", "The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form.", "Where proteins of the present invention are membrane bound.", "soluble forms of the proteins are also provided.", "In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which it is expressed.", "Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.", "The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention.", "By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence.", "Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.", "A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention.", "At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers.", "The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity.", "This technique is particularly useful in producing small peptides and fragments of larger polypeptides.", "Fragments are useful, for example, in generating antibodies against the native polypeptide.", "Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.", "The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein.", "As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level.", "One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.", "The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown.", "For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide.", "The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified.", "Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.", "In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein.", "One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention.", "These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography.", "See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology.", "Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.", "The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides.", "These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins.", "The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.", "In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.", "In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells.", "The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 4 or 6-10.The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.", "The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modifications are naturally provided or deliberately engineered.", "For example, modifications, in the peptide or DNA sequence, can be made by those skilled in the art using known techniques.", "Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.", "For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.", "Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat.", "No.", "4,518,584).", "Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.", "Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity.", "This type of analysis determines the importance of the substituted amino acid(s) in biological activity.", "Regions of the protein that are important for protein function may be determined by the eMATRIX program.", "Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein.", "Such modifications are encompassed by the present invention.", "The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.", "Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No.", "1555 (1987), incorporated herein by reference.", "As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.” The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.", "The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.", "The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.", "Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification.", "For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag.", "Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.", "), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively.", "The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.", "One such epitope (“FLAG®”) is commercially available from Kodak (New Haven, Conn.).", "Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein.", "Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.", "The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.” The polypeptides of the invention include analogs (variants).", "The polypeptides of the invention include CEA-like analogs.", "This embraces fragments of the CEA-like polypeptides of the invention, as well CEA-like polypeptides which comprise one or more amino acids deleted, inserted, or substituted.", "Also, analogs of the CEA-like polypeptides of the invention embrace fusions of the CEA-like polypeptides or modifications of the CEA-like polypeptides, wherein the CEA-like polypeptides or analogs are fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent.", "Such analogs may exhibit improved properties such as activity and/or stability.", "Examples of moieties which may be fused to the CEA-like polypeptides or an analogs include, for example, targeting moieties which provide for the delivery of polypeptide to neurons, e.g., antibodies to central nervous system, or antibodies to receptor and ligands expressed on neuronal cells.", "Other moieties which may be fused to CEA-like polypeptides include therapeutic agents which are used for treatment, for example anti-depressant drugs or other medications for neurological disorders.", "Also, CEA-like polypeptides may be fused to neuron growth modulators, and other chemokines for targeted delivery.", "5.6.1 Determining Polypeptide and Polynucleotide Identity and Similarity Preferred identity and/or similarity are designed to give the largest match between the sequences tested.", "Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec.", "Biol.", "215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res.", "vol.", "25, pp.", "3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp.", "Biol., vol.", "6, pp.", "219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, vol 4, pp.", "202-209, herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp.", "105-31 (1982), incorporated herein by reference).", "The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al.", "NCB NLM NIH Bethesda, Md.", "20894; Altschul, S., et al., J. Mol.", "Biol.", "215:403-410 (1990).", "5.7 Chimeric and Fusion Protiens The invention also provides chimeric or fusion proteins.", "As used herein, a “chimeric protein” or “fusion protein” comprises a polypeptide of the invention operatively linked to another polypeptide.", "Within a fusion protein the polypeptide according to the invention can correspond to all or a portion of a protein according to the invention.", "In one embodiment, a fusion protein comprises at least one biologically active portion of a protein according to the invention.", "In another embodiment, a fusion protein comprises at least two biologically active portions of a protein according to the invention.", "Within the fusion protein, the term “operatively linked” is intended to indicate that the polypeptide according to the invention and the other polypeptide are fused in-frame to each other.", "The polypeptide can be fused to the N-terminus or C-terminus, or to the middle.", "For example, in one embodiment a fusion protein comprises a polypeptide according to the invention operably linked to the extracellular domain of a second protein.", "In another embodiment, the fusion protein is a GST-fusion protein in which the polypeptide sequences of the invention are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.", "In another embodiment, the fusion protein is an immunoglobulin fusion protein in which the polypeptide sequences according to the invention comprise one or more domains fused to sequences derived from a member of the immunoglobulin protein family.", "The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand and a protein of the invention on the surface of a cell, to thereby suppress signal transduction in vivo.", "The immunoglobulin fusion proteins can be used to affect the bioavailability of a cognate ligand.", "Inhibition of the ligand/protein interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, e.g., cancer as well as modulating (e.g., promoting or inhibiting) cell survival.", "Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of a polypeptide of the invention with a ligand.", "A chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques.", "For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.", "In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.", "Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al.", "(eds.)", "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).", "Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).", "A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein of the invention.", "5.8 Gene Therapy Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein.", "The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention.", "Delivery of a functional gene encoding polypeptides of the invention to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).", "See.", "for example, Anderson, Nature, supplement to vol.", "392, no.", "6679, pp.", "25-20 (1998).", "For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992).", "Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression).", "Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.", "Treated cells can then be introduced in vivo for therapeutic purposes.", "Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states.", "It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.", "Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art.", "Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.", "The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.", "These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.", "Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide.", "Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels.", "The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein encoding sequences.", "See, for example, PCT International Publication No.", "WO 94/12650, PCT International Publication No.", "WO 92/20808, and PCT International Publication No.", "WO 91/09955.It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA.", "If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.", "In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination.", "As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods.", "Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences.", "Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting.", "These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.", "The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene.", "Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.", "Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements.", "Here, the naturally occurring sequences are deleted and new sequences are added.", "In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome.", "The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.", "Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.", "The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat.", "No.", "5,272,071 to Chappel; U.S. Pat.", "No.", "5,578,461 to Sherwin et al.", "; International Application No.", "PCT/US92/09627 (WO93/09222) by Selden et al.", "; and International Application No.", "PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.", "5.9 Transgenic Animals In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].", "Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.", "Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.", "Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.", "No.", "5,557,032, incorporated herein by reference.", "Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.", "Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.", "Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.", "No.", "5,489,743 and PCT Publication No.", "WO94/28122, incorporated herein by reference.", "Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention.", "Inactivation can be carried out using homologous recombination methods described above.", "Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.", "The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.", "The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express functional CEA-like polypeptide or that express a variant of CEA-like polypeptide.", "Such animals are useful as models for studying the in vivo activities of CEA-like polypeptide as well as for studying modulators of the CEA-like polypeptide.", "In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)].", "Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals.", "Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals.", "Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat.", "No.", "5,557,032, incorporated herein by reference.", "Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states.", "Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism.", "Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat.", "No.", "5,489,743 and-PCT Publication No.", "WO94/28122, incorporated herein by reference.", "Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention.", "Inactivation can be carried out using homologous recombination methods described above.", "Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.", "The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.", "5.10 Uses and Biological Activity of Human CEA-Like Polypetide The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein.", "Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA) The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment.", "Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity.", "Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.", "The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.", "5.10.1 Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes.", "The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response.", "Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.", "The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays, designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.", "Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.", "The polypeptides of the invention are also useful for making antibody substances that are specifically immunoreactive with CEA-like proteins.", "Antibodies and portions thereof (e.g., Fab fragments) which bind to the polypeptides of the invention can be used to identify the presence of such polypeptides in a sample.", "Such determinations are carried out using any suitable immunoassay format, and any polypeptide of the invention that is specifically bound by the antibody can be employed as a positive control.", "Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.", "Methods for performing the uses listed above are well known to those skilled in the art.", "References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.5.10.2 Nutritional Uses Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements.", "Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.", "In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.", "In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.", "Additionally, the polypeptides of the invention can be used as molecular weight markers, and as a food supplement.", "A polypeptide consisting of SEQ ID NO: 4, for example, has a molecular mass of approximately 47 kDa in its unprocessed and unglycosylated state.", "Protein food supplements are well known and the formulation of suitable food supplements including polypeptides of the invention is within the level of skill in the food preparation art.", "5.10.3 Cytokine nd Cell Proliferation/Differentation Activity A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.", "A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.", "Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.", "The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco.", "Therapeutic compositions of the invention can be used in the following: Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 3.In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.", "137:3494-3500, 1986; Bertagnolli et al., J. Immunol.", "145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.", "149:3778-3783,1992; Bowman et al., I. Immunol.", "152:1756-1761, 1994.Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology.", "J. E. e.a.", "Coligan eds.", "Vol 1 pp.", "3.12.1-3.12.14, John Wiley and Sons, Toronto.", "1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology.", "J. E. e.a.", "Coligan eds.", "Vol 1 pp.", "6.8.1-6.8.8, John Wiley and Sons, Toronto.", "1994.Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology.", "J. E. e.a.", "Coligan eds.", "Vol 1 pp.", "6.3.1-6.3.12, John Wiley and Sons, Toronto.", "1991; deVries et al., J. Exp.", "Med.", "173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6—Nordan, R. In Current Protocols in Immunology.", "J. E. Coligan eds.", "Vol 1 pp.", "6.6.1-6.6.5, John Wiley and Sons, Toronto.", "1991; Smith et al., Proc.", "Natl.", "Aced.", "Sci.", "U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J.", "In Current Protocols in Immunology.", "J. E. Coligan eds.", "Vol 1 pp.", "6.15.1 John Wiley and Sons, Toronto.", "1991; Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J.", "In Current Protocols in Immunology.", "J. E. Coligan eds.", "Vol 1 pp.", "6.13.1, John Wiley and Sons Toronto.", "1991.Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 77:6091-6095, 1980; Weinberger et al., Eur.", "J. Immun.", "11:405-411, 1981; Takai et al., J. Immunol.", "137:3494-3500, 1986; Takai et al., J. Immunol.", "140:508-512, 1988.5.10.4 Stem Cell Growth Factor Activity A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells.", "Administration of the polypeptide of the invention to stem cells in vivo or ex vivo may maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors.", "The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.", "It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).", "Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells.", "Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines is expected to enhance the survival and proliferation of the stem cell populations.", "This can be accomplished by direct administration of the polypeptide of the invention to the culture medium.", "Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo.", "Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat.", "No.", "5,690,926).", "Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention.", "This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types.", "These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments.", "These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.", "Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions.", "For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders.", "The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e.", "for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue.", "In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.", "Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types.", "A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker.", "The selectable marker allows only cells of the desired type to survive.", "For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin.", "Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds.", "Lanza et al., Academic Press (1997)).", "Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.", "In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity.", "Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al.", "Proc.", "Natl.", "Acad.", "Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines.", "The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g.", "as described by Bernstein et al., Blood, 77: 2316-2321 (1991).", "5.10.5 Hematopoiesis Regulating Activity A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders.", "Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.", "in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfisions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, a plastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.", "Therapeutic compositions of the invention can be used in the following: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.", "Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al.", "Cellular Biology 15:141-151, 1995; Keller et.", "al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells.", "R. I. Freshney, et al.", "eds.", "Vol pp.", "265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A.", "In Culture of Hematopoietic Cells.", "R. I. Freshney, et al.", "eds.", "Vol pp.", "23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells.", "R. I. Freshney, et al.", "eds.", "Vol pp.", "1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells.", "R. I. Freshney, et al.", "eds.", "Vol pp.", "163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J.", "In Culture of Hematopoietic Cells.", "R. I. Freshney, et al.", "eds.", "Vol pp.", "139-162, Wiley-Liss, Inc., New York, N.Y. 1994.5.10.6 Tissue Growth Activity A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.", "A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.", "Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints.", "De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.", "A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells.", "Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.)", "mediated by inflammatory processes may also be possible using the composition of the invention.", "Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation.", "Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.", "Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.", "De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.", "The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.", "The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.", "The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.", "The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e.", "for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue.", "More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.", "Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.", "Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.", "Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.", "Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.", "Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate.", "A polypeptide of the present invention may also exhibit angiogenic activity.", "A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.", "A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.", "Therapeutic compositions of the invention can be used in the following: Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No.", "WO95/16035 (bone, cartilage, tendon); International Patent Publication No.", "WO95/05846 (nerve, neuronal); International Patent Publication No.", "WO91/07491 (skin, endothelium).", "Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pp.", "71-112 (Maibach, H. I. and Rovee, D. T., eds.).", "Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.", "Invest.", "Dermatol 71:382-84 (1978).", "5.10.7 Immune Function Stimulating or Suppressing Activity A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.", "A polynucleotide of the invention can encode a polypeptide exhibiting such activities.", "A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.", "These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.", "More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp.", "and various fungal infections such as candidiasis.", "Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.", "Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.", "Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems.", "Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention.", "The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffinann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch.", "Toxocol.", "73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol.", "Environ.", "Health 53: 563-79).", "Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways.", "Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.", "The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.", "Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.", "Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.", "Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.", "Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).", "For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation.", "Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.", "The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant.", "Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.", "Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.", "To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.", "The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.", "Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc.", "Natl.", "Acad.", "Sci USA, 89:11102-11105 (1992).", "In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.", "846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.", "Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.", "Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.", "Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.", "Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process.", "Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.", "The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases.", "Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.", "840-856).", "Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.", "Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.", "For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.", "Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.", "Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.", "The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.", "A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.", "In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.", "Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell.", "Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.", "Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.", "The activity of a protein of the invention may, among other means be measured by the following methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.", "128:1968-1974, 1982; Handa et al., J. Immunol.", "135:1564-1572, 1985; Takai et al., I. Immunol.", "137:3494-3500, 1986; Takai et al., J. Immunol.", "140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.", "153:3079-3092, 1994.Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.", "144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology.", "J. E. e.a.", "Coligan eds.", "Vol 1 pp.", "3.8.1-3.8.16, John Wiley and Sons, Toronto.", "1994.Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.", "137:3494-3500, 1986; Takai et al., J. Immunol.", "140:508-512, 1988; Bertagnolli et al., J. Immunol.", "149:3778-3783, 1992.Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.", "134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc.", "Nat.", "Acad Sci.", "USA 88:7548-7551, 1991.5.10.8 Activin/Inhibin Activity A polypeptide of the present invention may also exhibit activin- or inhibin-related activities.", "A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics.", "Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the.", "release of follicle stimulating hormone (FSH).", "Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals.", "Administration of sufficient amounts of other inhibins can induce infertility in these mammals.", "Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.", "See, for example, U.S. Pat.", "No.", "4,798,885.A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.", "The activity of a polypeptide of the invention may, among other means, be measured by the following methods.", "Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 83:3091-3095, 1986.5.10.9 Chemotactic/Chemokinetic Activity A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.", "A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.", "Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action.", "Chemotactic or chemokinetic compositions (e.g.", "proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections.", "For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.", "A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.", "Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells.", "Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.", "Therapeutic compositions of the invention can be used in the following: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.", "Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al.", "J. Clin.", "Invest.", "95:1370-1376, 1995; Lind et al.", "APMIS 103:140-146, 1995; Muller et al Eur.", "J. Immunol.", "25:1744-1748; Gruber et al.", "J. of Immunol.", "152:5860-5867, 1994; Johnston et al.", "J. of Immunol.", "153:1762-1768, 1994.5.10.10 Hemostatic and Thermbolytic Activity A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis.", "A polynucleotide of the invention can encode a polypeptide exhibiting such attributes.", "Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.", "A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).", "Therapeutic compositions of the invention can be used in the following: Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin.", "Pharmacol.", "26:131-140, 1986; Burdick et al., Thrombosis Res.", "45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.5.10.11 Cancer Diagnosis and Therapy Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis.", "Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer.", "For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy.", "Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition.", "Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.", "Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness.", "Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma.", "basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.", "Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer.", "Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g.", "reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.", "The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail.", "An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery.", "The use of anti-cancer cocktails as a cancer treatment is routine.", "Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.", "In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer.", "There are hereditary conditions and/or environmental situations (e.g.", "exposure to carcinogens) known in the art that predispose an individual to developing cancers.", "Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.", "In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment.", "These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl.", "Can.", "Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl.", "J. Dev.", "Biol., 40: 1189-97 (1999) and Li et al., Clin.", "Exp.", "Metastasis, 17:423-9 (1999), respectively.", "Suitable tumor cells lines are available, e.g.", "from American Type Tissue Culture Collection catalogs.", "5.10.12 Receptor/Ligand Activity A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions.", "A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics.", "Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses.", "Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.", "A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.", "The activity of a polypeptide of the invention may, among other means, be measured by the following methods: Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub.", "Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 84:6864-6868, 1987; Bierer et al., J. Exp.", "Med.", "168:1145-1156, 1988; Rosenstein et al., J. Exp.", "Med.", "169:149-160 1989; Stoltenborg et al., J. Immunol.", "Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s).", "Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BlAcore assays, gel overlay assays, or other methods known in the art.", "Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands.", "The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, colorimetric molecules or a toxin molecules by conventional methods.", "(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol.", "182 (1990) Academic Press, Inc. San Diego).", "Examples of radioisotopes include, but are not limited to, tritium and carbon-14 .", "Examples of colorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules.", "Examples of toxins include, but are not limited, to ricin.", "5.10.13 Drug Screening This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques.", "The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.", "One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof.", "Drugs are screened against such transformed cells in competitive binding assays.", "Such cells.", "either in viable or fixed form, can be used for standard binding assays.", "One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.", "Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.", "Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.", "The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves.", "Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof.", "For a review, see Science 282:63-68 (1998).", "Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.", "Of particular interest are peptide and oligonucleotide combinatorial libraries.", "Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.", "For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr.", "Opin.", "Biotechnol.", "8:701-707 (1997).", "For reviews and examples of peptidomimetic libraries, see A1-Obeidi et al., Mol.", "Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1): 114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).", "Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to bind a polypeptide of the invention.", "The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art.", "In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.", "The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes.", "The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention.", "Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.", "5.10.14 Assay for Receptor Activity The invention also provides methods to detect specific binding of a polypeptide e.g.", "a ligand or a receptor.", "The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention.", "For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners.", "As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention.", "There are a number of different libraries used for the identification of compounds, and in particular small molecule, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention.", "Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not.", "The response of the two cell populations to the addition of ligands(s) are then compared.", "Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s).", "As still another example, BlAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.", "The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined.", "For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell.", "The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor.", "Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e.", "phosphorylation.", "Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.", "5.10.15 Anti-Inflammatory Activity Compositions of the present invention may also exhibit anti-inflammatory activity.", "The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.", "Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions, including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1.Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.", "Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.", "5.10.16 Leukemias Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention.", "Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.", "B. Lippincott Co., Philadelphia).", "5.10.17 Nervous System Disorders Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.", "Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis; (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis; (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.", "Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.", "For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention: (i) increased survival time of neurons in culture; (ii) increased sprouting of neurons in culture or in vivo; (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (iv) decreased symptoms of neuron dysfunction in vivo.", "Such effects may be measured by any method known in the art.", "In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al.", "(1990, J. Neurosci.", "10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al.", "(1980, Exp.", "Neurol.", "70:65-82) or Brown et al.", "(1981, Ann.", "Rev.", "Neurosci.", "4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.", "In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).", "5.10.18 Other Activities A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.", "5.10.19 Identification of Polymorphisims The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment.", "Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately.", "For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.", "Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA.", "For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced.", "Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides).", "In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed.", "Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms.", "The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention.", "In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.", "Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.", "5.10.20 Arthritis and Inflammation The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system.", "The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, orby B. Waksman et al., 1963, Int.", "Arch.", "Allergy Appl.", "Immunol., 23:129.Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).", "The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture.", "The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg.", "The control consists of administering PBS only.", "The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24.At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above.", "An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.", "5.11 Therapeutic Methods The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods.", "Examples of therapeutic applications include, but are not limited to, those exemplified herein.", "5.11.1 Example One embodiment of the invention is the administration of an effective amount of the CEA-like polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention.", "While the mode of administration is not particularly important, parenteral administration is preferred.", "An exemplary mode of administration is to deliver an intravenous bolus.", "The dosage of CEA-like polypeptides or other composition of the invention will normally be determined by the prescribing physician.", "It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient.", "Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight.", "For parenteral administration, CEA-like polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.", "Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin.", "The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient.", "The preparation of such solutions is within the skill of the art.", "5.12 Pharmaceutical Formulations and Routes of Administration A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders.", "Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.", "The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).", "The characteristics of the carrier will depend on the route of administration.", "The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.", "In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question.", "These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.", "The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment.", "Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects.", "Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents).", "A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.", "As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.", "As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site).", "Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.", "A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g.", "treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.", "When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone.", "When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.", "In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated.", "Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.", "When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.", "If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.", "5.12.1 Routes of Administration Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.", "Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.", "Intravenous administration to the patient is preferred.", "Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation.", "In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops.", "Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue.", "The liposomes will be targeted to and taken up selectively by the afflicted tissue.", "The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action.", "The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art.", "Preferably for wound treatment, one administers the therapeutic compound directly to the site.", "Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models.", "Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.", "5.12.2 Compositions/Formulations Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.", "These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.", "Proper formulation is dependent upon the route of administration chosen.", "When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.", "When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.", "The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention.", "When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.", "The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.", "When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.", "When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.", "The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.", "A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.", "The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.", "For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.", "For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art.", "For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.", "Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.", "Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.", "Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).", "If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.", "Dragee cores are provided with suitable coatings.", "For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.", "Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.", "Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.", "The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.", "In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.", "In addition, stabilizers may be added.", "All formulations for oral administration should be in dosages suitable for such administration.", "For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.", "For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.", "In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.", "Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.", "The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.", "Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.", "The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.", "Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.", "Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.", "Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.", "Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.", "Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.", "Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.", "The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.", "In addition to the formulations described previously, the compounds may also be formulated as a depot preparation.", "Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.", "Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.", "A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant.", "a water-miscible organic polymer, and an aqueous phase.", "The co-solvent system may be the VPD co-solvent system.", "VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.", "The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution.", "This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.", "Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.", "Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol., e.g.", "polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.", "Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed.", "Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.", "Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.", "Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.", "Various types of sustained-release materials have been established and are well known by those skilled in the art.", "Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.", "Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.", "The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.", "Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.", "Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions.", "Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.", "The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.", "The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.", "B lymphocytes will respond to antigen through their surface immunoglobulin receptor.", "T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.", "MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.", "The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.", "Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.", "The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.", "Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like.", "Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat.", "Nos.", "4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.", "The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.", "Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient.", "Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response.", "Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.", "It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight.", "For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.", "When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.", "Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage.", "Topical administration may be suitable for wound healing and tissue repair.", "Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.", "Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.", "Such matrices may be formed of materials presently in use for other implanted medical applications.", "The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties.", "The particular application of the compositions will define the appropriate formulation.", "Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.", "Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.", "Further matrices are comprised of pure proteins or extracellular matrix components.", "Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.", "Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.", "The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.", "Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.", "In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.", "A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).", "Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).", "The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.", "In further compositions, proteins or other active ingredient of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.", "These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).", "The therapeutic compositions are also presently valuable for veterinary applications.", "Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredient of the present invention.", "The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.", "The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.", "For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.", "Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.", "Polynucleotides of the present invention can also be used for gene therapy.", "Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject.", "Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).", "Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.", "Treated cells can then be introduced in vivo for therapeutic purposes.", "5.12.3 Efective Dosage Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose.", "More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated.", "Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.", "For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays.", "For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans.", "For example, a dose, can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein s biological activity).", "Such information can be used to more accurately determine useful doses in humans.", "A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.", "Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).", "The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.Compounds which exhibit high therapeutic indices are preferred.", "The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.", "The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.", "The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.", "The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.", "See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.", "1 p. 1.Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC).", "The MEC will vary for each compound but can be estimated from in vitro data.", "Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration.", "However, HPLC assays or bioassays can be used to determine plasma concentrations.", "Dosage intervals can also be determined using MEC value.", "Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.", "In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.", "An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children.", "Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.", "The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.", "5.12.4 Packaging The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.", "The pack may, for example, comprise metal or plastic foil, such as a blister pack.", "The pack or dispenser device may be accompanied by instructions for administration.", "Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.", "5.13 Antibodies Also included in the invention are antibodies to proteins, or fragments of proteins of the invention.", "The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.", "Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library.", "In general, an antibody molecule obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.", "Certain classes have subclasses as well, such as IgG1, IgG2, and others.", "Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.", "Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.", "An isolated related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.", "The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens.", "An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NO: 4, or 6-10, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.", "Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.", "Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.", "In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of CEA-like protein that is located on the surface of the protein, e.g., a hydrophilic region.", "A hydrophobicity analysis of the human related protein sequence will indicate which regions of a related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.", "As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation.", "See, e.g., Hopp and Woods, 1981, Proc.", "Nat.", "Acad.", "Sci.", "USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol.", "Biol.", "157: 105-142, each of which is incorporated herein by reference in its entirety.", "Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.", "A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.", "The term “specific for” indicates that the variable regions of the antibodies of the invention recognize and bind polypeptides of the invention exclusively (i.e., able to distinguish the polypeptide of the invention from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule.", "Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.", "For a comprehensive discussion of such assays, see Harlow et al.", "(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.Antibodies that recognize and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, full-length polypeptides of the invention.", "As with antibodies that are specific for full length polypeptides of the invention, antibodies of the invention that recognize fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.", "Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of a polypeptide of the invention), diagnostic purposes to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention.", "Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.", "In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.", "The invention further provides a hybridoma that produces an antibody according to the invention.", "Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.", "Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.", "Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.", "In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.", "The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed.", "The antibodies may also be used directly in therapies or other diagnostics.", "The present invention further provides the above-described antibodies immobilized on a solid support.", "Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®), acrylic resins and such as polyacrylamide and latex beads.", "Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth.", "Enzym.", "34 Academic Press, N.Y. (1974)).", "The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.", "Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference).", "Some of these antibodies are discussed below.", "5.13.1 Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing.", "An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.", "Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized.", "Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.", "The preparation can further include an adjuvant.", "Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.", "), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.", "Additional examples of adjuvants that can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).", "The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum.", "Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography.", "Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol.", "14, No.", "8 (Apr.", "17, 2000), pp.", "25-28).", "5.13.2 Monoclonal Antibodies The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.", "In particular, the complementary determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.", "MAbs thus contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.", "Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).", "In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.", "Alternatively, the lymphocytes can be immunized in vitro.", "The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.", "Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human manmmalian sources are desired.", "The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.", "59-103).", "Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.", "Usually, rat or mouse myeloma cell lines are employed.", "The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.", "For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.", "Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.", "More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.", "Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.", "51-63).", "The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.", "Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).", "Such techniques and assays are known in the art.", "The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal.", "Biochem., 107:220 (1980).", "Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.", "After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods.", "Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.", "Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.", "The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.", "The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat.", "No.", "4,816,567.DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).", "The hybridoma cells of the invention serve as a preferred source of such DNA.", "Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.", "The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat.", "No.", "4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.", "Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.", "5.13.3 Humanized Antibodies The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies.", "These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.", "Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.", "Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.", "(See also U.S. Pat.", "No.", "5,225,539).", "In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.", "Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.", "In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.", "The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.", "Op.", "Struct.", "Biol., 2:593-596 (1992)).", "5.13.4 Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes.", "Such antibodies are termed “human antibodies”, or “fully human antibodies” herein.", "Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.", "77-96).", "Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983.Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.", "77-96).", "In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol.", "Biol., 227:381 (1991); Marks et al., J. Mol.", "Biol., 222:581 (1991)).", "Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.", "Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.", "This approach is described, for example, in U.S. Pat.", "Nos.", "5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.", "(Bio/Technology 10, 779-783 (1992)); Lonberg et al.", "(Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern.", "Rev.", "Immunol.", "13 65-93 (1995)).", "Human antibodies may additionally be produced using transgenic nonhuman animals that are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.", "(See PCT publication WO94/02602).", "The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.", "The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.", "An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.", "The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells that secrete fully human immunoglobulins.", "The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.", "Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.", "An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat.", "No.", "5,939,598.It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.", "A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat.", "No.", "5,916,771.It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.", "The hybrid cell expresses an antibody containing the heavy chain and the light chain.", "In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an inmunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.5.13.5 Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat.", "No.", "4,946,778).", "In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.", "Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.", "5.13.6 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.", "In the present case, one of the binding specificities is for an antigenic protein of the invention.", "The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.", "Methods for making bispecific antibodies are known in the art.", "Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).", "Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure.", "The purification of the correct molecule is usually accomplished by affinity chromatography steps.", "Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., 1991 EMBO J., 10:3655-3659.Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences.", "The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.", "It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions.", "DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism.", "For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).", "According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.", "The preferred interface comprises at least a part of the CH3 region of an antibody constant domain.", "In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g.", "tyrosine or tryptophan).", "Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g.", "alanine or threonine).", "This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.", "Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g.", "F(ab′)2 bispecific antibodies).", "Techniques for generating bispecific antibodies from antibody fragments have been described in the literature.", "For example, bispecific antibodies can be prepared using chemical linkage.", "Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments.", "These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.", "The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.", "One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.", "The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.", "Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies.", "Shalaby et al., J. Exp.", "Med.", "175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule.", "Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.", "The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.", "Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described.", "For example, bispecific antibodies have been produced using leucine zippers.", "Kostelny et al., J. Immunol.", "148(5):1547-1553 (1992).", "The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.", "The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.", "This method can also be utilized for the production of antibody homodimers.", "The “diabody” technology described by Hollinger et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.", "The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain.", "Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.", "Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported.", "See, Gruber et al., J. Immunol.", "152:5368 (1994).", "Antibodies with more than two valencies are contemplated.", "For example, trispecific antibodies can be prepared.", "Tutt et al., J. Immunol.", "147:60 (1991).", "Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.", "Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g.", "CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64),Fc RII (CD32) andFc RIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.", "Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.", "These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.", "Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).", "5.13.7 Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.", "Heteroconjugate antibodies are composed of two covalently joined antibodies.", "Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat.", "No.", "4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).", "It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.", "For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.", "Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.", "No.", "4,676,980.5.13.8 Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.", "For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.", "The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).", "See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.", "Immunol., 148: 2918-2922 (1992).", "Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.", "Cancer Research, 53: 2560-2565 (1993).", "Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.", "See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).", "5.13.9 Immunoconjugates The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).", "Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above.", "Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.", "A variety of radionuclides are available for the production of radioconjugated antibodies.", "Examples include 212Bi, 13I, 131In, 90y, and 186Re.", "Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).", "For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987).", "Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.", "See WO94/11026.In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.", "5.14 Computer Readable Sequences In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media.", "As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer.", "Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.", "A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.", "As used herein, “recorded” refers to a process for storing information on computer readable medium.", "A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.", "A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention.", "The choice of the data storage structure will generally be based on the means chosen to access the stored information.", "In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium.", "The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like.", "A skilled artisan can readily adapt any number of data processor structuring formats (e.g.", "text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.", "By providing any of the nucleotide sequences SEQ ID NO: 1-3 or 5 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of the SEQ ID NO: 1-3 or 5 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes.", "Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.", "The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol.", "Biol.", "215:403410 (1990)) and BLAZE (Brutlag et al.", "Comp.", "Chem.", "17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence.", "Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.", "As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention.", "The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.", "A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention.", "As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means.", "As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.", "As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means.", "Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif.", "A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention.", "Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA).", "A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.", "As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids.", "A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database.", "The most preferred sequence length of a target sequence is from about 10 to 100 amino acids, or from about 30 to 300 nucleotide residues.", "However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.", "As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif.", "There are a variety of target motifs known in the art.", "Protein target motifs include, but are not limited to, enzyme active sites and signal sequences.", "Nucleic acid target motifs include but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).", "5.15 Triple Helix Formation In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA.", "Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.", "Acids Res.", "6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem.", "56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).", "Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.", "Both techniques have been demonstrated to be effective in model systems.", "Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.", "5.16 Diagnostic Asays and Kits The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.", "In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample.", "Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified.", "a polynucleotide of the invention is detected in the sample.", "In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.", "In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.", "Conditions for incubating a nucleic acid probe or antibody with a test sample vary.", "Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay.", "One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention.", "Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol.", "1 (1982), Vol.", "2 (1983), Vol.", "3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).", "The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine.", "The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.", "Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.", "In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.", "Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.", "In detail, a compartment kit includes any kit in which reagents are contained in separate containers.", "Such containers include small glass containers, plastic containers or strips of plastic or paper.", "Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.", "Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.", "), and containers which contain the reagents used to detect the bound antibody or probe.", "Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.", "One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.", "5.17 Medical Imaging The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection).", "See, e.g., Kunkel et al., U.S. Pat.", "No.", "5,413,778.Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.", "5.18 Screening Assays Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in the SEQ ID NO: 1-3 or 5, or bind to a specific domain of the polypeptide encoded by the nucleic acid.", "In detail, said method comprises the steps of: (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and (b) determining whether the agent binds to said protein or said nucleic acid.", "In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.", "Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.", "Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.", "Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).", "Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound).", "Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.", "The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents.", "The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.", "For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention.", "Alternatively, agents may be rationally selected or designed.", "As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein.", "For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides,” In Synthetic Peptides, A User's Guide, W. H. Freeman, N.Y. (1992), pp.", "289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.", "In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention.", "As described above, such agents can be randomly screened or rationally designed/selected.", "Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control.", "One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA.", "Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.", "Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl.", "Acids Res.", "6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem.", "56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).", "Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide.", "Both techniques have been demonstrated to be effective in model systems.", "Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.", "Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent.", "Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.", "5.19 Use of Nucleic Acids as Probes Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences.", "The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NO: 1-3 or 5.Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NO: 1-3 or 5 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.", "Any suitable hybridization technique can be employed, such as, for example, in situ hybridization.", "PCR as described in U.S. Pat.", "Nos.", "4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences.", "Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both.", "The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.", "Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes.", "Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides.", "The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences.", "The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques.", "These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like.", "The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data.", "Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f).", "Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease.", "The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.", "5.20 Preperation of Support Bound Oligonucleotides Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.", "Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon.", "One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers.", "Immobilization can be achieved using passive adsorption (Inouye & Hondo, 1990 J. Clin Microbiol 28(6) 1462-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, Mol.", "Cell Probes 1989 3(2) 189-207) or by covalent binding of base modified DNA (Keller et al., 1988; 1989); all references being specifically incorporated herein.", "Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker.", "For example, Broude et al.", "(1994) Proc.", "Natl.", "Acad.", "Sci USA 91(8) 3072-6 describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads.", "Streptavidin-coated beads may be purchased from Dynal, Oslo.", "Of course, this same linking chemistry is applicable to coating any surface with streptavidin.", "Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).", "Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used.", "Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH.", "CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling.", "CovaLink Modules may be purchased from Nunc Laboratories.", "DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussenet al., (1991) Anal Biochem 198(1) 138-42.The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., 1991).", "In this technology, a phosphoramidate bond is employed (Chu et al., 1983 Nucleic Acids 11 (18) 6513-29).", "This is beneficial as immobilization using only a single covalent bond is preferred.", "The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm.", "To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group.", "It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.", "More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min.", "at 95° C. and cooling on ice for 10 min.", "Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7.A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.", "Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylarninopropyl)-carbodiimide (EDC).", "dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well.", "The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).", "It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference.", "This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support.", "The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support.", "Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.", "An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed.", "For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al.", "(1991) Science 251(4995) 767-73, incorporated herein by reference.", "Probes may also be inmobilized on nylon supports as described by Van Ness et al.", "(1991) Nucleic Acids Res.", "19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal Biochem 169(1) 104-8; all references being specifically incorporated herein.", "To link an oligonucleotide to a nylon support, as described by Van Ness et al.", "(1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotideswith cyanuric chloride.", "One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) Proc.", "Natl.", "Acad.", "Sci USA 91(11) 5022-6.These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips).", "These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleosidephosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies.", "A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.", "5.21 Preperation of Nucleic Acid Fragments The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps.", "For example, Sambrook et al.", "(1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).", "DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods.", "Samples may be prepared or dispensed in multiwell plates.", "About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.", "The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al.", "(1989), shearing by ultrasound and NaOH treatment.", "Low pressure shearing is also appropriate, as described by Schriefer et al.", "(1990) Nucleic Acids Res.", "18(24) 7455-6.In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures.", "A lever device allows controlled application of low to intermediate pressures to the cell.", "The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.", "One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al.", "(1992) Nucleic Acids Res.", "20(14) 3753-62.These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.", "The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends.", "Atypical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC 19 (2688 base pairs).", "Fitzgerald et al.", "(1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector.", "Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.", "As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed).", "Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization.", "This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip.", "Phosphate groups must also be removed from genomic DNA by methods known in the art.", "5.22 Preperation of DNA Arrays Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane.", "Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane.", "By offset printing, a density of dots higher than the density of the wells is achieved.", "One to 25 dots may be accommodated in 1 mm2, depending on the type of label used.", "By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed.", "Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones.", "Each of the subarrays may represent replica spotting of the same samples.", "In one example, a selected gene segment may be amplified from 64 patients.", "For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample).", "A plate for each of the 64 patients is prepared.", "By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane.", "Subarrays may contain 64 samples, one from each patient.", "Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.", "Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g.", "a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips.", "A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.", "The present invention is illustrated in the following examples.", "Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention.", "Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples.", "The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention.", "Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments.", "Consequently,the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.", "All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.", "6.0 EXAMPLES Example 1 Isolation of SEQ ID NO: 1 from a cDNA Library of Rectum A plurality of novel nucleic acids were obtained from a cDNA library prepared from rectum (Hyseq clone identification numbers 15456780 (SEQ ID NO: 1)) using standard PCR, sequencing by hybridization sequence signature analysis, and Sanger sequencing techniques.", "The inserts of the library were amplified with PCR using primers specific for vector sequences flanking the inserts.", "These samples were spotted onto nylon membranes and interrogated with oligonucleotide probes to give sequence signatures.", "The clones were clustered into groups of similar or identical sequences, and single representative clones were selected from each group for gel sequencing.", "The 5′ sequence of the amplified inserts was then deduced using the reverse M13 sequencing primer in a typical Sanger sequencing protocol.", "PCR products were purified and subjected to fluorescent dye terminator cycle sequencing.", "Single-pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer.", "The insert was identified as a novel sequence not previously obtained from this library and not previously reported in public databases.", "The sequence was designated as SEQ ID NO: 1.Example 2 Assemblage of SEQ ID NO: 2 The nucleic acid of the present invention, designated as SEQ ID NO: 2 was assembled using SEQ ID NO:1 as a seed.", "Then a recursive algorithm was used to extend the seed into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 114, gb pri 114, and UniGene version 101) that belong to this assemblage.", "The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage.", "Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.", "The nearest neighbor result for the assembled contigs were obtained by a FASTA version 3 search against Genpept release 114, using FASTXY algorithm.", "FASTXY is an improved version of FASTA alignment which allows in-codon frame shifts.", "The nearest neighbor result showed the closest homologue for each assemblage from Genpept (and contains the translated amino acid sequences for which the assemblage encodes).", "The nearest neighbor results is set forth below: SEQ Accession Smith-Waterman % ID NO: No.", "Description Score Identity 2 AC005626 Homo sapiens 232 36.522 R29124_1 The nucleotide sequence within the assembled contigs that codes for signal peptide sequences and their cleavage sites can be determined from using Neural Network SignalP V1.1 program (from Center for Biological Sequence Analysis, The Technical University of Denmark).", "The process for identifying prokaryotic and eukaryotic signal peptides and their cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the publication “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites” Protein Engineering, vol.", "10, no.", "1, pp.", "1-6 (1997), incorporated herein by reference.", "A maximum S score and a mean S score, as described in the Nielson et al reference, are obtained for each assembled contig.", "Starting from the first amino acid of the predicted signal sequence, a sequence of 45 amino acids is described.", "Not all forty-five amino acids in the sequence may comprise the signal peptide.", "For SEQ ID NO: 2: Beginning and Amino acid segment containing signal peptide end nucleotide (A = Alanine, C = Cysteine, D = Aspartic Acid, E = Glutamic Acid, F = Phenylalanine, location G = Glycine, H = Histidine, I = Isoleucine, K = Lysine, L = Leucine, M = Methionine, corresponding N = Asparagine, P = Proline, Q = Glutamine, R = Arginine, S = Serine, T = Threonine, to amino acid Maximum mean S V = Valine, W = Tryptophan, Y = Tyrosine, X = Unknown, * = Stop Codon, segment S score score / = possible nucleotide deletion, \\ = possible nucleotide insertion) 55:190 0.964 0.902 MALTGYSWLLLSATFLNVGAEISITLEPAQPSEGDNVTLVVHGLS (SEQ ID NO: 10) Example 3 Assemblage of SEQ ID NOs: 3 and 4 Assembly of novel nucleotide sequence of SEQ ID NO: 3 was accomplished by using an EST sequence SEQ ID NO: 1 as a seed.", "The seed was extended by gel sequencing (377 Applied Biosystems (ABI) sequencer) using primers to extend the 3′ end (primer extension).", "A polypeptide (SEQ ID NO:4) was predicted to be encoded by SEQ ID NO:3 as set forth below.", "The polypeptide was predicted using a software program called BLASTX which selects 15 a polypeptide based on a comparison of translated novel polynucleotide to known polynucleotides.", "The initial methionine starts at position 55 of SEQ ID NO:3 and the putative stop codon, TAA, begins at position 1330 of the nucleotide sequence.", "FIG.", "1A shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4.FIG.", "1B shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) protein SEQ ID NO: 11, indicating that the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4.FIG.", "2A shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that the two sequences share 49% similarity over 342 amino acid residues of SEQ ID NO: 4 and 34% identity over the same 342 amino acid residues of SEQ ID NO: 4.FIG.", "2B shows the BLASTP amino acid sequence alignment between the protein encoded by SEQ ID NO: 3 (i.e.", "SEQ ID NO: 4) CEA-like polypeptide and human carcinoembryonic antigen CEA-(c) protein SEQ ID NO: 12, indicating that the two sequences share 52% similarity over 102 amino acid residues of SEQ ID NO: 4 and 37% identity over the same 102 amino acid residues of SEQ ID NO: 4.A predicted approximately twenty-residue signal peptide is encoded from approximately residue 1 to residue 20 of SEQ ID NO: 4 (SEQ ID NO: 8).", "The signal peptide region was predicted using the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol, 157, pp.", "105-31 (1982), incorporated herein by reference).", "Using eMATRIX software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.", "Biol., vol.", "6, pp.", "219-235 (1999), herein incorporated by reference), soluble CEA-like polypeptide is expected to have a CEA precursor amino-terminal domain at residues 363-407 of SEQ ID NO: 4 (SEQ ID NO: 6).", "Also, using eMATRIX software package (Stanford University, Stanford, Calif.) (Wu et al., J. Comp.", "Biol., vol.", "6, pp.", "219-235 (1999), herein incorporated by reference), soluble CEA-like polypeptide is expected to have a CEA precursor amino-terminal domain at residues 68-112 of SEQ ID NO: 4 (SEQ ID NO: 7).", "The domains corresponding to SEQ ID NO: 6-7 are as follows wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=Glutamic Acid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine, L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine, R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine: Carcinoembryonic antigen precursor amino-terminal domain.", "SQLPSGTWIAGPAHTGREVGFPNCSLLVQKLNLTDTGRYTLKTVT designated as SEQ ID NO: 6) p-value of 8.920e-1 5, DM00372B (identification number correlating to signature); located at residues 363-407 of SEQ ID NO: 4, Carcinoembryonic antigen precursor amino-terminal domain.", "YIVSTGDETPGPAHTGREAVRPDGSLDIQGILPRHSGTYILQTFN designated as SEQ ID NO: 7) p-value of 3.329e-12, DM00372B (identification number correlating to signature); located at residues 68-112 of SEQ ID NO: 4.Protein database search with Molecular Simulations Inc. GeneAtlas software (Molecular Simulations Inc., San Diego, Calif.) further show that the region of 32 through 120 residues of SEQ ID NO: 4 is structurally homologous to CD2 from human recombinant form expressed in Chinese Hamster (Cricetulus griseus) ovary, chain id=1 hnf with a verify score of 0.07 using SeqFold and PB90 methods.", "SEQ ID NO: 3 was determined to be present in the following tissues: rectum (Invitrogen) (Hyseq library name REC001) and fetal muscle (Invitrogen) (Hyseq library name FMS002).", "The tissue expression information was determined using the tissue source of the ESTs that comprise SEQ ID NO: 3 and the tissue sources of the other ESTs of the cluster to which those ESTs belong.", "Clusters were made depending on the sequence signature of each sequence as described in Example 1.Example 4 A.", "Expression of SEQ ID NO: 4 in cells Chinese Hamster Ovary (CHO) cells or other suitable cell types are grown in DMEM (ATCC) and 10% fetal bovine serum (FBS) (Gibco) to 70% confluence.", "Prior to transfection the media is changed to DMEM and 0.5% FCS.", "Cells are transfected with cDNAs for SEQ ID NO: 3 or 5 or with pBGal vector by the FuGENE-6 transfection reagent (Boehringer).", "In summary, 4 ill of FuGENE-6 is diluted in 100 μl of DMEM and incubated for 5 minutes.", "Then, this is added to 1 μg of DNA and incubated for 15 minutes before adding it to a 35 mm dish of CHO cells.", "The CHO cells are incubated at 37° C. with 5% CO2.After 24 hours, media and cell lysates are collected, centrifuged and dialyzed against assay buffer (15 mM Tris pH 7.6, 134 mM NaCl, 5 mM glucose, 3 mM CaCl2 and MgCl2.B.", "Expression Study Using SEO ID NO: 1-3 or 5 The expression of SEQ ID NO: 1-3 or 5 in various tissues is analyzed using a semi-quantitative polymerase chain reaction-based technique.", "Human cDNA libraries are used as sources of expressed genes from tissues of interest (adult bladder, adult brain, adult heart, adult kidney, adult lymph node, adult liver, adult lung, adult ovary, adult placenta, adult rectum, adult spleen, adult testis, bone marrow, thymus, thyroid gland, fetal kidney, fetal liver, fetal liver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).", "Gene-specific primers are used to amplify portions of the SEQ ID NO: 1-3 or 5 sequence from the samples.", "Amplified products are separated on an agarose gel, transferred and chemically linked to a nylon filter.", "The filter is then hybridized with a radioactively labeled (33P-dCTP) double-stranded probe generated from SEQ ID NO: 1-3 or 5 using a Klenow polymerase, random-prime method.", "The filters are washed (high stringency) and used to expose a phosphor imaging screen for several hours.", "Bands indicate the presence of cDNA including SEQ ID NO: 1-3 or 5 sequences in a specific library, and thus mRNA expression in the corresponding cell type or tissue." ] ]

[ [ "Novel Methods Using Pyridine Derivatives", "The invention provides methods of treating and preventing asthma, laryngitis, symptomatic gastroesophageal reflux disease, pregnancy-induced gastroesophageal reflux disease, noncardiac chest pains, coughing, apnea, dyspepsia, inflammatory bowel disease, irritable bowel syndrome, gastritis, stress ulcers, bleeding peptic ulcers, acute gastrointestinal bleeding, infectious enteritis, collagenous colitis, lymphocytic colitis, chronic diarrhea in immunocompromised patients, esophageal ulcers in immunocompromised patients, idiopathic gastric acid hypersecretion, gastroparesis, gastrointestinal motility disorders, Zollinger-Ellison syndrome, short bowel syndrome, emesis, regurgitation, early satiety, chronic sore throat, abdominal pain, abdominal bloating, nausea, sour stomach, diarrhea, constipation, bacterial infections, refractory ulcers, gastrointestinal disorders induced by NSAIDs, Barrett's esophagus, gastrointestinal disorders caused by steroids, gastrointestinal disorders induced by cholinergic compounds, and fungal or viral-induced ulcers in the gastrointestinal tract by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention also provides on demand relief of symptoms associated with gastroesophageal reflux disease (GERD), and provides relief from symptoms caused by the consumption of excessive amounts of food and/or alcohol by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention also provides methods for treating parasitic infections, such as malaria, by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "In preferred embodiments, the pyridine derivative of the invention is rabeprazole, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof." ], [ "1.A method for treating or preventing symptomatic gastroesophageal reflux disease in a patient in need thereof comprising administering a therapeutically effective amount of a compound of formula (C), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: 2.The method of claim 1, wherein the compound of formula (C) is rabeprazole sodium or a stereoisomer thereof.", "3.The method of claim 1, wherein the compound of formula (C) is a compound of formula (D) or a pharmaceutically acceptable salt thereof: 4.The method of claim 3, wherein the compound of formula (D) is a sodium salt.", "5.The method of claim 1, wherein the compound of formula (C) is a compound of formula (E) or a pharmaceutically acceptable salt thereof: 6.The method of claim 5, wherein the compound of formula (E) is a sodium salt.", "7.The method of claim 1, wherein the compound of formula (C), the pharmaceutically acceptable salt thereof or the stereoisomer thereof is orally administered.", "8.The method of claim 7, wherein the compound of formula (C), the pharmaceutically acceptable salt thereof or the stereoisomer thereof is orally administered in the form of a tablet.", "9.The method of claim 1, wherein the compound of formula (C), the pharmaceutically acceptable salt thereof or the stereoisomer thereof is administered in an amount of about 20 milligrams per day.", "10.A method for treating or preventing symptomatic gastroesophageal reflux disease in a patient in need thereof comprising administering a therapeutically effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: wherein R1 and R2 are each independently a hydrogen atom, a halogen atom, a lower alkyl, lower alkoxy, halogenated lower alkyl, lower alkoxycarbonyl or carboxyl group; X is —O—, —S— or ═N—R3, wherein R3 is a hydrogen atom or a lower alkyl, phenyl, benzyl or lower alkoxycarbonyl group; and Z is: (i) —O(CH2)p—O—R4 wherein p is an integer of 1 to 3 and R4 is hydrogen atom or a lower alkyl, aryl or aralkyl group, (ii) —O—(CH2)q—R5 wherein q is an integer of 1 to 3 and R5 is a halogen atom or an alkoxycarbonyl, aryl or heteroaryl group, (iii) —O—(CH2)r—O—(CH2)s—O—R6 wherein r and s are each independently an integer of 1 to 5 and R6 is a hydrogen atom or a lower alkyl group, wherein t is an integer of 0 to 2, and A is a lower alkyl, alkoxycarbonylmethyl, pyridyl, furyl, wherein B is —NH—, —O— or —S—, and w is an integer of 0 or 1; (viii) —N(R8)—CH2—C6H5 wherein R8 is an acetoxy or lower alkyl group; or (ix) —OR9 wherein R9 is a hydrogen atom, a lower alkyl or aryl group; n is an integer of 0 to 2; m is an integer of 2 to 10, and J and K are each independently a hydrogen atom or a lower alkyl group, with the proviso that when Z is —OR9, then R9 is a lower alkyl group and m is an integer of 3 to 10.11.The method of claim 10, wherein the compound of formula (I) is a compound of formula (A), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: wherein R1, R2, J, m and R9 are as defined above.", "12.The method of claim 5, wherein the compound of formula (I) is a compound of formula (B), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: wherein R1, R2, J, p, m and R4 are as defined above.", "13.The method of claim 12, wherein the compound of formula (I) is a compound of formula (C), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: 14.The method of claim 13, wherein the compound of formula (C) is rabeprazole sodium or a stereoisomer thereof.", "15.The method of claim 13, wherein the compound of formula (C) is R (+) rabeprazole or a pharmaceutically acceptable salt thereof.", "16.The method of claim 13, wherein the compound of formula (C) is S (−) rabeprazole or a pharmaceutically acceptable salt thereof.", "17.The method of claim 10, wherein the compound of formula (I), the pharmaceutically acceptable salt thereof, or the stereoisomer thereof is administered orally.", "18.The method of claim 12, wherein the compound of formula (I), the pharmaceutically acceptable salt thereof, or the stereoisomer thereof is orally administered as a solid dosage form or a liquid dosage form.", "19.The method of claim 18, wherein the solid dosage form is a capsule, a tablet, a sublingual tablet, a powder, a granule or a gel.", "20.The method of claim 10, wherein the compound of formula (I), the pharmaceutically acceptable salt thereof, or the stereoisomer thereof is administered in an amount of about 0.01 to about 200 milligrams per day.", "21.The method of claim 21, wherein the compound of formula (I), the pharmaceutically acceptable salt thereof, or the stereoisomer thereof is administered in an amount of about 0.1 to about 40 milligrams per day.", "22.The method of claim 21, wherein the compound of formula (I), the pharmaceutically acceptable salt thereof, or the stereoisomer thereof is administered in an amount of about 10 to about 30 milligrams per day." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Duodenal and gastric ulcers, known collectively as peptic ulcers, are localized erosions of the mucous membrane of the duodenum and stomach, respectively, which expose the underlying layers of the gut wall to the acid secretions of the stomach and to the proteolytic enzyme pepsin.", "They are believed to be caused by an imbalance between offensive factors, such as acid or pepsin, and defensive factors, such as resistance of the mucous membrane.", "Peptic ulceration is a common disease of the gastrointestinal tract and it is estimated that approximately 10 to 20% of the adult male population will experience peptic ulceration at some time in their lives.", "Proton pump inhibitors, such as ACIPHEX® (Eisai Inc., Teaneck, N.J.), have proven to be successful in treating peptic ulcers.", "ACIPHEX® is described in U.S. Pat.", "No.", "5,045,552, the disclosure of which is incorporated herein by reference in its entirety.", "There is a need in the art for new and improved treatments for other gastrointestinal diseases and disorders.", "The invention is directed to these, as well as other, important ends." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides methods of treating and preventing asthma in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing laryngitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing symptomatic gastroesophageal reflux disease in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing pregnancy-induced gastroesophageal reflux disease in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing noncardiac chest pains in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing coughing in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention also provides novel methods for treating and preventing bronchitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing apnea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing dyspepsia in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing inflammatory bowel disease, including ulcerative colitis and Crohn's disease, in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing irritable bowel syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastritis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastroesophageal reflux disease and peptic ulcers in infants and children by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing stress ulcers in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing bleeding peptic ulcers and for treating and preventing acute gastrointestinal bleeding in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing infectious enteritis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing collagenous colitis and lymphocytic colitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing chronic diarrhea in immunocompromised patients, including patients with transplants, AIDS or HIV infection by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing ulcers in the esophagus in immunocompromised patients, including patients with transplants, AIDS or HIV infection by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing idiopathic gastric acid hypersecretion in patients by administering at least one of the pyridine derivatives of the invention to a patient.", "The invention provides novel methods for treating and preventing gastroparesis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal motility disorders in patients by administering at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing Zollinger-Ellison syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating short bowel syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing emesis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing regurgitation in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing early satiety in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing chronic sore throat in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing abdominal pain in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing abdominal bloating in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing nausea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing sour stomach in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing diarrhea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing constipation in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing bacterial infections in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing refractory ulcers in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides on demand relief of symptoms associated with gastroesophageal reflux disease (GERD) in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides relief from symptoms caused by the consumption of excessive amounts of food and/or alcohol in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing Barrett's esophagus in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal disorders induced by NSAIDs in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal disorders caused by steroids in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention also provides novel methods for treating and preventing gastrointestinal disorders caused by cholinergic compounds in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing fungal-induced or viral-induced ulcers in the gastrointestinal tract in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides a novel diagnostic tool for suppressing gastric acid.", "The invention provides novel methods for treating parasitic infections, such as malaria, in patients and for modulating the growth of parasites by administering an effective amount of at least one of the pyridine derivatives of the invention.", "The invention is described in more detail below.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "RELATED APPLICATIONS The present application claims priority to U.S.", "Provisional Application No.", "60/243,278 filed Oct. 26, 2000, and U.S.", "Provisional Application No.", "60/212,637 filed Jun.", "19, 2000.FIELD OF THE INVENTION The invention provides safe and effective methods for treating and preventing gastrointestinal diseases and other disorders in patients by administering one or more pyridine derivatives.", "In preferred embodiments, the pyridine derivative is rabeprazole, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.", "In other preferred embodiments, the pyridine derivative is rabeprazole sodium or ACIPHEX®.", "BACKGROUND OF THE INVENTION Duodenal and gastric ulcers, known collectively as peptic ulcers, are localized erosions of the mucous membrane of the duodenum and stomach, respectively, which expose the underlying layers of the gut wall to the acid secretions of the stomach and to the proteolytic enzyme pepsin.", "They are believed to be caused by an imbalance between offensive factors, such as acid or pepsin, and defensive factors, such as resistance of the mucous membrane.", "Peptic ulceration is a common disease of the gastrointestinal tract and it is estimated that approximately 10 to 20% of the adult male population will experience peptic ulceration at some time in their lives.", "Proton pump inhibitors, such as ACIPHEX® (Eisai Inc., Teaneck, N.J.), have proven to be successful in treating peptic ulcers.", "ACIPHEX® is described in U.S. Pat.", "No.", "5,045,552, the disclosure of which is incorporated herein by reference in its entirety.", "There is a need in the art for new and improved treatments for other gastrointestinal diseases and disorders.", "The invention is directed to these, as well as other, important ends.", "SUMMARY OF THE INVENTION The invention provides methods of treating and preventing asthma in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing laryngitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing symptomatic gastroesophageal reflux disease in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing pregnancy-induced gastroesophageal reflux disease in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing noncardiac chest pains in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing coughing in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention also provides novel methods for treating and preventing bronchitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing apnea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing dyspepsia in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing inflammatory bowel disease, including ulcerative colitis and Crohn's disease, in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing irritable bowel syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastritis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastroesophageal reflux disease and peptic ulcers in infants and children by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing stress ulcers in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing bleeding peptic ulcers and for treating and preventing acute gastrointestinal bleeding in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing infectious enteritis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing collagenous colitis and lymphocytic colitis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing chronic diarrhea in immunocompromised patients, including patients with transplants, AIDS or HIV infection by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing ulcers in the esophagus in immunocompromised patients, including patients with transplants, AIDS or HIV infection by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing idiopathic gastric acid hypersecretion in patients by administering at least one of the pyridine derivatives of the invention to a patient.", "The invention provides novel methods for treating and preventing gastroparesis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal motility disorders in patients by administering at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing Zollinger-Ellison syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating short bowel syndrome in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing emesis in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing regurgitation in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing early satiety in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing chronic sore throat in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing abdominal pain in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing abdominal bloating in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing nausea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing sour stomach in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing diarrhea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing constipation in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing bacterial infections in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing refractory ulcers in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides on demand relief of symptoms associated with gastroesophageal reflux disease (GERD) in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides relief from symptoms caused by the consumption of excessive amounts of food and/or alcohol in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing Barrett's esophagus in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal disorders induced by NSAIDs in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing gastrointestinal disorders caused by steroids in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention also provides novel methods for treating and preventing gastrointestinal disorders caused by cholinergic compounds in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides novel methods for treating and preventing fungal-induced or viral-induced ulcers in the gastrointestinal tract in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides a novel diagnostic tool for suppressing gastric acid.", "The invention provides novel methods for treating parasitic infections, such as malaria, in patients and for modulating the growth of parasites by administering an effective amount of at least one of the pyridine derivatives of the invention.", "The invention is described in more detail below.", "DETAILED DESCRIPTION OF THE INVENTION “Patient” includes animals, preferably mammals, more preferably humans.", "“Patient” includes infants, children and adults, and includes males and females.", "“Gastroesophageal Reflux Disease” or “GERD” refers to a clinical syndrome involving the reflux of gastric contents into the esophagus, and is generally characterized by one or more symptoms of heartburn, coughing, wheezing, hoarseness, regurgitation, epigastric pain, dysphagia, and chest pain.", "The invention provides methods of treating and preventing asthma in infants, children and adults by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "GERD is common in patients with asthma, although no causal relationship between the two diseases has been proven.", "In other embodiments, the invention provides methods for treating and preventing asthma in patients who are also diagnosed with GERD.", "The invention provides methods for treating and preventing laryngitis by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "In other embodiments, the invention provides methods for treating and preventing laryngitis in patients who are also diagnosed with GERD.", "The invention also provides methods for preventing and treating laryngeal carcinoma by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides methods for treating and preventing Barrett's esophagus by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Barrett's esophagus is a condition in which the stratified squamous epithelium of the esophagus is replaced by a columnar epithelium with malignant potention.", "The invention provides methods for treating and preventing symptomatic gastroesophageal reflux disease (GERD) by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Symptomatic GERD is characterized by the presence of symptoms of GERD, most commonly heartburn, which are related to the reflux of gastric contents into the esophagus.", "Symptomatic GERD is distinguished from GERD (or erosive GERD) by the absence of erosions in the esophageal mucosa.", "The invention provides methods for preventing and treating pregnancy-induced gastroesophageal reflux disease by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a pregnant female patient in need thereof.", "The invention provides methods for treating and preventing noncardiac chest pains by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "In other embodiments, the invention provides methods for treating and preventing noncardiac chest pains in patients who are also diagnosed with GERD.", "The invention provides methods for treating and preventing coughing, preferably chronic coughing, by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "In other embodiments, the invention provides methods for treating and preventing coughing in patients who are also diagnosed with GERD.", "The invention also provides methods for treating and preventing bronchitis.", "The invention provides methods for treating and preventing apnea in patients by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The patient can be an infant, child or adult.", "In one embodiment, the patient is preferably an infant.", "The term “infant” includes neonates.", "The invention provides methods for treating and preventing dyspepsia, preferably non-ulcer or functional dyspepsia, by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Dyspepsia refers to upper abdominal pain or discomfort and can also include symptoms of nausea, early satiety and bloating.", "Dyspepsia can be episodic or chronic.", "The invention provides methods for treating and preventing inflammatory bowel disease by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Inflammatory bowel disease refers to chronic inflammatory disorders involving the gastrointestinal tract, and is characterized by symptoms of diarrhea, bloody diarrhea, perianal sepsis, and/or abdominal pain.", "Inflammatory bowel disease includes ulcerative colitis and Crohn's disease.", "Ulcerative colitis is an inflammatory reaction primarily involving the colonic mucosa, and is characterized by symptoms of bloody diarrhea, abdominal pain, fever, and/or weight loss.", "Crohn's disease is a chronic inflammation extending through all layers of the intestinal wall and involving the mesentery and regional lymph nodes and can involve the small bowel and/or colon.", "Crohn's disease is characterized by symptoms of fever, abdominal pain, diarrhea often without blood, fatigue, and/or weight loss.", "The invention provides methods for treating and preventing irritable bowel syndrome by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Irritable bowel syndrome is a common gastrointestinal disease characterized by three clinical variants: (i) chronic abdominal pain and constipation, (ii) chronic intermittent diarrhea, often without pain, and (iii) alternating constipation and diarrhea, with or without abdominal pain.", "The invention provides methods for treating and preventing gastritis by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "“Gastritis” refers to inflammation of the gastric mucosa.", "The term “gastritis” includes acute gastritis and chronic gastritis.", "The invention provides methods for treating and preventing gastroesophageal reflux disease and peptic (gastric and duodenal) ulcers in infants and children by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient who is an infant or child.", "The term “infants” includes neonates, and the term “children” includes adolescents.", "The invention provides methods for treating and preventing stress ulcers by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Stress ulcers are clinically distinct from peptic ulcers.", "Patients with stress ulcers often have multiple lesions in the acid-secreting portion of the stomach, in the antrum and/or the duodenum, and the lesions may be bleeding.", "Stress ulcers may be present in patients with severe injuries, burns, infections and/or shock.", "The invention provides methods for treating and preventing bleeding peptic ulcers and for treating and preventing acute gastrointestinal bleeding by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The bleeding peptic ulcers may be duodenal or gastric, or stomal ulcers.", "Gastrointestinal bleeding is a general term referring to bleeding from anywhere in the gastrointestinal tract.", "Many cases are due to peptic ulcers, but other causes are esophageal and intestinal bleeding.", "Bleeding is a potential complication of peptic ulcers, and is often associated with more severe ulcers.", "The invention provides methods for treating and preventing infectious enteritis by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Infectious enteritis refers to pathogen-induced diarrhea.", "Infectious enteritis can be caused by an infection from, for example, Campylobacter species, Shigella species, Yersinia species, such as Yersinia enterocolitica, Cryptosporidium species, Giardia species, such as Giardia lamblia, Salmonella species, Pseudomonas species, such as Pseudomonas aeruginosa, and the like.", "The invention provides methods for treating and preventing collagenous colitis and lymphocytic colitis by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Both conditions are characterized by signs of mucosal inflammation and symptoms of chronic watery diarrhea.", "The invention provides methods for treating and preventing chronic diarrhea and esophageal ulcers in immunocompromised patients, including patients with transplants, AIDS or HIV infection, by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "The invention provides methods for treating and preventing idiopathic gastric acid hypersecretion by administering at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides methods for treating and preventing gastroparesis by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Gastroparesis is delayed gastric emptying of either solids or liquids, and is accompanied by symptoms of postprandial nausea, epigastric pain/burning, bloating, early satiety, excessive eructation, anorexia and vomiting.", "The invention provides methods for treating and preventing gastrointestinal motility disorders by administering at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides methods for treating and preventing Zollinger-Ellison syndrome by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "Zollinger-Ellison syndrome refers to ulcer disease of the upper gastrointestinal tract, marked increases in gastric acid secretion, and/or nonbeta islet cell tumors of the pancreas.", "The invention provides methods for treating short bowel syndrome by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides methods for treating and preventing emesis in infants, children and adults by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention.", "In other embodiments, the invention provides methods for treating and preventing emesis in patients who are also diagnosed with GERD.", "In still other embodiments, the invention provides methods for treating and preventing emesis induced by chemotherapeutic agents.", "The invention provides methods for treating and preventing regurgitation, early satiety, chronic sore throat, abdominal pain, abdominal bloating, nausea, sour stomach, diarrhea or constipation by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides methods for treating and preventing gastrointestinal bacterial infections by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "In preferred embodiments, the gastrointestinal bacterial infection is caused by Helicobacter pylori.", "The invention provides methods for treating and preventing refractory ulcers by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The ulcers can be peptic ulcers (e.g., gastric ulcers and duodenal ulcers), and can be present in infants, children or adults.", "Refractory ulcers are generally defined as ulcers that fail to completely heal after daily treatment with 1 gram of cimetidine for three months.", "The invention provides on demand relief of symptoms associated with gastroesophageal reflux disease (GERD).", "Indications for the treatment of GERD with pyridine derivatives, such as rabeprazole or stereoisomers thereof, require daily administration for four to eight weeks.", "It has been unexpectedly discovered that pyridine derivatives, such as rabeprazole or stereoisomers thereof, can be administered on a one-time basis to treat occasional symptoms of GERD.", "For example, rabeprazole or stereoisomers thereof can be administered before, during or after a meal to provide relief from GERD symptoms caused by the meal.", "The invention provides relief from symptoms caused by the consumption of excessive amounts of food and/or alcohol by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The symptoms can be one or more of abdominal bloating, abdominal pain, regurgitation, emesis, nausea, sour stomach, and the like.", "The invention provides methods for treating and preventing gastrointestinal disorders (e.g., peptic ulcers) induced by NSAIDs (non-steroidal antiinflammatory drugs) by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "All NSAIDs have the potential to cause damage to the gastrointestinal tract, and have been associated with inducing peptic ulcers (e.g., gastric and duodenal ulcers) and gastrointestinal bleeding.", "NSAIDs cause gastrointestinal damage by two mechanisms: (1) a topical effect that is pH and pKa related, and (2) a systemic effect mediated by cyclooxygenase (COX) inhibition with a reduction in prostaglandin synthesis.", "Administering one or more of the pyridine derivatives of the invention can heal gastrointestinal ulcers caused by NSAIDs.", "The invention provides methods for treating and preventing gastrointestinal disorders (e.g., peptic ulcers) caused by steroids by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "There is a well known correlation between steroid therapy and peptic ulcer disease.", "Messer et al, N. Engl.", "J.", "Med., 309(1):21-24 (1983); Wolf et al, J. Pediatr.", "Gastroenterol.", "Nutr., 12(2):269-271 (1991).", "In other embodiments, the invention provides methods for treating peptic ulcers (e.g., gastric or duodenal) induced by corticosteroids.", "The invention also provides methods for treating and preventing gastrointestinal disorders (e.g., peptic ulcers) caused by cholinergic compounds (e.g., bethanecol, metoclopramide and the like) by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention also provides novel methods for treating fungal-induced or viral-induced ulcers in the gastrointestinal tract by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides a novel diagnostic tool for suppressing gastric acid.", "For example, the pyridine derivatives of the invention could be used in the diagnosis of GERD or non-cardiac chest pains.", "The invention also provides novel methods for treating parasitic infections by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The parasitic infection is preferably caused by a protozoan parasite, more preferably by Plasmodium falciparum.", "In other embodiments, the invention provides methods for treating malaria by administering a therapeutically effective amount of at least one of the pyridine derivatives of the invention to a patient in need thereof.", "The invention provides methods for modulating the growth of parasites by administering an effective amount of at least one of the pyridine derivatives of the invention.", "The parasite is preferably a protozoan, more preferably Plasmodium falciparum.", "“Modulating the growth of parasites” includes inhibiting the growth of parasites; reducing the rate at which the parasites reproduce or grow (i.e., compared to untreated parasites); and/or killing the parasites.", "The growth of parasites can be modulated in vitro or in vivo.", "The pyridine derivatives useful in the methods described herein are preferably compounds of formula (I), pharmaceutically acceptable salts thereof, or stereoisomers thereof: wherein R1 and R2 are each independently a hydrogen atom, a halogen atom, a lower alkyl, lower alkoxy, halogenated lower alkyl, lower alkoxycarbonyl or carboxyl group; X is —O—, —S— or ═N—R3, wherein R3 is a hydrogen atom or a lower alkyl, phenyl, benzyl or lower alkoxycarbonyl group; and Z is: 1.—O(CH2)p—O—R4 wherein p is an integer of 1 to 3 and R4 is hydrogen atom or a lower alkyl, aryl or aralkyl group, 2.—O—(CH2)q—R5 wherein q is an integer of 1 to 3 and R5 is a halogen atom or an alkoxycarbonyl, aryl or heteroaryl group, 3.—O—(CH2)r—O—(CH2)s—O—R6 wherein r and s are each independently an integer of 1 to 5 and R6 is a hydrogen atom or a lower alkyl group, wherein t is an integer of 0 to 2, and A is a lower alkyl, alkoxycarbonylmethyl, pyridyl, furyl, wherein B is —NH—, —O— or —S—, and w is an integer of 0 or 1; 8.—N(R8)—CH2—C6H5 wherein R5 is an acetoxy or lower alkyl group; 9.—OR9 wherein R9 is a hydrogen atom, a lower alkyl or aryl group; n is an integer of 0 to 2; m is an integer of 2 to 10, and J and K are each independently a hydrogen atom or a lower alkyl group, with the proviso that when Z is a group falling under the above category (9), then R9 is a lower alkyl group and m stands for an integer of 3 to 10, and pharmaceutically acceptable salts thereof.", "The same definitions for R1, R2, X, n, J, K, Z and m are used throughout the specification that follows and in the appended claims.", "Also disclosed are pharmaceutical compositions containing one or more of these compounds as the active ingredient(s) in a pharmaceutically acceptable carrier, adjuvant or vehicle.", "In the definition of the compounds of formula (I), the lower alkyl group defined with respect to R1, R2, R3, R4, R6, R7, R8, A, J and K may be a straight-chain or branched alkyl group having 1 to 6 carbon atoms.", "Examples include methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl, 1-methylpropyl, tert-butyl, n-pentyl, 1-ethylpropyl, isoamyl and n-hexyl groups, among which methyl and ethyl groups are most preferred.", "The lower alkoxy group and the lower alkoxy moiety of the lower alkoxycarbonyl group defined above with respect to R1 and R2 may be an alkoxy group derived form the above lower alkyl group.", "Methoxy and ethoxy groups are most preferred.", "The halogen atom defined above includes chlorine, bromine, iodine or fluorine.", "The aryl group defined above with respect to R4 and R5 may be phenyl, tolyl, xylyl, napthyl or the like, which may be substituted with a lower alkoxy or hydroxyl group, a halogen atom or the like.", "Examples of the arylalkyl defined above with respect to R4 include benzyl and phenethyl groups.", "Examples of the heteroaryl group defined above with respect to R5 include pyridyl and furyl groups.", "In the definition of Z in formula (I), groups 1, 2, 3, 4, 5 and 9 are preferred; and group 9 is the most preferred.", "As for R1 and R2, hydrogens for both and then a combination of a lower alkyl (e.g., methyl) for R1 and hydrogen for R2 are preferred.", "X is preferably ═NR3, where R3 is hydrogen.", "A preferred value for n is 1.The preferred substituents for J and K are both hydrogen or where J is lower alkyl (e.g., methyl), and K is hydrogen, or when J is hydrogen and K is lower alkyl (e.g., methyl).", "Thus, J or K are independently preferably hydrogen or methyl, most preferably J is methyl and K is hydrogen.", "A more preferred group of compounds falling within the scope of the compounds of formula (I) are compounds of formula (A), pharmaceutically acceptable salts thereof, or stereoisomers thereof: wherein R1, R2, J, m and R9 have the same meanings as defined above.", "In formula (A), the preferred R1 and R2 substituents are both hydrogen, or R1 is 5-lower alkoxy, 5-lower alkyl or 5-halogenated lower alkyl and R2 is hydrogen.", "The preferred substituent for J is hydrogen or methyl; the preferred value for m is in the range of 3 to 10, the most preferred being 3; and the preferred R9 substituent is lower alkyl (e.g., methyl), or aryl.", "Among these possibilities for the compounds of formula (A), the preferred combination is when R1 and R2 are both hydrogen, J is methyl, m is 3 and R9 is methyl.", "Another group of preferred compounds in formula (A) are combinations of the above substituents where both R1 and R2 are hydrogen, J is hydrogen, m is 3 and R9 is methyl.", "Another group of preferred compounds falling within formula (A) is when both R1 and R2 are hydrogen, J is methyl, m is 2 and R9 is benzyl.", "Another preferred group of compounds falling within the scope of formula (I) are compounds of formula (B), pharmaceutically acceptable salts thereof, or stereoisomers thereof: wherein R1, R2, J, p, m and R4 have the same meanings as given above.", "In formula (B), the preferred substituents for R1 and R2 are both hydrogen; or when R1 is 5-lower alkoxy, 5-lower alkyl or 5-halogenated lower alkyl, R2 is hydrogen.", "The preferred value of m is 2 or 3; the preferred value for p is 2 or 3; and the preferred substituent for R4 is methyl or benzyl.", "Of the above possibilities for formula (B), the most preferred combination is where R1 is 5-methyl, R2 is hydrogen, J is methyl, m is 2, p is 2 and R4 is methyl.", "In a most preferred embodiment, the compound of formula I is a compound of formula (C), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: Preferably, the compound of formula (C) is a sodium salt, which is known as rabeprazole sodium or ACIPHEX® (Eisai Inc., Teaneck, N.J.).", "Although the compounds of the invention may be present as a hydrate or as a stereoisomer, it is a matter of course that these hydrates and stereoisomers are also included in the scope of the invention.", "For example, the compound of formula (C) can be a compound of formula (D) or a pharmaceutically acceptable salt thereof (e.g., a sodium salt): The compound of formula (D) is also known as R (+) rabeprazole.", "Alternatively, the compound of formula (C) can be a compound of formula (E) or a pharmaceutically acceptable salt thereof (e.g., a sodium salt): The compound of formula (E) is also known as S (−) rabeprazole.", "As described above, the compounds of the invention can be administered as a pharmaceutically acceptable salt.", "Pharmaceutically acceptable salts are known in the art and include those of inorganic acids, such as hydrochloride, sulfate, hydrobromide, sulfate, and phosphate; those of organic acids, such as formate, acetate, maleate, tartrate, trifluoroacetate, methanesulfonate, benzenesulfonate and toluenesulfonate, and those of amino acids such as arginine, aspartic acid and glutamic acid.", "When certain substituents are selected, the compounds of the invention may form, for example, alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic amine salts, such as a salt with trimethyl-amine, triethylamine, pyridine, picoline, dicyclohexylamine or N,N′-dibenzylethylene-diamine.", "One skilled in the art will recognize that the compounds of the invention can be made in the form of any of these or of any other pharmaceutically acceptable salt.", "For example, compounds represented by formula (I), wherein X is ═N—R3 and R3 is a hydrogen atom, or compounds represented by formula (I), wherein Z is a group falling under the category 7 and B is a group of —NH—, can be present as a metal salt, such as Na, K, Mg or Ca.", "The pyridine derivatives of the invention can be prepared by processes that are known in the art and described, for example, in U.S. Pat.", "No.", "5,045,552, the disclosure of which is incorporated by reference herein in its entirety.", "Rabeprazole sodium, a preferred pyridine derivative for use in the methods described herein, is commercially available as ACIPHEX® from Eisai Inc., Teaneck, N.J. Methods for preparing R (+) rabeprazole are described in WO 99/55157, the disclosure of which is incorporated by reference herein in its entirety.", "Methods for preparing S (−) rabeprazole are described in WO 99/55158, the disclosure of which is incorporated by reference herein in its entirety.", "A therapeutically effective dosage regimen for treating the diseases described herein with the pyridine derivatives described herein is selected in accordance with a variety of factors, including the age, weight, sex, and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular pyridine derivative used, whether a drug delivery system is used and whether the pyridine derivative is administered as part of a drug combination.", "The pyridine derivatives described herein may be administered in amounts of about 0.01 to about 200 mg per day, preferably about 0.05 to about 50 mg per day, more preferably about 0.1 to about 40 mg per day, still more preferably about 10 to about 30 mg per day, most preferably about 20 mg per day.", "The compounds and/or compositions may be administered once a day or in divided doses, for example from 2 to 4 times a day, most preferably once per day.", "One skilled in the art will recognize that when the pyridine derivatives of the invention are administered to infants or children, the dose may be smaller than the dose administered to adults, and that the dose can be dependent upon the size and weight of the patient.", "In the methods for treating and preventing refractory ulcers, for treating and preventing Zollinger-Ellison syndrome, and for treating and preventing idiopathic gastric acid hypersecretion described herein, the patient may be administered at least one of the pyridine derivatives of the invention in amounts of about 1 to about 800 mg per day, preferably about 10 to about 300 mg per day, more preferably about 20 to about 200 mg per day, still more preferably about 40 to about 150 mg per day, most preferably about 60 to about 120 mg per day.", "In preferred embodiments of the methods described herein, rabeprazole sodium, which is commercially available as ACIPHEX® (Eisai Inc., Teaneck, N.J.), is administered as a delayed-release, enteric-coated tablet containing 20 milligrams rabeprazole sodium.", "The tablets can be administered one to about four times a day.", "In preferred embodiments, one 20 milligram ACIPHEX® tablet is administered once a day for the methods described herein.", "One skilled in the art will appreciate that when rabeprazole sodium is administered to infants or children, the dose may be smaller than the dose that is administered to adults.", "The pyridine derivatives of the invention can be administered orally, topically, parenterally, by inhalation (nasal or oral), or rectally in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.", "The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques.", "Preferably, the pyridine derivatives of the invention are orally administered as tablets.", "Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents, suspending agents (e.g., methylcellulose, Polysorbate 80, hydroxyethylcellulose, acacia, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate and the like), pH modifiers, buffers, solubilizing agents (e.g., polyoxyethylene hydrogenated castor oil, Polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, Macrogol, an ethyl ester of castor oil fatty acid, and the like), preservatives and/or stabilizers.", "The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.", "Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution.", "In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium.", "For this purpose any bland fixed oil may be used including synthetic mono- or diglycerides, in addition, fatty acids such as oleic acid find use in the preparation of injectables.", "The preparations can be lyophilized by methods known in the art.", "Solid dosage forms for oral administration may include capsules, tablets, sublingual tablets, powders, granules and gels; most preferably tablets.", "The solid dosage form may be a solid microencapsulated dosage, such as a microencapsulated powder, microencapsulated granules or a microencapsulated gel.", "A solid dosage form for oral administration can be prepared by mixing an active principle with filler and, if necessary, binder, disintegrating agent, lubricant, coloring agent, corrigent or the like and converting the obtained mixture into a tablet, coated tablet, granule, powder or capsule.", "Examples of the filler include lactose, corn starch, sucrose, glucose, sorbitol, crystalline cellulose and silicon dioxide, while those of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, acacia, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylstarch and polyvinylpyrrolidone.", "Examples of the disintegrating agent include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogencarbonate, calcium citrate, dextrin and pectin, while those of the lubricant include magnesium stearate, talc, polyethylene glycol, silica and hardened vegetable oils.", "The coloring agent may be any one which is permitted to be added to drugs.", "Examples of the corrigent include cacao powder, mentha herb, aromatic powder, mentha oil, borneol and powdered cinnamon bark.", "The tablets and granules may be, if necessary, coated with sugar, gelatin or the like.", "Preferably, the tablets have an enteric coating.", "In addition to the active ingredient, the tablets preferably comprise mannitol, hydroxypropyl cellulose, magnesium oxide, low-substituted hydroxypropyl cellulose, magnesium stearate, ethylcellulose, hydroxypropyl methylcellulose phthalate, diacetylated monoglycerides, talc, titanium dioxide, carnauba wax and a coloring agent, such as ferric oxide.", "In other embodiments, the solid dosage form can be packaged as granules or a powder in a pharmaceutically acceptable carrier, where the granules or powder are removed from the packaging and sprinkled on food or mixed with a liquid, such as water or juice.", "In this embodiment, the active compound can be mixed with flavoring or sweetening agents.", "The packaging material can be plastic, MYLAR® (DuPont), coated paper, or any material that prevents water or moisture from reaching the granules and/or powder.", "Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, and syrups containing inert diluents commonly used in the art, such as water.", "The liquid dosage form may be a microencapsulated liquid, including microencapsulated emulsions, microencapsulated solutions, microencapsulated suspensions and microencapsulated syrups.", "Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.", "For administration by oral or nasal inhalation, the compositions of the invention can be delivered from an insufflator, a nebulizer or a pressured pack or other convenient mode of delivering an aerosol spray.", "Pressurized packs can include a suitable propellant.", "Alternatively, for administration by oral or nasal inhalation, the compositions can be administered in the form of a dry powder composition or in the form of a liquid spray.", "Suppositories for rectal administration can be prepared by mixing one or more pyridine derivatives of the invention with suitable nonirritating excipients, such as cocoa butter and/or polyethylene glycols, that are solid at room temperature and that melt at body temperature.", "For topical administration to the epidermis, the pyridine derivatives of the invention can be formulated as ointments, creams or lotions, or as the active ingredient of a transdermal patch.", "Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.", "Lotions may be formulated with an aqueous or oily base and can also generally contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, and/or coloring agents.", "The pyridine derivatives can also be administered via iontophoresis.", "While the pyridine derivatives of the invention can be administered as the sole active pharmaceutical agent in the methods described herein, they can also be used in combination with one or more compounds which are known to be therapeutically effective against the specific disease that one is targeting for treatment.", "Each of the patents and publications cited herein are incorporated by reference herein in their entirety.", "It will be apparent to one skilled in the art that various modifications can be made to the invention without departing from the spirit or scope of the appended claims." ] ]

[ [ "Polymeric, fiber matrix delivery systems for bioactive compounds", "Multifunctional systems for delivery of bioactive compounds incorporated within or between polymeric fibers in a matrix are provided.", "Also provided are methods of delivering bioactive compounds via implementation, coating and/or wrapping of these systems and methods for modulating the rate of release of bioactive compounds from these delivery systems." ], [ "1.A system for delivery of bioactive compounds comprising a bioactive compound incorporated within or between polymeric fibers.", "2.The system of claim 1 which is biodegradable.", "3.The system of claim 1 which is nondegradable.", "4.The system of claim 1 wherein the fibers are arranged as a matrix or linear assembly, a film coating on a device, or a braided or woven structure.", "5.The system of claim 1 wherein particles of the bioactive compound are suspended in a polymer solution prior to electrospinning of the polymeric fibers so that the bioactive compound is incorporated between the polymeric fibers.", "6.The system of claim 1 wherein the bioactive compound is dissolved into a polymer solution prior to electrospinning of the polymeric fibers so that the bioactive compound is incorporated within the polymeric fibers.", "7.The system of claim 1 comprising more than one bioactive compound incorporated into a single or multiple layers of polymeric fibers for delivery of the bioactive compounds sequentially or in concert.", "8.A method for delivering bioactive compounds to a patient comprising incorporating a bioactive compound into a polymeric fiber matrix or linear assembly or a braided or woven structure and implanting the polymer fiber matrix or linear assembly or braided or woven structure into the patient.", "9.The method of claim 8 further comprising coating a device with the polymeric fiber matrix or linear assembly or braided or nonwoven structure and implanting the coated device into the patient for delivery of the bioactive compounds.", "10.The method of claim 9 wherein the device comprises a tissue engineering device and the bioactive compound enhances cell attachment and growth to the device.", "11.The method of claim 8 wherein the polymeric fiber matrix or linear assembly or a braided or woven structure is implanted directly on a wound of the patient to deliver the bioactive compound to the wound of the patient.", "12.The method of claim 8 wherein the polymeric fiber matrix or linear assembly or a braided or woven structure is implanted on the surface of an organ, tissue or vessel of the patient to deliver the bioactive compound to the organ, tissue or vessel of the patient.", "13.The method of claim 12 wherein the polymeric fiber matrix or linear assembly or a braided or woven structure is wrapped around the surface of an organ, tissue or vessel of the patient.", "14.A method for modulating rate of release of a bioactive compound from a delivery system for bioactive compounds comprising a bioactive compound incorporated within or between polymeric fibers, said method comprising modulating loading of the bioactive compound incorporated with or between polymeric fiber, selecting polymers to produce polymeric fibers which degrade at varying rates, varying diameter of the polymeric fibers, or varying polymeric concentration of the polymeric fibers." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>A number of polymer matrices for use in the controlled release and/or delivery of bioactive compounds, and for particular drugs, have been described.", "U.S. Pat.", "No.", "3,991,766 describes a medicament repository consisting of a surgical element in the form of tubes, sheets, sponges, gauzes or prosthetic devices of polyglycolic acid having incorporated therein an effective amount of a medicament.", "U.S. Pat.", "No.", "4,655,777 describes a method for producing a biodegradable prothesis or implant by encasing an effective amount of fibers of calcium phosphate or calcium aluminate in a matrix of polymer selected from the group consisting of polyglycolide, poly(DL-lactide), poly(L-lactide), polycaprolactone, polydioxanone, polyesteramides, copolyoxalates, polycarbonates, poly(glutamic-co-leucine) and blends, copolymers and terpolymers thereof to form a composite.", "U.S. Pat.", "No.", "4,818,542 discloses a method for preparing a spherical microporous polymeric network with interconnecting channels having a drug distributed within the channels.", "U.S. Pat.", "No.", "5,128,170 discloses a medical device and methods for manufacturing medical devices with a highly biocompatible surface wherein hydrophillic polymer is bonded onto the surface of the medical device covalently through a nitrogen atom.", "U.S. Pat.", "No.", "5,545,409 discloses a composition and method for controlled release of water-soluble proteins comprising a surface-eroding polymer matrix and water-soluble bioactive growth factors.", "U.S. Pat.", "No.", "5,769,830 discloses synthetic, biocompatible, biodegradable polymer fiber scaffolds for cell growth.", "Fibers are spaced apart by a distance of about 100 to 300 microns for diffusion and may comprise polyanhydrides, polyorthoesters, polyglycolic acid or polymethacrylate.", "The scaffolds may be coated withe materials such as agar, agarons, gelatin, gum arabic, basement membrane material, collagen type I, II, III, IV or V, fibronectin, laminin, glycosaminoglycans, and mixtures thereof.", "U.S. Pat.", "No.", "5,898,040 discloses a polymeric article for use in drug delivery systems which comprises a polymeric substrate with a highly uniform microporous polymeric surface layer on at least part of the substrate.", "Encapsulation of a bioactive compound within a polymer matrix has also been described.", "For example, WO 93/07861 discloses polymer microspheres of 50 to 100 microns comprising a compound contained in a fixed oil within the polymer microsphere.", "U.S. Pat.", "No.", "5,969,020 discloses a foam precursor comprising a crystalline thermoplastic polymer and solid crystalline additive for use in preparation of drug delivery systems.", "Recently, it has been shown that polymer fibers of nanometer diameter can be electrospun from sulfuric acid into a coagulation bath (Reneker, D. H. and Chun, I. Nanotechnology 1996 7:216).", "In these studies more than 20 polymers including polyethylene oxide, nylon, polyimide, DNA, polyaramide and polyaniline were electrospun into electrically charged fibers which were then collected in sheets or other useful geometrical forms.", "Electrospinning techniques have also been applied to the production of high performance filters (Doshi, J. and Reneker, D. H. Journal of Electrostatics 1995 35:151; Gibson et al.", "AIChE Journal 1999 45:190) and for scaffolds in tissue engineering (Doshi, J. and Reneker, D. H. Journal of Electrostatics 1995 35:151; Ko et al.", "“The Dynamics of Cell-Fiber Architecture Interaction,” Proceedings, Annual Meeting, Biomaterials Research Society, San Diego, Calif., April 1998; and WO 99/18893).", "WO 99/18893 describes a method for preparing nanofibrils from both nondegrading and biodegradable polymers for use as tissue engineering scaffolds.", "The present invention relates to delivery systems for the controlled release of bioactive compounds which comprise polymeric fibers and the bioactive compound." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>An object of the present invention is to provide a system for delivery of bioactive compounds comprising a bioactive compound incorporated within or between a polymeric fiber matrix or linear assembly, film coating or braided/woven structure.", "Another object of the present invention is to provide a method for delivering a bioactive compound to a patient for controlled release of the bioactive compound in the patient.", "In one embodiment of this method of the present invention, the bioactive compound is incorporated into a polymeric fiber matrix or linear assembly or a braided or woven structure and implanted into the patient.", "In another embodiment, the bioactive compound is incorporated into a polymeric fiber film used to coat implants, tissue engineering scaffolds and other devices such as pumps and pacemakers which are then implanted into the patient.", "In yet another embodiment, the bioactive compound is incorporated into a polymeric fiber film used to wrap organs, tissues or vessels in a patient.", "Another object of the present invention is to provide methods for modulating the rate of release of a bioactive compound from a delivery system for bioactive compounds comprising a bioactive compound incorporated within or between polymeric fibers.", "These methods include modulating loading of the bioactive compound incorporated with or between polymeric fiber, selecting polymers to produce the polymeric fibers which degrade at varying rates, varying polymeric concentration of the polymeric fibers and varying polymeric fiber diameter.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to delivery systems comprising polymeric fiber matrices, film coatings or braided/woven structures for the controlled release of bioactive compounds.", "The delivery systems of the present invention may be comprised of either biodegradable or nondegrading polymeric fibers.", "In one embodiment, these fibers have submicron and/or micron diameters.", "Bioactive compounds are included in the delivery system either by suspending the compound particles or dissolving the compound in the polymer solution used to produce the fibers.", "BACKGROUND OF THE INVENTION A number of polymer matrices for use in the controlled release and/or delivery of bioactive compounds, and for particular drugs, have been described.", "U.S. Pat.", "No.", "3,991,766 describes a medicament repository consisting of a surgical element in the form of tubes, sheets, sponges, gauzes or prosthetic devices of polyglycolic acid having incorporated therein an effective amount of a medicament.", "U.S. Pat.", "No.", "4,655,777 describes a method for producing a biodegradable prothesis or implant by encasing an effective amount of fibers of calcium phosphate or calcium aluminate in a matrix of polymer selected from the group consisting of polyglycolide, poly(DL-lactide), poly(L-lactide), polycaprolactone, polydioxanone, polyesteramides, copolyoxalates, polycarbonates, poly(glutamic-co-leucine) and blends, copolymers and terpolymers thereof to form a composite.", "U.S. Pat.", "No.", "4,818,542 discloses a method for preparing a spherical microporous polymeric network with interconnecting channels having a drug distributed within the channels.", "U.S. Pat.", "No.", "5,128,170 discloses a medical device and methods for manufacturing medical devices with a highly biocompatible surface wherein hydrophillic polymer is bonded onto the surface of the medical device covalently through a nitrogen atom.", "U.S. Pat.", "No.", "5,545,409 discloses a composition and method for controlled release of water-soluble proteins comprising a surface-eroding polymer matrix and water-soluble bioactive growth factors.", "U.S. Pat.", "No.", "5,769,830 discloses synthetic, biocompatible, biodegradable polymer fiber scaffolds for cell growth.", "Fibers are spaced apart by a distance of about 100 to 300 microns for diffusion and may comprise polyanhydrides, polyorthoesters, polyglycolic acid or polymethacrylate.", "The scaffolds may be coated withe materials such as agar, agarons, gelatin, gum arabic, basement membrane material, collagen type I, II, III, IV or V, fibronectin, laminin, glycosaminoglycans, and mixtures thereof.", "U.S. Pat.", "No.", "5,898,040 discloses a polymeric article for use in drug delivery systems which comprises a polymeric substrate with a highly uniform microporous polymeric surface layer on at least part of the substrate.", "Encapsulation of a bioactive compound within a polymer matrix has also been described.", "For example, WO 93/07861 discloses polymer microspheres of 50 to 100 microns comprising a compound contained in a fixed oil within the polymer microsphere.", "U.S. Pat.", "No.", "5,969,020 discloses a foam precursor comprising a crystalline thermoplastic polymer and solid crystalline additive for use in preparation of drug delivery systems.", "Recently, it has been shown that polymer fibers of nanometer diameter can be electrospun from sulfuric acid into a coagulation bath (Reneker, D. H. and Chun, I. Nanotechnology 1996 7:216).", "In these studies more than 20 polymers including polyethylene oxide, nylon, polyimide, DNA, polyaramide and polyaniline were electrospun into electrically charged fibers which were then collected in sheets or other useful geometrical forms.", "Electrospinning techniques have also been applied to the production of high performance filters (Doshi, J. and Reneker, D. H. Journal of Electrostatics 1995 35:151; Gibson et al.", "AIChE Journal 1999 45:190) and for scaffolds in tissue engineering (Doshi, J. and Reneker, D. H. Journal of Electrostatics 1995 35:151; Ko et al.", "“The Dynamics of Cell-Fiber Architecture Interaction,” Proceedings, Annual Meeting, Biomaterials Research Society, San Diego, Calif., April 1998; and WO 99/18893).", "WO 99/18893 describes a method for preparing nanofibrils from both nondegrading and biodegradable polymers for use as tissue engineering scaffolds.", "The present invention relates to delivery systems for the controlled release of bioactive compounds which comprise polymeric fibers and the bioactive compound.", "SUMMARY OF THE INVENTION An object of the present invention is to provide a system for delivery of bioactive compounds comprising a bioactive compound incorporated within or between a polymeric fiber matrix or linear assembly, film coating or braided/woven structure.", "Another object of the present invention is to provide a method for delivering a bioactive compound to a patient for controlled release of the bioactive compound in the patient.", "In one embodiment of this method of the present invention, the bioactive compound is incorporated into a polymeric fiber matrix or linear assembly or a braided or woven structure and implanted into the patient.", "In another embodiment, the bioactive compound is incorporated into a polymeric fiber film used to coat implants, tissue engineering scaffolds and other devices such as pumps and pacemakers which are then implanted into the patient.", "In yet another embodiment, the bioactive compound is incorporated into a polymeric fiber film used to wrap organs, tissues or vessels in a patient.", "Another object of the present invention is to provide methods for modulating the rate of release of a bioactive compound from a delivery system for bioactive compounds comprising a bioactive compound incorporated within or between polymeric fibers.", "These methods include modulating loading of the bioactive compound incorporated with or between polymeric fiber, selecting polymers to produce the polymeric fibers which degrade at varying rates, varying polymeric concentration of the polymeric fibers and varying polymeric fiber diameter.", "DETAILED DESCRIPTION OF THE INVENTION Electrospinning is a simple and low cost electrostatic self-assembly method capable of fabricating a large variety of long, meter-length, organic polymer fibers with micron or submicron diameters, in linear, 2-D and 3-D architecture.", "Electrospinning techniques have been available since the 1930's (U.S. Pat.", "No.", "1,975,504).", "In the electrospinning process, a high voltage electric field is generated between oppositely charged polymer fluid contained in a glass syringe with a capillary tip and a metallic collection screen.", "As the voltage is increased, the charged polymer solution is attracted to the screen.", "Once the voltage reaches a critical value, the charge overcomes the surface tension of the suspended polymer cone formed on the capillary tip of the syringe and a jet of ultrafine fibers is produced.", "As the charged fibers are splayed, the solvent quickly evaporates and the fibers are accumulated randomly on the surface of the collection screen.", "This results in a nonwoven mesh of nano and micron scale fibers.", "Varying the charge density (applied voltage), polymer solution concentration, solvent used, and the duration of electrospinning can control the fiber diameter and mesh thickness.", "Other electrospinning parameters which may be varied routinely to effect the fiber matrix properties include distance between the needle and collection plate, the angle of syringe with respect to the collection plate, and the applied voltage.", "In the present invention, electrospinning is used to produce polymeric fiber matrices with the capability of releasing bioactive compounds in a controlled manner over a selected period of time.", "In one embodiment, the delivery system of the present invention is used to maintain delivery of a steady concentration of bioactive compound.", "In another embodiment, the delivery system is used in pulsed delivery of the bioactive compound wherein the compound is released in multiple phases in accordance with either rapid or slow degradation of the polymer fibers or diffusion of the bioactive compound from the polymer fibers.", "In yet another embodiment, the delivery system is used to obtain a delayed release of a bioactive compound.", "For example, the bioactive compound-containing fiber polymer matrix can be coated with a layer of nonwoven polymer fiber matrix with no bioactive compound.", "In this embodiment, different polymers with different degradation times can be used to obtain the desired time delays.", "The delivery systems of the present invention can be used to deliver a single bioactive compound, more than one Ibioactive compound at the same time, or more than one bioactive compound in sequence.", "Thus, as used herein, the phrases “a bioactive compound” and “the bioactive compound”, are meant to be inclusive of one or more bioactive compounds.", "For purposes of the present invention by “fiber” it is meant to include fibrils ranging in diameter from submicron, i.e.", "approximately 1 to 100 nanometers (10−9 to 10−7 meters) to micron, i.e.", "approximately 1-1000 micrometers.", "The bioactive compound is incorporated within the polymeric fibers either by suspension of compound particles or dissolution of the compound in the solvent used to dissolve the polymer prior to electrospinning of the polymeric fibers.", "For purposes of the present invention, by “incorporated within” it is meant to include embodiments wherein the bioactive compound is inside the fiber as well as embodiments wherein the bioactive compound is dispersed between the fibers.", "The polymeric fibers comprising the bioactive compound can be arranged as matrices, linear assemblies, or braided or woven structures.", "In addition, the fibers which release a bioactive compound can serve as film coatings for devices such as implants, tissue engineering scaffolds, pumps, pacemakers and other composites.", "These fiber assemblies can be spun from any polymer which can be dissolved in a solvent.", "The solvent can be either organic or aqueous depending upon the selected polymer.", "Examples of polymers which can be used in production of the polymeric fibers of the present invention include, but are not limited to, nondegradable polymers such as polyethylenes, polyurethanes, and EVA, and biodegradable polymers such as poly(lactic acid-glycolic acid), poly(lactic acid), poly(glycolic acid), poly(glaxanone), poly(orthoesters), poly(pyrolic acid) and poly(phosphazenes).", "Examples of bioactive compounds which can be incorporated into the polymeric fibers include any drug for which controlled release in a patient is desired.", "Some examples include, but are not limited to, steroids, antifungal agents, and anticancer agents.", "Other bioactive compounds of particular use in the present invention include tissue growth factors, angiogenesis factors, and anti-clotting factors.", "If the bioactive compound is to reside within or inside the polymer fiber, selection of the polymer should be based upon the solubility of the bioactive compound within the polymer solution.", "Water soluble polymers such as polyethylene oxide can be used if the bioactive compound also dissolves in water.", "Alternatively, hydrophobic bioactive compounds which are soluble in organic solvent such as steroids can be dissolved in an organic solvent together with a hydrophobic polymer such as polylactic glycolic acid (PLAGA).", "If the bioactive compound is to reside between the polymer fibers, dissolution of the bioactive compound in the polymer solution is not required.", "Instead, the bioactive compound can be suspended in the polymer solution prior to electrospinning of the fibers.", "In one embodiment of the present invention, the bioactive compound-containing fibers can be splayed directly onto devices such as implants, tissue engineering scaffolds, pumps and pacemakers as a film coating.", "For implants and tissue engineering scaffolds, examples of preferred bioactive compounds include tissue growth factors and angiogenesis factors.", "For pumps or pacemakers, the bioactive compound may comprise an anti-clotting factor.", "The coated device is then implanted into a patient wherein the bioactive compound or compounds are released upon degradation of or by diffusion from, or combinations thereof, the polymeric fiber film.", "In another embodiment, a matrix or linear assembly of the bioactive compound-containing fibers is prepared.", "In this embodiment, the matrix or linear assembly of bioactive compound-containing fibers can be sandwiched between layers of polymer which contain no bioactive compound to decrease any burst effect and/or to obtain a delayed release.", "Alternatively, the matrix may comprise layers of fibers containing different bioactive compounds.", "The matrix or linear assembly is then implanted into a patient for controlled release of the bioactive compound as the polymeric fibers degrade or as the bioactive compound diffuses from the polymeric fibers.", "The time delay can be controlled by varying the choice of polymer used in the fibers, the concentration of polymer used in the fiber, the diameter of the polymeric fibers, and/or the amount of bioactive compound loaded in the fiber.", "For purposes of the present invention, by “implanting” or “implanted” as used herein, it is meant to be inclusive of placement of the delivery systems of the present invention into a patient to achieve systemic delivery of the bioactive compound, as well as placement of the delivery system into a patient to achieve local delivery.", "For example, the delivery systems of the present invention may be placed on the wound of a patient to enhance healing via release of the bioactive compound.", "Delivery systems may also be placed on the surface or wrapped around an organ, tissue or vessel for delivery of the bioactive compound to the organ tissue or vessel.", "In another embodiment of the present invention, a braided, knitted or woven structure of bioactive compound-containing fibers is prepared.", "These structures are prepared using an extension of the traditional 2-dimensional braiding technology in which fabric is constructed by the intertwining or orthogonal interlacing of yarns to form an integral structure through position displacement.", "A wide range of 3-dimensional structures comprising the bioactive compound-containing fibers can be fabricated in a circular or rectangular loom.", "In this embodiment, the structure may comprise only bioactive compound-containing fibers, bioactive compound-containing fibers sandwiched between polymeric fibers which contain no bioactive compound, or a mixtures of fibers containing different bioactive compounds.", "Like the matrix or linear assembly, this structure can be implanted into a patient for controlled release of the bioactive compound or compounds as the polymeric fibers degrade or as the bioactive compound diffuses from the polymeric fibers.", "Again, delivery rate of the bioactive compound can be controlled by varying the choice of polymer used in the fibers, the concentration of polymer used in the fiber, the diameter of the polymeric fibers, and/or the amount of bioactive compound loaded in the fiber.", "Accordingly, the present invention also relates to methods for modulating the rate of release of a bioactive compound from a delivery system for bioactive compounds comprising a bioactive compound incorporated within or between polymeric fibers.", "By “modulate” or “modulating”, it is meant that the rate or release of the bioactive compound incorporated within of between the polymeric fibers of the delivery system is increased or decreased.", "Methods for modulating the rate of release include increasing or decreasing loading of the bioactive compound incorporated within or between the polymeric fibers, selecting polymers to produce the polymeric fibers which degrade at varying rates, varying polymeric concentration of the polymeric fibers and/or varying diameter of the polymeric fibers varying one or more of these parameters can be performed routinely by those of skill in the art based upon teachings provided herein.", "The ability of systems of the present invention to release a bioactive compound in a controlled manner was demonstrated using polymeric fiber matrices containing fluorescently labeled bovine serum albumin (FITC-BSA) dispersed between the fibers of the matrix.", "To construct the bioactive compound-loaded matrices, various concentrations of finely ground FITC-BSA were suspended in biodegradable polymer polylactic glycolic acid in 50:50 dimethyl formamide:tetrahydrofuran.", "Suspensions contained in a glass syringe with a capillary tip were electrospun into approximately 500 nm diameter fibers via an electrostatic based self-assembly process in which a high voltage electric field was generated between the oppositely charged polymer and a metallic collection screen.", "At a critical voltage the charge overcomes the surface tension of the deformed polymer drop at the needle tip, producing an ultrafine jet.", "The similarly charged fibers are splayed and during their passage to the screen, the solvent quickly evaporates so that dry fibers accumulate randomly on the screen forming a mesh matrix.", "The material properties of this mesh matrix of bioactive compound-containing fibers were examined via standard electron microscopy and tensile testing.", "It was found that tensile strength and the release profiles were a function of protein loading.", "In vitro release of the FITC-BSA into an infinite sink of 37° C. phosphate buffered saline was also measured.", "This sink mimics in vivo conditions.", "While release in the first 24 hours after initiation was dominant, release to over 120 hours was observed with an increase in release at the point where the fibers started to breakdown.", "The following nonlimiting examples are provided to further illustrate the present invention.", "EXAMPLES Example 1 Preparation of Fiber Matrix Containing BSA-FITC A 25% (w/v) solution of polylactic glycolic acid was prepared in a 50:50 mixture of dimethylformamide and tetrahydrofuran.", "A mixture of FITC-BSA and BSA in the ratio of 1:5 was added to the solution in order to obtain 2% protein loading.", "A syringe containing 5 ml of the polymer plus bioactive compound mixture was placed at an angle of 45°.", "The syringe was fitted with a 16G needle with the tip of the needle at a distance of 24 cm from the metallic collection screen.", "A piece of nonwoven mat was placed on the metallic screen.", "A voltage of 20 kV was applied between the collection screen and the needle tip which resulted in fibers being sprayed into a nonwoven matrix on the metallic screen.", "The spraying was complete in about 4 hours.", "It was found that with this specific polymer solvent system, polymer concentrations lower than 25% resulted in fibers with beads of polymers.", "These beads were eliminated when the polymer concentration was increased to 25% or greater.", "However, as will be understood by the skilled artisan upon reading this disclosure, this concentration will vary for different polymer/solvent systems and different bioactive compounds.", "Example 2 In Vitro Release of Protein In vitro release of the FITC-BSA into an infinite sink of 37° C. phosphate buffered saline was measured.", "Pre-weighed pieces from different regions of the mat were placed into scintillation vials and 10 ml of phosphate buffered saline were added and the capped vials were placed on a rotary shaker at 37° C. The buffer was exchanged at different points in time in order to mimic infinite sink conditions.", "The amount of protein released was measured in the form of fluorescence of the FITC-BSA on a spectrophotofluorometer at an excitation wavelength of 495 nm and an emission wavelength of 513 nm." ] ]

[ [ "Method for transmitting short messages", "A method is provided for transmitting a short message in a telecommunication network to a number of communication stations, wherein address data for the transmission of the short message to more than one communication station are transmitted with a short message, wherein a first header information item is transmitted with a number of data fields in the data section of the short message, each data field including the address data of at least one communication station, and in the second header information item, which is arranged preceding the first header information item, the address data of a first communication station or the network unit are transmitted." ], [ "1-11.", "(canceled) 12.A method for transmitting a short message in a telecommunication network to a plurality of communication stations, the method comprising the steps of: providing the short message with a second header information item and a data section; and transmitting address data, for transmitting the short message to more than one communication station, with a short message, wherein in the data section of the short message a first header information item is transmitted with a plurality of data fields which include the address data of at least one communication station, and wherein in the second header information item, which is arranged to precede the first header information item, address data of one of a first communication station and a network unit are transmitted.", "13.A method for transmitting a short message in a telecommunication network as claimed in claim 12, the method further comprising the step of indicating a presence of the first header information item in the data section of the short message by a signaling entry in the preceding second header information item.", "14.A method for transmitting a short message in a telecommunication network as claimed in claim 12, wherein the telecommunication network is constructed as a GSM mobile radio network.", "15.A method for transmitting a short message in a telecommunication network as claimed in claim 12, wherein the telecommunication network is constructed as a UMTS mobile radio network.", "16.A method for transmitting a short message in a telecommunication network as claimed in claim 12, the method further comprising the step of transmitting, with the first header information item, at least one signaling identification data field which includes the signaling of the data fields.", "17.A method for transmitting a short message in a telecommunication network as claimed in claim 16, wherein the at least one signaling identification data field specifies a type of address data.", "18.A method for transmitting a short message in a telecommunication network as claimed in claim 12, wherein the at least one data field is transmitted in a data section of the short message preceding useful data of the short message.", "19.A method for transmitting a short message in a telecommunication network as claimed in claim 12, the method further comprising the step of transmitting, with the first header information item, at least one destination address data field which includes a destination address of the short message.", "20.A method for transmitting a short message in a telecommunication network as claimed in claim 19, the method further comprising the step of transmitting an address identification data field preceding each destination address data field which specifies a type of the destination address.", "21.A method for transmitting a short message in a telecommunication network as claimed in claim 19, the method further comprising the step of transmitting at least one length data field preceding each destination address data field in the first header information item.", "22.A method for transmitting a short message in a telecommunication network as claimed in claim 16, wherein for a plurality of destination address data fields, exactly one signaling identification data field is transmitted with the first header information item.", "23.A method for transmitting a short message in a telecommunication network as claimed in claim 12, wherein the short message is first transmitted to a network unit of the telecommunication network, and an address of at least one of the communication stations is extracted from the first header information item of the short message in order to send its content at least partially to the address." ], [ "PRIOR ART The invention is based on a method for transmitting short messages of the generic type of the main claim.", "Methods for transmitting short messages are known, for example, for the GSM (Global System for mobile Communications) mobile radio network and are implemented there as SMS (Short Message Service).", "In the SMS short message service, only a single receiver can be specified for sending out a short message.", "If the same short message is also to be sent out to other receivers, it must be sent out several times by the sender.", "ADVANTAGES OF THE INVENTION By comparison, the method for transmitting short messages according to the invention, having the features of the main claim, has the advantage that address data for transmitting the short message to more than one communication station are transmitted with a short message.", "In this manner, the short message can be sent simultaneously to a number of communication stations or receivers so that time and effort is saved by the sender.", "Advantageous developments and improvements of the method specified in the main claim are possible by means of the measures listed in the subclaims.", "It is particularly advantageous that exactly one signaling identification data field is transmitted with the first header information item for a number of destination address data fields.", "In this manner, the volume of data used for the first header information item is reduced in the data section so that a greater data acapacity is available for the useful data in the short message.", "DRAWING Exemplary embodiments of the invention are shown in the drawing and explained in greater detail in the description following.", "FIG.", "1 shows a block diagram for the transmission of short messages via a first telecommunication network and a second telecommunication network, FIG.", "2 shows the basic structure of a short message, FIG.", "3 shows the structure of a first header information item with data fields for the transmission of address data, FIG.", "4 shows a first exemplary embodiment of the structure of the first header information item, FIG.", "5 shows a second exemplary embodiment of the structure of the first header information item and FIG.", "6 shows a third exemplary embodiment of the structure of the first header information item.", "DESCRIPTION OF THE EXEMPLARY EMBODIMENTS In FIG.", "1, 125 designates a transmitter which transmits a short message to a first network unit 140 via a first telecommunication network 100.The first network unit 140 forwards the short message via the first telecommunication network 100 to a second network unit 150 which represents both a network unit of the first telecommunication network 100 and a network unit of a second telecommunication network 200.From the second network unit 150, the short message can thus be transmitted via the first telecommunication network 100 to a first telecommunication station 175, on the one hand, and via the second telecommunication network 200 to a second communication station 176.According to FIG.", "1, the first network unit 140 and the second network unit 150 are constructed as two separate units but they can be integrated into one common unit.", "In FIG.", "2, 5 designates a short message.", "The short message 5 is generated by the transmitter 125 of the first telecommunication network 100 and, as described, transmitted via the first network unit 140 of the first telecommunication network 100 to the second network unit 150 via the first telecommunication network l00.For this purpose, a so-called short message service is set up in the first telecommunication network 100 and in the second telecommunication network 200 for transmitting such short messages 5.Such a short message service is in existence, for example, in a telecommunication network constructed as mobile radio network.", "By way of example, it is to be assumed in the text which follows that the first telecommunication network 100 and the second telecommunication network 200 are in each case constructed as mobile radio network.", "Such a mobile radio network can be, for example, a mobile radio network according to the GSM (Global System for Mobile communications) Standard or according to the UMTS (Universal Mobile Telecommunications System) Standard.", "In the GSM mobile radio network, the so-called SMS (Short Message Service) is specified which provides the sending of SMS short messages between a transmitter and a receiver, the SMS short messages being restricted to text messages of a maximum text length of, at present, 160 characters.", "If larger text messages are to be transmitted, the SMS short message service provides the possibility of concatenating a number of SMS short messages, i.e.", "the short message to be transmitted is distributed over a number of SMS short messages.", "In principle, the short message 5 shown in FIG.", "2 comprises a second header information item 12 and a data section 50.The second header information item 12 comprises signaling entries and a first destination address for the short message 5 to be sent and, respectively, a source address for the short message 5 to be received.", "The first destination address identifies the second network unit 150 as receiver of the short message 5 in the first telecommunication network 100 and the source address identifies the transmitter 125 of the short message 5 in the first telecommunication network 100.The data section 50 comprises the message actually to be transmitted, and thus the so-called useful data which are identified by the reference symbol 1 in FIG.", "2.In the SMS short message service, the source address and, respectively, the first destination address identified via a so-called MSISDN (Mobile Subscriber Integrated Services Digital Network) number according to the publications “Technical Realisation of the Short Message Service (SMS); Point to Point (PP)” GSM 03.40 V 7.1.0, (1998-11) and “Technical Realisation of the Short Message Service (SMS); Point to Point (PP)” 3G23.040 V 3.2.0 (1999-10).", "When the short message 5 is transmitted in the first telecommunication network l00, the first network unit 140, to which the short message 5 was initially transmitted via the first telecommunication network 100 as predetermined, addresses the second network unit 150 by means of the first destination address and replaces it with the source address of the transmitter 125 in the second header information item 12.According to the standard, the second header information item 12 thus contains exactly one source address or exactly one first destination address.", "According to FIG.", "2, the short message 5 comprises a first header information item 11 which is arranged preceding the useful data 1 in the data section 50 of the short message 5.In the SMS short message service, the first header information item 11 is called “User Data Header”.", "The presence of the first header information item 11 in the data section 50 is indicated by a corresponding signaling entry in the second header information item 12.In the SMS short message service, various types of user data header are specified in accordance with the said publications “Technical Realisation of the Short Message Service (SMS); Point-to-Point (PP)”.", "They are distinguished by a first signaling identification data field 15 in the first header information item 11 according to FIG.", "2, FIG.", "2 already showing the structure of the first header information item 11 in principle.", "The useful data 1 transmitted in the data section 50 of the short message 5 will now be transmitted in each case in the form of a short message to a number of communication stations, to the first communication station 175 and to the second communication station 176 in this example.", "The signaling entry in the second header information item 12 additionally specifies that the useful data 1 contained in the short message 5 are to be transmitted to a number of communication stations.", "This signaling is done in that the first header information item 11 in the second data section 50 is referred to in the second header information item 12.Using the first destination address in the second header information item 12, the short message 5 is first transitted, as described, via the first telecommunication network 100 to the first network unit 140 which can be constructed as service telecommunication network 100.The first network unit 140 evaluates the first destination address in the second header information item 12 and sends the short message 5 to the second network unit 150 associated with the first destination address.", "Using the evaluation of the signaling entry in the second information item 12, the second network unit 150 detects the presence of the first header information item 11 in the data section 50.The second network unit 150 has the task of evaluating this first header information item 11.This first header information item 11 contains the destination addresses of the communication stations 175, 176 to which the useful data 1 of the short message 5 are in each case to be transmitted again in a separate short message.", "In this process, these communication stations 175, 176 can be reached via the first telecommunication network 100, and thus the same telecommunication network as the transmitter 125, or via other telecommunication networks such as, for example, the second telecommunication network 200.According to FIG.", "1, the first telecommunication station 175 is reached via the first telecommunication network and the second telecommunication station 176 is reached via the second telecommunication network 200 in this example.", "The second network unit 150 extracts the receiver addresses of these telecommunication stations 175, 176 from the first header information item 11.Depending on the range of functions of the second network unit 150, this unit can transmit the useful data 1 in the form of in each case a short message directly to the individual telecommunication stations 175, 176, either via the first telecommunication network 100 or via the second telecommunication network 200.Otherwise, the second network unit 150 causes the first network unit 140 to send the useful data to the communication stations 175, 176 with the addresses, extracted from the first header information item 11, of the telecommunication stations 175, 176.The sending to the second communication station 176 via the second telecommunication network 200 can again take place via the second network uit 150 since the latter is also a party in the second telecommunication network 200, in contrast to the first network unit 140.The corresponding short message can be sent to the first communication station 175 from the first network unit 140 directly via the first telecommunication network 100, which is not shown in FIG.", "1 for reasons of clarity.", "With the data section 50, a number of data fields 20, 25, 30 according to FIG.", "3 are now transmitted which comprise address data for the transmission of the useful data 1 to the first communication station 175 and to the second communication station 176.After evaluation of these address data in the second network unit 150, the useful data 1 can then be forwarded to the first communication station 175 and the second communication station 176 in the manner described.", "For this purpose, the second network unit 150 must be informed about how these data fields 20, 25, 30 are structured or whether there are such data fields 20, 25, 30 at all in the data field 50.This is done by means of a signaling identification data field 15 in the first header information item 11.There, a so-called identifier specifies the manner in which the address data are present in the data section 50 and thus also signals that the data fields 20, 25, 30 exist at all.", "For example, the identifier can specify that the data fields 20, 25, 30 in the data section 50 comprise a number of addresses of communication stations for sending out the useful data 1.According to FIG.", "2 and FIG.", "3, the signaling identification data field 15 of the first header information item 11 is preceded by a first length data field 40 which specifies the length UDHL (User Data Header Length) of the first header information item 11.The first length data field 40 is then followed by the signaling identification data field 15, already described, with the identifier IEI (Information item Element Identifier), which, in this case, specifies that the data fields 20, 25, 30 comprise address data for a number of communication stations 175, 176.The signaling identification data field 15 is followed by a second length data field 45 which specifies the length IEIDL (Information item Element Identifier Data Length) of the actual address data 55 according the FIG.", "2 in the first header information item 11.According to FIG.", "3, the actual address data 55 are formed by the data fields 20, 25, 30.The addresses of the communication stations 175, 176 can be coded, for example, in accordance with the said publications “Technical Realisation of the Short Message Service (SMS); Point-to-Point (PP)”.", "The address data 55 of the first header information item 11 of the short message 5 are in each case formed by the three data fields 20, 25, 30, as described, a first data field 20, as length data field, specifying the length of the address, which can be present, for example, in the form of a telephone number, in half-octets, or so-called semioctets which, when the address is structured as telephone number, corresponds to the number of digits in the telephone number.", "The first data field 20 is already shown in FIG.", "3.It is followed by a second data field 25 which specifies the type of address as is specified, for example, in the standard ITU-T E.164/E163 or ITU-T X.121.The second data field 25 of the address data 55 in FIG.", "3 is then followed by a third data field 30 which contains the address or, respectively, the telephone number itself.", "The first header information item 11 can also comprise a number of identical or different types of user data header according to the said publications “Technical Realisation of the Short Message Service (SMS); Point-to-Point (PP)”.", "The arrangement, described according to FIG.", "2 and FIG.", "3, in the first header information item 11, consisting of the first length data field 40, the signaling identification data field 15, the second length data field 45 and the address data 55, may be repeated several times in the same order, as shown in FIG.", "4.In the text which follows, an example of the structure of the first header information item 11 is described.", "Firstly, the second header information item 12 contains the signaling entry TP-UDHT (Transfer Protocol User Data Header Identification) which specifies whether the first header information item 11, and thus a number of address data 55, 56, 57, are present in the short message 5.If this is the case, TP-UDHI is set to 1 and, if not, TP-UDHI is set to 0.The first header information item 11 can then be structured, for example, as shown in FIG.", "4: In the first length data field 40, the length UDHL of the first header information item 11 is set to 1E in the hexadecimal system.", "In the signaling identification data field 15, which will be called the first signaling identification data field in the text which follows, the identiifier IEI is set to 25 in the hexadecimal system and references with this value the addressing to a number of addresses of communication stations 175, 176, as described.", "In the second length data field 45, the length IEIDL for the address data 55, which will also be called first address data in the text which follows, and thus the length of the data fields 20, 25, 30 of the first address data 55 of the first header information item 11 is set to 8 in the hexadecimal system.", "According to FIG.", "4, the second length data field 45 is followed by the first data field 20 of the first address data 55 which references the value 0c in the hexadecimal system, and thus a length of the first address data 55 of 12 half-octets of 6 bytes, i.e.", "a telephone number of 12 digits.", "The first data field 20 of the first address data 55 is followed by the second data field 25 of the first address data 55 which references the address type of an international directory number according to the ISDN (Integrated Services Data Network) Standard with the value 91 in the hexadecimal system according to the said publications “Technical Realisation of the Short Message Service (SMS); Point-to-Point (PP)”.", "The second data field 25 of the first address data 55 is followed by the third data field 30 of the first address data 55 which, in this example, comprises the following telephone number as address of the first telecommunication station 175 of the first telecommunication network 100: +45 170 812 7112.According to the address type specified in the second data field 25 of the first address data 55, this address is structured as follows, according to FIG.", "4: “547180201721”.", "One digit of the telephone number corresponds to one half-octet, i.e.", "to a total length of 6 bytes.", "Thus, the value 8 is obtained for the second length data field 45.To specify the addresses of further communication stations, use is made of the possibility that the first header information item 11 can contain a number of elements, and thus addresses of a number of communication stations.", "For the present example, this means that the first address data 55 are followed by a second signaling identification data field 15 with the identifier IEI=25 in the hexadecimal system which thus corresponds to the identifier IEI of the first signaling identification field 15.The second signaling identification data field 16 is then followed by a third length data field 46 wich references the length IEIDL-=8 like the second length data field 45.The third length data field 46 is then followed by second address data 56.A first data field 21 of the second address data 56 references the value 0c in the hexadecimal system, and thus an address data length of 12 half-octets, as length data field for the second address data 56 and the first data field 20 of the first address data 55.This is followed by a second data field 26 of the second address data 56 which references the value 91 for the type of the address transmitted with the second address data 56 like the second data field 25 of the first address data 55.This is followed by a third data field 31 of the second address data 56 which contains the telephone number: =49 172 802 7222 as address of the second communication station 176 of the second telecommunication network 200 and is structured as follows according to the type referenced in the second data field 21 of the second address data 56: “947182202722”.", "For the case, not shown in FIG.", "1, of a third communication station which can be connected to the second network unit 150 via a third telecommunication network and to which the useful data 1 of the short message 5 are also to be transmitted in the form of a short message, the second address data 56 can be followed , according to FIG.", "4, by a third signaling identification data field 17 which contains the identifier IEI=25 in the hexadecimal system like the first signaling identification data field 15.The third signaling idenitification data field 17 is then followed by a fourth length data field 47 which references the length IEIDL=8 in the hexadecimal system like the second length data field 45.This is followed by third address data 57.These comprise a first data field 22 which references the value 0c in the hexadecimal system as length data field of the third address data 57 like the first data field 20 of the first address data 55.This is followed by a second data field 27 of the third address data 57 which references the value 91 like the second data field 25 of the first address data 55, and thus the same address type as the second data field 25 of the first address data 55.This is followed by a third data field 32 of the third address data 57 which, in the present example, comprises the address or, respectively, the telephone number of the third communication station, which is structured as follows: =49 177 802 7128.According to the type specified in the second data field 27 of the third address data 57, it is structured as followed: “947187201781”.", "The first address data 55, the second address data 56 and the third address data 57 thus correspond to each other in the type of the address or telephone number transmitted with them, and the data volume or length claimed in each case in the first header information item 11.In the exemplary embodiment described in FIG.", "4, the coding of the addresses or of the directory numbers of the communication stations 175, 176 corresponds to the standard described in the siad pubications “Technical Realisation of the Short Message Service (SMS); Point-to-Point (PP)”.", "Using the signaling identification data fields 15, 16, 17 in the first header information item 11, the second network unit 150 an identify the type of address data 55, 56, 57.Using the length data fields 40, 45, 46, 47 in the first header information item 11, the second network unit 150 can locate the address data 55, 56, 57 in the first header information item 11.Using the first data fields 20, 21, 22 in the first header information item 11, the second network unit 150 can locate the third data fields 30, 31, 32 and thus the telephone numbers of the communication stations in the area of the address data 55, 56, 57.Using the second data fields 25, 26, 27 in the first header information item 11, the second network unit 150 can identify the type of the addresses or telephone numbers stored in the third data fields 30, 31, 32.The second network unit 150 is thus able to extract the addresses or directory numbers stored in the third data fields 30, 31, 32 fromthe first header information item 11.The second network unit 150 can then form a new short messasge for each of the communication stations addressed, and copy the telephone number or address extracted for the respective communication station into the second header information item of the newly formed respective short message, this newly formed short message also comprising the useful data 1 in its data section but in each case only being transmitted to the communication station which is addressed by the respective extracted telephone number in the second header information item 12 of the newly formed short message.", "A first header information item is not required in a short message newly formed in this manner.", "In the exemplary embodiment described according to FIG.", "4, two length data fields are in each case allocated to the address data 55, 56, 57.The second length data field 45 and the first data field 20 of the first address data 55 are allocated as length data field to the first address data 55.This correspondingly applies to the second address data 56 and the third afdress data 57.Thus, one of the two length data fields allocated to the respective address data 55, 56, 57 is redundant.", "To save transmission capacity and to increase the volume of data available for the useful data 1 in the short message 5, the first data first 20, 21, 22 of the address data 55, 56, 57 can be omitted.", "The values of the first data fields 20, 21, 22 can then be calculated on the basis of the remaining length data fields 45, 46, 47 in the second network unit 150.FIG.", "5 shows a second exemplary embodiment of the first header information item 11 in which the first data fields 20, 21, 22 have been omitted and which otherwise corresponds to the first exemplary embodiment according to FIG.", "4, the difference being that the first length data field 40 now has the value 1b, reduced by three, for the parameter UDHL and the second length data field 45, the third length data field 46 and the fourth length data field 47 in each case have the value 7, reduced by 1, for the parameter IEIDL.", "This is due to the fact that in each case the octet for the first data field 20, 21, 22 is saved for the address data 55, 56, 57.Both in the first exemplary embodiment according to FIG.", "4 and in the second exemplary embodiment according to FIG.", "5, in each case an identically named length data field 45, 46, 47 and an identically named signaling identification data field 15, 16, 17 are allocated to the address data 55, 56, 57 so that here, too, there is redundancy and data capacity can be saved in favor of the useful data 1 as is shown by the third exexmplary embodiment according to FIG.", "6.In this firgure, only a single signaling identification data field in the form of the first signaling identification data field 15 is usd which follows the first length data field 40.In addition, apart from the first length data field 40, only the second length data field 45 is used which follows the irst signaling identification data field 15 and has the value 18 in the hexadecimal system according to FIG.", "6.The further length data fields 46, 47 and the further signaling identification data fields 16, 17 can then be omitted, according to FIG.", "6, so that the address data 55, 56, 57 follow one another directly and directly adjoin the second length data field 45.In this arrangement, however, the first data field 20, 21, 22 with the value 0c must again be introduced for the address data 55, 56, 57 in order to again ensure that the third data fields 30, 31, 32 can be located in the second network unit 150 since, of course, the third length data fuekd 46 and the fourth length data field 47 have been omitted.", "Thus, the second length data field 45 now specifies the length of all three address data 55, 56, 57 with the value 18 in the hexadecimal system for the length IEIDL.", "As can be seen in FIG.", "6, the length of the first header information item 11 can be produced again by 1 in this manner since the value UDHL of the first length data field 40 is now 1a in the hexadecimal system.", "As described, the first data fields 20, 21, 22 specify the address data length of the address or, respectively, telephone number stored in the respective associated third data field 30, 31, 32.Since the value in the first data fields 20, 21, 22 specifies the number of half-octets, this value must be divided by two in each case in order to obtain the number if octets in the respective third data field 30, 31, 32.If another two octets for the respective data field 20, 21, 22 and the respective second data field 25, 26, 27 are added to the number of octets of the respective third data field 30, 31, 32, the number of octets for the respective address data 55, 56, 57 is obtained.", "Multiplying this value by three provides the total number of octets for all address data 55, 56, 57 in the first header information item 11.According to FIG.", "6, a total of 24 octets, which are referenced by the value 18 in te hexadecimal system for the parameter IEIDL of the second length data field 45, is provided for the address data 55, 56, 57.Due to this known relationship between the value IEIDL in the second length data field 45 and the length values in the first data fields 20, 21, 22, the address data 55, 56, 57 can be separated from one another without problems in the second network unit 150.Hitherto, it has been assumed that the short message 5, with a number of addresses of communication stations 175, 176 is conducted via the first network unit 140 initially to the second network unit 150 which then evaluates the first header information item 11 and after that distributes the useful data 1 to the communication stations 175, 176 addressed.", "Even if the two network units 140, 150 can be combinecd, it is necessary to specify the address of the second network unit 150 as the first destination address in the second header information item 12.As an alternative, however, it is also possible that the first destination address in the second header information item 12 already represents one of the addresses of the communication stations 175, 176 and all other addresses of the remaining communication stations are specified in the first header information item 11 as in the three exemplary embodiments described.", "In this case, the first network unit 140 must take over the functions of the second network unit 150 and itself check every arriving short message for the first header information item 11 or, respectively, the signaling identification data fields 15, 16, 17 present there, and extract the addresses of the additionally required communication stations in order to be able to also send the useful data 1 to these with the aid of short messages to be newly formed.", "In the exemplary embodiments described, it as assumed that there are three different communication stations, two of which are whown in FIG.", "1, and to which the address data 55, 56, 57 in the first header information item 11 are allocated.", "In the first header information item 11, however, it is also possible to transit more or less than three address data in the manner described in accordance with one of the three exemplary embodiments.", "In addition, it can be proved that address data for a communication station of a data network, for example the Internet, are also contained in the first header information item 11, the type of which, naturally, differs from the address data described and must be identified by a corresponding signaling identification data field in the first header information item 11.The address of such a communication station of a data network can then be specified, for example, in the form of an internet address in the associated third data field.", "The second network unit 150 will then send the useful data 1 to such a communication station as Internet-E-Mail with the extracted internet address if it is connected to such a data network." ] ]

[ [ "Method of obtaining peptides with tissue-specific activity and pharmaceutical compositions on their basis", "The invention refers to the field of chemistry and concerns the method of obtaining peptides with tissue-specific activity by targeted chemical synthesis.", "This invention can be employed in medicine to obtain peptide-based pharmaceuticals normalising the functions of various organs and functions.", "The method of obtaining peptides proposed in this patent claim embraces quantitative amino acid analysis of acetic extracts from tissues, selection on its basis of two amino acids (Glu and Asp) prevailing in the studied tissue, synthesis of the central link from these amino acids and attachment to its N- and C-ends of the amino acids prevailing among the remaining amino acids in the studied tissue.", "The peptides obtained by the claimed method possess a tissue-specific activity.", "There is proposed a pharmaceutical composition possessing a tissue-specific activity and containing as its active base one of the peptides obtained by the claimed method or its salts and a pharmaceutically admissible carrier." ], [ "1.A method of obtaining a peptide with tissue-specific activity comprising amino acid analysis of an acetic extract from an animal tissue, selection of amino acids prevailing in the studied tissue, synthesis of a central link of the peptide and attachment to its both ends of amino acids prevailing among the remaining amino acids in the studied tissue.", "2.The method of claim 1, wherein the amino acid analysis is conducted on an acetic extract from epiphysis tissue.", "3.The method of claim 1, wherein the amino acid analysis is conducted on an acetic extract from cerebral cortex tissue.", "4.The method of claim 1, wherein the amino acid analysis is conducted on an acetic extract from liver tissue.", "5.The method of claim 1, wherein the central link is presented by glutamic (Glu) and aspartic acids (Asp).", "6.The method of claim 1, wherein the amino acids are joined to the central link a N- and C-ends.", "7.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 1.8.The composition of claim 7, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation.", "9.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 2.10.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 3.11.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 4.12.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 5.13.A pharmaceutical composition possessing a tissue-specific activity and containing an active base and a pharmaceutically admissible carrier, wherein the composition contains as its active base an effective quantity of a peptide obtained by the method of claim 6.14.The composition of claim 9, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation.", "15.The composition of claim 10, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation.", "16.The composition of claim 11, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation.", "17.The composition of claim 12, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation.", "18.The composition of claim 13, which contains a salt of the amino group, of carboxyl group, or a salt of an organic or inorganic cation." ], [ "<SOH> BACKGROUND ON THE INVENTION <EOH>There are known the methods of obtaining complex peptide substances with tissue-specific activity: Thymalin (1), Epithalamin (2), Prostatilen (3), Cortexin (4) and Retinalamin (5).", "These substances are complexes of low-molecular polypeptides obtained from animal organs and tissues by extraction in 3% acetic add with chlorous zinc and further treatment of the supraprecipitation fluid with acetone (6).", "The given method of obtaining peptide substances is marked by considerable variability of physical and chemical properties of the exacted peptides and presence of ballast components in them.", "Limited reserves of required organic raw material and high labour and energy consumption by production are the drawbacks of the said method of obtaining complex peptide substances, which impede their industrial production.", "There are known the methods of obtaining peptides by classical peptide synthesis in a solution (7) on the basis of active fractions isolated from Thymalin—Glu-Trp (Thymogen) (8), Thymopoietin II—Arg-Lys-Asp-Val-Tyr (Thymopentin) (9), Splenin—Arg-Lys-Glu-Val-Tyr (Splenopentin) (10), Immunoglobulin G—Thr-Lys-Pro-Arg (Taphcin) (11) and other.", "However, the substances obtained by the given method primarily possess a singly directed spectrum of biological activity (immunoregulatory), instability of the substances in a solution and high doses of application.", "There are known methods of obtaining peptides of modified structure by classical peptide synthesis in a solution (7): Arg-α-Asp-Lys-Val-Tyr-Arg (Immunophan) (12) structurally distinguished from Thymopentin by the presence of amino acid substitutes with the chain elongated by end Arginine; γ-Glu-Trp (Bestim) (13) structurally distinguished from Thymogen by γ-bond.", "These substances are also characterised by their narrowly directed immunobiological activity and complicated procedure of chemical synthesis.", "There is also known a synthetic polymer (Cop 1) inhibiting cellular immune response, which is obtained by chemical synthesis and contains amino acids L-Ala, L-Glu, L-Lys, L-Tyr in the following molar correlation: 6.0:1.9:4.7:1.0 (14).", "This method of obtaining active substances is not widespread and concerns only the design of polymer Cop 1." ], [ "FIELD OF THE INVENTION The invention refers to the field of chemistry and concerns the method of obtaining peptides with tissue-specific activity by targeted chemical synthesis.", "The invention can be employed in medicine to obtain pharmaceuticals based on these peptides normalising the functions of various organs and tissues.", "BACKGROUND ON THE INVENTION There are known the methods of obtaining complex peptide substances with tissue-specific activity: Thymalin (1), Epithalamin (2), Prostatilen (3), Cortexin (4) and Retinalamin (5).", "These substances are complexes of low-molecular polypeptides obtained from animal organs and tissues by extraction in 3% acetic add with chlorous zinc and further treatment of the supraprecipitation fluid with acetone (6).", "The given method of obtaining peptide substances is marked by considerable variability of physical and chemical properties of the exacted peptides and presence of ballast components in them.", "Limited reserves of required organic raw material and high labour and energy consumption by production are the drawbacks of the said method of obtaining complex peptide substances, which impede their industrial production.", "There are known the methods of obtaining peptides by classical peptide synthesis in a solution (7) on the basis of active fractions isolated from Thymalin—Glu-Trp (Thymogen) (8), Thymopoietin II—Arg-Lys-Asp-Val-Tyr (Thymopentin) (9), Splenin—Arg-Lys-Glu-Val-Tyr (Splenopentin) (10), Immunoglobulin G—Thr-Lys-Pro-Arg (Taphcin) (11) and other.", "However, the substances obtained by the given method primarily possess a singly directed spectrum of biological activity (immunoregulatory), instability of the substances in a solution and high doses of application.", "There are known methods of obtaining peptides of modified structure by classical peptide synthesis in a solution (7): Arg-α-Asp-Lys-Val-Tyr-Arg (Immunophan) (12) structurally distinguished from Thymopentin by the presence of amino acid substitutes with the chain elongated by end Arginine; γ-Glu-Trp (Bestim) (13) structurally distinguished from Thymogen by γ-bond.", "These substances are also characterised by their narrowly directed immunobiological activity and complicated procedure of chemical synthesis.", "There is also known a synthetic polymer (Cop 1) inhibiting cellular immune response, which is obtained by chemical synthesis and contains amino acids L-Ala, L-Glu, L-Lys, L-Tyr in the following molar correlation: 6.0:1.9:4.7:1.0 (14).", "This method of obtaining active substances is not widespread and concerns only the design of polymer Cop 1.DISCLOSURE OF THE INVENTION This invention operates the following specific terminology accepted in this field.", "The notion “small regulatory peptides” under this patent claim implies the presence of endogenous small peptides formed in the process of proteolysis and their synthetic analogues of the known amino acid sequence (up to 10 amino acids), which reveal physiologically active properties (7, 15).", "The notion “tissue-specific activity” of peptides under this patent claim implies the impact of peptides on the very tissues, whose amino acid composition serves the basis for their acquisition (6, 16, 17).", "The notion “pharmaceutical composition” under this patent claim implies an active peptide obtained by the claimed method or its salts of the amino group, of carboxyl groups, salts of organic and inorganic cations and a pharmacologically admissible carrier.", "The notion “effective quantity” under this patent claim implies the employment of such an amount of the active base, which, in compliance with the quantitative indices of its activity and toxicity, as well as with respect to the knowledge available, shall be effective in a given drug form.", "The purpose of this patent claim consists in creating a method of obtaining peptides with tissue-specific activity by targeted chemical synthesis, which enables its industrial employment at minimal costs, as well as in creating pharmaceutical compositions exerting a tissue-specific effect and based on the peptides obtained by the claimed method.", "The claimed method embraces quantitative amino acid analysis of acetic extracts from animal tissues, selection on its basis of two amino acids (Glu and Asp) prevailing in the studied tissue, synthesis of the central link from these amino acids and attachment to its N- and C-ends of the amino acids prevailing among the remaining amino acids in the studied tissue.", "Peptides obtained by the claimed method possess a tissue-specific activity, i.e.", "they exert an activity upon the very tissues whose amino acid composition serves the basis for their acquisition.", "This patent claim describes a pharmaceutical composition of tissue-specific activity containing as its active base an effective quantity of one of the peptides obtained by the claimed method or its salts of the amino acid group, of carboxyl groups, salts of organic and inorganic cations and a pharmaceutically admissible carrier, for example, isotonic sodium chloride solution.", "To obtain pharmaceutical compositions meeting the invention, the proposed peptides or their pharmaceutically admissible carriers in the form of salts are blended as active bases with a pharmaceutically admissible carrier according to the methods of compounding accepted in pharmaceutics.", "The carrier may have various forms depending on the drug form preferable for administration to a body.", "INDUSTRIAL APPLICATION The proposed invention is illustrated by the examples of: amino acid analysis of acetic extracts from the tissues of the epiphysis, cerebral cortex, brain and liver (Example 1); synthesis of Ala-Glu-Asp-Gly tetrapeptide (Example 2); effect of Ala-Glu-Asp-Gly tetrapeptide on the growth of brain subcortical structure explants (Example 3) clearly demonstrating the revealed tissue-specific properties; synthesis of Ala-Glu-Asp-Pro tetrapeptide (Example 4); effect of Ala-Glu-Asp-Pro tetrapeptide on the growth of cerebral cortex explants (Example 5) clearly demonstrating the revealed tissue-specific properties; synthesis of Lys-Glu-Asp-Ala tetrapeptide (Example 6); effect of Lys-Glu-Asp-Ala tetrapeptide on the protein synthesis intensity in a monolayer hepatocyte culture of rats of different age (Example 7) clearly demonstrating the revealed tissue-specific properties; effect of Lys-Glu-Asp-Ala tetrapeptide on the growth of liver explants (Example 8) clearly demonstrating the revealed tissue-specific properties.", "EXAMPLE 1 Amino Acid Analysis of Acetic Extracts from the Tissues of Epiphysis, Cerebral Cortex and Liver TABLE 1 Amino acid analysis of acetic extracts from the tissues of epiphysis, cerebral cortex and liver Relative content Amino acids Epiphysis Cerebral cortex Liver Alanine 1.42** 1.90** 2.31** Arginine 0.84 1.15 0.75 Asparagine — — — Aspartic acid 1.66* 2.70* 2.45* Valine 1.29 1.01 1.28 Histidine 0.61 0.62 0.81 Glycine 1.39*** 1.38 1.65 Glutamine — — — Glutamic acid 2.96* 4.37* 2.73* Isoleucin 0.36 0.60 0.32 Leucin 1.06 1.00 1.25 Lysine 1.29 1.77 2.19** Methionine 0.22 0.37 0.34 Proline 0.95 2.07*** 1.04 Serine 1.05 1.33 1.20 Threonine 0.68 0.82 0.78 Tryptophan — — — Tyrosine 0.36 0.60 0.33 Phenyl alanine 0.46 0.54 0.61 Cyctein — — — *amino acids prevailing in the studied tissue, of which the peptide central link is synthesised; **amino acids joined to the central link at the N-end; ***amino acids joined to the central link at the C-end.", "Amino acid analysis of acetic extracts from animal tissues enabled to reveal four prevailing amino acids for each tissue.", "The choice of this very number of amino acids was determined by the fact that lower number of amino acids (e.g., two or three) would inevitably result in an amino acid set with low specificity to the given tissue.", "Thus, the four chosen amino acids appeared sufficient to assess the minimal variability of the composition.", "When modelling the proposed tissue-specific tetrapeptides we also took into account the location of hydrophobic and hydrophilic amino acid residues in a number of known tissue oligopeptides (18) corresponding to the general formula H-X-Glu-Asp-Y-OH.", "In this case, the partial negative charge attains its maximal possible concentration in the molecule centre.", "If X and Y are aliphatic hydrophobic amino acids (Ala, Gly, Pro) this site will also be the most hydrophilic.", "This conforms to the data evidencing that intracellular proteinases are marked by a certain preference of the bonds formed by hydrophobic amino acids (19).", "Another extreme case is provided for X-Lys variant.", "Here, the partially positive charge reaches its maximal possible concentration at the N-end of the molecule (pointing at increased hydrophilic nature as well).", "At the same time, this secures the potentiality of forming intramolecular quasi-cyclic structures due to electrostatic interaction of ionised γ- and β-COO(−) groups of Glu or Asp, respectively, with ε- or α-NH3(+)- group of Lys.", "Presence of Pro at the C-end considerably raises the hydrophobic property of this site in comparison with Gly or Ala variants.", "Consequently, selected were the following sequences of amino acids, which made a part of the tetrapeptides designed on the basis of amino aced analysis of acetic extracts from the tissues of the epiphysis, cerebral cortex and liver: H-Ala-Glu-Asp-Gly-OH; H-Ala-Glu-Asp-Pro-OH; H-Lys-Glu-Asp-Ala-OH.", "EXAMPLE 2 Synthesis of Ala-Glu-Asp-Gly Tetrapeptide 1.Product name: alanyl-glutamyl-aspartyl-glycine.", "2.Structural formula: H-Ala-Glu-Asp-Gly-OH.", "3.Molecular formula without ion pair: C14H22N4O9.4.Molecular weight without ion pair: 390.35.5.Ion pair: acetate.", "6.Appearance: white amorphous powder without smell 7.Method of synthesis: the peptide is obtained by a classical method of synthesis in a solution by scheme A: Z -benzyloxycarbonyl group; BOc -tert.butyloxycarbonyl group; OSu -N-oxysuccinimide ester; OBzl -benzyl ester; DCC -N,N′-dicyclohexylcarbodiimide; HOBT -N-oxybenzotriazol.", "N,N′-dimethylformamide was used as a solvent.", "When adding aspartic acid, the defence of α-COOH group was applied by salification with triethylamine.", "BOC-protecting group was removed with trifluoracetic acid (TFA) solution and Z-protecting group—with catalytic hydrogenation.", "The product was extracted and purified by the method of preparative high-performance liquid chromatography (HPLC) on a reversed phase column.", "Properties of the finished product: amino acid analysis Glu Asp Ala Gly 1.02 1.00 1.01 1.00 peptide content 98.45% (by HPLC, 220 nm); thin layer chromatography (TLC)—individual, Rf=0.73 (acetonitrile acetic acid-water 5:1:3); moisture content: 5%; pH of 0.001%-solution: 4.37; specific rotary power [α]D22 : −32° (c=1, H2O) “Polamat A”, Carl Zeiβ Jena.", "Example of Synthesis: 1.BOC-Glu (OBzl)-Asp(OBzl)-OH (I), N-tert-butyloxycarbonyl-(γ-benzyl)glutamyl-(β-benzyl)aspartate.", "4.34 g (0.0100 mole) of N-oxysuccinimide ester of N-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid (BOC-Glu (OBzl)-OSu) is dissolved in 20 ml of dimethylformamide; and added 1.72 ml (0.0125 mole) of triethylamine and 2.80 g (0.0125 mole) of β-benzyl aspartate.", "The mixture is stirred for 24 hours at room temperature.", "Afterwards the product is precipitated with 0.5N sulphuric acid solution (150 ml), extracted by ethyl acetate (3×30 ml), washed in 0.5N sulphuric acid solution (2×20 ml), water, 5% sodium bicarbonate solution (1×20 ml), water, 0.5N sulphuric acid solution (2×20 ml), water.", "The product is dried over anhydrous sodium sulphate.", "Ethyl acetate is filtered out and removed in vacuo at 40° C. The residue is dried in vacuo over P2O5.5.68 g (≈100%) of oil is obtained.", "Rf=0.42 (benzene-acetone 2:1; Sorbfil plates, Silicagel 8-12 μm, development by UV and chlorine/benzidine).", "2.TFA.H-Glu(OBzl)-Asp(OBzl)-OH (II), trifluoracetate of (γ-benzyl)-glutamyl-(β-benzyl)aspartate.", "5.68 g (≈0.01 mole) of N-tert.butyloxycarbonyl-(γ-benzyl)-glutamyl-(β-benzyl) aspartate (I) is dissolved in 20 ml of dichlormethan-trifluoracetic acid mixture (3:1).", "Two hours later the solvent is removed in vacuo at 40° C. The removal is repeated with another portion of dichlormethan (2×10 ml).", "The residue is dried in vacuo over NaOH.", "5.80 g (≈100%) of oil is obtained.", "Rf=0.63 (n-butanol-pyridine-acetic acid-water, 15:10:3:12).", "3.Z-Ala-Glu-(OBzl)-Asp(OBzl)-OH (III), N-carbobenzoxyalanyl-(γ-benzyl)-glutamyl-(β-benzyl)aspartate.", "5.65 g (0.01 mole) of trifluoracetate of (γ-benzyl)-glutamyl-(β-benzyl)aspartate (II) is dissolved in 10 ml of dimethylformamide and added 2.80 ml (0.02 mole) of triethylamine and 4.14 g (0.013 mole) N-oxysuccinimide ester of N-carbobenzoxyalanine.", "The mixture is stirred for 24 hours at room temperature.", "The product is precipitated with 0.5N sulphuric acid solution (150 ml), extracted by ethyl acetate (3×30 ml), washed in 0.5N sulphuric acid solution (2×20 ml), water, 5% sodium bicarbonate solution (1×20 ml), water, 0.5N sulphuric acid solution (2×20 ml), water.", "The product is dried over anhydrous sodium sulphate.", "Ethyl acetate is filtered out and removed in vacuo at 40° C. The residue is crystallised in the ethyl acetate/hexane system.", "The product is filtered and dried in vacuo over P2O5.The yield is 4.10 g (66%).", "The temperature of melting (Tml) equals 154° C. Rf=0.48 (benzene-acetone, 1:1), Rf=0.72 (N-butanol-pyridine-acetic acid-water, 15:10:3:12).", "4.Z-Ala-Glu-(OBzl)-Asp(OBzl)Gly-OBzl (III), N-carbobenzoxyalanyl-(γ-benzyl)glutamyl-(β-benzyl)aspartylglycine benzyl ester.", "1.01 g (3 mmole) of glycine benzyl ether tosylate (TosOH.H-Gly-OBzl) is suspended in 15 ml of tetrahydrofuran and added 0.4 ml (3 mmole) of triethylamine while stirring.", "In 5 minutes 1.28 g (2 mmole) of N-carbobenzoxyalanine-(γ-benzyl)glutamyl-(β-benzyl) aspartate (III) and 0.27 g (2 mmole) of N-oxybenzotriazo, are added and the mixture is cooled down to 0° C. 0.42 g (2 mmole) of N,N′-dicyclohexylcarbodiimide solution cooled down to 0° C. is added in 5 ml of tetrahydrofuran.", "The mixture is stirred at this temperature for 2 hours and left to blend for a night at room temperature.", "Precipitate of dicyclohexylurea is filtered out, the solvent is removed in vacuo and the residue is dissolved in 30 ml of ethyl acetate.", "The product is washed in 1N hydrochloric acid solution, water, 5% sodium bicarbonate solution, water, 1N hydrochloric acid solution, water and dried over anhydrous sodium sulphate.", "The solvent is removed in vacuo and the product is crystallised in the ethyl acetate/hexane system.", "The yield is 1.30 g (82%).", "Tml=146-148° C. Rf=0.75 (benzene-acetone, 2:1).", "5.H-Ala-Glu-Asp-Gly-OH (IV), alanyl-glutamyl-aspartyl-glycine.", "1.25 g of benzyl ester of N-carbobenzoxyalanyl-(γ-benzyl)glutamyl-(β-benzyl)aspartylglycine (III) is hydrogenated in the methanol/water/acetic acid system (3:1:1) over Pd/C catalyst.", "Completeness of the deblocking reaction is monitored by TLC in the benzene/acetone (2:1) and acetonitrile/acetic acid/water (5:1:3) systems.", "When the reaction is over the catalyst is filtered out, the filtrate is removed in vacuo and the residue is crystallised in the water/methanol system.", "The product is dried in vacuo over KOH.", "The yield is 520 mg (95%).", "Rf=0.73 (acetonitrile-acetic acid-water, 5:1:3).", "For purification, 390 mg of the preparation is dissolved in 4 ml of 0.01% trifluoracetic acid and subjected to HPLC on a reversed phase column measuring 50×250 mm (Diasorb-130-C16T, 7μ).", "The employed chromatograph is Beckman System Gold, 126 Solvent Module, 168 Diode Array Detector Module.", "Conditions of chromatography A: 0.1% of TFA; B: 50% of MeCN/0.1% of TFA, grad.", "B 0→5% in 80 min.", "Sample volume is 5 ml, detection is conducted by 215 nm, scanning—by 190-600 nm, flow rate equals 10 ml/rin.", "The fraction is selected within 54.0-66.0 min.", "The solvent is removed in vacuo at a temperature not exceeding 40° C. The removal is multiply repeated (5 times) with 10 ml of 10% acetic acid solution.", "The residue is finally dissolved in 20 ml of deionised water and lyophilised.", "290 mg of purified preparation in the form of amorphous odourless white powder is obtained.", "The obtained peptide in the form of acetate is converted into a free form by treating it with IRA anionite or its analogue in (OH−)-form.", "Afterwards salts of the amino acid group are obtained by adding an equivalent of the respective acid hydrochloric or oxalic).", "The obtained aqueous solution is lyophilised and analysed as a finished product.", "In order to obtain corresponding salts of carboxyl groups, the free tetrapeptide is added a calculated quantity of the aqueous solution of a corresponding metal hydroxide (NaOH, KOH, Zn(OH)2, LiOH, Ca(OH), Mg(OH)2, NH4OH).", "To obtain triethylammonium salt, the processing is carried out similarly, triethylamine being used as the base.", "6.Analysis of the Finished Product.", "Content of the active base (peptide) is defined by HPLC on Supelco LC-18-DB column, 4.6×250 mm, grad.", "LC-18-DB.", "A: 0.1% of TFA; B: 50% of MeCN/0.1% of TFA; grad.", "B 0→20% in 20 min.", "The flow rate equals 1 ml/min.", "Detection by 220 nm, scanning—by 190600 nm, the sample volume is 20 μl.", "Peptide content—98.5%.", "The amino acid content is defined on an analyser after 24-hour hydrolysis in 6N HCl at 125° C. Glu Asp Ala Gly 1.02 1.00 1.01 1.00 TLC: individual, Rf=0.73 (acetonitrile-acetic acid-water, 5:1:3).", "Sorbfil plates, 8-12 μm Silicagel, developing in chlorine/benzidine.", "Moisture content: 5% (gravimetrically, according to the mass loss by drying, −20 mg at 100° C. pH of 0.001% solution: 4.37 (potentiometrically).", "Specific rotary power: [α]D22: −32° (c=1, H2O) “Polamat A”, Carl Zeiβ Jena.", "The pharmaceutical peptide substance in injection form containing the tetrapeptide as its active base is obtained the following way: the tetrapeptide obtained by the above-described method is dissolved in 0.9% isotonic sodium chloride solution.", "One vial contains 1 ml of the tetrapeptide solution in the concentration of 10 μg/ml.", "EXAMPLE 3 Effect of Ala-Glu-Asp-Gly Tetrapeptide on the Growth of Brain Subcortical Structure Explants Experiments were conducted on 69 fragments of brain subcortical structures of 10-11-days' old chicken embryos.", "The nutrient medium for cultivation consisted of 35% Eagle's solution, 25% calf foetal serum, 35% Hanks' solution, 5% chicken embryonic extract.", "The medium was also added glucose (0.6%), insulin (0.5 unit/ml), penicillin (100 unit/ml), glutamine (2 mM).", "Fragments of the brain subcortical structures were placed in this medium and cultivated in Petri's dishes in a thermostat at 36.7° C. for 48 hours.", "The experimental medium was added Ala-Glu-Asp-Gly tetrapeptide and Epithalamin in the concentrations of 2, 10, 20, 50, 100, 200, 400 ng/ml.", "Square index (SI) considered the criterion of biological activity.", "It was counted as the ratio between the total explant square including the growth zone and the initial square of the subcortical structure fragment Significance of differences between the average values of SI was assessed by Student's t-criterion.", "ST values were expressed percent, the control SI value being taken for 100%.", "The growth zone of the control explants of the brain, subcortical structures included short neurites, migrating glia and fibroblast-resembling cells.", "Immediate influence of the substances upon the fragments of the brain subcortical structures was investigated in the following experimental series.", "The nutrient medium of the explants of chicken embryonic subcortical structures was added Epithalamin in various concentrations.", "On the third day of cultivation, in the concentrations of 20 and 200 ng/ml a significant rise in the explant SI was noted in comparison with the control SI values (by 20% and 26% respectively).", "FIG.", "1 demonstrates the effect of Ala-Glu-Asp-Gly tetrapeptide on the growth of the brain subcortical structure explants.", "No significant values of the subcortical structure SI were registered in other Epithalamin concentrations.", "Pronounced stimulation of the development of the brain subcortical structure explants was revealed by applying Ala-Glu-Asp-Gly tetrapeptide in the concentration of 100 ng/ml when SI of the experimental explants was higher by 24% than that of the control fragments.", "Investigation of the subcortical structure explants by longer periods of cultivation—up 5 to 7 days—demonstrated analogous neurite stimulating effects in the same concentrations.", "In certain cases, a statistically insignificant decrease in explant SI was noted, probably, due to the reckon of nerve fibres by longer cultivation periods.", "EXAMPLE 4 Synthesis of Ala-Glu-Asp-Pro Tetrapeptide 1.Product name: L-alanyl-L-glutamyl-L-aspartyl-L-proline.", "2.Structural formula: H-Ala-Glu-Asp-Pro-OH.", "3.Gross formula without ion pair: C17H26N4O9.4.Molecular weight without ion pair: 430.41.5.Ion pair: acetate.", "6.Appearance: white amorphous odourless powder.", "7.Method of synthesis: the peptide is obtained by a classical method of synthesis in a solution by Scheme B.", "Z -benzyloxycarbonyl group; BOC -tert.butyloxycarbonyl group; OSu -N-oxysuccinimide ester, OBzl -benzyl ester; DCC -N,N′-dicyclohexylcarbodiimide; HOBT -N-oxybenzotriazol.", "N,N′-dimethylformamide was used as a solvent.", "When adding aspartic acid, the defence of α-COOH group was applied by salification with triethylamine.", "BOC-protecting group was removed with trifluoracetic acid (TFA) solution and Z-protecting group—with catalytic hydrogenation.", "The product was extracted and purified by the method of preparative high-performance liquid chromatography (HPLC) on a reversed phase column.", "Specification of the finished product: Amino acid assay: Glu Asp Ala Pro 1.10 1.01 1.00 1.10 peptide content: 98.56% (by HPLC, 200 nm); thin layer chromatography (TLC)—individual; Rf=0.67 (acetonitrile-acetic acid-water 5:1:3); moisture content: 7%; pH of 0.001% solution: 4.24; specific rotary power [α]D25:−78.9° (c=1.09, H2O), “Polamat A”, Carl Zeiss Jena.", "Synthesis is performed according to Example 2 and distinguished by the fact that at the C-end of the molecule benzyl ether of proline is used instead of benzyl ether of glycine.", "Pharmaceutical composition is obtained according to Example 2.EXAMPLE 5 Effect of Ala-Glu-Asp-Pro Tetrapeptide on the Growth of Cerebral Cortex Explants The experiments are conducted on 73 fragments of cerebral cortex explants of 10-11-days' chicken embryos.", "The nutrient medium for cultivation consisted of 35% Eagle's solution, 25% calf foetal serum, 35% Hanks' solution, 5% chicken embryonic extract.", "The medium was also added glucose (0.6%), insulin (0.5 unit/ml), penicillin (100 unit/ml), glutamine (2 mM).", "Fragments of the cerebral cortex were placed in this medium and cultivated in Petri's dishes in a thermostat at 36.7° C. for 48 hours.", "The experimental medium is was added Ala-Glu-Asp-Pro tetrapeptide and Cortexin in the concentrations of 2, 10, 20, 50, 100, 200, 400 ng/ml.", "Square index (SI) was considered the criterion of biological activity.", "It was counted as the ratio between the total explant square including the growth zone and the initial square of the cerebral cortex fragment and.", "Significance of differences between the average values of SI was assessed by Student's t-criterion.", "SI values were expressed per cent, the control SI value being taken for 100%.", "The growth zone of the control cerebral cortex explants included short neurites, migrating glia and fibroblast cells.", "Immediate influence of the substances upon the cerebral cortex fragments was investigated in the following experimental series.", "The nutrient medium of the explants of chicken embryonic cerebral cortex was added Cortexin in various concentrations.", "On the third day of cultivation, in the concentrations of 100 ng/ml a significant rise in the explant SI by 30±2% was noted in comparison with the control SI values.", "FIG.", "2 exhibits the effect of Ala-Glu-Asp-Pro tetrapeptide on the growth of the cerebral cortex explants.", "No significant values of the cerebral cortex SI were registered in other Cortexin concentrations.", "Pronounced stimulation of the development of the cerebral cortex explants was revealed by applying Ala-Glu-Asp-Pro tetrapeptide in the concentration of 20 ng/ml when SI of the experimental explants was higher by 40±7% than that of the control cerebral cortex fragments.", "Investigation of the cerebral cortex explants by longer periods of cultivation—up to 7 days—demonstrated analogous neurite-stimulating effects in the same concentrations.", "In certain cases, a statistically insignificant decrease in explant SI was noted, probably, due to the retraction of nerve fibres by longer cultivation periods.", "Thus, regarding the cerebral tissues, a decrease in the threshold of effective concentrations of Ala-Glu-Asp-Pro tetrapeptide was registered in comparison with Cortexin.", "For instance, Cortexin stimulated the fragments of cultivated cerebral cortex in the concentration of 100 ng/ml, while the tetrapeptide—in the concentration of 20 ng/ml.", "This evidences a more expressed and directed action of Ala-Glu-Asp-Pro tetrapeptide upon the cerebral cortex neurones.", "The performed studies and experiments suggest that the peptides obtained by the proposed method and pharmaceutical compositions on their basis possess a tissue-specific activity, i.e.", "they exert an effect upon the very tissues whose amino acid composition serves as the basis for their acquisition.", "EXAMPLE 6 Synthesis of Lys-Glu-Asp-Ala Tetrapeptide 1.Product name: lysyl-glutamyl-aspartyl-alanine.", "2.Structural formula: H-Lys-Glu-Asp-Ala-OH 3.Molecular formula without ion pair: C18H31N5O9.4.Molecular weight without ion pair 461.48.5.Ion pair: acetate.", "6.Appearance: white amorphous powder without smell.", "7.Method of synthesis: the peptide is obtained by a classical method of synthesis in a solution by the following scheme: Z -benzyloxycarbonyl group; BOC -tert.butyloxycarbonyl group; OSu -N-oxysuccinimide ester; OBzl -benzyl ester; DCC -N,N′-dicyclohexylcarbodiimide; HOBT -N-oxybenzotriazol.", "N,N′-dimethylformamide was used as a solvent.", "When adding aspartic acid, the defence of α-COOH group was applied by salification with triethylamine.", "BOC-protecting group was removed with trifluoracetic acid (TFA) solution and Z-protecting groups—with catalytic hydrogenation.", "The product was extracted and purified by the method of preparative high-performance liquid chromatography (HPLC) on a reversed phase column.", "Properties of the finished product: amino acid analysis Lys Glu Asp Ala 0.97 1.02 1.01 1.00 peptide content 98.75% (by HPLC, 220 nm); thin layer chromatography (TLC)—individual, Rf=0.71 (acetonitrile-water 1:1); moisture content: 7%; pH of 0.001%-solution: 5.54; specific rotary power [α]D23: −28.0° (c=1.0; H2O), “Polamat A”, Carl Zeiβ Jena.", "Synthesis is performed according to Example 2 and distinguished by the fact that at the C-end of the molecule benzyl ether of alanine is used and at the N-end of the molecule oxysuccinimide ester of Nα,Nα-dibenzyloxycarbonyl lysine is used instead of oxysuccinimide ester of N-benzyloxycarbonyl alamine.", "Pharmaceutical composition is obtained according to Example 2.EXAMPLE 7 Effect of Lys-Glu-Asp-Ala Tetrapeptide on the Intensity of Protein Synthesis in a Monolayer Hepatocyte Culture of Rats of Different Age Intensity of the protein synthesis was investigated in the hepatocyte culture of rats aged 4, 8 and 18 months.", "To isolate hepatocytes, rat liver was perfused with calcium-free Hanks' solution added 0.5 mM of EDTA and then 0.05% collagenase solution in Medium 199.The cellular suspension was filtered out and centrifuged.", "The hepatocyte suspension in the concentration of 5×105 was introduced into Petri's dishes, their bottoms surfaced with collagen-covered glass.", "Applied Medium 199 contained no bovine serum but was added 0.2 mg/ml of albumen and 5 μg/ml of insulin.", "The dishes with glass-covered bottoms were placed in a thermostat at 37° C., aerated and added CO2.In 2 hours, the glasses with adhered cells were washed and the medium was changed for a similar one.", "Twenty-four hours later and after washing the cultures, protein synthesis in them was investigated.", "Within 24 hours monolayer cultures with densely seated hepatocytes were formed in the cellular suspension at the above given concentration.", "Protein synthesis was assessed by [3H]-leucin inclusion regarding the standard errors for a free marked leucin pull in the same culture.", "The molar activity of the applied leucin equalled 150 Ci/mM.", "Incubation with marked leucin took 10 minutes.", "After the incubation the cultures containing marked leucin were washed with the medium and treated with cold (4° C.) sulphuric acid for 90 minutes to isolate non-included leucin.", "The same culture was rinsed with ethyl alcohol, after which proteins were dissolved with hyamine.", "Radioactivity of the pull of free intracellular leucin and cellular proteins (in hyamine fraction) after adding the corresponding scintillators was measured on a radioactivity counter SL-30.The intensity of protein synthesis was calculated by the formula Icorr=Ii×Pav/Pi(cpm), where Icorr—inclusion of leucin regarding the standard errors of free leucin pull, Ii—measured radioactivity of the proteins for i-culture, Pav—mean radioactivity of proteins and pull for the cultures studied in this experiment, Pi—total radioactivity of proteins and pull of the same culture.", "Hepatocyte cultures were incubated with Lys-Glu-Asp-Ala tetrapeptide in the concentration of 0.005 μg/ml during 4 hours.", "FIG.", "3(a, b, c) demonstrates the effect of Lys-Glu-Asp-Ala tetrapeptide on the protein synthesis kinetics in hepatocyte monolayer culture of rats of different age.", "The level of protein synthesis in the hepatocyte cultures was found to decrease with age (FIG.", "3a, b, c).", "Addition of Lys-Glu-Asp-Ala tetrapeptide to the culture raised the level of protein synthesis in hepatocytes of rats of different age.", "Thereby, the strongest effect was observed in the cells of older animals.", "Besides, the amplitude of synthesis oscillations increased considerably in the hepatocytes of older rats, which enabled a conclusion on a raised degree of the cell population activity synchronisation (FIG.", "3a, b, c).", "EXAMPLE 8 Effect of Lys-Glu-Asp-Ala Tetrapeptide on the Development of Liver The experiments were carried out in 53 liver fragments of 10-11 days old chicken embryos.", "Nutrient medium for the explant cultivation consisted of 35% of Eagle's solution, 25% of foetal calf serum, 35% of Hank's solution and 5% of chicken embryonic extract.", "The mixture is added glucose (0.6%), insulin (0.5 unit/ml), penicillin (100 unit/m) and glutamine (2 mM).", "The liver fragments were placed in this medium and cultivated in Petri's dishes in a thermostat at 36.7° C. during 48 hours.", "Lys-Glu-Asp-Ala tetrapeptide was added to the experimental medium in the concentrations of 2, 10, 20, 50, 100, 200 and 400 ng/mL Square index (SI) was taken for a biological activity criterion and calculated as a correlation of the total explant square including the growth zone to the initial square of a liver fragment.", "The SI values were expressed percent, the control SI value taken for 100%.", "FIG.", "4 demonstrates the effect of Lys-Glu-Asp-Ala tetrapeptide on the development of liver explants.", "In 24 hours of cultivation, the explants on a collagen lining were found to lie flat Proliferating and migrating cells started to move along the explant periphery.", "By the tetrapeptide concentration of 20 ng/ml on the third day of the cultivation, a significant increase in the explant SI by 24% was observed as compared to the control value (FIG.", "4).", "In case of longer terms of the liver explant cultivation (up to 7 days), an analogous stimulating effect of the tetrapeptide in the same concentration was revealed.", "Consequently, Lys-Glu-Asp-Ala tetrapeptide exerted a tissue-specific effect upon the liver tissue expressed in the explant growth stimulation.", "The conducted studies and experimental series enable the following conclusion: the peptides obtained by the claimed method and pharmaceutical compositions containing as their active bases these peptides or their salts possess a tissue-specific activity, i.e.", "they exert an action on the very tissues, whose amino acid composition serves the basis for their acquisition.", "Moreover, it is possible to create pharmaceuticals normalising the functions of various organs and tissues and containing the peptides with tissue-specific activity obtained by the claimed method.", "REFERENCES 1.Patent of the Russian Federation No.", "1112606 “Method of obtaining an immunomodulator from the thymus”.—1982.2.Patent of the Russian Federation No.", "944191 “Method of obtaining a substance possessing an antitumour effect”.—1980.3.Patent of the Russian Federation No.", "1417244 “Method of obtaining a substance restoring the prostate function”.—1986.4.Patent of the Russian Federation No.", "2104702 “Method of obtaining from animal raw material of a complex of biologically active polypeptides normalising the brain functions, a pharmacological composition and its application”.—1996.5.Patent of the Russian Federation No.", "2073518 “Substance restoring the retinal function”.—1993.6.Morazov V. G., Khavinson V. Kh.", "Peptide bioregulators (25-years' experience of experimental and clinic studies).—Nauka, St. Petersburg.—1996.—74 p. 7.Yakubke Kh.-D., Eshkeit Kh.", "Amino acids, peptides, proteins: translated from German.—Mir, Moscow.—1985.—456 p. 8.Khavinson V.", "Kh., Zhukov V. V., Deigin V. I., Korotkov A. M. Effect of Thymalin and synthetic peptide of the thymus on the activity of the enzymes of purine nucleotide metabolism in thymocytes.//Abstr.", "Conference “Biochemistry—to medicine”.—Leningrad.—1988.—PP.", "198-199.9.Schlesinger D. H., Goldstein G. The amino acid sequence of thymopoietin II//Cell.—1975.—Vol.", "5, No.", "4.—PP.", "361-365.10.Audbya T., Scheid M. P., Goldstein G. Contrasting biological activities of thymopoietin and splenin, two closely related polypeptide products of thymus and spleen//Proc.", "Natl.", "Acad.", "Sci USA.—1984.—Vol.", "81, No.", "9.—PP.", "2847-2849.11.Fridkin M., Najjar V. A Tuftsin: its chemistry, biology, and clinical potential//Crit.", "Rev.", "Biochem.", "Mol.", "Biol.—1989.—Vol.", "24, No.", "1.—PP.", "140.12.Pokrovsky V. I., Suleimanov A. K., Lebedev V. V. et al.", "Immunorehabilitating effect of thymogexin in the treatment of patients with chronic brucellosis.//Therapeutic archive.—1992.—Vol.", "64, No.", "11.—PP.", "22-26.13.Pigareva N. V., Kalinin N. M, Simbirtsev A. S. Characteristic of the immunologic action of synthetic peptide <<bestim>>//Medical immunology.—1999.—Vol.", "1, No.", "34.—127 p. 14.Teitelbaum D., Aharoni R., Arnon R., Sela M. Specific inhibition of the T-cell response to myelin basic protein by the synthetic copolymer Cop 1//Proc.", "Natl.", "Acad.", "Sci.", "USA.—1988.—Vol.", "85, No 24.—PP.", "9724-9728." ] ]

[ [ "Packaging of a microchip device", "A method of packaging a microchip device, an interposer for packaging, and a packaged microchip device.", "An interposer (7) is placed on microchip devices (1).", "The interposer (7) comprises an aperture (11) which extends from the interposer surface where external electrical contacts (9) are located to the surface of the microchip devices (1).", "Electrical contacts (3) on the microchip device surface are accessible through the aperture (11) in order to electrically connect the electrical contacts (3) with the external electrical contacts (9) of the interposer (7).", "The aperture (11) is divided into at least two openings or aperture regions, separated by a bridge (18).", "This facilitates the handling of the interposer (7)." ], [ "1.A method of packaging a microchip device (1), said method comprising: providing a microchip device (1) having a plurality of first electrical contacts (3); providing an interposer (7) with a plurality of second electrical contacts (9) on an outer side of the interposer (7) and with an aperture (11) extending from the outer side through the interposer (7), wherein the aperture (11) is divided into at least two openings (15, 16, 17) by a bridge (18) which connects opposite aperture edges (13); arranging the interposer (7) adjacent to the microchip device (1) so that the first electrical contacts (3) are accessible from the outer side through at least a first of the openings (15, 16, 17); and making electrical connections (5) between respective ones of the first electrical contacts (3) and the second electrical contacts (9).", "2.A method according to claim 1, wherein, after making the electrical connections (5), at least the first opening (15) is filled with electrically insulating material by transfer moulding.", "3.A method according to claim 1 or 2, wherein at least one of the electrical connections (5) is connected so as to extend from the microchip device (1) through one of the openings (15) and to further extend outside of the opening (15) on the outer side of the interposer (7) and wherein the connection (5) is encapsulated at least outside the opening (15) with insulating material (25) by transfer moulding.", "4.A method according to one of claims 1 to 3, wherein the first opening (15) defines a connecting area to be used for locating the electrical connections (5) and wherein the bridge (18) and the nearest one of the electrical connections (5) in the connecting area are located so that their distance is smaller than three times, preferably two times, the average distance between the connections (5) in the connecting area.", "5.An interposer (7) for packaging a microchip device (1), comprising: a plurality of electrical contacts (9) on an outer side of the interposer (7), for electrically contacting the packaged microchip device and to be electrically connected with the microchip device (1), an aperture (11) extending from the outer side into the interposer (7), wherein the aperture (11) is divided into at least two openings (15, 16, 17) by a bridge (18) which connects opposite aperture edges (13) and wherein at least a first of the openings extends from the outer side through the interposer (7) in order to allow connection to the microchip device (1) to be made.", "6.An interposer according to claim 5, wherein at least the first opening (15, 16) is a frame or window like opening.", "7.An interposer according to claim 5 or 6, wherein at least one of the openings which extends from the outer side through the interposer has an open side where the edge defining the opening's circumference is open.", "8.A packaged microchip device, comprising: a microchip device (1) having a plurality of first electrical contacts (3); an interposer (7) with a plurality of second electrical contacts (9), with an aperture (11) extending from an outer side through the interposer (7) wherein the aperture (11) is divided into at least two openings (15, 16, 17) by a bridge (18); and electrical conductors (5) which electrically connect the first electrical contacts (3) with corresponding ones of the second electrical contacts (9); wherein the interposer (7) is attached to the microchip device (1); at least one of the conductors (5) extends within the aperture (11); and the aperture (11) and the separate opening (16, 17) are at least partly filled with an electrically insulating material (25) and thereby the at least one conductor (5) is fixed to the interposer (7).", "9.A device according to claim 8 with a plurality of packaged microchips wherein the outlines of the microchips (1) each define a package area in which the interposer (7) covers the surface of the corresponding microchip (1), except for the package area regions of the aperture (11); and wherein the bridge (18) extends substantially parallel to the microchip surface from the outside of one of the package areas into this package area.", "10.A device according to claim 9, wherein the bridge (18) connects opposite aperture edges located in different package areas." ], [ "This invention relates generally to the field of semiconductor device manufacture and more particularly to the packaging of microchip devices with integrated circuit chips to be used in an electronic assembly, such as a printed circuit board.", "In the electronics industry, the quest for smaller electronic devices which perform better at a lower cost is ongoing.", "This is especially true for the portable electronics and wireless communications sector which has undergone recent rapid growth.", "In particular, as a result of the decreasing size of microchips and the increasing density of electronic functions per chip area, there is a strong demand for very small microchip packages.", "Examples for conventional packaging of microchip devices are thin small outline package (TSOP) and quad flat pack package (QFP).", "These packages have fixed package sizes with respect to the number of external electrical contacts for contacting the packed microchip device with its periphery.", "With reduced sizes of microchips, the technical requirements for microchip packaging have also been increased.", "In particular, the electrical signal path length should be as short as possible to avoid delay in the transportation of high speed electrical signals.", "Usually, the microchip device comprises very small electrical pads located on the surface of the microchip.", "It is known to connect these electrical pads with the external contacts of the package using very small wires (“wire bonding”) or ribbons (e.g.", "tape automatic bonding, TAB).", "In both cases these conductors are very-delicate and should be protected against mechanical defects.", "Further, it is known to provide external electrical contacts in the shape of pins or balls so as to allow electrical contact to be made to the packaged microchip from the outside of package.", "In certain packaging systems, the electrical pads are contacts on the microchip surface arranged in a certain layout and thereby adapted to a specific packaging method.", "A class of packaging methods which covers such techniques is called Chip Size Packaging (CSP).", "According to the definition of CSP as given by the Institute for Interconnecting and Packaging Electronic Circuits (IPC) the resulting package surface area is not greater than 1.5 times the microchip surface area.", "Further, the resulting package can easily be contacted via exposed external contacts on the package surface.", "In a new subclass of CSP, an interposer with a plurality of external contacts is used for packaging.", "The external contacts are located on an outer side of the interposer which has an aperture extending from the outer side through the interposer.", "In particular, the aperture has the shape of a window or frame (window chip scale packaging, wCSP), but it may also be open on one or more than one of its lateral sides.", "The interposer is arranged adjacent or in the vicinity of the microchip device so that the electrical contacts on the microchip surface are accessible from the outer side through the aperture.", "After that, the contacts on the microchip surface are electrically connected with the external contacts on the outer side of the interposer using electrical conductors.", "At least some of the conductors will extend within the aperture and will be encapsulated afterwards by an electrically insulating material.", "This new subclass of CSP has the following advantages: The conductor length for connecting the electrical contacts on the microchip surface with the external contacts can be kept very short, especially if the edge of the aperture is located close to the contacts on the microchip device surface.", "The external contacts on the outside of the interposer will be connected from the same side as the contacts on the microchip surface during the connecting process.", "Therefore, the arrangement can be very compact.", "The conductors are protected by the encapsulating material.", "The external contacts of the package can directly be connected to contacts of a board.", "Further, the microchip device is located on or near the opposite surface of the package so as to allow the maximum possible heat dissipation to the ambient.", "However, the positioning of the interposer relative to the microchip device must be performed very precisely.", "Otherwise, very little deviations from the desired position may result in contact failure and/or damage the conductors.", "Further, if transfer moulding is used for encapsulating the conductors with electrically insulating material, the handling of the interposer and the microchip device in order to prepare the transfer moulding may result in contact failure or damaging.", "It is an object of the present invention to provide a method of packaging a microchip device which facilitates the handling of the microchip device and/or the interposer and/or the packaged microchip device and allows for small reject rates in mass production.", "It is a further object of the invention to provide a corresponding interposer and a corresponding packaged microchip device.", "Accordingly, the following method of packaging a microchip device is proposed.", "The method comprises: providing a microchip device having a plurality of first electrical contacts; providing an interposer with a plurality of second electrical contacts on an outer side of the interposer and with an aperture extending from the outer side through the interposer, wherein the aperture is divided into at least two openings by a bridge which connects opposite aperture edges; arranging the interposer adjacent to the microchip device so that the first electrical contacts are accessible from the outer side through at least a first of the openings; and making electrical connections between respective ones of the first electrical contacts and the second electrical contacts.", "This solution facilitates the handling of the interposer and of the microchip device, since the bridge strengthens the interposer in the region of the aperture.", "At least in the region next to the bridge, the dimensions of the at least two openings are stable and do not depend on the handling of the interposer.", "Especially if both openings are used to connect the first electrical contacts with the second electrical contacts, the stability is significantly higher compared to a non-divided aperture with the same or a similar cross-sectional area or dimensions.", "Also, the dimensions of the second electrical contacts on the outer side of the interposer are kept stable.", "Therefore, the failure rate for contacting a packaged microchip device to external devices, such as electrical boards, can be decreased.", "Preferably, the bridge is located as near as possible to the first electrical contacts.", "Therefore, in a preferred embodiment, wherein the first opening defines a connecting area to be used for locating the electrical conductors, the bridge and the nearest one of the electrical conductors in the connecting area are located so that their distance is smaller than 3 times, preferably 2 times, the average distance between the conductors in the connecting area.", "Preferably, after the contacting of the first electrical contacts, at least the first opening is filled with electrically insulating material by transfer moulding.", "In a preferred embodiment, at least one of the electrical conductors is connected so as to extend from the microchip device through the first opening and to further extend outside the opening on the outer side of the interposer.", "In this embodiment, the conductor is encapsulated at least outside the opening with insulating material by transfer moulding.", "During the transfer moulding the interposer may be at least partly covered by a mould so as to form a cavity for the insulating material which is to be injected into the cavity.", "Preferably, the cavity will comprise the opening and additionally comprise a space on said outer side of the opening so as to provide sufficient space to entirely encapsulate the at least one conductor.", "A separate opening may be provided, which may be a recess that does not extend through the interposer or may extend through the interposer wherein the insulating material is supplied from a direction so as to pass the region of the separate opening before it reaches the aperture.", "The separate opening stabilises the transfer moulding process with regard to different aspects.", "First, the separate opening acts as a reservoir for the electrically insulating material.", "As a result, there will be sufficient material to fill the aperture when a first amount of material has entered the aperture.", "Especially if the insulating material is a cohesive material, the material which has already entered the aperture can attract material which is located in the separate opening.", "Thus, there will be a sufficient flow into the aperture, even if the flow from a material source is temporarily not sufficient.", "Second, the separate opening acts like a reflection shield, if the insulating material is injected with a velocity which is too high or is injected from a direction which would result in a reverse flow and therefore prevent the succeeding material to enter or fill the aperture.", "One explanation for this effect is that, first, the insulating material will pass the separate opening and at least a part of the insulating material is stored temporarily in the separate opening.", "Afterwards, when additional material has arrived, at least a part of the stored material escapes from the separate opening and joins the succeeding material.", "This results in a flow with satisfactory flux density but with moderate velocity.", "Apart from that, the temporary storage effect allows the filling of other cavities or spaces with insulating material wherein these additional cavities or spaces may be located on side paths of the material flow.", "In a preferred embodiment such additional cavities or spaces to be filled comprise the exterior region of the interposer and/or the microchip devices, especially lateral regions along outer edges of the interposer and/or the microchip device.", "This allows for encapsulating at least a part of the interposer and/or the microchip device.", "Preferably, in this case, the separate opening is dimensioned and arranged so that, during the transfer moulding, the insulating material flows along the exterior of the interposer and/or the microchip device before it reaches the aperture.", "The cross-section of the separate opening may have any shape, for example the shape of a circle, slot, square or rectangle.", "Further, the separate opening can be sub-divided and/or a plurality of separate openings can be provided.", "Preferably, the separate opening or openings are shaped and located so that the aperture, and preferably also an adjacent part of the same cavity to be filled, is/are filled completely with the insulating material.", "Two major advantages of the proposed method of providing the separate opening are that the performing of the transfer moulding and therefore the handling of the interposer and the microchip device are facilitated, since the separate opening stabilises the flow of insulating material, and that, as a result of satisfactory transfer moulding, the final package is protected against damage and can be handled easily.", "In a preferred embodiment, there are plural separate apertures or separate aperture regions to be filled with the electrically insulating material, wherein the apertures or regions are aligned in series so as to form a passageway to be passed by the electrically insulating material during the transfer moulding and wherein a further separate opening is arranged in between two of the apertures or regions along the passageway.", "The further separate opening stabilises the flow of insulating material into the second aperture in a similar way as described above.", "In a further preferred embodiment, the aperture forms at least one passageway extending along the outer side of the interposer (i.e.", "the side from where the aperture extends through the interposer).", "The passageway allows the insulating material to be supplied to or through the interior of the aperture.", "In this embodiment, the separate opening is located next to the beginning of the passageway.", "This location allows for a particularly stable flow into the aperture.", "In a further embodiment, there are a plurality of separate openings, each located next to the beginning or to the end of the passageway or of one of plural passageways.", "A separate opening next to the end of the passageway also contributes to a complete filling of the aperture or the cavity.", "One explanation for this is that the material entering that separate opening acts as a reservoir and deflects the succeeding material.", "Thus, the velocity of the succeeding material at the end of the passageway will be decreased and the deposition of material in that region will be enhanced.)", "According to the invention, it is further proposed to provide an interposer for packaging a microchip device comprising: a plurality of electrical contacts on an outer side of the interposer, for electrically contacting the packaged microchip device and to be electrically connected with the microchip device, an aperture extending from the outer side into the interposer, wherein the aperture is divided into at least two openings by a bridge which connects opposite aperture edges and wherein at least a first of the openings extends from the outer side through the interposer in order to allow connection to the microchip device to be made.", "In particular, at least the first opening is a frame or window like opening.", "It is further preferred that there are a plurality of the apertures, wherein the apertures are aligned in series so as to form a passageway for allowing the passage of electrically insulating material during a transfer moulding process.", "An opening which is located in between two of the apertures along the passageway may be provided, wherein the opening extends from the outer side into the interposer.", "According to the invention, there is further proposed to provide a packaged microchip device, comprising: a microchip device having a plurality of first electrical contacts; an interposer with a plurality of second electrical contacts, with an aperture extending from an outer side through the interposer, wherein the aperture is divided into at least two openings by a bridge; and electrical conductors which electrically connect the first electrical contacts with corresponding ones of the second electrical contacts; wherein the interposer is attached to the microchip device; at least one of the conductors extends within the aperture; and the aperture and the separate opening are at least partly filled with an electrically insulating material and thereby the at least one conductor is fixed to the interposer.", "In particular, the device may comprise a plurality of packaged microchips wherein the outlines of the microchips each define a package area in which the interposer covers the surface of the corresponding microchip, except for the package area regions of the aperture; and wherein the bridge extends substantially parallel to the microchip surface from the outside of one of the package areas into this package area.", "The present invention will now be described by way of non-limitative example only, with reference to the accompanying schematic drawings, in which: FIG.", "1 to 3 show cross-sections through different packaged microchip devices; FIG.", "4 shows in perspective view a part of an interposer positioned on a plurality of microchip devices; FIG.", "5 shows a view onto the contact surface of an interposer which is arranged next to two microchip devices; FIG.", "6 shows a cross-section along line VI-VI through the arrangement of FIG.", "5.FIG.", "7 shows schematically an arrangement for encapsulating material to form microchip packages by transfer moulding; and FIG.", "8 is a cross-sectional view of a part of the arrangement of FIG.", "7.In FIGS.", "1 to 8 the same reference numerals are used for parts and features having the same or similar functions.", "FIG.", "1 shows a cross-section through an arrangement with a microchip device 1 with an interposer 7 and with encapsulating resin 25.The interposer 7 is fixed to the microchip device 1 with a layer of adhesive 27.The width of the microchip device 1 is greater than the width of the interposer 7 so that the edges of the microchip device 1 project outwards on both sides of the microchip device/interposer arrangement.", "On the surface of the microchip device 1 which faces to the interposer 7 chip pads 3 are provided where the interposer 7 does not cover the surface.", "These chip pads 3 act as electrical contacts in order to allow the contact and electrical connection of the microchip device 1 to the interposer 7.There is at least one electrical contact (not shown) on the interposer surface (on the “outer side” 10 of the interposer 7) for each chip pad 3.These contacts are located on the surface of the interposer which faces downwards in FIG.", "1.This means that the chip pads 3 and the contacts on the interposer surface are facing towards the same direction.", "Each chip pad 3 is electrically connected to one of these contacts by a conductor wire 5.Further, each of the contacts is electrically connected with a contact ball 9 on the same interposer surface by an electrical connection (not shown).", "These electrical connections are parts of the interposer 7.The contact balls 9 act as electrical contacts to allow the electrical connection of the packaged microchip device 1.The conductor wires 5 are encapsulated in the resin 25 which can be provided by any suitable process, especially by potting, dispensing, printing and/or transfer moulding.", "The resin 25 encapsulates not only the conductor wires 5 but also the edges of the microchip device 1 and of the interposer 7 so as to mechanically stabilise the package and so as to cover and electrically insulate the chip pads 3 as well as the contacts (not shown) on the interposer surface next to the side edges.", "The package design according to FIG.", "1 represents a class of CSP (Chip Scale Packaging) which is called fan-in design.", "The design according to the arrangement shown in FIG.", "2 represents another class of CSP, called fan-out design.", "The fan-in design differs from the fan-out design with regard to the arrangement of the contacts on the surfaces of the microchip device 1 and the interposer 7 which are to be electrically connected by the conductor wires 5 or by other appropriate means.", "In the fan-in design, both groups of contacts are distributed over the surface areas next to the side edges of the microchip device 1 or the interposer 7.In the fan-out design, these contacts are placed on a surface region of the interposer 7 or the microchip device 1 which are located in the central area of the cross-section.", "Combinations of the fan-in design and the fan-out design are possible.", "FIG.", "3 shows a variant of the fan-out design in which both the side edges and the back surface (facing upwards in FIG.", "3) of the microchip device 1 are covered by the resin 25.Therefore, the microchip device 1 is encapsulated by the resin 25.This design results in particularly stable packages.", "In contrast to this, the resin 25 according to the design of FIG.", "2 does not cover the back surface of the microchip device 1.Further, the side edge of the peripheral resin 25 is aligned with the side edge of the interposer 7 in the embodiment of FIG.", "2.The invention is not limited to the designs which have been described with reference to FIG.", "1 to 3.FIG.", "4 shows a perspective view onto an interposer 7 which is located adjacent to a plurality of microchip devices 1 in order to allow the packaging of the these microchip devices 1 at the same time or in parallel in one process.", "The arrangement according to FIG.", "4 comprises at least nine microchip devices 1 to be packaged as indicated by the dotted package outlines 8.The part on the right hand side of the arrangement has been cut off in order to show the profile of the arrangement.", "The interposer 7 of the arrangement of FIG.", "4 is a single part.", "This facilitates the handling of the interposer and accelerates the production of a plurality of packages.", "However, an interposer consisting of a plurality of parts can be used instead.", "The interposer 7 comprises an aperture 11 with several aperture regions, wherein each aperture region corresponds to the package area of one of the microchip devices 1.There is an elongate first opening 15 in each package area with a cross-section elongate rectangle with rounded corners.", "The first openings 15 extend from the outer side (the top side in FIG.", "4) through the interposer 7 to the surface of the corresponding microchip device 1.As an example for all microchip devices 1 and for other regions on the surface of this particular microchip device 1, eight chip pads 3 are shown on the lefthand side of the package area of the central package in FIG.", "4.The interposer 7 is located on the microchip device surface so as to allow the access to the chip pads 3 from the outer side (top side) through the first opening 15.After arranging the interposer 7 adjacent to the microchip devices 1 conductor wires 5, or other appropriate electrical conductor means, are provided and are connected to the chip pads 3 and to corresponding electrical interposer pads 6 so as to electrically connect each one of the chip pads 3 with one of the interposer pads 6.The interposer pads 6 are located on a surface region of the interposer 7 which faces to said outer side.", "Each one of the interposer pads 6 is electrically connected (connections not shown) to each one of the contact balls 9 on the interposer surface.", "In the embodiment shown in FIG.", "4, the aperture 11 or the aperture regions are stepped so that the surface regions where the interposer pads 6 are located and the surface regions where the contact balls 9 are located are on different levels.", "In particular, the level of the interposer pads 6 is lower so that the aperture 11 can be filled up to the level of the surface region of the contact balls 9.However, the invention is not limited to this particular embodiment.", "Rather, other stepped profiles of the interposer so that there are different surface levels are possible.", "It is also possible to provide an interposer where the interposer pads or contacts are located on the same surface level as the contact balls or other types of external contacts (as for example shown in the designs according to FIG.", "1 to 3).", "The different first openings 15 of the aperture regions are separated by a bridge 18 between each two of the first openings 15.Each bridge 18 comprises two tie bar 19 which extend from one side of the aperture 11 to the opposite side so as to connect the opposite aperture edges 13 that extend along the first opening 15.Between the two tie bars 19 of each bridge 18, there is a second opening 16 which extends also from the outer side of the interposer 7 through the interposer 7 to the surface level of the microchip devices 1.In an alternative embodiment at least one of these second openings does not extend through the interposer, but only extends from the outer side into the interposer.", "In both embodiments the second opening 16 act as stabilising means in order to stabilise the flow of liquid or molten insulating material which is transferred to at least partly fill the aperture 11 or aperture regions.", "Further, the tie bars 19 act as cross struts for the aperture 11.Therefore, the dimensions of the first openings 15 and the distance D between the contact balls 9 on opposite sides of the aperture 11 are stabilised and the handling of the interposer 7 is facilitated.", "The aperture 11 forms a passageway for supplying encapsulating material or filling material to the aperture regions.", "The passageway is elongate and composed of a series of succeeding aperture regions according to the embodiment of FIG.", "4.Another embodiment of an interposer/microchip device arrangement is shown in FIGS.", "5 and 6.FIG.", "5 shows a top view onto the contact surface of the arrangement where the contact balls 9 are located.", "The interposer 7 consists of a single part and is provided for the packaging of two microchip devices 1.The arrangement is similar to the fan-in design, but the resulting two packages of the microchip devices 1 will be divided after the packaging by separating them along the package outline 8.The aperture 11 is elongate and extends along the edges in the central area of the interposer 7 where the interposer pads 6 are located.", "The aperture 11 is subdivided by a tie bar 19 which is located half way between the open end of the aperture 11 (at the top and bottom in FIG.", "5 and in the front and back in FIG.", "6).", "The tie bar 19 does not extend from the surface level of the microchip device 1 to the surface level on the outer side of the interposer 7 where the contact balls 9 are located (as can be seen best from FIG.", "6).", "Rather, the tie bar 19 extends from the surface level of the microchip device 1 to a level which is at about two thirds of the level difference to the surface level on the outer side of the interposer 7.This allows insulating material or other filling or encapsulating material to pass the tie bar 19 when the material is supplied from one of the open ends of the aperture 11.Further, the region of the aperture 11 at the open end, through which the material is supplied, acts like one of the second openings 16 of the embodiment of FIG.", "4, i.e.", "it stabilises the flow of the material to the aperture region on the other side of the tie bar 19.FIG.", "7 shows schematically a process of transfer moulding in order to perform the packaging of four microchip devices 1.Each two of the microchip devices 1 are aligned in series on a supply path of the material to be supplied during the transfer moulding, two on the right hand side and two on the left hand side of FIG.", "7.The flow direction of the supplied material is indicated by several arrows in FIG.", "7.From a material source 22, the material is supplied through two supply paths 23 each leading to one of the two areas where each two of the microchip devices 1 are aligned in series.", "A sectional view of a part of such an area is shown in FIG.", "8 with some details in the region where the supply path 23 ends.", "The arrangement comprises several cavities to be filled completely with encapsulating material: the aperture 11 which is similarly subdivided as in the embodiment of FIG.", "4, but does not have the stepped profile; peripheral cavities 21 on both sides of each package so as to allow the encapsulation of the lateral edges of the packages similar to the embodiment of FIGS.", "1 and 2; and front and back side cavities 24,26 located in the front and back of the packages in the supply direction of the material to be supplied.", "The front and back side cavities 24,26 allow for the encapsulation of the front and back sides of the packages.", "For the transfer moulding process, a mould or moulds (not shown) can be provided which cover at least the surface region of interposers 7 where contact balls 9 are located as external contacts of the packages.", "Each interposer 7 is elongate and extends from the end region of the supply path 23 through the first and second package area of the two aligned microchip devices.", "It comprises each one first opening 15 per microchip device package.", "The first openings extend from the outer side of the interposer 7 through the interposer 7 to the surface of the corresponding microchip device 1.Further, the interposer 7 comprises altogether three separate openings 17 located in supply direction in front of the first microchip device package, in between the two microchip device packages, and behind the second microchip device package.", "All separate openings 17 extend from the outer side of the interposer 7 through the interposer 7.The separate openings 17 stabilise the flow of material which is supplied from the material source 22 through the supply path 23.Further, the handling and the dimensions of the interposer 7 are stabilised by four tie bars 19 in total, located each between one of the first openings 15 and one of the separate openings 17.When the material from the material source 22 is supplied through the supply path 23, the material reaches the separate opening 17 which is located next to the end of the supply path 23 before it reaches the first front side cavity 24 and before it reaches the first opening 15.Therefore, the filling material partly fills this separate opening 17 and allow other filling material to reach the peripheral cavities 21 before the first opening 15 is completely filled.", "Therefore, the peripheral cavities 21 can be provided with sufficient material to encapsulate the microchip device 1 and the interposer 7 on their lateral sides during the transfer moulding process.", "A similar effect is achieved by the separate opening 17 between the two aligned microchip devices 1.However, it should be noted that the length of this particular separate opening 17 is greater than the length of the separate opening 17 in front of the first microchip device 1 and behind the second microchip device 1.By adapting the length of the separate openings 17 and optionally by adapting also or alternatively the length of the first openings 15 the filling of all cavities 15,21,24,26 to be filled can be controlled to achieve satisfactory filling results." ] ]

[ [ "Cover for golf club protection", "A head cover for protection a golf club and a shaft, more particularly to a cover for golf club, of which the golf club can be inserted into or taken out through the opening part of the head cover, then the upper side is open, so that the club can be inserted into or taken out and connecting ribs are formed as one with plastic supporting ribs like umbrella ribs for obtaining the enough space, then after cover sheets are formed inside and outside of the supporting ribs, they are combined with the head cover, which is open upside and then a protection pipe is combined with the connecting ribs." ], [ "1.A head cover for protection a golf club wherein its improvement comprising, a golf club which can be inserted into or taken out through the opening part of the head cover.", "2.A head cover for protection a golf club as claimed in claim 1 wherein, a few of supporting ribs that are spread upward and the connecting ribs that are extended downward are combined with a protection pipe.", "3.A head cover for protection a golf club as claimed in claim 1 and 2 wherein, the cover sheets are formed inside and outside of the supporting ribs.", "4.A head cover for protection a golf club as claimed in claim 2 wherein, the above supporting ribs is formed as a barrel.", "5.A head cover for protection a golf club as claimed in claim 1 wherein, the elasticity frame, which has an elasticity power, is formed on the boundary of the opening part in the head cover.", "6.A head cover for protection a golf club as claimed in claim 1 wherein, after the cover sheets is formed and combined with the protection pipe (3), the ornamental cover (8) is formed.", "7.A head cover for protection a golf club as claimed in claim 1 wherein, the sleeve, which has a wing that becomes narrow, is formed on the connecting ribs.", "8.A head cover for protection a golf club as claimed in claim 5 wherein, the center opening part of the elasticity frame is open and closed using a string or a patch." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to a head cover for protecting a golf club and a shaft, more particularly to a cover for golf club, of which the golf club can be inserted into or taken out through the opening part of the head cover.", "Usually the golf club is kept in a bag that is divided into two or three equal parts by a partition, so the golf clubs are moving in a disordered way, which causes tangling among the golf clubs and makes inconvenient for inserting into or taking out and causes damage to the shaft and the grip of the golf clubs.", "Therefore, in order to protect the golf clubs, usually a cover, which is produced with textiles that have a spandex quality, protects by covering the head.", "However, this kind of cover is inconvenient to use, because it is covered form upside and after covering, it should be pulled out.", "In addition, when the clubs are not placed vertically in the bottom, they are to be tangled with each other, because the grip's diameter is usually bigger than the shaft's, 2.Discussion of Related Art Thus, the present invention is designed in order to solve the above-mentioned problem, the upper side is open, so that the club can be inserted into or taken out and connecting ribs are formed as one with plastic supporting ribs like umbrella ribs for obtaining the enough space.", "And after cover sheets are formed inside and outside of the supporting ribs, they are combined with the head cover, which is open upside and then a protecting pipe is combined with the connecting ribs." ], [ "<SOH> BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS <EOH>FIG.", "1 is a sectional view showing that a protecting pipe is combined with the connecting ribs which are formed supporting ribs; FIG.", "2 is a sectional view showing another embodiment of a cover sheet; FIG.", "3 is a sectional view showing a state attaching an ornamental cover; FIG.", "4 is a state view showing that the cover sheet covers a supporting ribs; FIG.", "5 is a perspective view showing that a barrel is used instead supporting ribs; FIG.", "6 is a state view showing that the cover sheet covers the barrel; FIG.", "7 is a sectional view showing of the embodiment of the present invention used.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to a head cover for protecting a golf club and a shaft, more particularly to a cover for golf club, of which the golf club can be inserted into or taken out through the opening part of the head cover.", "Usually the golf club is kept in a bag that is divided into two or three equal parts by a partition, so the golf clubs are moving in a disordered way, which causes tangling among the golf clubs and makes inconvenient for inserting into or taking out and causes damage to the shaft and the grip of the golf clubs.", "Therefore, in order to protect the golf clubs, usually a cover, which is produced with textiles that have a spandex quality, protects by covering the head.", "However, this kind of cover is inconvenient to use, because it is covered form upside and after covering, it should be pulled out.", "In addition, when the clubs are not placed vertically in the bottom, they are to be tangled with each other, because the grip's diameter is usually bigger than the shaft's, 2.Discussion of Related Art Thus, the present invention is designed in order to solve the above-mentioned problem, the upper side is open, so that the club can be inserted into or taken out and connecting ribs are formed as one with plastic supporting ribs like umbrella ribs for obtaining the enough space.", "And after cover sheets are formed inside and outside of the supporting ribs, they are combined with the head cover, which is open upside and then a protecting pipe is combined with the connecting ribs.", "BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS FIG.", "1 is a sectional view showing that a protecting pipe is combined with the connecting ribs which are formed supporting ribs; FIG.", "2 is a sectional view showing another embodiment of a cover sheet; FIG.", "3 is a sectional view showing a state attaching an ornamental cover; FIG.", "4 is a state view showing that the cover sheet covers a supporting ribs; FIG.", "5 is a perspective view showing that a barrel is used instead supporting ribs; FIG.", "6 is a state view showing that the cover sheet covers the barrel; FIG.", "7 is a sectional view showing of the embodiment of the present invention used.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENT The following is the detailed description of the most desirable embodiment of the present invention.", "As shown in FIG.", "1, for the structure of the present invention, a few of supporting ribs (1) that are spread upward and connecting ribs (2) that are extended downward are combined with a protecting pipe (3) and the said supporting ribs (1) and connecting ribs (2) are covered by cover sheets (4)(4a) inside and outside, then the upper part is combined with a head cover (5), when it is covered as above.", "At this point, if a sleeve (7), which has a wing (6) that becomes narrow, is formed on the connecting ribs (2) and the protection pipe (3), the shaft of the golf club is placed vertically and it makes easy the shaft to go in and out as well as it prevents from friction at the same time that it prevents from moving freely.", "In addition, the above-described sleeve (7) can be placed on the bottom of the protection pipe (3) or various places.", "Also, the said supporting ribs (1) is composed of about 6 to 8 ribs as umbrella ribs, so that it can be bent easily, which makes convenient the cover sheet (4) to be covered.", "Although various textiles can be applied for the cover sheets (4)(4a) that covers the supporting ribs (1) inside and outside, the textile (4a) that covers the inside part, namely, lining, is made of velvet or a carpet, so that the club is damaged to the minimum using a buffer such as a sponge.", "Moreover, the above-mentioned supporting ribs (1) can be formed as a barrel when it is pliable and not a rib and this kind of barrel can be manufactured as one by a brow forming.", "Furthermore, for the cover sheets (4)(4a) that cover inside and outside part of the supporting ribs (1), in cased of that the protection pipe (3) is combined with the in the outside, after the cover sheets (4a) that covers inside, is pulled to the outside of the connecting ribs (2) and fix it, an ornamental cover (8) is formed at the outside of the protection pipe (3) and fix it, then the ornamental cover (8) covers the cover sheets (4)(4a) neatly.", "Also, the protection pipe (3) can be made transparent and colored or separate textiles can be used for this.", "Besides, a round board (10), which is formed a lot of holes (9) that the protection pipe (3) are inserted into, is prepared and a protrusion (11) that a sill (12) is formed on the forward part of the holes (9) of the round board (10) vertically.", "After a spring (13) is placed on the sill (12) of the said protrusion (11), the protection pipe (3) is placed on the upper part of the spring (13), so that in case that the golf clubs, which are kept in the cover of the bag, are taken out of the bag, if the golf cover is lowered, then the golf club is exposed to the outside.", "In addition, a cover (15), which is bent, is formed at the opening part of the head cover (5), so when it is not used for a while, the cover (15) can close the opening part (16) preventing from inserting some extraneous matters such as dust.", "At this point, Velcro or zipper can fix the cover (15).", "Furthermore, when the above cover (15) is not used, the upper part can be tighten, so that it prevents from inserting some extraneous matters such as dust.", "An elasticity frame (17) is formed on the boundary of the opening part in the head cover (5), so that the golf club can be inserted into or taken out easily and it is not carelessly slipped out.", "And when it is used continuously, the club is located on the upper part of the elasticity frame, (17) and the said elasticity frame (17) is formed with two steps.", "As comprised above, because the present invention is open upside of the head cover (5), the golf club can be inserted into or taken out easily.", "In some part of the protection pipe (3), the grip can be exactly moved to up and down by the sleeve (7) of which mouth is formed narrow and the shaft is not moved." ] ]

[ [ "Multilayered packaging for greasy products", "The invention relates to a multilayered packaging for greasy products or part of said packaging, comprising a carrier layer made of a polymer material as main component and at least one layer placed on said carrier layer that does not form the outer side of the packaging, said layer containing a starch derivative as main component.", "The invention is characterized in the starch derivative is a starch derivative modified with a C2-C6-alkylene oxide.", "The invention also relates to the use of a C2-C6-alkylene oxide derivatized starch as main component of a layer of a multilayered packaging, which is placed on a carrier layer made of a polymer material in said packaging with the aim of rendering said multilayered packaging grease-tight." ], [ "1.Multilayer packaging for greasy products or part of said packaging, comprising a carrier layer made of a polymer material as the main component, and at least one layer applied onto said carrier layer and not forming the outer side of said packaging, said layer containing a starch derivative as the main component, characterized in that said starch derivative is a starch derivative modified with a C2-C6 alkylene oxide 2.Multilayer packaging in accordance with claim 1, characterized in that said starch derivative is a starch derivative modified with a C2-C4 alkylene oxide.", "3.Multilayer packaging or part of said packaging in accordance with claim 1, characterized in that said C2-C6 alkylene oxide is propylene oxide.", "4.Multilayer packaging or part of said packaging in accordance with any of the aforementioned claims, characterized in that said starch derivative was obtained by modification of corn, wheat, potato, HA pea, or tapioca starch which was optionally partly degraded.", "5.Multilayer packaging or part of said packaging in accordance with any of the aforementioned claims, characterized in that said starch derivative has a degree of derivatization of from 0.1 to 1, more preferably of from 0.1 to 0.4.6.Multilayer packaging or part of said packaging in accordance with any of the aforementioned claims, characterized in that the polymer material of said carrier layer is a polymer occurring in nature, preferably cellulose.", "7.Multilayer packaging or part of said packaging in accordance with any of the aforementioned claims, characterized in that said layer containing a starch derivative as the main component contains additional components selected from among pigments, softeners, agents increasing the long-term stability, agents increasing the water stability, and agents affecting the elasticity.", "8.Multilayer packaging or part of said packaging, preferably in accordance with claim 4, characterized in that additional components are selected from among glycerol, urea, borax or glyoxal.", "9.Use of a starch derivatized with a C2-C6 alkylene oxide as the main component of a layer of a multilayer packaging, said layer being applied onto a carrier layer of said packaging which is made of a polymer material, with the aim of rendering said multilayer packaging greaseproof.", "10.Use in accordance with claim 9, characterized in that said C2-C6 alkylene oxide is propylene oxide.", "11.Use in accordance with any of claims 9 and 10, characterized in that said starch derivative was obtained by modification of optionally partly degraded corn, wheat, potato, HA pea, or tapioka starch and optionally has a degree of derivatization of from 0.1 to 1, more preferably of from 0.1 to 0.4.12.Use in accordance with any of claims 9 to 11, characterized in that said layer contains additional components selected from among pigments, softeners, agents increasing the long-term stability, agents increasing the water stability, agents increasing the KIT value, and agents affecting the elasticity, preferably selected from among glycerol, urea, borax, or glyoxal." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The invention relates to multilayer greaseproof packaging materials having a carrier layer which is made of paper/cardboard or other suitable substances on a polymer basis.", "2.Description of the Related Art It has been known for a long time to provide paper and cardboard containers with coatings having a barrier effect for flavors or humidity/liquids.", "DE 41 09 983 A1 describes a flexible packaging container comprising a composite made of a paper layer and a thermoplastic layer or film.", "The thermoplastic layer or film material consists of starch, a synthetic, non-polyolefinic polymer containing hydroxyl groups, e. g. an oxygenated polymer, and softeners of natural origin, e. g. polyalcohols derived from starch.", "Said material can be melted by supplying heat and therefore, it is extrudable.", "DE 41 37 802 A1 proposes to laminate a cardboard with a coated paper web to obtain a rottable, liquid-repellent product.", "The coating of the paper web shall be effected on a starch basis, e. g. on potato starch basis.", "DE 42 94 110 discloses a coating dispersion which is prepared from copolymers of oxidized starch and styrene, butadiene, acrylic acid or similar polymerizable molecules.", "Said dispersion reduces the gas and water permeability of cardboard or paper.", "However, it is often necessary to provide packaging materials which have a high resistance to grease.", "Thus, food for animals, bread and confectionery, sweets and chocolate require a packaging having a particularly high resistance to grease which is for example indicated by the so-called KIT numbers exhibiting values between 8 and 12.High KIT numbers represent high resistances to grease, values as from 6 already represent a good resistance to grease.", "Corresponding commercially offered paper/cardboard packaging has usually been subjected to a grease-repellent surface and/or mass treatment.", "At present, mainly fluoropolymers are used for said mass treatment or surface treatment, up to 5% by weight of coating material being applied onto the material.", "Grease resistances of >6 to 8 can only be obtained by combining layer and mass treatment, grease resistances with KIT numbers of >12 cannot be guaranteed with the present systems.", "For example, packing dry food for animals with a low grease content (<10%) requires a mass treatment of the backside, in case of higher grease contents, a barrier is realized by mass treatment in combination with a surface coating.", "Waste paper, paperboard and cardboard packaging are regularly disposed via the waste paper circuit.", "Thus, via the pulping process, the halogen polymers used as grease barrier either arrive at the virgin paper product or in the waste water of the process.", "Starch ethers are known as auxiliary agents and starting materials in the paper industry.", "The properties used are described in detail in the pertinent literature.", "They are used in surface coating and coating, respectively, and in pigmented paper coatings.", "In accordance with the BGVV (Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin), paper, cardboard, and paperboard admitted for food contact may also contain starch ethers (e. g. hydroxyethyl ether and hydroxypropyl ether).", "Further, starch ethers are used as a component of adhesives because of their good film-forming property and their water bonding capacity.", "Respective literature is to be found for example in Ullmanns Enzyklopäidie der technischen Chemie; W. Baumann/B.", "Herberg: Papierchemikalien—Fakten zum Umweltschutz (Springer-Verlag); O.", "B: Wüirzburg: Modified Starches: Properties and Uses (CRC Press).", "Further, it is known that starch ether derivatives can be processed to foils/films from an aqueous solution, particularly using casting technology.", "When preparing the starch ethers in accordance with the so-called Slurry method, the aqueous starch suspension is derivatized under alkaline conditions at temperatures of up to 50° C. The degree of substitution (DS) is substantially around 0.2.The preferred derivatization at the C 2 atom is characteristic for said methods.", "Another method which is substantially known from scientific examinations (autoclave method) is based on alkaline-activated starch and realizes more homogeneous derivatizations at lower TS (i. e. dry substance) concentrations, the degree of substitution (DS), however, being adjusted similarly.", "Proceeding in accordance with said strategy is described in DE 42 23 471 A1, the starch ethers obtained according to this document being intended to be used for the preparation of films, particularly for use as overhead, copying, and printing films or for the surface finishing of special papers, and as packaging material.", "Further, it is indicated in said publication that the ether derivative films mentioned therein can be used in combination with other materials." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The object of the present invention is to provide packaging materials which are admissible according to food law, and which are greaseproof.", "Surprisingly, it was found that substrates which themselves do not provide sufficient resistance to grease, such as paper, cardboard, paperboard, or other materials which are made of or contain cellulose, are greaseproof when coated with alkylene-oxide-derivatized starch.", "Therefore, the present invention provides multilayer packagings for greasy products or parts of said packagings, said packagings comprising a carrier layer made of a polymer material as the main component, and at least one layer applied onto said carrier layer and not forming the outer side of said packaging, said layer applied onto said carrier layer containing an alkyene-oxide-derivatized starch as the main component.", "The alkylene oxide used for this purpose can suitably be a C 2 -C 6 alkylene oxide.", "C 2 -C 4 alkylene oxides are preferred.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "This application claims benefit of priority of PCT Application No.", "PCT/EP01/07456, filed Jun.", "29, 2001 and published on Jan. 10, 2002, as WO 02/02412, and German Application Nos.", "100 32 252.2, filed Jul.", "3, 2000, and 100 49 665.2, filed Oct. 6, 2000.BACKGROUND OF THE INVENTION 1.Field of the Invention The invention relates to multilayer greaseproof packaging materials having a carrier layer which is made of paper/cardboard or other suitable substances on a polymer basis.", "2.Description of the Related Art It has been known for a long time to provide paper and cardboard containers with coatings having a barrier effect for flavors or humidity/liquids.", "DE 41 09 983 A1 describes a flexible packaging container comprising a composite made of a paper layer and a thermoplastic layer or film.", "The thermoplastic layer or film material consists of starch, a synthetic, non-polyolefinic polymer containing hydroxyl groups, e. g. an oxygenated polymer, and softeners of natural origin, e. g. polyalcohols derived from starch.", "Said material can be melted by supplying heat and therefore, it is extrudable.", "DE 41 37 802 A1 proposes to laminate a cardboard with a coated paper web to obtain a rottable, liquid-repellent product.", "The coating of the paper web shall be effected on a starch basis, e. g. on potato starch basis.", "DE 42 94 110 discloses a coating dispersion which is prepared from copolymers of oxidized starch and styrene, butadiene, acrylic acid or similar polymerizable molecules.", "Said dispersion reduces the gas and water permeability of cardboard or paper.", "However, it is often necessary to provide packaging materials which have a high resistance to grease.", "Thus, food for animals, bread and confectionery, sweets and chocolate require a packaging having a particularly high resistance to grease which is for example indicated by the so-called KIT numbers exhibiting values between 8 and 12.High KIT numbers represent high resistances to grease, values as from 6 already represent a good resistance to grease.", "Corresponding commercially offered paper/cardboard packaging has usually been subjected to a grease-repellent surface and/or mass treatment.", "At present, mainly fluoropolymers are used for said mass treatment or surface treatment, up to 5% by weight of coating material being applied onto the material.", "Grease resistances of >6 to 8 can only be obtained by combining layer and mass treatment, grease resistances with KIT numbers of >12 cannot be guaranteed with the present systems.", "For example, packing dry food for animals with a low grease content (<10%) requires a mass treatment of the backside, in case of higher grease contents, a barrier is realized by mass treatment in combination with a surface coating.", "Waste paper, paperboard and cardboard packaging are regularly disposed via the waste paper circuit.", "Thus, via the pulping process, the halogen polymers used as grease barrier either arrive at the virgin paper product or in the waste water of the process.", "Starch ethers are known as auxiliary agents and starting materials in the paper industry.", "The properties used are described in detail in the pertinent literature.", "They are used in surface coating and coating, respectively, and in pigmented paper coatings.", "In accordance with the BGVV (Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin), paper, cardboard, and paperboard admitted for food contact may also contain starch ethers (e. g. hydroxyethyl ether and hydroxypropyl ether).", "Further, starch ethers are used as a component of adhesives because of their good film-forming property and their water bonding capacity.", "Respective literature is to be found for example in Ullmanns Enzyklopäidie der technischen Chemie; W. Baumann/B.", "Herberg: Papierchemikalien—Fakten zum Umweltschutz (Springer-Verlag); O.", "B: Wüirzburg: Modified Starches: Properties and Uses (CRC Press).", "Further, it is known that starch ether derivatives can be processed to foils/films from an aqueous solution, particularly using casting technology.", "When preparing the starch ethers in accordance with the so-called Slurry method, the aqueous starch suspension is derivatized under alkaline conditions at temperatures of up to 50° C. The degree of substitution (DS) is substantially around 0.2.The preferred derivatization at the C2 atom is characteristic for said methods.", "Another method which is substantially known from scientific examinations (autoclave method) is based on alkaline-activated starch and realizes more homogeneous derivatizations at lower TS (i. e. dry substance) concentrations, the degree of substitution (DS), however, being adjusted similarly.", "Proceeding in accordance with said strategy is described in DE 42 23 471 A1, the starch ethers obtained according to this document being intended to be used for the preparation of films, particularly for use as overhead, copying, and printing films or for the surface finishing of special papers, and as packaging material.", "Further, it is indicated in said publication that the ether derivative films mentioned therein can be used in combination with other materials.", "SUMMARY OF THE INVENTION The object of the present invention is to provide packaging materials which are admissible according to food law, and which are greaseproof.", "Surprisingly, it was found that substrates which themselves do not provide sufficient resistance to grease, such as paper, cardboard, paperboard, or other materials which are made of or contain cellulose, are greaseproof when coated with alkylene-oxide-derivatized starch.", "Therefore, the present invention provides multilayer packagings for greasy products or parts of said packagings, said packagings comprising a carrier layer made of a polymer material as the main component, and at least one layer applied onto said carrier layer and not forming the outer side of said packaging, said layer applied onto said carrier layer containing an alkyene-oxide-derivatized starch as the main component.", "The alkylene oxide used for this purpose can suitably be a C2-C6 alkylene oxide.", "C2-C4 alkylene oxides are preferred.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS By coating a carrier material with the functional layer of a “starch derivative”, a greaseproof composite system is realized.", "The starch component is responsible for the resistance to grease and additionally has the property of being biologically degradable.", "Moreover, such a starch can be suitably incorporated into coating masses for the purpose indicated, since—in contrast to native starch—it is not susceptible to re-aggregation (retro gradation).", "The packagings according to the invention are not limited to specific embodiments.", "An exemplary, preferred field of application comprises packagings of foodstuffs and food for animals which simultaneously have a low water content and a high grease content, particularly folding boxes.", "Respective examples are packagings for biscuits or cookies, chocolates, other sweets, dry food for animals for which a particularly good barrier against the passage of water vapor is not required.", "In addition, packagings for fat-containing non-foods (e.g.", "cosmetics, oil-containing color pigments, or the like) can be embodied in accordance with the invention.", "Further possibilities of applications are the coating of polymers other than cellulose with the material proposed according to the invention (e. g. other packaging plastics) for similar packaging requirements.", "Another possible use is the coating of paper intended as a wrapping paper.", "With the packaging materials according to the present invention, high resistances to grease are realized corresponding to KIT numbers in the range of from 6, particularly at least of from 8, but usually substantially higher.", "Thus, resistances having a KIT number of 10 or 12, in extreme cases of even more than 21, can be achieved which cannot be guaranteed with the previous, non-bio-compatible and non-bio-degradable systems.", "Further advantages of the packagings according to the invention are compatibility of their production with usual methods of the paper and cardboard production and biological degradability; said packagings, in comparison with usual systems, being classified as particularly advantageous with regard to the economic and/or ecologic evaluation, particularly to the coating costs, including process costs, and the compatibility with the paper recycling process.", "Due to their bio-degradability, the packagings according to the invention ensure a good compatibility with the recycling and waste water purification possibilities of waste paper disposal facilities.", "The degradation behavior in the paper circuit represents a decisive advantage in the sense of avoiding the introduction of additional interfering substances.", "Particularly suitable materials for the carrier layer are paper, paperboard or cardboard, if desired in mixture with other suitable materials or materials usual or admissible in the packaging technology of foodstuffs, such as binders or colorants.", "But also other materials, preferably materials on the basis of natural polymers, such as cellulose or the like, or plastics can be used.", "Starch derivatives which are suitable according to the invention are starch derivatives which have been derivatized with an alkylene oxide, such as ethylene oxide or propylene oxide, or with a longer-chain alkylene oxide.", "The attached radicals enlarge the distances between the molecular chains and thus increase their mobility.", "The inner softening effect thus provided can only be undone by destroying the chemical bonds.", "The starch derivative should preferably form a continuous film on the carrier layer.", "If this is the case, already very thin layers with a weight per unit area as from 6 g/m2 can be greaseproof, provided that the carrier material has a relatively high evenness.", "The coating can be provided as a surface layer of the inner side of the packaging and/or as an intermediate layer, optionally having the function of an adhesive layer between paper or cardboard layers or the like.", "Also several coatings applied one directly onto the other can be advantageous.", "Further, a so-called prime-coating (e. g. with usual paper coating substances, like kaolin or starch) can be applied prior to the coating with the starch derivative, said prime-coating serving the purpose of calendering or polishing (smoothing) the surface in advance.", "Weights per unit area for achieving defect-free layers can thus be reduced.", "The layer containing the starch derivative can optionally be placed onto the carrier layer by applying a self-supporting layer made of this material.", "Preferably, however, a solution or a suspension of the starch derivative is prepared using a suitable quantity of dry substance, and is applied onto the carrier material; it is preferably an aqueous solution or a suspension.", "A well-suited quantity of dry substance (TS) of the starch derivative is in the range of about 5 to about 50% by weight, preferably in the range of from about 10 to about 40% by weight, the quantity to be actually selected depending on the intended application method.", "Thus, in some cases, a quantity of down to 4% by weight may be sufficient.", "The application can for example be effected with a knife (or “doctor blade”), by spraying or by roller application, also by “pressure casting” of a more concentrated solution, and by application of a thermo-plasticized melt (“extrusion”) to the surface of the carrier.", "In any case, the water content of the starch derivative should be reduced to preferably <25% by weight after application onto the carrier material (e. g. by infrared or convection drying).", "Besides the starch derivative, the layer to be applied onto the carrier layer may contain additional additives.", "On the one hand, the addition of pigments (as it is customary in the paper industry) offers itself, on the other hand, glycerol, urea, borax, glyoxal or other additives having similar properties and effects can be added in order to achieve desired values with respect to elasticity, water stability, and long-term stability.", "Also, the KIT value can sometimes be influenced positively by adding such substances, e. g. by adding glycerol or curing agents (e. g. glyoxal).", "The proportion of starch derivative should preferably be in an amount to ensure the formation of a defect-free film.", "Preferably, native starches, such as corn (maize), wheat, pea, potato, or tapioca starch, are used as starting materials.", "The modification is for example effected with a C2 or C3 alkylene oxide.", "Propylene oxide is preferred.", "Since the starch is suitably modified in the presence of a base, although the mass provided for the coating should advantageously react substantially neutrally, which implies that normally, neutralization with acid has to be effected, the modified starch is usually highly charged with salts.", "Advantageously, said salt content should not be too high, particularly when using the particularly preferred corn or wheat starch.", "Therefore, it is recommended that the coating mass in the concentration provided for the application should have a conductivity of not more than 4,000 to 5,000 μS/cm, preferably of <2,000 μS/cm.", "The addition of acids and bases should be effected under the aspect that the occurring salt is generally recognized as safe with regard to food law.", "Suitable acids are phosphoric acids, a suitable base is sodium hydroxide.", "The desalination can for example be effected by dialysis.", "Coatings with higher-derivatized starches present more favorable KIT numbers than those with lower degrees of derivatization.", "However, it is not necessary to achieve high substitution rates, since already low rates can have positive effects.", "These are moreover dependent on the origin of the starch used.", "Whereas in general, a degree of derivatization of from 0.05 to 1.5 can be useful, ranges of between 0.1 to 1.0, particularly of between 0.1 and 0.3 are preferred.", "The preparation of a starch ether solution suitable as a coating material or casting solution or the like for the present invention can for example be effected as follows: The starch (e. g. wheat, corn, tapioca, potato, or HA pea starch, e. g. from peas) is stirred for several hours in approximately twice its weight of water and subsequently roughly released from water, e. g. by sucking-off.", "The starch absorbs approximately its own weight of water, so that it contains about 40 to 60% of dry substance.", "Subsequently, it is re-suspended in about 1.5 times of its wet weight and disintegrated by addition of the same quantity of about a 10% base or sodium hydroxide.", "Immediately thereafter, within several minutes up to about one hour, alkylene oxide, preferably propylene oxide, in a quantity of from about 25 to 75% by weight relative to the basic weight of the dry starch is added, maintaining mild temperatures.", "Room temperature is well-suited.", "The mixture is stirred for several hours and subsequently rested for about 20 hours; thereupon, it is neutralized with acid.", "In case desalination is to be carried out, dialysis against water is for example used.", "If desired, the desalinated solution is carefully reconcentrated.", "In case propylene oxide is used in a quantity of about 50% by weight, the degree of derivatization of the starches is about 0.2, in other cases it is correspondingly higher or lower.", "A desalination or separation of interfering inhomogeneities can for example also be effected by ultrafiltration.", "Should the concentration of the product be too high, a dilution with deionized water can be effected.", "After addition of possibly desired additives (e. g. preserving agents, fillers, antistatic agents, elasticity-improving agents, curing agents), a mechanical separation using filters or a centrifuge can be carried out, if required, which simultaneously will accomplish degasification of the solution to be processed.", "A coating solution which is particularly suitable for the purposes of the invention has the following rheological properties.", "A dynamic viscosity of from 0.1 Pas to 40 Pas at a temperature of 25° C. and a shearing speed of 30.7 s−1.Viscoelastic properties of the polymer solution, the ratio of viscous to elastic proportion having Tan κ values of between 1 and 10 (50 at maximum) at a temperature of 25° C. and a shearing speed of 30.7 s−1.Such values can be obtained without difficulties when using the method mentioned as an example.", "Further, the method offers the advantage that the starch is reacted and processed particularly carefully and continuously at relatively low temperatures (<60° C.) or completely at room temperature, which has positive effects on the coating of the carrier material.", "Due to the solubility in cold water after neutralization, separation, desalination and reconcentration, the starch can be processed so carefully that degradation reactions do not occur at all or only to an insignificant extent.", "The aqueous casting solution can preferably be applied onto the material web (paper) at room temperature or at slightly increased temperatures, using a suitable application system (e. g. a knife).", "The use of hydroxypropyl ether starches prepared according to the autoclave method, particularly from wheat starch, corn starch, or HA pea starch, has been proven to be particularly favorable, said starches being used as solutions having TS contents of from 12 to 20% by weight and degrees of derivatization (DS) of preferably from 0.1 to 1.0, more preferably of up to 0.4.In comparison with commercial samples (coated with fluorocarboxylic acids), said starches show markedly better resistances to grease, particularly also in edge portions which are particularly critical when folding boxes are used.", "Compared to the previously mentioned coatings using commercial starch derivatives according to the invention, the weights per unit area used for the coatings with said starches can be reduced.", "Example: 713 g wheat starch is stirred for four hours in 1.3 1 distilled water and subsequently filtered by sucking.", "The wet starch is stirred up with 1.824 1 water and mixed with 1.811 g of 10% sodium hydroxide which was obtained by mixing 376 g 50% NaOH with 1.505 g water.", "Derivatization is effected using 323 g propylene oxide at 23° C. which is added under stirring within 20 minutes.", "The mixture is stirred for further 4 hours and rested for 20 hours.", "Neutralization is effected with 40% phosphoric acid (about 700 g).", "Subsequently, the solution is filled into dialysis tubes and dialyzed for about 4 days, the water being exchanged daily.", "The product is reconcentrated to more than 20% of dry substance using a vacuum rotation evaporator.", "The starch ether obtained has a degree of derivatization of about 0.2.The conductivity of the coating mass is about 1100 μS/cm.", "The below-mentioned coating masses were prepared similar to said example and applied with a knife onto a unilaterally coated chromo duplex cardboard (GD2), 310 g/m2, thickness about 420 μm.", "After the first coating had dried (finger-dry, duration about 2 h), a second layer was applied and dried at room temperature and at about 50% room moisture for about 1 week or longer, if desired.", "Three cardboard sheets of the coated samples were used to determine the coated mass by weighing (according to DIN 53 104: test of paper and cardboard, determination of the weight per unit area, September 1971) and the thickness with a caliper gauge (caliper: even/bulged, 30 SKT, MB=1 μm).", "Further, the KIT number for nonpolar substances was determined according to the 3M KIT test.", "Solvent mixtures from castor oil, toluene and heptane serve as test liquids.", "The KIT solution which has the highest number and stands on the sample for 15 sec.", "without penetrating or causing a discoloration, is the characterizing KIT number.", "Derivatization with PPO (in % Conductivity Surface by weight (optionally application Layer relative to the after weight thickness KIT- Raw material raw material) desaltation) [g/m2] [μm] number Wheat starch 50 1100 21.5 33.24 >21 (Kröner) 50 8180 32.6 29.6 8 75 1600 11.2 25 9 25 5150 31 34.8 13 25 10700 21.1 27.1 9 Corn starch 50 1800 21.5 34.3 15 (Cerestar) 50 6600 16.3 20.4 14 25 4400 15 25.9 14 75 1340 4.9 18.6 >21 Corn starch + 50 1800 15.6 23.1 19 5% by weight glycerol Corn starch + 50 1800 16.6 34.7 15 2% by weight curing agent Potato starch 50 870 21 18.7 9 Amylex 20/20 50 3700 18.1 18.4 6 (Südstärke) Tapioka 50 840 8.6 221 8 starch (Cerestar) 50 12200 11.3 23 8 HA pea ˜50 >16 starch, 18% by weight TS Multilayer Packaging for Greasy Products The present invention relates to a multilayer packaging for greasy products or part of said packaging, comprising a carrier layer made of a polymer material as the main component, and at least one layer applied onto said carrier layer and not forming the outer side of said packaging, said layer containing a starch derivative as the main component, characterized in that said starch derivative is a starch derivative modified with a C2-C6 alkylene oxide.", "The invention further relates to the use of a starch derivatized with a C2-C6 alkylene oxide as the main component of a layer of a multilayer packaging said layer being applied onto a carrier layer of said packaging which is made of a polymer material, with the aim of rendering said multilayer packaging greaseproof." ] ]

[ [ "Automotive trim component having an elastomeric skin with a foam core and method for making same", "A mold assembly for manufacturing a trim component of an automotive vehicle.", "The mold assembly comprises a first mold half having a contoured first mold surface and an outer peripheral edge and a second mold half having a contoured second mold surface and an outer peripheral edge.", "The mold halves pivotal about a hinge between an open position providing access to the respective mold surface and a closed position with the outer peripheral edge of the first mold half aligned with an abutting the outer peripheral edge of the second mold surface.", "A cutting ridge is formed around the outer peripheral edge of the first mold half for engaging a bulbous projection formed around the outer peripheral edge of the second mold halves in the closed position whereby upon the injection of a urethane material onto the mold halves, the cutting ridge abuts against the projection and perforates any oversprayed material extending outside of the mold surfaces when the mold halves are moved to the closed position." ], [ "1.A mold assembly for manufacturing a trim component of an automotive vehicle comprising: a first mold half having a contoured mold surface and an outer peripheral edge; a second mold half having a contoured mold surface and an outer peripheral edge; said mold halves movable between an open position providing access to said respective mold and a closed position with said outer peripheral edge of said first mold half aligned with and abutting said outer peripheral edge of said second mold surface; and a cutting ridge formed around said outer peripheral edge of one of said mold halves for engaging said other of said mold halves in said closed position whereby upon the injection of a urethane material onto at least one of said mold halves said cutting ridge perforates any oversprayed material extending outside of said mold surfaces when said mold halves are moved to said closed position.", "2.A mold assembly as set forth in claim 1 further including a projection formed around said outer peripheral edge of said other of said mold halves opposite said cutting ridge for abutting with said cutting ridge with said mold halves in said closed position to thereby cut any oversprayed material therebetween.", "3.A mold assembly as set forth in claim 2 further including a mold cavity defined between said mold surfaces of said first and second mold halves in said closed position.", "4.A mold assembly as set forth in claim 3 wherein said cutting ridge includes a substantially triangular barb having a sharp leading edge extending upwardly adjacent said outer peripheral edge of said first mold half.", "5.A mold assembly as set forth in claim 4 wherein said projection includes a generally bulbous semi-circular configuration extending upwardly adjacent said outer peripheral edge of said second mold half for abutting with said sharp leading edge of said cutting ridge with said mold halves in said closed position.", "6.A mold assembly as set forth in claim 5 further including a hinge interconnecting said first and second mold halves for providing pivotal movement of said mold halves between said open and closed positions.", "7.A method for manufacturing a trim component for an automotive vehicle with a mold assembly having a first mold half having a first mold surface and a cutting ridge surrounding the first mold surface and a second mold half having a second mold surface and a projection surrounding the second mold surface, wherein the mold halves are moveable relative to each other between an open position having the cutting ridge disengaged from the projection and a closed position having the cutting ridge engage the projection; said method comprising the steps of: applying an in-mold coat to each of the mold halves when the mold halves are in the open position; applying a skin coat to each in-mold coat when the mold halves are in the open position, whereby the skin coat in the first mold half bonds with the respective in-mold coat to form a first structural skin and the skin coat in the second mold half bonds with the respective in-mold coat to form a second structural skin; moving the mold halves to the closed position whereby the cutting ridge abuts the projection to cut any overspray of in-mold coat and skin coat extending outside of the first and second mold surfaces; injecting a foam into the mold halves after applying the skin coats to expand and cure between the first and second structural skins forming the trim component; moving the mold halves to the open position after forming the trim component; and removing the trim component from the mold assembly after moving the mold halves to the open position.", "8.A method as set forth in claim 7 further including bonding the first structural skin to the second structural skin by allowing the skins to at least partially harden to a non-liquid state prior to moving the mold halves to the closed position and then abutting the first and second structural skin around the periphery of the first and second mold surfaces.", "9.A method as set forth in claim 8 further including venting gas from a mold cavity formed between the mold halves in the closed position as the foam is injected between the first and second structural skins.", "10.A method as set forth in claim 9 further including inserting at least one elongated hollow armature between the first and second mold halves in the closed position and injecting the foam into the mold cavity to cure around the armature as part of the trim component.", "11.A method as set forth in claim 10 further including injecting the foam through the hollow armature into the mold cavity.", "12.A method as set forth in claim 10 further including venting the gas in the mold cavity through the hollow armature during the injecting of the foam between the mold halves.", "13.A method as set forth in claim 7 further including injecting the foam into at least one of the mold halves against the in-mold coat and skin coat prior to moving the mold halves to the closed position.", "14.A method as set forth in claim 13 further including cutting any overspray of in-mold coat and skin coat and any excess foam outside the periphery of the first and second mold surfaces by abutting the cutting ridge against the projection with the mold halves in the closed position.", "15.A method as set forth in claim 7 further including injecting the foam between the first and second structural skins after the mold halves are moved to the closed position and the structural skins are bonded around the periphery of the mold surfaces." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1) Field of the Invention The subject invention relates to an automotive trim component, such as a headrest, armrest or the like.", "Further, the subject invention also relates to a mold assembly and method for manufacturing the aforementioned automotive trim component.", "2) Description of the Related Art Automotive trim components, such as headrests and armrests, and their general method of manufacture are known in the art.", "For example, a conventional method for making headrests includes the following steps.", "Initially, a plurality of trim cover pieces are manually, either by machine or by hand, sewn together to form a trim cover envelope, also known in the art as an outer shell of the headrest.", "The outer shell includes sewn seams throughout.", "Next, the sewn outer shell is positioned into cavity of a mold assembly.", "Foam is then injected into the sewn outer shell while in the mold assembly to form the headrest.", "Such conventional methods for making headrests have a number of deficiencies.", "In particular, upon injecting foam into the sewn outer shell, foam may leak through the sewn seams in the headrest and result in a defective headrest.", "Further, automotive seating headrests with the sewn seams are prone to tearing, ripping, snagging, and opening during repeated use over the life of an automobile.", "Illustrative of another example, an alternative method of making a headrest is disclosed in U.S. Pat.", "No.", "5,116,557 wherein the outer shell or layer of the headrest is synthetically made of an elastomer within a mold tool.", "The mold tool includes a pair of mold halves which form a hermetic seal when closed together.", "The method as disclosed in the '557 patent requires that the elastomer be in a substantially liquid state during the closing of the mold halves in order to press the elastomer which may have been oversprayed onto the mold halves out from between the mold halves.", "This method is overly sensitive to the specific curing times of the elastomer and outside factors such as humidity, temperature and the like." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>According to one aspect of the invention, a mold assembly is provided for manufacturing a trim component of an automotive vehicle.", "The mold assembly comprises a first mold half having a contoured mold surface and an outer peripheral edge and a second mold half having a contoured mold surface and an outer peripheral edge.", "The mold halves are movable between an open position providing access to the respective mold surfaces and a closed position with the outer peripheral edge of the first mold half aligned with and abutting the outer peripheral edge of the second mold surface.", "A cutting ridge is formed around the outer peripheral edge of one of the mold halves for engaging the other of the mold halves in the closed position whereby upon the injection of a urethane material onto at least one of the mold halves, the cutting ridge perforates any oversprayed material extending outside of the mold surfaces when the mold halves are moved to the closed position.", "According to another aspect of the invention, a method is provided for manufacturing a trim component for an automotive vehicle with a mold assembly having a first mold half having a first mold surface and a cutting ridge surrounding the first mold surface and a second mold half having a second mold surface and a projection surrounding the second mold surface, wherein the mold halves are moveable relative to each other between an open position having the cutting ridge disengaged from the projection and a closed position having the cutting ridge engage the projection.", "The method comprises the steps of applying an in-mold coat to each of the mold halves when the mold halves are in the open position; applying a skin coat to each in-mold coat when the mold halves are in the open position, whereby the skin coat in the first mold half bonds with the respective in-mold coat to form a first structural skin coat in the second mold half bonds with the respective in-mold coat to form a second structural skin; moving the mold halves to the closed position whereby the cutting ridge abuts the projection to cut any overspray of in-mold coat and skin coat extending outside of the first and second mold surfaces; injecting a foam into the mold halves after applying the skin coat to expand and cure between the first and second structural skins forming the trim component; moving the mold halves to the open position after forming the trim component; and removing the trim component from the mold assembly after moving the mold halves to the open position." ], [ "BACKGROUND OF THE INVENTION 1) Field of the Invention The subject invention relates to an automotive trim component, such as a headrest, armrest or the like.", "Further, the subject invention also relates to a mold assembly and method for manufacturing the aforementioned automotive trim component.", "2) Description of the Related Art Automotive trim components, such as headrests and armrests, and their general method of manufacture are known in the art.", "For example, a conventional method for making headrests includes the following steps.", "Initially, a plurality of trim cover pieces are manually, either by machine or by hand, sewn together to form a trim cover envelope, also known in the art as an outer shell of the headrest.", "The outer shell includes sewn seams throughout.", "Next, the sewn outer shell is positioned into cavity of a mold assembly.", "Foam is then injected into the sewn outer shell while in the mold assembly to form the headrest.", "Such conventional methods for making headrests have a number of deficiencies.", "In particular, upon injecting foam into the sewn outer shell, foam may leak through the sewn seams in the headrest and result in a defective headrest.", "Further, automotive seating headrests with the sewn seams are prone to tearing, ripping, snagging, and opening during repeated use over the life of an automobile.", "Illustrative of another example, an alternative method of making a headrest is disclosed in U.S. Pat.", "No.", "5,116,557 wherein the outer shell or layer of the headrest is synthetically made of an elastomer within a mold tool.", "The mold tool includes a pair of mold halves which form a hermetic seal when closed together.", "The method as disclosed in the '557 patent requires that the elastomer be in a substantially liquid state during the closing of the mold halves in order to press the elastomer which may have been oversprayed onto the mold halves out from between the mold halves.", "This method is overly sensitive to the specific curing times of the elastomer and outside factors such as humidity, temperature and the like.", "SUMMARY OF THE INVENTION According to one aspect of the invention, a mold assembly is provided for manufacturing a trim component of an automotive vehicle.", "The mold assembly comprises a first mold half having a contoured mold surface and an outer peripheral edge and a second mold half having a contoured mold surface and an outer peripheral edge.", "The mold halves are movable between an open position providing access to the respective mold surfaces and a closed position with the outer peripheral edge of the first mold half aligned with and abutting the outer peripheral edge of the second mold surface.", "A cutting ridge is formed around the outer peripheral edge of one of the mold halves for engaging the other of the mold halves in the closed position whereby upon the injection of a urethane material onto at least one of the mold halves, the cutting ridge perforates any oversprayed material extending outside of the mold surfaces when the mold halves are moved to the closed position.", "According to another aspect of the invention, a method is provided for manufacturing a trim component for an automotive vehicle with a mold assembly having a first mold half having a first mold surface and a cutting ridge surrounding the first mold surface and a second mold half having a second mold surface and a projection surrounding the second mold surface, wherein the mold halves are moveable relative to each other between an open position having the cutting ridge disengaged from the projection and a closed position having the cutting ridge engage the projection.", "The method comprises the steps of applying an in-mold coat to each of the mold halves when the mold halves are in the open position; applying a skin coat to each in-mold coat when the mold halves are in the open position, whereby the skin coat in the first mold half bonds with the respective in-mold coat to form a first structural skin coat in the second mold half bonds with the respective in-mold coat to form a second structural skin; moving the mold halves to the closed position whereby the cutting ridge abuts the projection to cut any overspray of in-mold coat and skin coat extending outside of the first and second mold surfaces; injecting a foam into the mold halves after applying the skin coat to expand and cure between the first and second structural skins forming the trim component; moving the mold halves to the open position after forming the trim component; and removing the trim component from the mold assembly after moving the mold halves to the open position.", "BRIEF DESCRIPTION OF THE DRAWINGS Advantages of the present invention will be readily appreciated as the same becomes understood by reference to the following detailed description when considered in connection with the accompanying wherein: FIG.", "1 is a sectional view of a mold assembly for manufacturing a trim component shown in an open position; and FIG.", "2 is a sectional view of the mold assembly shown in a closed position.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to the Figures, wherein like numerals indicate like or corresponding parts throughout the several views, a mold assembly for making an automotive trim component is generally shown at 10 in FIGS.", "1 and 2.For illustrative purposes only, the mold assembly 10 is depicted as a mold assembly for making a headrest.", "It should be appreciated that the mold assembly 10 may be configured to make any other suitable trim components, such as armrests or the like.", "Referring to FIG.", "1, the mold assembly 10 includes a first mold half 11 and a second mold half 12.The mold halves 11, 12 are movable relative to each other between an open position, as shown in FIG.", "1, and a closed position, as shown in FIG.", "2.The first and second mold halves 11, 12 include first and second contoured mold surfaces 14, 16, respectively.", "The mold surfaces 14, 16 are concave and complimentary to each other such that the molded surfaces 14, 16 define a substantially enclosed mold cavity 18 when the mold assembly 10 is in the closed position.", "The surfaces of the mold surfaces 14, 16 defining the mold cavity 18 correspond to an outer contour of the headrest (not shown in the Figures).", "The mold surfaces 14, 16 preferably include a textured surface that forms a final surface texture of the headrest.", "It should be appreciated that while the mold surfaces 14, 16 are illustrated as concave, the mold surfaces 14, 16 may be of any suitable shape so as to define a cavity commensurate with the desired shape of the headrest.", "The mold halves 11, 12 further include first and second receiving recessed channels 15, 17, respectively, and a hinge 19, such as a piano-type hinge, interconnecting the mold halves 11, 12 opposite the channels 15, 17 for providing pivotal movement of the mold halves 11, 12 between the open and closed positions.", "The operation of these components will be described further hereinbelow.", "Additionally, a filler port 9, 13 is disposed within each of the first 11 and second 12 mold halves, respectively, to receive an injection funnel 40, as will be described in greater detail below.", "The second mold half 12 includes a peripheral cutting ridge 21 surrounding the circumference of the opening defined by the second mold surface 16.The size of the cutting ridge 21 is exaggerated in the Figures.", "The cutting ridge 21 is shown as a substantially triangular barb having a sharp leading edge.", "Alternatively, the cutting ridge 21 may have any shape or configuration suitable for performing a cutting operation described below.", "The first mold half 11 includes a projection or lip 26 extending from and surrounding the circumference of the opening defined by the first mold surface 14.The projection 26 acts as cutting board for the cutting ridge 21 as further described below.", "Additionally, the projection 26 provides an indentation in the completed headrest so to convey the appearance of headrest being formed of two pieces.", "Preferably, the first mold surface 14 is slightly deeper than the second mold surface 16 such that the projection 26 is centered on the completed headrest.", "In other words, the first 14 and second 16 mold surfaces are preferably designed such that when the mold halves 11, 12 are closed, the projection 26 is in the center of the headrest.", "The projection 26 is shown as having a bulbous semi-circular configuration.", "It should be appreciated that the projection 26 may have any suitable configuration depending upon the desired effect for the trim component being manufactured.", "In forming the headrest, the mold assembly 10 is initially placed in the open position as shown in FIG.", "1.An in-mold coat 20 is then applied to each of the mold surfaces 14, 16.It is optional that a mold-release coat is applied to each of the mold surfaces 14, 16 prior to the in-mold coating 20 to promote efficient removal of the headrest from the mold assembly 10 after the forming process is complete.", "The in-mold coat 20 is preferably applied in a generally uniform manner to promote consistent gloss, texture, and color of the outer contour of the seating headrest.", "Preferably, the in-mold coat 20 is a waterborne urethane coating, which can be applied by an air-atomized coating applicator such as a spray gun or bell applicator.", "Alternatively, the composition and means of applying the in-mold coat 20 may be of any suitable type.", "A skin coat 22 is next applied to the in-mold coat 20 in a manner similar to application of the in-mold coat 20.The composition of the skin coat 22 is preferably a two component polyurethane elastomer including a polyol component and an isocyanate component as disclosed and described in U.S. Pat.", "No.", "5,885,662 which is incorporated herein by reference in its entirety.", "The in-mold coat 20 applied to each mold half 11, 12 bonds to the respective skin coat 22 to form a structural skin 29.During the application of the in-mold coat 20 and the skin coat 22, a certain amount of overspray 24 can accumulate on the mold halves 11, 12 outside of the mold surfaces 14, 16 and outside of the cutting ridge 21 and projection 26.For illustrative purposes, the overspray 24 is exaggerated in the Figures.", "A pair of elongated headrest armatures 30, which function as a support of the headrest to an automotive seat, are then inserted into the respective receiving channels 15, 17 of a designated one of the mold halves 11, 12 such that the armature 30 extend into the mold cavity 18.The headrest armatures 30 are preferably connected to a cross member 31 to form a U-shaped support.", "The U-shaped support may be an integral U-shaped tube having a solid or hollow construction.", "Alternatively, the U-shaped support may have a pair of separate armature posts 30 connected to a separate cross member by any suitable attachment means.", "In addition, there may be two separated armatures 30 without a cross member or even a single armature 30.Each armature 30 has at least one air channel 32 formed longitudinally therein that is in fluid communication with the mold cavity 18 for venting the mold cavity 18 as further discussed below.", "The air channels 32 may be notches formed in the outer surfaces of the armatures 30 or may be vent holes formed in armatures 30 that are hollow.", "The armatures 30 are held in place by a clamp, toggle, or the like.", "It should be appreciated that the armatures 30 may be placed in the designated mold half 11, 12 prior application of the mold-release coat, the in-mold coat 20 or the skin coat 22.Once the in-mold 20 and skin coat 22 are applied and with the armatures 30 held in the respective receiving channels 15, 17, the injection funnel 40 is then inserted into the filler port 9, 13 of a designated one of mold halves 11, 12.The mold assembly 10 is then placed in the closed position as shown in FIG.", "2 by rotating the second mold half 12 about the hinge 19 (the movement of which is represented by Arrow A).", "As the mold assembly 10 is placed in the closed position, the armatures 30 and the funnel 40 are received in the other receiving channel 15, 17 and the other filler port 9, 13, respectively, thereby enabling complete closure of the second mold half 12 relative to the first mold half 11.It should be appreciated that the hinge 19 is optional, and that the mold assembly 10 may alternatively be placed in the closed position by moving the mold halves 11, 12 relative to each other in any suitable manner including linear movement, arcuate movement or a combination linear and arcuate movements.", "As the mold halves 11, 12 are moved to the closed position, the peripheral cutting ridge 21 cuts through the overspray 24 of the coats 20, 22 on the mold halves 11, 12 and into abutment with the projection 26.The cutting ridge 21 and projection 26 extend around the entire perimeter of the mold cavity 18 such that any overspray 24 on the mold halves 11, 12 will be cut by the ridge 21.When the cutting ridge 21 abuts the projection 26, the mold assembly 10 is in the closed position.", "Before the mold halves 11, 12 are moved to the closed position, the coats 20, 22 are allowed to at least partially harden to a non-liquid or gel state.", "The non-liquid or gel state of the coats 20, 22 promotes the cutting of the overspray 24 during the closing of the mold halves 11, 12.Once the mold halves 11, 12 have reached, or just prior to reaching, the closed position, the mold halves 11, 12 are mechanically clamped or locked together to ensure that the mold assembly is properly placed and held in the closed position.", "When in the closed position, the coats 20, 22 of the first mold half 11 bond with the coats 20, 22 of the second mold half 12 at the junction between the mold halves 11, 12.That is, the portion of the coats 20, 22 around the perimeter the first mold surface 14 bond with the portion of the coats 20, 22 around the perimeter of the second mold surface 16.The bond between the coats 20, 22 of the mold halves 11, 12 is more durable than a conventional headrest having sewn seams because the bonding of the subject invention is particularly resistant to tearing, ripping, snagging and opening during repeated use over the life of the headrest.", "A cellular foam filler 42 is then injected through the funnel 40 placed in fluid communication with the mold cavity 18.Alternatively, provided that the armatures 30 are hollow, the foam filler 42 may be injected through an opening formed in either or both of the armatures 30.An advantage of injecting the foam filler 42 through the armatures 30 is that the foam filler 42 has additional time to blend into a proper mixture.", "The cellular foam filler 42 expands in the mold cavity 18 and bonds with the armatures 30 and the structural skin 29, formed by the coats 20, 22, to form the headrest.", "The foam filler 42 is preferably urethane and may be manufactured of a color that is complementary to the color of the structural skin 29, which provides the advantage of partially concealing any tears, cuts or breakage in the structural skin 29 that may occur during the life of the headrest.", "The air channels 32 formed in the armatures 30 allow gases to escape from the cavity 18 as the foam filler 42 occupies the cavity 18.Alternatively, gases may be vented through one or more vent holes formed in the armatures 30.As a further alternative, provided that the armatures 30 utilize the U-shaped cross member 31 that is hollow, one of the armatures 30 may have inlet opening external to the cavity 18 and one or more fill holes in fluid communication with the cavity 18 for facilitating the injection of the foam filler 42.The other armature 30 may have an outer opening external to the cavity 18 and one or more vent holes in fluid communication with the cavity 18 for facilitating the venting of gases.", "In this embodiment, the foam filler 42 is injected into the cavity 18 trough the inlet opening via the fill holes, and gases escape the cavity 18 through outlet opening via the vent holes.", "It should be further appreciated that the injecting and venting may be further facilitated through apertures formed in the cross member having a hollow construction.", "Preferably, the mold halves 11, 12 are manipulated 90 degrees before the injection of the foam filler 42 such that the armature 30 extends upwardly (as shown in FIG.", "2).", "The 90 degree rotation assists in venting the mold cavity 18 during the foam injection.", "The structural skin 29 is allowed to expand in the mold cavity 18 and cure and then the mold halves 11, 12 are opened to remove the completed headrest.", "Finally, if necessary, the subject invention may include a step of deflashing excess structural skin 29 at the bonding line created between the mold halves 11, 12.As stated above, the completed headrest conveys the appearance of being formed of two pieces as provided by the indentation formed by the projection 26.Such a headrest may be regarded as a more aesthetically pleasing than a conventional headrest having sewn seams.", "An alternative embodiment of the subject invention proceeds as follows.", "With the mold halves 11, 12 in the open position, the in-mold coat 20 and the skin coat 22 are applied as introduced and described above.", "Further, the armature 30 is inserted into the receiving channel 15 of the first mold half 11 as described above.", "However, prior to rotating the second mold half 12 about the hinge 19 to close upon the first mold half 11, the cellular foam filler 42 is injected over the skin coat 22 and the in-mold coat 20 in the first mold half 11.That is, the semi-rigid cellular foam filler 42 is injected into one of the mold halves in the open position.", "Next, the second mold half 12 is rotated about the hinge 19 to close upon the first mold half 11, and the first 11 and second 12 mold halves are locked together.", "In this alternative embodiment of the subject invention, all reactions described above occur simultaneously.", "That is, the in-mold coat 20 and the skin coat 22 bond to establish the structural skin 29 of the headrest, and simultaneously, the cellular foam filler 42 expands in the structural skin 29 to bond with the structural skin 29 thereby forming the complete headrest.", "In this embodiment, the cutting ridge 21 may remove excess, or overspilled, foam filler 42 as well as the oversprayed coats 24.As appreciated, the use of the funnel 40 is eliminated from this alternative embodiment.", "One disadvantage to using the funnel 40 is that residue or overspill can be formed on the exterior of the headrest when the funnel 40 is removed.", "This alternative embodiment therefore eliminates any residue or overspill occurring in this manner.", "The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.", "Many modifications and variations of the present invention are possible in light of the above teachings.", "It is, therefore, to be understood that the invention may be practiced otherwise as specifically described." ] ]

[ [ "Device for combating targets", "An ammunition unit, for combating targets (10) with or without using shaped charge effect, that incorporates an openable or removable and replaceable hull or outer casing (2).", "The explosive charge in the ammunition unit comprises a charge section (3) with shaped charge function arranged to be able to assume either of two freely selectable positions in the ammunition unit, the first of which enables the charge to exert its shaped charge function and the second of which disables the shaped charge function and enables a different effect to be triggered." ], [ "1.A device for combating targets (10, 12) with or without using shaped charge effect wherein an ammunition unit (1), for example in the form of a round, shell, projectile, etc., is designed with an openable or removable and replaceable hull or outer casing (2) and whereby the ammunition unit charge comprises an explosive charge section (3) with shaped charge function such that the said charge section is arranged to be indexible between two freely selectable positions, in the first of which the shaped charge function is enabled and in the second of which the shaped charge function is disabled and the charge thus exerts a different function such as a pressure generating and/or fragmentation effect function.", "2.A device as claimed in claim 1 wherein the explosive charge section (3) is arranged to be indexible from the first mode/position to the second, or vice versa, by indexing/reversing the main charge 180° in the ammunition unit, such indexing preferably being performed manually.", "3.A device as claimed in claim 1 or 2 wherein the initial position of the explosive charge section in the ammunition unit is the position that enables the shaped charge function and the said explosive charge section can be indexed to the second position after opening or removing the hull or outer casing or parts thereof, or can be indexed from second position to initial position in a similar manner.", "4.A device as claimed in claim 1, 2 or 3 wherein the explosive charge section (3), herein called the first charge section in the ammunition unit, is interactable with another charge section herein called the second charge section (4).", "5.A device as claimed in claim 4 wherein in the first position the conical shaped charge liner (3a, 3b) faces forwards and the rear face (3c) of the first charge section (3) abuts on the front face (4a) of the second charge section (4).", "6.A device as claimed in claim 5 wherein the first charge section (3) displays a flat rear face (3c) that in the first position interacts with the corresponding flat front face (4a) of the second charge section (4) while in the second position the first charge section rests against the periphery of the flat front face (4a) of the second charge section (4) to form an empty triangular space (11).", "7.A device as claimed in any of the previous claims wherein the hull or casing is openable or removable via a screw joint (9, 9′) arranged so that at least the front section (2a) of the hull or casing (2) is removable to enable the first charge section (3) to be indexed 180°.", "8.A device as claimed in any of the previous claims wherein the conical front and rear cavities of the first charge section are arranged so that the distance (a) between the innermost points of the said cavities is relatively small.", "9.A device as claimed in any of the previous claims wherein the shaped charge liner (3a, 3b) extends to the lateral surface (3d) of the first charge section (3) thereby forming an extension of the said lateral surface." ], [ "The present invention relates to a device for combating targets with or without using shaped charge effect.", "In cases where the above mentioned effect is not used the target is combated using pure pressure effect, for example.", "Units of ammunition, e.g.", "in the form of rounds, shells, projectiles, etc., with or without shaped charge effect exist in extremely large numbers and in a multiplicity of designs.", "Shaped charge ammunition is used to engage a specific type of target in which a hole needs to be achieved to enable devastating penetration.", "Units of ammunition with only propelling charge effect or pressure effect are very common and are normally used to combat various types of target.", "There is a major need to be able to reduce the wide assortment of natures of ammunition while still being able to fire on different types of target with the desired optimal effect.", "Thus there is even a desire to be able to combat the said types of targets despite the fact that they require an advanced type of ammunition to achieve at least one of the desired effects in target, namely shaped charge effect.", "The purpose of the present invention is to propose a device that resolves the above mentioned problem by proposing that the ammunition in question be easy to adapt to achieve the desired optimal effect in both cases.", "There is thus an inherent requirement that shaped charge effect, for example, shall not be impaired but shall function equally well as in the case with more dedicated ammunition.", "The present invention also resolves this problem.", "One of the characteristic features of the device in the present invention is that the unit of ammunition, e.g.", "in the form of a round, shell, projectile, etc., incorporates an openable or removable and closeable hull or outer casing, and that the charge comprises a charge section with shaped charge function.", "Another feature is that the said charge section is arranged to have two selectable modes in the ammunition unit of which the first mode is to enable the shaped charge function and the second mode is to disable this function.", "The second mode enables a function to be triggered that is divorced from the shaped charge function and that can consist of a pressure generating function and/or fragmentation effect.", "In the designs in the invention concept the charge section in question is arranged to be indexible from the first mode/position to the second, or vice versa, by reversing the charge section 180° in the ammunition unit.", "In an initial mode for the ammunition unit the charge section can thus assume a shaped charge function and be arranged so that the second mode can be enabled after opening or removal of the hull or outer casing as stated above.", "Furthermore, the charge section, hereinafter called the first charge section, can interact with another charge section in the ammunition unit hereinafter called the second charge section.", "In additional designs of the invention concept it is proposed how the interacting faces of the first and second charge sections shall be designed.", "Reference is hereby made to the subsequent patent claims and the description.", "The above proposal enables a number of advantages.", "The ammunition unit can be switched from one type of ammunition to another, such as for delivery by firing or by dropped release.", "The ammunition unit can be supplied with a function that is the most common, and can be switched to the other function only when an engagement situation that requires it is encountered.", "The technical and economic benefits can be retained since switchability between two different engagement situations and indexibility as such do not, as a whole, need to make manufacture, handling and service more expensive.", "A currently proposed design for a device as claimed in the present invention is described below with reference to the appended FIGS.", "1-4 in which FIGS.", "1 and 2 show an example of an ammunition unit, such as a fin-stabilised or spin controlled projectile, in longitudinal section comprising first and second charge sections where the first section is indexible 180° to enable a shaped charge function in mode one as illustrated in FIG.", "1, and to enable a pure pressure effect function as illustrated in FIG.", "2, i.e.", "the shaped charge function is disabled for when the ammunition unit is used, and FIGS.", "3 and 4 show another design in longitudinal section of a projectile with two charge sections where the first charge section is in principle indexible 180° for the same purpose as the design illustrated in FIGS.", "1 and 2.In FIG.", "1 the ammunition unit is in the form of a shell designated 1.The ammunition unit comprises, in a commonly known manner, a casing 2 containing a charge arrangement with a first charge section 3 and a second charge section 4.This unit is fin- or spin-stabilised by aft fins 5 and is equipped—in an already known manner—with propulsion and guidance devices 6 as well as initiation and detonation devices 7.In the present design example the first charge section 3 consists of a shaped charge.", "The shaped charge liner designated 3a, 3b in the present case is in the form of a conical cavity, and the present invention functions in principle for other directions of the said liner 3a, 3b.", "Depending on the type of charge (explosive) and the liner 3a, 3b a forwards directed jet effect can be achieved when the ammunition unit is triggered, which jet effect generally coincides with the longitudinal axis 8 in the direction of fire of the ammunition unit.", "The second charge section 4 comprises an explosive charge corresponding to the explosive charge in the first charge section 3.The said casing 2 shall be openable or removable by unscrewing to enable the first charge section 3 to be removed from its position/mode shown in FIG.", "1 and then to be indexed/reversed 180° to be re-installed in the position/mode shown in FIG.", "2.The said openability or removability function can be designed in various ways.", "In the present invention it is proposed that a screw joint 9 be arranged between front and aft sections, 2a and 2b respectively, of the casing 2.The screw joint can be comprised of an internal thread in one section and an external thread on the other section, or vice versa.", "Alternatively, the openability or removability function can be located somewhere else along the length of the casing 2.Openability or removability can also be arranged in some other way, such as by means of a bayonet connector or snap-catch, etc.", "As illustrated in FIG.", "1 the ammunition unit is fired in a conventional manner and activated, also conventionally, in its trajectory adjacent to a target symbolised by 10.When triggered the shaped charge function is activated in an already known manner and attacks the target using the said jet, pressure and/or fragmentation effect.", "As illustrated in FIG.", "1 the first charge section 3 has a rear face 3c that is flat and extends perpendicular to the plane of the figure in FIG.", "1.The second charge section 4 has a similar flat, front face 4a extending likewise at right angles to the plane of the figure.", "In the mode illustrated for the charge sections 3 and 4 in FIG.", "1 the sections abut on each other via the faces 3c and 4a.", "In this way the effect from the explosive in the second charge section 4 can be easily transmitted to the explosive in the first charge section 3.In FIG.", "2 the said face 4a of the first charge section 3 abuts on the outer extremities of the said liner 3a, 3b.", "The said liner 3a, 3b incorporates lateral surfaces 3a′ and 3b′ which, in principle, coincide with or comprise an extension of lateral surface 3d.", "Because the first charge section has been indexed/reversed the shaped charge function has been disabled for when the ammunition unit 1 is triggered.", "In this case the ammunition unit operates with the pressure or fragmentation effect obtained when the explosive in the first and second charge sections is initiated.", "This pressure effect can be used to combat a target like type 12 that is different from target type 10 illustrated in FIG.", "1.In the case illustrated in FIG.", "2 there is a rear conical space 11 between first and second charge sections 3 and 4 formed by the conical liner 3a, 3b.", "The detonation, activation and triggering functions operate in principle in the same way in the different application cases for the first and second charge sections 3 and 4 in FIGS.", "1 and 2.In the version illustrated in FIGS.", "3 and 4 the front face 4a in FIGS.", "1 and 2 has been modified to comprise the conical face designated 4a′, 4a″ that interfaces with the cavity formed by the conical liner designated 3a″, 3b″.", "This eliminates the rear space 11 illustrated in FIG.", "2.The screw joint 9′ or equivalent has been re-located further forwards in relation to the design illustrated in FIG.", "1.The rear face of the first charge section 3′ similarly comprises a conical cavity designated 3d′, 3d″ which, as shown in FIG.", "4, interfaces with the conical front face designated 4a′, 4a″ of the second charge section 4′.", "FIG.", "3 illustrates a case where the shaped charge function is disabled and the ammunition unit 1 combats the target by pressure effect (and/or fragmentation effect)—cf.", "the case illustrated in FIG.", "2.FIG.", "4 illustrates a case when the shaped charge function is enabled, i.e.", "facing forwards to enable shaped charge effect when the ammunition unit is triggered.", "In this case the first charge section 3′ is arranged with a distance designated a between the cavity formed by conical face 3d′ and 3d″ and the cavity formed by liner 3a″, 3b″.", "The said distance a is filled with the explosive in the first charge section.", "The effects achieved by the different designs in FIGS.", "1 and 2 contra 3 and 4 are essentially the same with the difference being the effect of rear space 11 shown in FIG.", "2.The indexibility/reversibility of the first charge section can, in principle, be arranged in some other way.", "Manual indexing/reversing has been proposed in the above, but automation of this function is feasible.", "The above mentioned rear space 11 becomes front space 11′ after indexing, as shown in FIG.", "3.The present invention is not limited to the design examples illustrated above, but can be subjected to modifications within the framework of the subsequent patent claims and the invention concept." ] ]

[ [ "Camera-based touch system", "A camera-based touch system (50) includes a passive touch surface (60) and at least two cameras (63) associated surface.", "The at least two cameras (63) have overlapping fields of view (FOV) encompassing the touch surface.", "The at least two cameras (63) acquire images of the touch surface from different locations and generate image data.", "A processor (54) receives and processes image data generated by the at least two cameras to determine the location of the pointer relative to the touch surface when the pointer is captured in images acquired by the at least two cameras.", "Actual pointer contact with the touch surface sand pointer hover above the touch surface can be determined." ], [ "1.A camera-based touch system comprising: at least two cameras associated with a passive touch surface and having overlapping fields of view encompassing said touch surface, said at least two cameras acquiring images of said touch surface from different locations and generating image data; and a processor receiving and processing image data generated by said at least two cameras to determine the location of a pointer relative to said touch surface when said pointer is captured in images acquired by said at least two cameras.", "2.A touch system according to claim 1 wherein said at least two cameras are digital cameras having fields of view looking generally along the plane of said touch surface.", "3.A touch system according to claim 2 wherein the image data generated by each digital camera includes a pointer median line x and a pointer tip location z.", "4.A touch system according to claim 3 wherein each of said digital cameras includes a pixel array having selectable pixel rows, pixel intensities of pixels in said selectable pixel rows being used during generation of said image data.", "5.A touch system according to claim 4 wherein pixel intensities of pixels in a region of interest within said selectable pixel rows are used during generation of said image data.", "6.A touch system according to claim 5 wherein each of said digital cameras packages said image data into packets for transmission to said processor to provide bandwidth economy.", "7.A touch system according to claim 6 wherein each of said digital cameras includes a CMOS image sensor and a digital signal processor, said digital signal processor receiving image output from said image sensor and executing a find pointer routine to determine if a pointer is in each image acquired by said digital camera, and if so the median line x of said pointer.", "8.A touch system according to claim 7 wherein during said find pointer routine, said digital signal processor builds a vertical intensity histogram including columns of pixel intensities representing differences between an acquired image and a background image, the column of said vertical intensity-histogram having the largest pixel intensity above a threshold level being used to define the center of said region of interest, the width of said region of interest being defined by columns of said vertical intensity histogram on opposite sides of the column defining the center of said region of interest that have pixel intensities greater than said threshold level.", "9.A touch system according to claim 8 wherein the digital signal processor of each digital camera analyses the pixels in said region of interest to locate the pixel row where the pointer tip is located and determine said pointer tip location z.", "10.A touch system according to claim 9 wherein the digital signal processor of each digital camera creates a mask in said region of interest with white pixels representing said pointer and black pixels representing background to enable said median line x and pointer tip location to be calculated.", "11.A touch system according to claim 10 wherein the digital signal processor of each digital camera further executes an update background image routine to update the background image after each image is acquired.", "12.A touch system according to claim 11 wherein during said update background image routine, the digital signal processor of each digital camera uses the equation: Bn+1(i,j)=(1-a)Bn(i,j)+aI(i,j) where: Bn+1 is the new background image; Bn is the current background image; I is the current acquired image; i,j are the row and column coordinates of the background image pixels being updated; and a is a number between 0 and 1 that indicates the degree of learning that should be taken from the current acquired image I.", "13.A touch system according to claim 12 wherein the digital signal processor of each digital camera further determines the differences between each acquired image and the background image to detect changing light conditions.", "14.A touch system according to claim 13 wherein each digital camera transmits light condition information to said processor, said processor using said light condition information to adjust the exposure of each said digital camera.", "15.A touch system according to claim 14 wherein the digital signal processor of each digital camera further executes a create packet routine to package said image data and light condition information into said packets.", "16.A touch system according to claim 3 wherein for image data received from each digital camera, said processor calculates an angle φcam using the equation: tan ⁢ ⁢ ϕ cam = 2 ⁢ ( x a ) ⁢ tan ⁢ FOV 2 1 - ( 2 ⁢ x a - 1 ) ⁢ tan 2 ⁢ FOV 2 where: x is the number representing the median line or tip of the pointer; and a is the total length enclosed by the field of view (FOV) of the digital camera at a distance from the camera; said processor using the calculated angles to determine the position of the pointer relative to said touch surface using triangulation.", "17.A touch system according to claim 16 wherein said calculated angles are adjusted to take into account digital camera offsets prior to determination of said pointer position.", "18.A touch system according to claim 17 wherein said digital camera offsets are determined during execution of a digital camera calibration routine.", "19.A touch system according to claim 17 including at least three digital cameras, said processor determining the pointer position using triangulation for pairs of digital cameras and averaging the determined pointer positions.", "20.A touch system according to claim 19 wherein said processor further executes a touch surface determination routine to calculate the orientation of the touch surface as seen by each digital camera to determine when the pointer is in contact with said touch surface and when said pointer is hovering above said touch surface.", "21.A touch system according to claim 20 wherein said processor further calculates the velocity of a pointer as said pointer moves toward said touch surface within the fields of view of said digital cameras.", "22.A touch system according to claim 20 wherein said processor tracks said pointer within the fields of view of said digital cameras.", "23.A touch system according to claim 21 wherein said processor tracks said pointer using at least one Kalman filter.", "24.A touch system according to claim 19 further including a computer coupled to said processor, said computer receiving said pointer location from said processor.", "25.A camera-based touch system comprising: a generally rectangular passive touch surface on which contacts are made using a pointer; a digital camera mounted adjacent each corner of said touch surface, said digital cameras having overlapping fields of view encompassing said touch surface, said digital cameras acquiring images of said touch surface and generating image data that includes the median line x and pointer tip location z of a pointer when said pointer is captured in images acquired by said digital cameras; and a processor receiving and processing image data generated by said digital cameras to determine the location of said pointer relative to said touch surface and whether said pointer is in contact with said touch surface.", "26.A touch system according to claim 25 wherein each of said digital cameras includes a pixel array with selectable pixel rows, pixel intensities of pixels in said selectable pixel rows being used during generation of said image data.", "27.A touch system according to claim 26 wherein pixel intensities of pixels in a region of interest within said selectable pixel rows are used during generation of said image data.", "28.A touch system according to claim 27 wherein each of said digital cameras includes a CMOS image sensor and a digital signal processor, said digital signal processor receiving image output from said image sensor and executing a find pointer routine to determine if a pointer is in each image acquired by said digital camera, and if so the median line x of said pointer.", "29.A touch system according to claim 28 wherein during said find pointer routine, said digital signal processor of each digital camera builds a vertical intensity histogram including columns of pixel intensities representing differences between an acquired image and a background image, the column of said vertical intensity histogram having the largest pixel intensity above a threshold level being used to define the center of said region of interest, the width of said region of interest being defined by columns of said vertical intensity histogram on opposite sides of the column defining the center of said region of interest that have pixel intensities greater than a threshold level.", "30.A touch system according to claim 29 wherein the digital signal processor of each digital camera analyses the pixels in said region of interest to locate the pixel row where the pointer tip is located and determine said pointer tip location z.", "31.A touch system according to claim 30 wherein the digital signal processor of each digital camera creates a mask in said region of interest with white pixels representing said pointer and black pixels representing background to enable said median line x and pointer tip location to be calculated.", "32.A touch system according to claim 31 wherein the digital signal processor of each digital camera further executes an update background image routine to update the background image after each image is acquired.", "33.A touch system according to claim 32 wherein during said update background image routine, the digital signal processor of each digital camera uses the equation: Bn+1(i,j)=(1-a)Bn(i,j)+aI(i,j) where: Bn+1 is the new background image; Bn is the current background image; I is the current acquired image; i,j are the row and column coordinates of the background image pixels being updated; and a is a number between 0 and 1 that indicates the degree of learning that should be taken from the current acquired image I.", "34.A touch system according to claim 33 wherein the digital signal processor of each digital camera further determines the differences between each acquired image and the background image to detect changing light conditions.", "35.A touch system according to claim 34 wherein each digital camera transmits light condition information to said processor, said processor using said light condition information to adjust the exposure of each said digital camera.", "36.A touch system according to claim 3 5 wherein the digital signal processor of each digital camera further executes a create packet routine to package said image data and light condition information into packets for transmission to said processor.", "37.A touch system according to claim 26 wherein for image data received from each digital camera, said processor calculates an angle φcam using the equation: tan ⁢ ⁢ ϕ cam = 2 ⁢ ( x a ) ⁢ tan ⁢ FOV 2 1 - ( 2 ⁢ x a - 1 ) ⁢ tan 2 ⁢ FOV 2 where: x is the number representing the median line or tip of the pointer; and a is the total length enclosed by the field of view (FOV) of the digital camera at a distance from the camera; said processor using the calculated angles to determine the position of the pointer relative to said touch surface using triangulation.", "38.A touch system according to claim 37 wherein said calculated angles are adjusted to take into account digital camera offsets prior to determination of said pointer position.", "39.A touch system according to claim 37 wherein said processor further executes a touch surface determination routine to calculate the orientation of the touch surface as seen by each digital camera to determine when the pointer is in contact with said touch surface and when said pointer is hovering above said touch surface.", "40.A touch system according to claim 39 wherein said processor further calculates the velocity of a pointer as said pointer moves toward said touch surface within the fields of view of said digital cameras.", "41.A touch system according to claim 40 wherein said processor track said pointer within the fields of view of said digital cameras.", "42.A touch system according to claim 41 wherein said processor tracks said pointer using at least one Kalman filter.", "43.A touch system according to claim 26 wherein said touch surface is bordered by a frame and wherein each of said digital cameras is mounted on a frame assembly positioned at a corner of said touch surface that is coupled to a frame, each digital camera being oriented so that the field of view thereof looks downward and generally along the plane of said touch surface.", "44.A touch system according to claim 26 further including a computer coupled to said processor, said computer receiving pointer location information from said processor and using said pointer location information to update an applications program executed thereby.", "45.A touch system according to claim 44 wherein computer display information is presented on said touch surface.", "46.A method of detecting the position of a pointer relative to a touch surface comprising the steps of: acquiring images of said touch surface from different locations using cameras having overlapping fields of view and generating image data; and processing said image data to detect the existence of a pointer within said acquired images and to determine the location of said pointer relative to said touch surface.", "47.The method of claim 46 wherein during the processing step, the location of said pointer relative to said touch screen is determined using triangulation.", "48.The method of claim 47 wherein during said processing step, the images are processed to determine when said pointer is in contact with said touch surface and when said pointer is hovering above said touch surface.", "49.The method of claim 48 wherein said processing step further includes the step of tracking the pointer as the pointer approaches the touch surface." ], [ "<SOH> BACKGROUND ART <EOH>Touch systems are well known in the art and typically include a touch screen having a touch surface on which contacts are made using a pointer in order to generate user input.", "Pointer contacts with the touch surface are detected and are used to generate corresponding output depending on areas of the touch surface where the contacts are made.", "There are basically two general types of touch systems available and they can be broadly classified as “active” touch systems and “passive” touch systems.", "Active touch systems allow a user to generate user input by contacting the touch surface with a special pointer that usually requires some form of on-board power source, typically batteries.", "The special pointer emits signals such as infrared light, visible light, ultrasonic frequencies, electromagnetic frequencies, etc.", "that activate the touch surface.", "Passive touch systems allow a user to generate user input by contacting the touch surface with a passive pointer and do not require the use of a special pointer in order to activate the touch surface.", "A passive pointer can be a finger, a cylinder of some material, or any suitable object that can be used to contact some predetermined area of interest on the touch surface.", "Passive touch systems provide advantages over active touch systems in that any suitable pointing device, including a user's finger, can be used as a pointer to contact the touch surface.", "As a result, user input can easily be generated.", "Also, since special active pointers are not necessary in passive touch systems, battery power levels and/or pointer damage, theft, or pointer misplacement are of no concern to users.", "Passive touch systems have a number of applications relating to computer operation and video display.", "For example, in one interactive application, as is disclosed in U.S. Pat.", "No.", "5,448,263 to Martin, assigned to the assignee of the present invention, a passive touch system is coupled to a computer and the computer display is presented on the touch surface of the touch screen.", "The coordinates representing specific locations on the touch surface are mapped to the computer display.", "When a user contacts the touch surface, the coordinates of the contact position are fed back to the computer and mapped to the computer display thereby allowing the user to operate the computer in a manner similar to using a computer mouse simply by contacting the touch surface.", "Furthermore, the coordinates fed back to the computer can be recorded in an application and redisplayed at a later time.", "Recording contact coordinates is typically done when it is desired to record information written or drawn on the touch surface by the user.", "The resolution of a passive touch screen determines if the touch system is suitable for recording information written or drawn on the touch screen or only useful for selecting areas on the touch screen mapped to regions on the computer or video display in order to manipulate the computer or video display.", "Resolution is typically measured in dots per inch (DPI).", "The DPI is related to the size of the touch screen and the sampling ability of the touch system hardware and software used to detect contacts on the touch surface.", "Low-resolution passive touch screens only have enough DPI to detect contacts on the touch surface within a large group of pixels displayed by the computer or video display.", "Therefore, these low-resolution passive touch screens are useful only for manipulating the computer or video display.", "On the other hand, high-resolution passive touch screens have sufficient DPI to detect contacts that are proportional to a small number of pixels or sub-pixels of the computer or video display.", "However, a requirement for high-resolution touch screens is the ability to detect when the pointer is in contact with the touch surface.", "This is necessary for writing, drawing, mouse-click operations, etc.", "Without the ability to detect pointer contact with the touch screen, writing and drawing would be one continuos operation, and mouse clicks would not be possible thereby making computer display manipulation virtually impossible.", "A secondary requirement is the ability to detect when the pointer is “hovering” above the touch surface.", "Although not required for writing or drawing, today's computer operating systems are increasingly using hover information to manipulate computer or video displays or pop-up information boxes.", "Passive touch screens are typically either of the analog resistive type, surface acoustic wave (SAW) type or capacitive type.", "Unfortunately, these touch screens suffer from a number of problems or shortcomings as will be described.", "Analog resistive touch screens typically have a high-resolution.", "Depending on the complexity of the touch system, the resolution of the touch screen can produce 4096×4096 DPI or higher.", "Analog resistive touch screens are constructed using two flexible sheets that are coated with a resistive material and arranged as a sandwich.", "The sheets do not come into contact with each other until a contact has been made.", "The sheets are typically kept separated by insulating microdots or by an insulating air space.", "The sheets are constructed from ITO, which is mostly transparent.", "Thus, the touch screen introduces some image distortion but very little parallax.", "During operation of an analog resistive passive touch screen, a uniform voltage gradient is applied in one direction along a first of the sheets.", "The second sheet measures the voltage along the first sheet when the two sheets contact one another as a result of a contact made on the touch surface.", "Since the voltage gradient of the first sheet can be translated to the distance along the first sheet, the measured voltage is proportional to the position of the contact on the touch surface.", "When a contact coordinate on the first sheet is acquired, the uniform voltage gradient is then applied to the second sheet and the first sheet measures the voltage along the second sheet.", "The voltage gradient of the second sheet is proportional to the distance along the second sheet.", "These two contact coordinates represent the X-Y position of the contact on the touch surface in a Cartesian coordinate system.", "Unfortunately, since mechanical pressure is required to bring both sheets into contact, analog resistive touch screens can only detect contact when there is sufficient pressure to bring the two sheets together.", "Analog resistive passive touch screens also cannot sense when a pointer is hovering over the touch surface.", "Therefore, in the case of analog resistive touch screens contact events and positions can only be detected when actual contacts are made with the touch surface.", "Surface acoustic wave (SAW) touch screens typically provide for medium resolution and are not suitable for recording good quality writing.", "SAW touch screens employ transducers on the borders of a glass surface to vibrate the glass and produce acoustic waves that ripple over the glass surface.", "When a contact is made on the glass surface, the acoustic waves reflect back and the contact position is determined from the signature of the reflected acoustic waves.", "Unfortunately, SAW touch screens exhibit noticeable parallax due to the thickness of the vibrating glass that is placed over the surface of the video or computer display.", "Also, contact events and positions can only be detected when actual contacts are made with the glass surface.", "Furthermore, SAW touch screens do not scale beyond a few feet diagonal.", "Capacitive touch screens provide for low resolution because contacts can only be determined in large areas (approximately ½″×½″).", "As a result, capacitive touch screens cannot be used for recording writing or drawing but are suitable for selecting areas on the touch screen corresponding to computer generated buttons displayed on the video or computer display.", "Capacitive touch screens also suffer disadvantages in that they are sensitive to temperature and humidity.", "Similar to analog resistive touch screens and SAW touch screens, capacitive touch screens can also only detect contact events and positions when actual contacts are made with the touch surface.", "Scalability of passive touch screens is important since the demand for larger electronic digitizers is increasing.", "Where digitizers were once small desktop appliances, today they have found there way onto electronic whiteboarding applications.", "The need to build a passive touch sensitive “wall” has become a requirement for new touch screen applications.", "Existing passive touch screens of the types discussed above are all limited in the maximum size where they are still functional.", "As will be appreciated, improvements to passive touch systems are desired.", "It is therefore an object of the present invention to provide a novel camera-based touch system." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Embodiments of the present invention will now be described more fully with reference to the accompanying drawings in which: FIG.", "1 is a schematic diagram of a camera-based touch system in accordance with the present invention; FIG.", "2 is an isometric view of a touch screen forming part of the touch system of FIG.", "1 ; FIG.", "3 is an isometric view of a corner portion of the touch screen of FIG.", "2 ; FIG.", "4 is a schematic diagram of a digital camera forming part of the touch screen of FIG.", "2 ; FIG.", "5 is a schematic diagram of a master controller forming part of the touch system of FIG.", "1 ; FIG.", "6 is a flowchart showing the steps performed during execution of a processFrame routine; FIG.", "7 is a flowchart showing the steps performed during execution of a segmentPointer routine; FIG.", "8 is a flowchart showing the steps performed during execution of a findPointer routine; FIG.", "9 shows an image acquired by a digital camera and a pixel subset of the image that is processed; FIG.", "10 shows a region of interest (ROI) within the pixel subset of FIG.", "9 ; FIG.", "11 shows triangulation geometry used to calculate a pointer contact position on the touch surface of the touch screen illustrated in FIG.", "2 ; FIG.", "12 shows an image acquired by a digital camera including the pointer tip and its median line; FIG.", "13 shows pointer contact and pointer hover for different orientations of the pointer; FIG.", "14 is an image of the touch surface of the touch screen as seen by a digital camera; FIGS.", "15 and 16 show the results of a Matlab simulation of pointer tracking using a Kalman filter; and FIGS.", "17 a to 17 d show the results of another Matlab simulation of pointer tracking using a Kalman filter.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates generally to touch systems and in particular to a camera-based touch system.", "BACKGROUND ART Touch systems are well known in the art and typically include a touch screen having a touch surface on which contacts are made using a pointer in order to generate user input.", "Pointer contacts with the touch surface are detected and are used to generate corresponding output depending on areas of the touch surface where the contacts are made.", "There are basically two general types of touch systems available and they can be broadly classified as “active” touch systems and “passive” touch systems.", "Active touch systems allow a user to generate user input by contacting the touch surface with a special pointer that usually requires some form of on-board power source, typically batteries.", "The special pointer emits signals such as infrared light, visible light, ultrasonic frequencies, electromagnetic frequencies, etc.", "that activate the touch surface.", "Passive touch systems allow a user to generate user input by contacting the touch surface with a passive pointer and do not require the use of a special pointer in order to activate the touch surface.", "A passive pointer can be a finger, a cylinder of some material, or any suitable object that can be used to contact some predetermined area of interest on the touch surface.", "Passive touch systems provide advantages over active touch systems in that any suitable pointing device, including a user's finger, can be used as a pointer to contact the touch surface.", "As a result, user input can easily be generated.", "Also, since special active pointers are not necessary in passive touch systems, battery power levels and/or pointer damage, theft, or pointer misplacement are of no concern to users.", "Passive touch systems have a number of applications relating to computer operation and video display.", "For example, in one interactive application, as is disclosed in U.S. Pat.", "No.", "5,448,263 to Martin, assigned to the assignee of the present invention, a passive touch system is coupled to a computer and the computer display is presented on the touch surface of the touch screen.", "The coordinates representing specific locations on the touch surface are mapped to the computer display.", "When a user contacts the touch surface, the coordinates of the contact position are fed back to the computer and mapped to the computer display thereby allowing the user to operate the computer in a manner similar to using a computer mouse simply by contacting the touch surface.", "Furthermore, the coordinates fed back to the computer can be recorded in an application and redisplayed at a later time.", "Recording contact coordinates is typically done when it is desired to record information written or drawn on the touch surface by the user.", "The resolution of a passive touch screen determines if the touch system is suitable for recording information written or drawn on the touch screen or only useful for selecting areas on the touch screen mapped to regions on the computer or video display in order to manipulate the computer or video display.", "Resolution is typically measured in dots per inch (DPI).", "The DPI is related to the size of the touch screen and the sampling ability of the touch system hardware and software used to detect contacts on the touch surface.", "Low-resolution passive touch screens only have enough DPI to detect contacts on the touch surface within a large group of pixels displayed by the computer or video display.", "Therefore, these low-resolution passive touch screens are useful only for manipulating the computer or video display.", "On the other hand, high-resolution passive touch screens have sufficient DPI to detect contacts that are proportional to a small number of pixels or sub-pixels of the computer or video display.", "However, a requirement for high-resolution touch screens is the ability to detect when the pointer is in contact with the touch surface.", "This is necessary for writing, drawing, mouse-click operations, etc.", "Without the ability to detect pointer contact with the touch screen, writing and drawing would be one continuos operation, and mouse clicks would not be possible thereby making computer display manipulation virtually impossible.", "A secondary requirement is the ability to detect when the pointer is “hovering” above the touch surface.", "Although not required for writing or drawing, today's computer operating systems are increasingly using hover information to manipulate computer or video displays or pop-up information boxes.", "Passive touch screens are typically either of the analog resistive type, surface acoustic wave (SAW) type or capacitive type.", "Unfortunately, these touch screens suffer from a number of problems or shortcomings as will be described.", "Analog resistive touch screens typically have a high-resolution.", "Depending on the complexity of the touch system, the resolution of the touch screen can produce 4096×4096 DPI or higher.", "Analog resistive touch screens are constructed using two flexible sheets that are coated with a resistive material and arranged as a sandwich.", "The sheets do not come into contact with each other until a contact has been made.", "The sheets are typically kept separated by insulating microdots or by an insulating air space.", "The sheets are constructed from ITO, which is mostly transparent.", "Thus, the touch screen introduces some image distortion but very little parallax.", "During operation of an analog resistive passive touch screen, a uniform voltage gradient is applied in one direction along a first of the sheets.", "The second sheet measures the voltage along the first sheet when the two sheets contact one another as a result of a contact made on the touch surface.", "Since the voltage gradient of the first sheet can be translated to the distance along the first sheet, the measured voltage is proportional to the position of the contact on the touch surface.", "When a contact coordinate on the first sheet is acquired, the uniform voltage gradient is then applied to the second sheet and the first sheet measures the voltage along the second sheet.", "The voltage gradient of the second sheet is proportional to the distance along the second sheet.", "These two contact coordinates represent the X-Y position of the contact on the touch surface in a Cartesian coordinate system.", "Unfortunately, since mechanical pressure is required to bring both sheets into contact, analog resistive touch screens can only detect contact when there is sufficient pressure to bring the two sheets together.", "Analog resistive passive touch screens also cannot sense when a pointer is hovering over the touch surface.", "Therefore, in the case of analog resistive touch screens contact events and positions can only be detected when actual contacts are made with the touch surface.", "Surface acoustic wave (SAW) touch screens typically provide for medium resolution and are not suitable for recording good quality writing.", "SAW touch screens employ transducers on the borders of a glass surface to vibrate the glass and produce acoustic waves that ripple over the glass surface.", "When a contact is made on the glass surface, the acoustic waves reflect back and the contact position is determined from the signature of the reflected acoustic waves.", "Unfortunately, SAW touch screens exhibit noticeable parallax due to the thickness of the vibrating glass that is placed over the surface of the video or computer display.", "Also, contact events and positions can only be detected when actual contacts are made with the glass surface.", "Furthermore, SAW touch screens do not scale beyond a few feet diagonal.", "Capacitive touch screens provide for low resolution because contacts can only be determined in large areas (approximately ½″×½″).", "As a result, capacitive touch screens cannot be used for recording writing or drawing but are suitable for selecting areas on the touch screen corresponding to computer generated buttons displayed on the video or computer display.", "Capacitive touch screens also suffer disadvantages in that they are sensitive to temperature and humidity.", "Similar to analog resistive touch screens and SAW touch screens, capacitive touch screens can also only detect contact events and positions when actual contacts are made with the touch surface.", "Scalability of passive touch screens is important since the demand for larger electronic digitizers is increasing.", "Where digitizers were once small desktop appliances, today they have found there way onto electronic whiteboarding applications.", "The need to build a passive touch sensitive “wall” has become a requirement for new touch screen applications.", "Existing passive touch screens of the types discussed above are all limited in the maximum size where they are still functional.", "As will be appreciated, improvements to passive touch systems are desired.", "It is therefore an object of the present invention to provide a novel camera-based touch system.", "DISCLOSURE OF THE INVENTION According to one aspect of the present invention there is provided a camera-based touch system comprising: at least two cameras associated with a passive touch surface and having overlapping fields of view encompassing said touch surface, said at least two cameras acquiring images of said touch surface from different locations and generating image data; and a processor receiving and processing image data generated by said at least two cameras to determine the location of a pointer relative to said touch surface when said pointer is captured in images acquired by said at least two cameras.", "Preferably the at least two cameras are digital cameras having fields of view looking generally along the plane of the touch surface.", "The image data generated by each digital camera includes a pointer median line x and a pointer tip location z.", "Each of the digital cameras includes a pixel array having selectable pixel rows.", "Pixel intensities of pixels in the selectable pixel rows are used during generation of the image data.", "Preferably, pixel intensities of pixels in a region of interest within the selectable pixel rows are used during generation of the image data.", "In a preferred embodiment, each of the digital cameras includes a CMOS image sensor and a digital signal processor.", "The digital signal processor receives image output from the image sensor and executes a find pointer routine to determine if a pointer is in each image acquired by the digital camera and if so, the median line of the pointer.", "It is also preferred that the digital signal processor of each digital camera executes an update background image routine to update the background image after each image is acquired.", "Preferably, the digital signal processor of each digital camera further determines the differences between each acquired image and the background image to detect changing light conditions.", "According to another aspect of the present invention there is provided a camera-based touch system comprising: a generally rectangular passive touch surface on which contacts are made using a pointer; a digital camera mounted adjacent each corner of said touch surface, said digital cameras having overlapping fields of view encompassing said touch surface, said digital cameras acquiring images of said touch surface and generating image data that includes the median line x and pointer tip location z of a pointer when said pointer is captured in images acquired by said digital cameras; and a processor receiving and processing image data generated by said digital cameras to determine the location of said pointer relative to said touch surface and whether said pointer is in contact -with said touch surface.", "According to yet another aspect of the present invention there is provided a method of detecting the position of a pointer relative to a touch surface comprising the steps of: acquiring images of said touch surface from different locations using cameras having overlapping fields of view and generating image data; and processing said image data to detect the existence of a pointer within said acquired images and to determine the location of said pointer relative to said touch surface.", "The present invention provides advantages in that the passive touch system is of high resolution and allows actual pointer contacts with the touch surface as well as pointer hovers above the touch surface to be detected and corresponding output generated.", "Also, the present passive touch system provides advantages in that it does not suffer from parallax, image distortion, pointer position restrictions, image projection and scalability problems that are associated with prior art passive touch systems.", "Furthermore, the present invention provides advantages in that since CMOS digital cameras are used, arbitrary pixel rows in the digital camera pixel arrays can be selected.", "This enables the frame rates of the digital cameras to be increased significantly.", "Also, since the pixel rows can be arbitrary selected, the pixel arrays can be exposed for greater durations for given digital camera frame rates allowing for good operation in dark rooms as well as well lit rooms.", "BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the present invention will now be described more fully with reference to the accompanying drawings in which: FIG.", "1 is a schematic diagram of a camera-based touch system in accordance with the present invention; FIG.", "2 is an isometric view of a touch screen forming part of the touch system of FIG.", "1; FIG.", "3 is an isometric view of a corner portion of the touch screen of FIG.", "2; FIG.", "4 is a schematic diagram of a digital camera forming part of the touch screen of FIG.", "2; FIG.", "5 is a schematic diagram of a master controller forming part of the touch system of FIG.", "1; FIG.", "6 is a flowchart showing the steps performed during execution of a processFrame routine; FIG.", "7 is a flowchart showing the steps performed during execution of a segmentPointer routine; FIG.", "8 is a flowchart showing the steps performed during execution of a findPointer routine; FIG.", "9 shows an image acquired by a digital camera and a pixel subset of the image that is processed; FIG.", "10 shows a region of interest (ROI) within the pixel subset of FIG.", "9; FIG.", "11 shows triangulation geometry used to calculate a pointer contact position on the touch surface of the touch screen illustrated in FIG.", "2; FIG.", "12 shows an image acquired by a digital camera including the pointer tip and its median line; FIG.", "13 shows pointer contact and pointer hover for different orientations of the pointer; FIG.", "14 is an image of the touch surface of the touch screen as seen by a digital camera; FIGS.", "15 and 16 show the results of a Matlab simulation of pointer tracking using a Kalman filter; and FIGS.", "17a to 17d show the results of another Matlab simulation of pointer tracking using a Kalman filter.", "BEST MODE FOR CARRYING OUT THE INVENTION Turning now to FIG.", "1, a camera-based touch system in accordance with the present invention is shown and is generally identified by reference numeral 50.As can be seen, touch system 50 includes a touch screen 52 coupled to a digital signal processor (DSP) based master controller 54.Master controller 54 is also coupled to a computer 56.Computer 56 executes one or more application programs and provides display output that is presented on the touch screen 52 via a projector 58.The touch screen 52, master controller 54, computer 56 and projector 58 form a closed-loop so that user contacts with the touch screen 52 can be recorded as writing or drawing or used to control execution of application programs executed by the computer 56.FIGS.", "2 to 4 better illustrate the touch screen 52.Touch screen 52 includes a touch surface 60 bordered by a rectangular frame 62.Touch surface 60 is in the form of a rectangular planar sheet of passive material.", "DSP-based CMOS digital cameras 63 are associated with each corner of the touch screen 52.Each digital camera 63 is mounted on a frame assembly 64.Each frame assembly 64 includes an angled support plate 66 on which the digital camera 63 is mounted.", "Supporting frame elements 70 and 72 are mounted on the plate 66 by way of posts 74 and secure the plate 66 to the frame 62.Each digital camera 63 includes a two-dimensional CMOS image sensor and associated lens assembly 80, a first-in-first-out (FIFO) buffer 82 coupled to the image sensor and lens assembly 80 by a data bus and a digital signal processor (DSP) 84 coupled to the FIFO 82 by a data bus and to the image sensor and lens assembly 80 by a control bus.", "A boot EPROM 86 and a power supply subsystem 88 are also included.", "In the present embodiment, the CMOS camera image sensor is a Photobit PB300 image sensor configured for a 20×640 pixel subarray that can be operated to capture image frames at rates in excess of 200 frames per second since arbitrary pixel rows can be selected.", "Also, since the pixel rows can be arbitrarily selected, the pixel subarray can be exposed for a greater duration for a given digital camera frame rate allowing for good operation in dark rooms as well as well lit rooms.", "The FIFO buffer 82 is manufactured by Cypress under part number CY7C4211V and the DSP 84 is manufactured by Analog Devices under part number ADSP2185M.", "The DSP 84 provides control information to the image sensor and lens assembly 80 via the control bus.", "The control information allows the DSP 84 to control parameters of the image sensor and lens assembly 80 such as exposure, gain, array configuration, reset and initialization.", "The DSP 84 also provides clock signals to the image sensor and lens assembly 80 to control the frame rate of the image sensor and lens assembly 80.The angle of the plate 66 is selected so that the field of view (FOV) of each digital camera 63 extends beyond a designated peripheral edge of the touch surface 60 as shown in FIG.", "11.In this way, the entire touch surface 60 is within the fields of view of the digital cameras 63.Master controller 54 is best illustrated in FIG.", "5 and includes a DSP 90, a boot EPROM 92, a serial line driver 94 and a power supply subsystem 95.The DSP 90 communicates with the DSPs 84 of the digital cameras 63 over a data bus via a serial port 96 and communicates with the computer 56 over a data bus via a serial port 98 and the serial line driver 94.In this embodiment, the DSP 90 is also manufactured by Analog Devices under part number ADSP2185M.", "The serial line driver 94 is manufactured by Analog Devices under part number ADM222.The master controller 54 and each digital camera 63 follow a communication protocol that enables bi-directional communications via a common serial cable similar to a universal serial bus (USB).", "The transmission bandwidth is divided into thirty-two (32) 16-bit channels.", "Of the thirty-two channels, six (6) channels are assigned to each of the DSPs 84 in the digital cameras 63 and to the DSP 90 in the master controller 54 and the remaining two (2) channels are unused.", "The master controller 54 monitors the twenty-four (24) channels assigned to the DSPs 84 while the DSPs 84 monitor the six (6) channels assigned to the DSP 90 of the master controller 54.Communications between the master controller 54 and the digital cameras 63 are performed as background processes in response to interrupts.", "The general operation of the touch system 50 will now be described.", "Each digital camera 63 acquires images of the touch surface 60 within the field of view of its image sensor and lens assembly 80 at a desired frame rate and processes each acquired image to determine if a pointer is in the acquired image.", "If a pointer is in the acquired image, the image is further processed to determine characteristics of the pointer contacting or hovering above the touch surface 60.Pointer information packets (PIPs) including pointer characteristics, status and/or diagnostic information are then generated by the digital cameras 63 and the PIPs are queued for transmission to the master controller 54.The digital cameras 63 also receive and respond to command PIPs generated by the master controller 54.The master controller 54 polls the digital cameras 63 for PIPs.", "If the PIPs include pointer characteristic information, the master controller 54 triangulates pointer characteristics in the PIPs to determine the position of the pointer relative to the touch surface 60 in Cartesian rectangular coordinates.", "The master controller 54 in turn transmits calculated pointer position data, status and/or diagnostic information to the personal computer 56.In this manner, the pointer position data transmitted to the personal computer 56 can be recorded as writing or drawing or can be used to control execution of application programs executed by the computer 56.The computer 56 also updates the display output conveyed to the projector 58 so that information presented on the touch surface 60 reflects the pointer activity.", "The master controller 54 also receives commands from the personal computer 56 and responds accordingly as well as generates and conveys command PIPs to the digital cameras 63.Specifics concerning the processing of acquired images and the triangulation of pointer characteristics in PIPs will now be described with particular reference to FIGS.", "6 to 8.Initially, a camera offset angle calibration routine is performed to determine the offset angle δ of each digital camera 63 (see FIG.", "11) so that the contact or hover position of a pointer relative to the touch surface 60 can be accurately determined.", "Details of the camera offset angle calibration are described in Applicants' co-pending U.S. application entitled “Calibrating Camera Offsets to Facilitate Object Position Determination Using Triangulation” filed on Jun.", "1, 2001, the content of which is incorporated herein by reference.", "Following the camera offset angle calibration routine, a surface detection routine is performed to enhance determination as to whether a pointer is in contact with the touch surface 60 at a given point or hovering above the touch surface.", "With rectangular coordinates of a pointer in the plane of the touch surface 60 accurately known from the camera offset angle calibration, the orientation of the touch surface 60 as seen by each digital camera 63 can be determined.", "This is necessary due to the fact that the digital cameras do not just see along the plane of the touch surface 60 but also in a direction perpendicular to it.", "To some degree, each digital camera 63 looks downward into the touch surface 60.FIG.", "14 generally shows the shape of the touch surface 60 as seen by a digital camera 63.Because of this, it is desired to define a “vertical” coordinate z which describes the touch surface location as a function of rectangular coordinates x and y.", "The z coordinate of the pointer can be measured from a digital camera image, and hence, z coordinates for pointer positions on the touch surface 60 can be determined.", "This vertical calibration becomes a matter of fitting the z coordinate data for given rectangular coordinates x and y.", "The vertical calibration can be described as a surface of the form: z(x,y)=Ax+By+Cx2+Dy2+Exy+F (0.1) Note that if the coefficients C, D, and E are zero, this becomes a plane.", "The fit is easily computed as equation (0.1) represents a linear least-squares problem.", "The corresponding matrix takes the form: [ x 1 y 1 x 1 2 y 1 2 x 1 ⁢ y 1 1 x 2 y 2 x 2 2 y 2 2 x 2 ⁢ y 2 1 ⋮ ⋮ ⋮ ⋮ ⋮ ⋮ x N y N x N 2 y N 2 x N ⁢ y N 1 ] ⁡ [ A ⁢ B C D E ] = [ z 1 z 2 ⋮ z N ] In order to fit the rectangular coordinates x and y to the equation (0.1) to determine the coefficients A to E, the Moore-Penrose pseudo-inverse method that is based on singular value decomposition (SVD) is used to determine a minimum-norm least squares solution.", "As will be appreciated, a matrix can always be decomposed in the following way: A=USVT (0.2) Matrix A can have any shape.", "The matrices U and V are orthogonal matrices, meaning that: UTU=I=VT V The diagonal matrix S is composed entirely of the singular values of matrix A, which are related to the squares of the eigenvalues of matrix A.", "The importance of the singular value decomposition (SVD) lies in the fact that with it, the inverse of matrix A can always be computed.", "Moreover, it is possible to control this inversion when a poorly determined problem is encountered.", "Consider the system of linear equations: A ⁢ x V = b V whose solution would be: {right arrow over (x)}=A−1{right arrow over (b)} SVD allows the inverse of matrix A to be written as: A−1=VS−1UT (0.3) since both matrices U and V are orthogonal.", "In a poorly determined situation, some of the singular values will be very small, so that when matrix S−1 is formed, large values will be produced, which is not desirable.", "In this case, the inverses of the smallest singular values are set to zero.", "This has the effect of eliminating the poorly determined part of the solution.", "For least-squares problems, this is a powerful tool.", "The usual normal equations method for least-squares problems is based on solving: A T ⁢ A ⁢ x V = A T ⁢ b V ⁢ ⁢ x V = ( A T ⁢ A ) - 1 ⁢ A T ⁢ b V ( 0.4 ) in the over-determined case, and solving: x V = A T ⁡ ( AA T ) - 1 ⁢ b V ( 0.5 ) in the under-determined case.", "As will be appreciated, during fitting of the system of equations to equation (0.1), the same method is used as is used during determination of the camera offset angles δ.", "Since the same procedure is used, memory usage and processing speed is maintained at desired levels.", "With the coefficients A through E known, the z coordinate for any given (x,y) point on the touch surface can be calculated and thus, a determination can be made as to whether a pointer is contacting the touch surface 60 or hovering above it.", "With the touch system 50 calibrated, during operation each digital camera 63 acquires images of the touch surface 60 within its field of view.", "The images are acquired by the image and lens assembly 80 at intervals in response to the clock signals received from the DSP 84.Each image acquired by the image and lens assembly 80 is sent to the FIFO buffer 82.The DSP 84 in turn reads each image from the FIFO buffer 82 and processes the image.", "To avoid processing significant numbers of pixels containing no useful information, only a subset of the pixels in the acquired image are processed as is shown in FIG.", "9.During processing of an image acquired by a digital camera 63, the DSP 84 executes a processFrame routine as shown in FIG.", "6.When an image is available for processing (step 120), a check is made to determine if the image has been captured for the purpose of adjusting the digital camera 63 (step 122).", "If the image has been acquired for the purpose of exposure adjustment, an exposureControl routine is called (step 124) to adjust the exposure of the digital camera 63.Following this, the DSP 84 awaits receipt of the next image available for processing.", "At step 122, if the image has not been captured for the purpose of adjusting the exposure of the digital camera 63, a check is made to determine if the image has been captured for the purpose of replacing a background image (step 126).", "If the image has been acquired for the purpose of background image replacement, a captureBackground routine is called (step 128) and the acquired image is used as the background image.", "This is done if a digital camera acquires an image and sends a PIP to the master controller indicating that a pointer is in the image when it is actually noise.", "Replacing the background image effectively inhibits the digital camera from falsely identifying a pointer in future PIPs.", "Following this, the DSP 84 awaits receipt of the next image available for processing.", "At step 126, if the image has not been captured for the purpose of background image replacement, a copyICur routine is called by the DSP 84 (step 130).", "During this routine, the current acquired image is copied into memory and is used to update the background image as well as to form a difference image representing the differences between the current acquired image and the background image.", "After completion of the copyICur routine, a segmentPointer routine is called (step 132) to determine if a pointer is in the acquired image and if so to determine the location of the pointer relative to the touch surface 60 and whether the pointer is in contact with the touch surface 60 or hovering above it.", "The segmentPointer routine 132 also allows changing light conditions to be detected.", "Following the segmentPointer routing 132, the DSP 84 calls a fillPIP routine (step 134) to place the pointer and light condition information into a PIP for transmission to the master controller 54.Thereafter, the DSP 84 awaits receipt of the next image available for processing.", "FIG.", "7 illustrates the steps performed by the DSP 84 during execution of the segmentPointer routine 132.As can be seen, when the DSP 84 executes the segmentPointer routine, the DSP 84 calls a findPointer routine to determine if a pointer is in the acquired image and if so, the position of the pointer in the current acquired image (step 140).", "Upon completion of the findPointer routine 140, the DSP 84 calls an updateBackground routine to update the background image thereby to deal with changes in lighting conditions (step 142).", "During execution of the updateBackground routine, the DSP 84 continuously updates the background image using the equation: Bn+1(i,j)=1-a)Bn(i,j)+aI(i,j) (0.6) where: Bn+1 is the new background image; Bn is the current background image; I is the current acquired image; i,j are the row and column coordinates of the background image pixels being updated; and a is a number between 0 and 1 that indicates the degree of learning that should be taken from the current acquired image I.", "The larger the value of a, the faster the background image is updated.", "After the updateBackground routine 142 has been executed, the intensity difference between the current acquired image and the background image is calculated by the DSP 84.This information is sent to the master controller 54 to enable the master controller to determine if the digital camera 63 needs to be re-exposed.", "This would be required if a drastic change in lighting conditions occurred (i.e.", "environment lighting was switched on or off).", "When re-exposure of the digital camera 63 is required, the master controller 54 sends a command PIP to the digital camera 63 instructing the digital camera to acquire an image for exposure adjustment.", "FIG.", "8 illustrates the steps performed by the DSP 84 during execution of the findPointer routine 140.As can be seen, when the DSP 84 executes the findPointer routine 140, the DSP 84 clears pointer location and pointer tip parameters x and z respectfully (step 150).", "Thereafter a vertical intensity histogram is built (step 152).", "During this stage, the difference image representing differences between the current image and background image is formed and pixel intensities in the difference image are summed by column.", "In this manner a 640×1 vector is formed that represents the sum of each column in the 640×20 difference image.", "Thus, the first element in the 640×1 vector represents the sum of the 20 pixels in the first column of the 640×20 difference image, the second element in the 640×1 vector represents the sum of the 20 pixel in the second column of the 640×20 difference image and so on.", "Further specifics of this process can be found in the article entitled “A smart camera application: DSP—based people detection and tracking” authored by V. Cheng et al and published in the SPIE Journal of Electronic Imaging July, 2000.Following the creation of the vertical intensity histogram at step 152, the pointer location parameter x is determined by finding the column in the vertical intensity histogram with the highest intensity above a noise threshold (step 154).", "The column is used as the center of a region of interest (ROI) to be processed with the width of the ROI being equal to the base of the peak formed by the vertical intensity histogram (see FIG.", "10).", "If no column has an intensity above the noise threshold, it is assumed no pointer is within the acquired image.", "When a pointer location parameter x is determined, the DSP 84 analyses the ROI to determine the pixel row where the pointer tip is located and determine whether that row represents a touch surface contact or hover (step 156).", "Specifically, the DSP 84 creates a binary mask in the ROI so that white pixels represent the pointer and black pixels represent the background as shown in FIG.", "12.From the mask, the medium line of the pointer and the pointer tip location z can be easily calculated.", "During the fillPIP routine 134, the DSP 84 uses the pointer and light condition information acquired during execution of the segmentPointer routine 132 and creates a PIP to reduce the acquired image to a small set of data thereby to provide bandwidth economy.", "The PIP is in the form of a six (6) word packet, with each word in the packet being sixteen (16) bits.", "The PIP typically takes the form: Header Data Checksum The header portion of the PIP is typically sixteen (16) bits and includes a determination/source field, a data type field, an image frame number field, a sequence number field and a packet number field.", "The destination/source field identifies the PIP destination and the PIP source.", "If the PIP is generated by the master controller 54, the destination may be a single digital camera 63 or all digital cameras.", "The data type indicates whether the PIP relates to pointer information or other information such as status and diagnostic information.", "The image frame number field stores a number so that images from each digital camera 63 are processed by the master controller 54 in sequence.", "The sequence number field stores a number that relates the PIP to other PIPs.", "The packet number field stores a number identifying the packet.", "The data portion of the PIP is typically sixty-four (64) bits and includes a pointer ID field, a pointer location parameter field, a pointer tip parameter field, a contact state field and a goodness of pointer field.", "The pointer ID field stores an identifier for the pointer to allow multiple pointers to be tracked.", "The pointer location parameter field stores the x-value calculated by the DSP 84.The pointer tip parameter field stores the z-value calculated by the DSP 84.The contact state field stores a value that indicates whether the pointer is in contact, out of contact or possibly in contact with the touch surface 60.The goodness of pointer field stores a statistical value on the likelihood that a detected pointer is real.", "The checksum portion of the PIP is used to ensure PIP transmission integrity.", "If PIP checksum errors are infrequent, the PIPs exhibiting checksum errors are ignored by the destination device.", "Status PIPs that do not relate to pointer information have a different form then the above-identified described PIPs.", "For PIPs of this nature, the data portion includes an instruction type field, an instruction code field and a data field.", "The instruction type field identifies whether the instruction type is an instruction to be performed or a status request.", "The instruction code field stores the actual instruction or status request identifier.", "The data field stores data that varies depending on the type of instruction.", "Examples of status PIPs include frame header PIPs, command PIPs and error message PIPs.", "A frame header PIP typically includes the number of pointer PIPs that are to follow for a current acquired image with statistics for the current image such as intensity variance between the current acquired image and a previous image.", "A command PIP issued by the master controller 54 may instruct a digital camera to adjust one or more of its settings such as exposure or capture an image to be used as a new background image.", "An error PIP may pass an error condition from a digital camera 63 to the master controller 54 for storage in an error log.", "Each digital camera 63 processes each image it acquires in the manner described above in response to each clock signal generated by its DSP 84.The PIPs created by the DSPs 84 are only sent to the master controller 54 when the digital cameras 63 are polled by the master controller 54.When the master controller 54 polls the digital cameras 63, frame sync pulses are sent to the digital cameras 63 to initiate transmission of the PIPs created by the DSPs 84.Upon receipt of a frame sync pulse, each DSP 84 transmits the PIP to the master controller 54 over the data bus.", "The PIPs transmitted to the master controller 54 are received via the serial port 96 and auto-buffered into the DSP 90.After the DSP 90 has polled the digital cameras 63 and has received PIPs from each of the digital cameras 63 that include pointer information, the DSP 90 processes the PIPs using triangulation to determine the location of the pointer relative to the touch surface 60 in (x,y) coordinates.", "Specifically, the PIPs from pairs of digital cameras 63 are processed using triangulation.", "FIG.", "11 shows that two angles φcam1 and φcam2 are needed to triangulate the position (x0, y0) of a pointer relative to the touch screen 60.The PIPs generated by each digital camera 63 include a number θ (see FIG.", "12) identifying the median line or tip of the pointer.", "When the master controller 54 receives a PIP from a digital camera 63, the master controller uses the number representing the median line or tip of the pointer and the field of view of the digital camera to calculate an angle φcam , using the equation: tan ⁢ ⁢ ϕ cam = 2 ⁢ ( x a ) ⁢ ⁢ tan ⁢ ⁢ FOV 2 1 - ( 2 ⁢ ⁢ x a - 1 ) ⁢ ⁢ tan 2 ⁢ ⁢ FOV 2 ( 0.7 ) where: x is the number representing the median line or tip of the pointer; and a is the total length enclosed by the field of view (FOV) of the digital camera at a distance from the camera.", "The calculated angle φcam is equal to the angle formed between the extremity of the field of view extending beyond the designated peripheral edge of the touch surface 60 of the digital camera 63 that generated the PIP and a line extending from the optical axis of the digital camera that intersects the pointer within the acquired image.", "Preferably, the extremity of the field of view extends beyond the designated peripheral edge (i.e.", "in this case the x-axis) of the touch surface 60 within the field of view by a known amount.", "However, in almost all cases the angular offset δcam scan of each digital camera 63 is different and unknown.", "Once the master controller 54 calculates the angle φcam, the master controller 54 uses the camera offset angle δcam determined during the camera offset calibration to adjust the angle φcam.", "With the two angles available and with the angles φcam adjusted, the master controller 54 uses the angles φcam to determine the position of the pointer relative to the touch surface 60 using triangulation.", "In this embodiment, since the touch screen 52 includes four digital cameras 63, six pairs of digital cameras can be used for triangulation.", "The following discussion describes how a pointer position is determined by triangulation for each pair of the digital cameras 63.In order to determine a pointer position using the PIPs received from the digital cameras 63 along the left side of the touch screen 52, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ0 and φ1 for the upper and lower digital cameras: x 0 = h w × 1 tan ⁡ ( ϕ 0 ) + tan ⁡ ( ϕ 1 ) ( 0.8 ) y 0 = tan ⁡ ( ϕ 0 ) tan ⁡ ( ϕ 0 ) + tan ⁡ ( ϕ 1 ) ( 0.9 ) where: h is the height of the touch screen 52 i.e.", "the vertical distance from digital camera focal point-to-focal point; w is the width of the touch screen 52 i.e.", "the horizontal distance from digital camera focal point-to-focal point; and φi is the angle with respect to the horizontal, measured using digital camera i and equation (0.7).", "For the digital cameras 63 along on the right side of the touch screen 52, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ2 and φ3 for the upper and lower digital cameras: x 0 = 1 - h w × 1 tan ⁡ ( ϕ 2 ) + tan ⁡ ( ϕ 3 ) ( 0.10 ) y 0 = 1 - tan ⁡ ( ϕ 2 ) tan ⁡ ( ϕ 2 ) + tan ⁡ ( ϕ 3 ) ( 0.11 ) The similarity between equations (0.8) and (0.10), i.e.", "equation (0.10)−1−equation (0.8) once angles φ2 and φ3 have been substituted into equation (0.8) for angles φ1 and φ2 respectively should be apparent.", "Equations (0.9) and (0.11) are related in a similar manner.", "In order to determine a pointer position using the digital cameras 63 along the bottom of the touch screen 52, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ0 and φ3 for bottom left and bottom right digital cameras: x 0 = tan ⁡ ( ϕ 3 ) tan ⁡ ( ϕ 0 ) + tan ⁡ ( ϕ 3 ) ( 0.12 ) y 0 = w h × tan ⁡ ( ϕ 3 ) tan ⁡ ( ϕ 0 ) + tan ⁡ ( ϕ 3 ) × tan ⁡ ( ϕ 0 ) ( 0.13 ) ⁢ = w h × x 0 × tan ⁡ ( ϕ 0 ) In order to determine a pointer position using the digital cameras 63 along the top of the touch screen 52, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ1 and φ2 for the top left and top right digital cameras: x 0 = tan ⁡ ( ϕ 2 ) tan ⁡ ( ϕ 1 ) + tan ⁡ ( ϕ 2 ) ( 0.14 ) y 0 = 1 - w h × tan ⁡ ( ϕ 2 ) tan ⁡ ( ϕ 1 ) + tan ⁡ ( ϕ 2 ) × tan ⁡ ( ϕ 1 ) ( 0.15 ) ⁢ = 1 - w h × x 0 × tan ⁡ ( ϕ 1 ) The similarity between equations (0.12) and (0.14), i.e.", "equation (0.14)=equation (0.12) once angles φ1 and φ2 have been substituted into equation (0.12) for angles φ0 and φ3 should be apparent.", "Equations (0.13) and (0.15) have the following relationship: equation (0.15)=1−equation (0.13) once angles φ1 and φ2 have been substituted into equation (0.13) for angles φ0 and φ3 respectively.", "In order to determine a pointer position using the digital cameras 63 across the bottom left to top right corner diagonal, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ0 and φ2 for bottom left and top right digital cameras: x 0 = h w - tan ⁡ ( ϕ 2 ) tan ⁡ ( ϕ 0 ) - tan ⁡ ( ϕ 2 ) ( 0.16 ) y 0 = 1 - w h - tan ⁡ ( ϕ 2 ) tan ⁡ ( ϕ 0 ) - tan ⁡ ( ϕ 2 ) × tan ⁡ ( ϕ 0 ) ( 0.17 ) In order to determine a pointer position using the digital cameras 63 across the bottom right to top left diagonal, the following equations are used to determine the (x0, y0) coordinates of the pointer position given the angles φ1 and φ3 for the bottom right and top left digital cameras: x 0 = h w - tan ⁡ ( ϕ 3 ) tan ⁡ ( ϕ 1 ) - tan ⁡ ( ϕ 3 ) ( 0.18 ) y 0 = 1 - 1 - w h - tan ⁡ ( ϕ 3 ) tan ⁡ ( ϕ 1 ) - tan ⁡ ( ϕ 3 ) × tan ⁡ ( ϕ 1 ) ( 0.19 ) The similarity between equations (0.16) and (0.18), i.e.", "equation (0.18)=equation (0.16) once angles φ1 and φ3 have been substituted into equation (0.16) for angles φ0 and φ2 should be apparent.", "Equations (0.17) and (0.19) have the following relationship: equation (0.19)=1−equation (0.17) once angles φ1 and φ3 have been substituted into equation (0.17) for angles φ0 and φ2 respectively.", "As will be appreciated, the above equations generate the coordinates x0 and y0 on a scale of [0, 1].", "Therefore, any appropriate coordinate scale can be reported by multiplying x0 and y0 by the maximum X and maximum Y values respectively.", "In the present embodiment, the DSP 90 calculates the pointer position using triangulation for each digital camera pair excluding the diagonal pairs.", "The resulting pointer positions are then averaged and the resulting pointer position coordinates are queued for transmission to the personal computer 56 via the serial port 98 and the serial line driver 94.With the (x,y) position of a pointer known by triangulation, using the coefficients A to E calculated during the surface detection calibration, the z coordinate corresponding to the (x,y) position can be determined using equation (0.1).", "Calculating the z coordinate and comparing the z coordinate with the z parameter in the PIP provides an indication as to whether the pointer is hovering above the touch surface 60 or is in actual contact with the touch surface.", "If desired, pointer velocity v and angle can be calculated by the DSP 90 as shown in FIG.", "13.The velocity of the pointer is calculated by examining the changes in the z-position (or x-intercept) of the pointer in successive PIPs and knowing the camera frame rate.", "For example, if the camera frame rate is 200 frames per second and the z-position changes by 1 pixel row per frame, the pointer velocity is 200 pixels per second.", "The angle of the pointer can be determined due to the fact that the PIP includes the x-intercept at pixel rows 0 and 19 of the median line.", "Since the x distance (the difference between x-intercepts) and the y distance (the number of pixel rows) are known, all of the information necessary to calculate the pointer angle is available.", "If desired, a Kalman filter (essentially a recursive least-squares method) may be used to effectively “track” the pointer when it is within a certain distance of the touch surface 60.To do this, it is necessary to define a system equations or model to be used in the filter.", "Since the master controller 54 is able to provide both the position z and velocity v of the pointer, the following description can be used: z=z0+vt v=v The second of these equations is required as the filter has to know what to do with the velocity, and also since both z and v are measurable.", "Define the state vector as: [z v]T To relate the state of the system at two successive times n and n+1, write the system equations as a matrix difference equation: [ z v ] n + 1 = [ 1 dt 0 1 ] ⁡ [ z v ] n + [ σ z σ v ] or in matrix notation, {circumflex over (x)}n+1=A{circumflex over (x)}n+σ Here, dt denotes the time interval between successive time steps.", "Also introduced here on the RHS is the “process noise” term.", "It is purely formal, but part of the Kalman filter method.", "It is also necessary to specify how a measurement is introduced into the procedure.", "This is done via the matrix equation: zn=Hxn+w where zn is a measurement of position and velocity, H is a “measurement matrix” which is taken to be an identity matrix, xn is the state vector and w is measurement noise.", "Essentially, it is assumed that the measurements are noisy versions of the state vector.", "It is also necessary to define a covariance matrix associated with w. If the measurement error in z is 0.5 pixel, then the covariance matrix is: R = ( 0.5 ) 2 ⁡ [ 1 0 0 1 ] A similar matrix Q is required for the process noise introduced above, but as it is somewhat arbitrary, it may be treated as a tuning parameter for the filter.", "In this example, the matrix Q is taken to be an identity matrix multiplied by a factor of order unity or less.", "With the above established, there is sufficient information to start the filter process.", "The first (prediction) step is: {circumflex over (x)}k+1(−)=A{circumflex over (x)}k(+) Pk(−)=APk−1(+)AT+Qk−1 Here, the (−) notation implies that a measurement has not yet been made while (+) does (but in this case the (+) refers to the previous step).", "Also, the matrix equation for matrix P predicts a covariance matrix.", "The next step is the filter gain computation: Kk=Pk(−)HkT[HkPk(−)HkT+Rk]−1 Once a measurement is made, the state estimate and its covariance can be updated: {circumflex over (x)}k(+)={circumflex over (x)}k(−)+Kk[zl−Hkxk(−)] Pk(+)=[Pk−1(−)+HkTRk−1Hk]−1 It is this estimate of the state x that is used to determine whether or not contact with the touch surface has occurred.", "Note here that the matrices H and R are both constant with time, and that only matrices K and P change (in fact, P approaches a constant matrix).", "An additional simplification occurs in that there is no control process involved.", "The results of a Matlab simulation of a Kalman filter using a set of measurements representing a pointer approaching the touch surface 60 at a constant velocity was performed.", "FIGS.", "15 and 16 illustrate the simulation, with a time step dt of 0.1 sec and a measurement precision of 0.5 pixel.", "The open symbols represent the data, and the lines the state estimate from the Kalman filter.", "Clearly, the state estimate follows the data quite well.", "A second Matlab simulation was performed to take into account both vertical (z) and horizontal (x) motion of a pointer.", "This simulation is basically two similar Kalman filters operating together in a “parallel” fashion.", "The formulation is exactly the same, except twice the number of variables need to be considered.", "FIGS.", "17a to 17d show the results of the simulation and represent movement of a pointer towards the touch surface 60 at constant velocity and at a slowly-varying x position (i.e.", "the person's hand is unsteady).", "Although the touch system 50 has been described as including a projector to present images on the touch screen, those of skill in the art will appreciate that this is not required.", "The touch screen 52 may be transparent or translucent and placed over a display unit so that the display presented on the display unit is visible through the touch screen.", "Also, the touch screen need not be a rectangular sheet of material bordered by a frame.", "The touch screen may in fact be virtually any surface within overlapping fields of view of two or more digital cameras.", "Also, although the touch system 50 is described as including a master controller separate from the digital cameras, if desired one of the digital cameras can be conditioned to function as both a camera and the master controller and poll the other digital cameras for PIPs.", "In this case, it is preferred that the digital camera functioning as the master controller includes a faster DSP 84 than the remaining digital cameras.", "In addition, although the surface detection routine is described as determining the coefficients A to E to be used with equation (0.1) to calculate the z coordinates of the pointer at a given point (x,y) relative to the touch screen, during the surface detection routine, the master controller 54 can be programmed to calculate a z coordinate for unique (x,y) regions of the touch surface and store the z coordinates in a look-up table (LUT).", "In this instance; when a pointer appears in images captured by the digital cameras and the (x,y) position of the pointer relative to the touch surface is determined, a decision can be made as to whether the pointer is in contact with the touch surface by comparing the z coordinate in the LUT corresponding with the (x,y) region in which the pointer is located, with the pixel row of the image sensor and lens assembly at which the pointer tip is located.", "As described above, the master controller 54 calculates or looks up the z coordinates of the touch surface for each digital camera and compares the z coordinates with the pointer tip location z to determine if the pointer is in actual contact with the touch surface.", "However, those of skill in the art will appreciate that the DSPs 84 in the digital cameras may include image processing software to determine if the pointer is in actual contact with the touch surface.", "This image processing can be preformed in conjunction with or instead of the master controller pointer contact determination.", "Although a preferred embodiment of the present invention has been described, those of skill in the art will appreciate that variations and modifications may be made without departing from the spirit and scope thereof as defined by the appended claims." ] ]

[ [ "Equipment and method for enhancing combustion and heat transfer in a boiler by using sound", "The invention concerns equipment and a method for enhancing the combustion event and heat transfer in a heating boiler.", "According to the invention, the space above the major combustion zone in the combustion space of the heating boiler is equipped with sound sources, which are used to generate an acoustic field to enhance the combustion event and to achieve more complete combustion." ], [ "1.Equipment for enhancing the combustion event and heat transfer in a heating boiler, said equipment comprising sound sources (S) in a space above the major combustion zone in a combustion space (11) of the heating boiler (10), which sound sources (S) generate an acoustic field in order to enhance the combustion event and a more complete combustion, characterised in that the sound sources (S) are placed in such a way that the acoustic pressure patterns generated by the sound sources (S) meet at angles of 20-90° sideways and/or vertically and that the sound sources (S) are fitted into the heating boiler (10) in such a way that the generated acoustic field is rotating.", "2.Equipment as defined in claim 1, characterised in that the acoustic field generated by the sound sources (S) is continuous.", "3.Equipment as defined in claim 1 or 2, characterised in that the acoustic pressure level generated by the sound sources (S) is no less than 130 dB at the place where the acoustic pressure patterns meet.", "4.Equipment as defined in any one of claims 1-3, characterised in that the frequency of the sound generated by the sound sources (S) is in a range of 20-1000 Hz.", "5.Equipment as defined in any one of claims 1-4, characterised in that the secondary and/or tertiary air of the heating boiler (10) or a part of that air has been supplied into the boiler through the sound sources (S).", "6.Equipment as defined in any one of claims 1-5, characterised in that the sound sources (S) are fitted into the heating boiler (10) in such a way that the generated rotating acoustic field rotates in the direction of the positive acoustic pressure.", "7.Equipment as defined in any one of claims 1-6, characterised in that the sound sources (S) are fitted into the heating boiler (10) in such a way that the sound frequency increases towards the top part of the heating boiler (10).", "8.Equipment as defined in any one of claims 1-7, characterised in that the sound sources (S) are acoustic horns.", "9.Equipment as defined in any one of claims 1-7, characterised in that the sound sources (S) are pneumatically operated continuous sirens.", "10.Equipment as defined in any one of claims 1-7, characterised in that the sound sources (S) are sound sources (SP) based on a pulse burner.", "11.Equipment as defined in claim 10, characterised in that the fuel of the pulse burner (SP) functioning as sound source essentially includes a gaseous inflammable matter and an oxidiser.", "12.Equipment as defined in claim 10 or 11, characterised in that the pulse burner (SP) functioning as sound source includes an antechamber (SP1), into which air under pressure is arranged to be supplied, a set of valves (V) opened and closed by pressure and separating the space between the antechamber (SP1) and the combustion chamber (SP2), and into which combustion chamber (SP2) supply of fuel is arranged, and a sound horn (SP3).", "13.Equipment as defined in claim 12, characterised in that a continuous supply of air under pressure is arranged into the antechamber (SP1) of the pulse burner (SP) through an assembly (L1).", "14.Equipment as defined in claim 12 or 13, characterised in that the supply of fuel into the combustion chamber (SP2) of the pulse burner (SP) is arranged through an assembly (L2).", "15.Equipment as defined in any one of claims 12-14, characterised in that the pressure of the combustion chamber (SP2) of the pulse burner (SP) discharges into the combustion space (11) of the heating boiler (10).", "16.Method for enhancing the combustion event and heat transfer in a heating boiler, in which method an acoustic field is generated with sound sources (S) in a space above the major combustion zone in a combustion space (11) of the heating boiler (10), characterised in that a rotating acoustic field is generated by sound sources (S), which are located on various sides and/or at different elevations of the heating boiler (10) in such a way that the acoustic pressure patterns generated by the sound sources (S) meet at angles 20-90° sideways and/or vertically.", "17.Method as defined in claim 16, characterised in that sound sources (SP) based on a pulse burner are used as sound sources (S)." ], [ "The invention concerns equipment and a method for enhancing the combustion event and heat transfer in a heating boiler.", "The invention relates to combustion taking place in power plants, heating furnaces, heating boilers and other such combustion spaces and to enhancement of such combustion.", "In the following, the name of heating boiler will be used for these application objects of the invention.", "Combustion is a complex event, which depends on several different factors, such as the particle size of the fuel, the combustion temperature and the structure of the heating boiler.", "The fuel used in the combustion event becomes oxidized, whereby heat will result.", "At typical combustion temperatures, combustion of hot solid matter depends on the speed at which oxygen is diffused into the surface of the matter.", "As the combustion proceeds, the particle size and particle mass are reduced.", "When burning carbon, the combustion results in carbon monoxide, which in the continued combustion will become carbon dioxide.", "It is a known method to enhance the combustion event by causing turbulence to occur in the combustion space to enhance movement of the matter and heat transfer in the combustion.", "However, in known solutions problems still occur in the completion of combustion taking place in power plants, heating furnaces and heating boilers and other such installations.", "It is difficult to make turbulence generators, such as agitators, to reach high temperatures, which are typically in a range of 600-1000° C. In particular, problems occur in the combustion of non-volatile carbon particles.", "Enhancing of combustion is also associated with cleaning of the heat transfer surfaces of the heating boiler to remove ash particles and unburnt carbon particles from them.", "Acoustic cleaning is a known cleaning method, wherein sound sources are placed in the heat transfer parts of the heating boiler and sound is produced periodically at intervals of about 2-15 minutes for a few seconds each time.", "Acoustic cleaning equipment is known e.g.", "from publications WO-82/01328 and WO-82/03803.The purpose of the invention is to bring about equipment and a method for enhancing the combustion event in such a way that the combustion is more complete and emissions are reduced.", "Another purpose of the invention is to bring about equipment of a new kind, wherein a sound source based on a pulse burner is used as the sound source.", "The equipment according to the invention for enhancing the combustion event and heat transfer in a heating boiler is mainly characterised in that the space above the major combustion zone in the combustion space of the heating boiler is equipped with sound sources to generate an acoustic field in order to enhance the combustion event and to achieve a more complete combustion, and that the sound sources are placed in such a way that the acoustic pressure patterns generated by the sound sources meet at angles of 20-90° sideways and/or vertically.", "The method according to the invention for its part is characterised in that in a space above the major combustion zone in the combustion space of the heating boiler a rotating acoustic field is generated by sound sources, which are located on various sides and/or at different elevations of the heating boiler.", "In the arrangement according to the invention, the combustion event is enhanced in such a way in a heating boiler that an acoustic field is applied to a space above the actual major combustion zone.", "Matter volatilising at this location has already burnt out for the most part, but some carbon still remains in the form of small particles, which have not yet burnt The acoustic field is preferably continuous and it is generated by suitable sound sources, one or more, which are placed in the combustion space of the heating boiler.", "The sound source is preferably a sound source based on a pulse burner.", "By the arrangement according to the invention, the combustion event is enhanced in such a way that more heat is obtained from the fuel used and less combustion residue will result.", "At the same time, a cleaner combustion is also achieved, which means that harmful emissions are reduced.", "By using the sound source according to the invention, which is based on a pulse burner, that problem is solved, which is associated with pneumatically operated sound sources in that due to the higher pressure existing inside the combustion space dust or other impurities will drift from the combustion space into the sound source.", "No such problem occurs in the sound source based on a pulse burner.", "In the following, the invention will be described with reference to the figures shown in the appended drawing, but the intention is not to limit the invention only to the embodiments shown in the figures.", "FIG.", "1 shows a heating boiler and the location therein of the equipment according to the invention.", "FIG.", "2 shows an example of the acoustic field according to the invention.", "FIG.", "3A shows the sound source according to the invention.", "FIG.", "3B shows a valve in the sound source according to the invention.", "FIG.", "3C is a cross-sectional view of the sound source according to the invention.", "FIG.", "3D is another cross-sectional view of the sound source according to the invention.", "FIG.", "1 is a schematic view of a heating boiler, wherein the method according to the invention for enhancing the combustion event is applied.", "Sound sources S are located in combustion space 11 of heating boiler 10.The place of location of sound sources S is a space above the major combustion zone before cooling of the combustion gases.", "The sound sources used are preferably such sound horns known in acoustic cleaning, which produce a sound volume of 130-170 dB at a frequency of 20-1000 Hz.", "The sound source used may also be pneumatically operated continuous sirens or the sound source according to the invention shown in FIGS.", "3A-D, which is based on a pulse burner.", "Due to the good penetration depth, the acoustic frequency range chosen is preferably used explicitly in order to enhance the combustion event.", "The power and frequency of the sound sources S may be controlled within the chosen range according to the type and size of boiler and the locations of sound sources S. The sound volume is chosen so that the acoustic pressure level generated by sound sources S is preferably no less than 130 dB at the place where acoustic pressure patterns meet, where the temperature in the combustion space 11 is typically over 800° C. Sound sources S are placed in combustion space 11 in such a way that the acoustic pressure patterns which they generate will meet at angles of 20-90° C. sideways and/or vertically.", "In an advantageous embodiment of the invention, sound sources S are placed at different elevations in the heating boiler 10, and when moving upwards in the combustion space 11 the frequency of the sound source S is increased as the diameter and mass of solid particles diminish further ahead in combustion space 11.Hereby the desired effect enhancing the combustion event will remain at an optimum.", "In another advantageous embodiment of the invention, sound sources S are placed in such a way in heating boiler 10 that the resulting acoustic field is rotating.", "FIG.", "2 illustrates such a rotating acoustic field, which is generated by locating in the combustion space 11 of a heating boiler, which as regards its diameter is of a rectangular shape, four sound sources S, which are used to generate the acoustic pressure direction pattern shown in FIG.", "2.A rotating acoustic field of a corresponding kind can also be generated in a heating boiler of some other shape.", "The number and locations of sound sources S may be different from those shown in this example, when generating a rotating acoustic field.", "In an advantageous embodiment of the invention a part of the secondary and/or tertiary air needed in the combustion event is supplied through the sound sources S placed in heating boiler 10.This supplied air at the same time functions as cooling air for sound sources S. FIG.", "3A shows a sound source SP according to the invention, which is based on a pulse burner.", "FIG.", "3B shows an example of the structure of a valve V of sound source SP, FIG.", "3C is a cross-sectional view of sound source SP along line A-A′ indicated in FIG.", "3A and FIG.", "3D is a cross-sectional view of sound source SP along line B-B′ indicated in FIG.", "3A.", "The sound source used is a pulse burner, which preferably operates with the combustion reaction between a gaseous or gasified fuel and air and functions on the “pulse jet” principle known as such.", "In such an embodiment the sound source SP includes an antechamber SP1, into which air under pressure is supplied through assembly L1, a combustion chamber SP2, into which a gaseous inflammable matter is supplied through assembly L2, and a sound horn SP3.Between antechamber SP1 and combustion chamber SP2 there is a valve/set of valves V, whose structure is shown by the cross-sectional view in FIG.", "3B.", "In addition, sound source SP includes one or more igniters I located in combustion chamber SP2.Combustion chamber SP2 is surrounded by cooling devices C including cooling fins c1, .", ".", ".", ", cn.", "Into cooling devices C a cooling medium, preferably cooling air or water, is conducted by way of assembly L3.After the combustion event, the pressure of combustion chamber SP2 is released into the space to be cleaned, that is, into combustion space 11.The igniting device/devices I used are preferably spark plugs or a hot shoe functioning as a constantly glowing igniting component The pulse burner operating as sound source SP preferably uses hydrocarbon gas and air in starting.", "In addition, in one embodiment of the invention a gasified gas is used, which is taken from that part of the combustion space, where no secondary or tertiary air has yet been introduced.", "The sound source according to the invention forms a continuous acoustic pressure pulse, with which a continuous quick pulse-like acoustic pressure can be driven into the combustion space of the boiler.", "The invention may be applied in all types of power plants, heating furnaces, heating boilers and other such installations.", "The sound sources used are sound horns known in acoustic cleaning, but also sound sources of other kinds may be used.", "The following is a presentation of the claims, but the invention is not intended to be limited only to the different embodiments presented in the claims." ] ]

[ [ "Internal Combustion Engine", "The invention relates to internal combustion engines, more exactly reciprocating piston engines and it may be utilized for example both in V-engines and one-row single-cylinder or two-cylinder engines.", "The internal combustion engine is comprised of block head housing (1), at least one cylinder (2), piston (3), ignition plug (8), combustion chamber (5) sleeves (6) of gas distribution mechanism, intake and exhaust channels (7, 13) whereas the intake channel (7) and the exhaust channel (13) are connected with the corresponding sleeve (6) of the gas distribution mechanism, the gas distribution mechanism sleeves (6) are positioned at an equal angle of the axis of the cylinder and on the same plane the mentioned plane being at a 40-50° angle of the axis of the cylinder (2)." ], [ "1.An internal combustion engine that is comprised of block head housing (1), at least one cylinder (2), piston (3), ignition plug (8), combustion chamber (5), sleeves (6) of gas distribution mechanism, intake and exhaust channels (7, 13) whereas the intake channel (7) and the exhaust channel (13) are connected with the corresponding sleeve (6) of the gas distribution mechanism, the gas distribution mechanism sleeves (6) are positioned at an equal angle of the axis of the cylinder and on the same plane, characterized in that the mentioned plane is at a 40-50° angle of the axis of the cylinder (2)." ], [ "The invention relates to internal combustion engines, more exactly slide valve engines and it may be utilized for example both in V-engines and one-row single-cylinder or two-cylinder engines.", "Piston engines used in airplanes are known form prior art, with the block and the head being a monolithic construction, since a gasket would crack in the event of overheating.", "All these engines have been built with valves and they pollute the environment due to incomplete combustion of fuel.", "Examples of such engines are the AM38 of IL-2, AM105 of JAK and AM100 of MIG.", "Internal combustion engines where valves are used in gas distribution mechanism, fuel injection in order to reduce fuel consumption, and where the intake channels and the exhaust ejection channels are positioned at a 20-30° (preferably 25°) angle of the axis of the cylinder, have been described in the international patent application PCT/SE99/00827.The primary aim of the brought solution is to minimize fuel consumption and therefore to achieve a combustible fuel mix primarily in the vicinity of the ignition plug by the means of creating two vortexes.", "In order to create suitable vortexes there is a recess at the injection jet end, and a slanted mound at the opposite end with top almost on the same line with the axis of the cylinder.", "The drawback of this solution in comparison with the aims of the present invention is the summation of the two different vortexes only during the compression phase After the combustion phase the exhaust of the burnt fuel mix from the cylinder will not form an even vortex because of the recess and the mound at end surface of the piston head, and therefore part of the exhaust fumes will remain in the cylinder during the next operation cycle.", "In order to facilitate the ejection of exhaust fumes from the cylinder it is necessary to maintain the vortex movement of the gases also during the operation combustion phase and the exhaust phase.", "The classical valve system has a potential for over-consuming resources.", "37% of the fuel will be ejected together with the exhaust fumes and 3% will be mixed with un-ejected processed gas.", "This will dilute the fresh fuel mix, there will be over-consumption of fuel and the performance of the engine will drop.", "In a valve engine a persistent dynamic “gas cap” is formed under the valve hindering the introduction of fresh fuel mix and causing loss of fuel.", "The more valves are used, the bigger is the loss of fuel.", "Also a slide valve internal combustion engine, mentioned also as valveless engine is known from EP-0773352-A1 that is comprised by block head housing, at least one cylinder, piston, ignition plug, combustion chamber, intake and exhaust channel, the intakes and exhaust channel have been connected with a corresponding sleeve of gas distribution mechanism whereas the sleeves of the gas distribution mechanism are located at equal angels to the axis of the cylinder and on the same plane.", "The drawback of the mentioned engine with piston distribution mechanism is as well over-consumption of fuel that is caused by the V-shaped positioning of the intake channels causing uneven creation of fuel mix and slow formation of fuel mix, since the projection of the intake channel on the side surface of the cylinder is located below the connection of the cylinder and the block head, the fuel mix enters the cylinder so that a vortex to guarantee fast and even blending of fuel mix and air is not created.", "An analogical process takes place during the exhaust stroke and the burnt exhaust fumes exit the cylinder as an uneven vortex and therefore with insufficient speed.", "The aim of the invention is to increase the productiveness of the engine by more economical usage of fuel.", "This will also facilitate decrease of environmental pollution.", "In order to reach this aim the plane, where the sleeves of gas distribution pistons are positioned, has been set at 40-50° angle of the axis of the cylinder.", "Thanks to the circumstance that the sleeves of the gas distribution mechanism intake channel and exhaust channel are positioned on the same plane that has been set at a 40-50° angle of the axis of the cylinder the fuel mix will enter the cylinder through the intake channel and the sleeve of the gas distribution mechanism, fluidly collides with the cylinder wall and both obtains spiral motion and heats burning up completely in a shorter period of time and there is no necessity of dosing oxygen into the exhaust collector to ensure full combustion of exhaust fumes.", "When the angle is less than 40° then the fuel mix will slide directly down the cylinder side surface and the vortex is not created.", "When, the angle is bigger than 50° then a vortex of sufficient speed is created but the cylinder will not be filled with sufficient speed.", "In an analogical manner the exhaust fumes will exit during the exhaust stroke in a vortex form and therefore faster.", "The construction of the engine and its working principles are described in more detail in the following sample embodiment with the aid of figures.", "FIG.", "1 illustrates the block head housing removed from the crankcase with the piston.", "FIG.", "2 illustrates the block head housing from the side of the intake channel.", "FIG.", "3 is the top view of the block head.", "The internal combustion engine is comprised of block head housing 1, cylinder 2 of which sleeve is seen, piston 3, cooling jacket 4, combustion chamber 5, sleeve 6 of gas distribution mechanism, intake channel 7, plug 8, channels of cooling mix 9, fixation nut of the cooling jacket 10 and check-nut 11, piston valve 12, exhaust channel 13, mounting flange 14 of the block and stud bolt 15 that fastens the block head to the crankcase.", "On FIG.", "2 the block head is depicted from the side of the intake channel and the cylinder sleeve 2, case of the cooling jacket 4, apertures of the intake channel 7 and exhaust channel 13 are seen, as well as the relative positioning of the sleeves 6 of the gas distribution mechanism.", "The work channels of the gas distribution mechanism or the sleeves 6 have been positioned side by side on the same plane that is set at angle a of the axis of the cylinder.", "On FIG.", "3 the relative positions of the cylinder sleeve 2, intake channel 7, plug 8 and exhaust channel 13 can be seen.", "The block head with slide valve gear is suitable for any engine.", "The operating system of the piston valves is launched by a processor that is controlled through all parameters of the engine.", "The construction of the engine brought in the invention allows solving the problem of using different engine fuels.", "The piston valves 12 have the capability of inward pumping since the piston itself will raise the level of pressure so that the engine can operate besides gasoline also on gas, spirit and solid fuel.", "In case of direct injection into the cylinder the engine can operate on kerosene and diesel fuel.", "Normal operation of the engine with different fuels is facilitated by adjusting the ignition angle and pressure level.", "As and example a solution has been brought with the phases of gas distribution in a 4-stroke gasoline engine that corresponds to the invention.", "Intake phase: The intake channel 7 is opened 8° before the top dead center (TDC).", "At the same time the exhaust channel 13 is closed.", "Fresh fuel mix enters the cylinder 2 through intake channel 7 and sleeve 6 of the gas distribution mechanism that is positioned at a 40-50% angle of the cylinder axis, the fuel mix collides against the wall of the cylinder 2 and acquires spiral motion as well as heats, at the same time washing the walls of the cylinder.", "Compression phase: 49° after the bottom dead center (BDC) the intake channel 7 is closed.", "The fuel mix is compressed.", "Combustion phase: 6° before TDC ignition is performed.", "Since the fuel mix will maintain spiral motion during combustion it is combusted in a shorter time than in a valve engine.", "Ignition is accelerated by using a circular side electrode on plugs 8.Exhaust phase: 30° before-the BDC the exhaust channel 13 is opened.", "The fuel mix will rapidly exit in a spiral motion and the cycle is concluded." ] ]

[ [ "Method for therapy of neurodegenerative disease of the brain", "A specific clinical protocol for use toward therapy of defective, diseased and damaged cholinergic neurons in the mammalian brain, of particular usefulness for treatment of neurodegenerative conditions such as Alzheimer's disease.", "The protocol is practiced by delivering a definite concentration of recombinant neurotrophin into, or within close proximity of, identified defective, diseased or damaged brain cells.", "Using a viral vector, the concentration of neurotrophin delivered as part of a neurotrophic composition varies from 1010 to 1015 neurotrophin encoding viral particles/ml of composition fluid.", "Each delivery site receives form 2.5 μl to 25 μl of neurotrophic composition, delivered slowly, as in over a period of time ranging upwards of 10 minutes/delivery site.", "Each delivery site is at, or within 500 μm of, a targeted cell, and no more than about 10 mm from another delivery site.", "Stable in situ neurotrophin expression can be achieved for 12 months, or longer." ], [ "1.A method for delivery of a therapeutic neurotrophin to targeted defective, diseased or damaged cholinergic neurons in the mammalian brain, the method comprising delivering a neurotrophic composition, comprising a neurotrophin encoding transgene, into one or more delivery sites within a region of the brain containing targeted neurons; wherein the transgene is expressed in, or within 500 μm from, a targeted cell, and no more than about 10 mm from another delivery site; and wherein further contact with the neurotrophin ameliorates the defect, disease or damage.", "2.The method according to claim 1, wherein the transgene is expressed by a viral expression vector.", "3.The method according to claim 2, wherein the viral expression vector is an adenovirus.", "4.The method according to claim 2, wherein the viral expression vector is an adeno-associated virus.", "5.The method according to claim 2, wherein the viral expression vector is a lentivirus.", "6.The method according to claim 2, wherein the viral expression vector is HIV-1.7.The method according to claim 2, wherein the neurotrophic composition is a fluid having a concentration of neurotrophin encoding viral particles in the range from 1010 to 1015 particles per ml of neurotrophic composition.", "8.The method according to claim 7, wherein from 2.5 μl to 25 μl of the neurotrophic composition is delivered to each delivery site.", "9.The method according to claim 8, wherein delivery to each delivery site is accomplished over a period of time greater than or equal to 3 minutes.", "10.The method according to claim 9, wherein delivery to each delivery site is accomplished over a period of time less than or equal to 10 minutes.", "11.The method according to claim 1 wherein the treated mammal is a human and the transgene encodes a human neurotrophin.", "12.The method according to claim 11 wherein the neurotrophin is human beta nerve growth factor (β-NGF).", "13.The method according to claim 11 wherein the neurotrophin is human neurotrophin 3 (NT-3).", "14.The method according to claim 1 wherein the delivery sites are intraparenchymal.", "15.The method according to claim 1 wherein the delivery sites are within the Ch4 region of the cholinergic basal forebrain.", "16.The method according to claim 1 wherein the transgene is expressed by a non-viral expression vector.", "17.The method according to claim 1 wherein the ameliorated disease is Alzheimer's disease." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The invention relates to methods for treatment of neurodegenerative disease and methods for delivery of therapeutic neurotrophins into the mammalian brain." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a clinically useful protocol for delivery of neurotrophins into the mammalian brain.", "The invention is particularly useful in treating neurodegenerative conditions in primates, in whom neurotrophins delivered according to the invention stimulate growth of neurons and recovery of neurological function.", "More specifically, the invention consists of methods for intraparenchymal delivery of neurotrophins to defective, diseased or damaged cells in the mammalian brain.", "In one aspect, the invention provides a specific protocol for use in genetically modifying target cholinergic neurons (“target cells”) to produce a therapeutic neurotrophin.", "The genetic modification of target cells is achieved by in vivo transfection of neurons targeted for treatment, or by transfection of cells neighboring these target neurons (neurons or glia), with a recombinant expression vector for expression of the desired neurotrophin in situ.", "The location for delivery of individual unit dosages of neurotrophin into the brain is selected for proximity to previously identified defective, diseased or damaged target cells in the brain.", "To intensify exposure of such target cells to the endogenous growth factors, each delivery site is situated no more than about 500 μm from a targeted cell and no more than about 10 mm from another delivery site.", "The total number of sites chosen for delivery of each unit dosage of neurotrophin will vary with the size of the region to be treated.", "Optimally, for delivery of neurotrophin using a viral expression vector, each unit dosage of neurotrophin will comprise 2.5 to 25 μl of an expression vector composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid (“neurotrophic composition”) and provides from 10 10 up to 10 15 NGF expressing viral particles per ml of neurotrophic composition.", "According to the method, neurotrophic composition is delivered to each delivery site in the brain by injection through a surgical incision, with delivery to be completed within about 5-10 minutes, depending on the volume of neurotrophic composition to be provided.", "This targeted, regionally specific protocol for nervous system growth factor delivery avoids limitations imposed by diffusion of substances across the blood-brain barrier and through central nervous system (CNS) parenchyma, while avoiding potential adverse effects of neurotrophic factors delivered intact in a non-directed manner to the CNS." ], [ "RELATED U.S. PATENT APPLICATIONS This is a continuation-in-part of, and claims the priority of, U.S. patent application, Ser.", "No.", "09/060,543, which was filed on Apr.", "15, 1998, and is pending.", "FIELD OF THE INVENTION The invention relates to methods for treatment of neurodegenerative disease and methods for delivery of therapeutic neurotrophins into the mammalian brain.", "HISTORY OF THE RELATED ART Neurotrophins play a physiological role in the development and regulation of neurons in mammals.", "In adults, basal forebrain cholinergic neurons, motor neurons and sensory neurons of the CNS retain responsiveness to neurotrophic factors and can regenerate after loss or damage in their presence.", "For this reason, neurotrophins are considered to have great promise as drugs for the treatment of neurodegenerative conditions such as Alzheimer's Disease (AD), Parkinson's Disease (PD), amyotrophic lateral sclerosis (ALS), peripheral sensory neuropathies and spinal cord injuries.", "Clinical trials for the use of neurotrophins in the treatment of AD, ALS and sensory neuropathies are underway.", "However, the search for a protocol for delivery of neurotrophins to target tissues with minimal side effects (e.g., from diffusion to non-targeted cells or immune reaction to the delivery vehicle) and sufficient penetration of the CNS (e.g., bypassing the blood-brain barrier and achieving chronic delivery of neurotrophin to target cells) has not yet revealed a clear path for clinical administration of neurotrophins.", "In particular, effective delivery methods and dosing parameters have not yet been identified, although several methods have been proposed.", "Therefore, although the prospects for therapy of neurodegenerative disease of the brain and CNS are believed to be bright, a successful clinical protocol remains elusive.", "SUMMARY OF THE INVENTION The invention provides a clinically useful protocol for delivery of neurotrophins into the mammalian brain.", "The invention is particularly useful in treating neurodegenerative conditions in primates, in whom neurotrophins delivered according to the invention stimulate growth of neurons and recovery of neurological function.", "More specifically, the invention consists of methods for intraparenchymal delivery of neurotrophins to defective, diseased or damaged cells in the mammalian brain.", "In one aspect, the invention provides a specific protocol for use in genetically modifying target cholinergic neurons (“target cells”) to produce a therapeutic neurotrophin.", "The genetic modification of target cells is achieved by in vivo transfection of neurons targeted for treatment, or by transfection of cells neighboring these target neurons (neurons or glia), with a recombinant expression vector for expression of the desired neurotrophin in situ.", "The location for delivery of individual unit dosages of neurotrophin into the brain is selected for proximity to previously identified defective, diseased or damaged target cells in the brain.", "To intensify exposure of such target cells to the endogenous growth factors, each delivery site is situated no more than about 500 μm from a targeted cell and no more than about 10 mm from another delivery site.", "The total number of sites chosen for delivery of each unit dosage of neurotrophin will vary with the size of the region to be treated.", "Optimally, for delivery of neurotrophin using a viral expression vector, each unit dosage of neurotrophin will comprise 2.5 to 25 μl of an expression vector composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid (“neurotrophic composition”) and provides from 1010 up to 1015 NGF expressing viral particles per ml of neurotrophic composition.", "According to the method, neurotrophic composition is delivered to each delivery site in the brain by injection through a surgical incision, with delivery to be completed within about 5-10 minutes, depending on the volume of neurotrophic composition to be provided.", "This targeted, regionally specific protocol for nervous system growth factor delivery avoids limitations imposed by diffusion of substances across the blood-brain barrier and through central nervous system (CNS) parenchyma, while avoiding potential adverse effects of neurotrophic factors delivered intact in a non-directed manner to the CNS.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a reprint of the nucleotide sequence coding for human beta nerve growth factor as shown in GENBANK Accession No.", "X52599.FIG.", "2 is a reprint of the nucleotide sequence coding for human NT-3 as shown in GENBANK Accession No.", "E07844.DETAILED DESCRIPTION OF THE INVENTION I.", "Target Tissues for Treatment of Neurodegenerative Disorders According to the Invention The invention identifies and defines the required parameters of a method for successful regeneration of neurons in the brain with neurotrophins, especially the neurons whose loss is associated with neurodegenerative conditions with impairment of cognition such as AD.", "The first method parameter defined by the invention is selection of a suitable target tissue.", "A region of the brain is selected for its retained responsiveness to neurotrophic factors.", "In humans, CNS neurons which retain responsiveness to neurotrophic factors into adulthood include the cholinergic basal forebrain neurons, the entorhinal cortical neurons, the thalamic neurons, the locus coeruleus neurons, the spinal sensory neurons and the spinal motor neurons.", "Abnormalities within the cholinergic compartment of this complex network of neurons have been implicated in a number of neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease).", "The cholinergic basal forebrain (particularly, the Ch4 region of the basal forebrain) is a particularly suitable target tissue.", "Within the primate forebrain, magnocellular neurons Ch1-Ch4 provide cholinergic innervation to the cerebral cortex, thalamus and basolateral nucleus of the amygdala.", "In subjects with neurodegenerative diseases such as AD, neurons in the Ch4 region (nucleus basalis of Meynert) which have nerve growth factor (NGF) receptors undergo marked atrophy as compared to normal controls (see, e.g., Kobayashi, et al., Mol.", "Chem.", "Neuropathol., 15:193-206 (1991)).", "In normal subjects, neurotrophins prevent sympathetic and sensory neuronal death during development and prevents cholinergic neuronal degeneration in adult rats and primates (Tuszynski, et al., Gene Therapy, 3:305-314 (1996)).", "The resulting loss of functioning neurons in this region of the basal forebrain is believed to be causatively linked to the cognitive decline experienced by subjects suffering from neurodegenerative conditions such as AD (Tuszynski, et al., supra and, Lehericy, et al., J. Comp.", "Neurol., 330:15-31 (1993)).", "In human AD, basal forebrain neuronal loss occurs over an intraparenchymal area of approximately 1 cm in diameter.", "To treat affected neurons over such a large region, treatment with vector composition at upwards of 10 separate in vivo gene vector delivery sites is desirable.", "However, in treating localized injuries to the basal forebrain, the affected areas of the brain will likely be smaller such that selection of fewer delivery sites (e.g., 5 or fewer) will be sufficient for restoration of a clinically significant number of cholinergic neurons.", "Importantly, specific in vivo gene delivery sites are selected so as to cluster in an area of neuronal loss.", "Such areas may be identified clinically using a number of known techniques, including magnetic resonance imaging (MRI) and biopsy.", "In humans, non-invasive, in vivo imaging methods such as MRI will be preferred.", "Once areas of neuronal loss are identified, delivery sites are selected for stereotaxic distribution so each unit dosage of NGF is delivered into the brain at, or within 500 μm from, a targeted cell, and no more than about 10 mm from another delivery site.", "II.", "Dosing Requirements and Delivery Protocol for Treatment of Neurodegenerative Disorders According to the Invention A further parameter defined by the invention is the dosage of neurotrophin to be delivered into the target tissue.", "In this regard, “unit dosage” refers generally to the concentration of neurotrophin/ml of neurotrophic composition.", "For viral vectors, the neurotrophin concentration is defined by the number of viral particles/ml of neurotrophic composition.", "Optimally, for delivery of neurotrophin using a viral expression vector, each unit dosage of neurotrophin will comprise 2.5 to 25 μl of a neurotrophic composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid and provides from 1010 up to 1015 NGF expressing viral particles per ml of neurotrophic composition.", "The neurotrophic composition is delivered to each delivery cell site in the target tissue by microinjection, infusion, scrape loading, electroporation or other means suitable to directly deliver the composition directly into the delivery site tissue through a surgical incision.", "The delivery is accomplished slowly, such as over a period of about 5-10 minutes (depending on the total volume of neurotrophic composition to be delivered).", "Those of skill in the art will appreciate that the direct delivery method employed by the invention obviates a limiting risk factor associated with in vivo gene therapy; to wit, the potential for transfection of non-targeted cells with the vector carrying the NGF encoding transgene.", "In the invention, delivery is direct and the delivery sites are chosen so diffusion of secreted NGF takes place over a controlled and pre-determined region of the brain to optimize contact with targeted neurons, while minimizing contact with non-targeted cells.", "Startlingly, in primates, a viral vector (AAV) with an operable neurotrophin encoding transgene has been shown to express human neurotrophin after delivery to the brain and to the CNS for up to 12 months.", "As such, the invention provides a chronically available source for neurotrophin in the brain.", "III.", "Materials for Use in Practicing the Invention Materials useful in the methods of the invention include in vivo compatible recombinant expression vectors, packaging cell lines, helper cell lines, synthetic in vivo gene therapy vectors, regulatable gene expression systems, encapsulation materials, pharmaceutically acceptable carriers and polynucleotides coding for nervous system growth factors of interest.", "A. Neurotrophins Known nervous system growth factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5.", "(NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), the fibroblast growth factor family (FGF's 1-15), leukemia inhibitory factor (LIF), certain members of the insulin-like growth factor family (e.g., IGF-1), the neurturins, persephin, the bone morphogenic proteins (BMPs), the immunophilins, the transforming growth factor (TGF) family of growth factors, the neuregulins, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and others.", "NGF and NT-3 in particular have been tested with promising results in clinical trials and animal studies (see, e.g., Hefti and Weiner, Ann Neurol., 20:275-281 (1986); Tuszynki and Gage, Ann.", "Neurol., 30:625-636 (1991); Tuszynski, et al., Gene Therapy, 3:305-314 (1996) and Blesch and Tuszynski, Clin.", "Neurosci., 3:268-274 (1996)).", "Of the known nervous system growth factors, NGF and NT-3 (for treatment of the Ch4 region, as in AD) are preferred for use in the invention.", "Human (h) NGF and hNT3 are preferred for use in therapy of human disease according to the invention due to their relatively low immunogenicity as compared to allogenic growth factors.", "However, other nervous system growth factors are known which may also be suitable for use in the invention with adequate testing of the kind described herein.", "Coding polynucleotides for hNGF and hNT3 are known, as are coding sequences for neurotrophins of other mammalian species (e.g., mouse, in which the coding sequence for NGF is highly homologous to the human coding sequence).", "For example, a cDNA including the coding sequence for hNGF is reported in GenBank at E03015 (Kazuo, et al., Japanese Patent Application No.", "JP19911175976-A, while the nucleotide sequence of genomic hNGF (with putative amino acid sequence) is reported in GenBank at HSBNGF (Ullrich, Nature, 303:821-825 (1983)) and the mRNA sequence is reported in GenBank at HSBNGFAC (Borsani, et al., Nucleic Acids Res., 18:4020 (1990)).", "The genomic nucleotide sequence of hNT3 is reported in GenBank at E07844 (Asae, et al., JP Patent Application No.", "1993189770-A4).", "These references are incorporated herein to illustrate knowledge in the art concerning nucleotide and amino acid sequences for use in synthesis of neurotrophins.", "Exemplary reprints of nucleotide sequences coding for NGF and NT-3 obtained from the GENBANK nucleotide database are provided in, respectively, FIGS.", "1 and 2.B.", "Recombinant Expression Vectors The strategy for transferring genes into target cells in vivo includes the following basic steps: (1) selection of an appropriate transgene or transgenes whose expression is correlated with CNS disease or dysfunction; (2) selection and development of suitable and efficient vectors for gene transfer; (3) demonstration that in vivo transduction of target cells and transgene expression occurs stably and efficiently; (4) demonstration that the in vivo gene therapy procedure causes no serious deleterious effects; and (5) demonstration of a desired phenotypic effect in the host animal.", "Although other vectors may be used, preferred vectors for use in the methods of the present invention are viral and non-viral vectors.", "The vector selected should meet the following criteria: 1) the vector must be able to infect targeted cells and thus viral vectors having an appropriate host range must be selected; 2) the transferred gene should be capable of persisting and being expressed in a cell for an extended period of time (without causing cell death) for stable maintenance and expression in the cell; and 3) the vector should do little, if any, damage to target cells.", "Because adult mammalian brain cells are non-dividing, the recombinant expression vector chosen must be able to transfect and be expressed in non-dividing cells.", "At present, vectors known to have this capability include DNA viruses such as adenoviruses, adeno-associated virus (AAV), and certain RNA viruses such as HIV-based lentiviruses and feline immunodeficiency virus (FIV).", "Other vectors with this capability include herpes simplex virus (HSV).", "For example, a HIV-based lentiviral vector has recently been developed which, like other retroviruses, can insert a transgene into the nucleus of host cells (enhancing the stability of expression) but, unlike other retroviruses, can make the insertion into the nucleus of non-dividing cells.", "This lentiviral vector has been shown to stably transfect brain cells after direct injection, and stably express a foreign transgene without detectable pathogenesis from viral proteins (see, Naldini, et al., Science, 272:263-267 (1996), the disclosure of which is incorporated by reference).", "Following the teachings of the researchers who first constructed the HIV-1 retroviral vector, those of ordinary skill in the art will be able to construct lentiviral vectors suitable for use in the methods of the invention (for more general reference concerning retrovirus construction, see, e.g., Kriegler, Gene Transfer and Expression, A Laboratory Manual, W. Freeman Co. (NY 1990) and Murray, E J, ed., Methods in Molecular Biology, Vol.", "7, Humana Press (NJ 1991)).", "Adenoviruses and AAV have been shown to be quite safe for in vivo use and have been shown to result in long-term gene expression in vivo; they are therefore preferred choices for use in the methods of the invention, where safety and long-term expression of nervous system growth encoding transgenes (persisting for longer than necessary to stimulate regrowth of injured or diseased neurons) is necessary.", "Those of ordinary skill in the art are familiar with the techniques used to construct adenoviral and AAV vectors and can readily employ them to produce vector compositions useful in the claimed invention (for reference, see, e.g., Straus, The Adenovirus, Plenum Press (NY 1984), pp.", "451-496; Rosenfeld, et al., Science, 252:431-434 (1991); U.S. Pat.", "No.", "5,707,618 [adenovirus vectors for use in gene therapy]; and U.S. Pat.", "No.", "5,637,456 [method for determining the amount of functionally active adenovirus in a vector stock], the contents of each of which is incorporated herein to illustrate the level of skill in the art).", "Lentiviral-based vectors such as HIV and FIV are currently at earlier stages of development but also are attractive candidates for in vivo gene therapy based upon stability of expression in vivo and safety profiles.", "Herpes viruses, alpha viruses and pox viruses are also well-characterized virus vectors which may be applied to the methods of the invention.", "Of these vectors, adeno-associated vectors are an especially attractive choice for their lack of pathogenicity and ability to insert a transgene into a host genome.", "Non-viral delivery methods are also an option for use in the methods of the invention.", "In particular, the plasmid (in a “naked” or lipid-complexed form), lipoplexes (liposome complexed nucleic acids), amino acid polymer complexes with nucleic acids and artificial chromosomes are all non-viral gene delivery agents which are demonstrably able to transduce cells and deliver a foreign transgene.", "Synthetic in vivo gene therapy vectors are also an option for use in the methods of the invention.", "Construction of vectors for recombinant expression of nervous system growth factors for use in the invention may be accomplished using conventional techniques which do not require detailed explanation to one of ordinary skill in the art.", "For review, however, those of ordinary skill may wish to consult Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (NY 1982).", "Briefly, construction of recombinant expression vectors employs standard ligation techniques.", "For analysis to confirm correct sequences in vectors constructed, the ligation mixtures may be used to transform a host cell and successful transformants selected by antibiotic resistance where appropriate.", "Vectors from the transformants are prepared, analyzed by restriction and/or sequenced by, for example, the method of Messing, et al., (Nucleic Acids Res., 9:309, 1981), the method of Maxam, et al., (Methods in Enzymology, 65:499, 1980), or other suitable methods which will be known to those skilled in the art.", "Size separation of cleaved fragments is performed using conventional gel electrophoresis as described, for example, by Maniatis, et al., (Molecular Cloning, pp.", "133-134, 1982).", "Expression of a gene is controlled at the transcription, translation or post-translation levels.", "Transcription initiation is an early and critical event in gene expression.", "This depends on the promoter and enhancer sequences and is influenced by specific cellular factors that interact with these sequences.", "The transcriptional unit of many prokaryotic genes consists of the promoter and in some cases enhancer or regulator elements (Banerji et al., Cell 27:299 (1981); Corden et al., Science 209:1406 (1980); and Breathnach and Chambon, Ann.", "Rev.", "Biochem.", "50:349 (1981)).", "For retroviruses, control elements involved in the replication of the retroviral genome reside in the long terminal repeat (LTR) (Weiss et al., eds., The molecular biology of tumor viruses: RNA tumor viruses, Cold Spring Harbor Laboratory, (NY 1982)).", "Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV) LTRs contain promoter and enhancer sequences (Jolly et al., Nucleic Acids Res.", "11:1855 (1983); Capecchi et al., In: Enhancer and eukaryotic gene expression, Gulzman and Shenk, eds., pp.", "101-102, Cold Spring Harbor Laboratories (NY 1991).", "Other potent promoters include those derived from cytomegalovirus (CMV) and other wild-type viral promoters.", "Promoter and enhancer regions of a number of non-viral promoters have also been described (Schmidt et al., Nature 314:285 (1985); Rossi and de Crombrugghe, Proc.", "Natl.", "Acad.", "Sci.", "USA 84:5590-5594 (1987)).", "Methods for maintaining and increasing expression of transgenes in quiescent cells include the use of promoters including collagen type I (1 and 2) (Prockop and Kivirikko, N. Eng.", "J. Med.", "311:376 (1984); Smith and Niles, Biochem.", "19:1820 (1980); de Wet et al., J. Biol.", "Chem., 258:14385 (1983)), SV40 and LTR promoters.", "In addition to using viral and non-viral promoters to drive transgene expression, an enhancer sequence may be used to increase the level of transgene expression.", "Enhancers can increase the transcriptional activity not only of their native gene but also of some foreign genes (Armelor, Proc.", "Natl.", "Acad.", "Sci.", "USA 70:2702 (1973)).", "For example, in the present invention collagen enhancer sequences are used with the collagen promoter 2(I) to increase transgene expression.", "In addition, the enhancer element found in SV40 viruses may be used to increase transgene expression.", "This enhancer sequence consists of a 72 base pair repeat as described by Gruss et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 78: 943 (1981); Benoist and Chambon, Nature 290:304 (1981), and Fromm and Berg, J. Mol.", "Appl.", "Genetics, 1:457 (1982), all of which are incorporated by reference herein.", "This repeat sequence can increase the transcription of many different viral to and cellular genes when it is present in series with various promoters (Moreau et al., Nucleic Acids Res.", "9:6047 (1981).", "Transgene expression may also be increased for long term stable expression using cytokines to modulate promoter activity.", "Several cytokines have been reported to modulate the expression of transgene from collagen 2(I) and LTR promoters (Chua et al., connective Tissue Res., 25:161-170 (1990); Elias et al., Annals N.Y. Acad.", "Sci., 580:233-244 (1990)); Seliger et al., J. Immunol.", "141:2138-2144 (1988) and Seliger et al., J. Virology 62:619-621 (1988)).", "For example, transforming growth factor (TGF), interleukin (IL)-1, and interferon (INF) down regulate the expression of transgenes driven by various promoters such as LTR.", "Tumor necrosis factor (TNF) and TGF1 up regulate, and may be used to control, expression of transgenes driven by a promoter.", "Other cytokines that may prove useful include basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).", "Collagen promoter with the collagen enhancer sequence (Coll(E)) can also be used to increase transgene expression by suppressing further any immune response to the vector which may be generated in a treated brain notwithstanding its immune-protected status.", "In addition, anti-inflammatory agents including steroids, for example dexamethasone, may be administered to the treated host immediately after vector composition delivery and continued, preferably, until any cytokine-mediated inflammatory response subsides.", "An immunosuppression agent such as cyclosporin may also be administered to reduce the production of interferons, which downregulates LTR promoter and Coll(E) promoter-enhancer, and reduces transgene expression.", "C. Pharmaceutical Preparations To form a neurotrophic composition for use in the invention, neurotrophin encoding expression vectors (including, without limitation, viral and non-viral vectors) may be placed into a pharmaceutically acceptable suspension, solution or emulsion.", "Suitable mediums include saline and liposomal preparations.", "More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions.", "Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.", "Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.", "Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.", "Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.", "Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.", "Further, a composition of neurotrophin transgenes may be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.", "A colloidal dispersion system may also be used for targeted gene delivery.", "Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.", "Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo.", "It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macro molecules.", "RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem.", "Sci., 6:77, 1981).", "In addition to mammalian cells, liposomes have been used for delivery of operatively encoding transgenes in plant, yeast and bacterial cells.", "In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes encoding the antisense polynucleotides at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).", "The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol.", "Other phospholipids or other lipids may also be used.", "The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.", "Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.", "Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.", "Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.", "The targeting of liposomes can be classified based on anatomical and mechanistic factors.", "Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.", "Mechanistic targeting can be distinguished based upon whether it is passive or active.", "Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.", "Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.", "The surface of the targeted gene delivery system may be modified in a variety of ways.", "In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.", "Various linking groups can be used for joining the lipid chains to the targeting ligand.", "IV.", "Methods for Delivery of Vector Composition Following the protocol defined by the invention, direct delivery of a neurotrophic composition may be achieved by means familiar to those of skill in the art, including microinjection through a surgical incision (see, e.g., Capecchi, Cell, 22:479-488 (1980)); electropotation (see, e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988)); infusion, chemical complexation with a targeting molecule or co-precipitant (e.g., liposome, calcium), and microparticle bombardment of the target tissue (Tang, et al., Nature, 356:152-154 (1992)).", "V. Animal Models and Clinical Evaluation In non-human primate subjects (Example III), the process of aging simulates the neurological changes in the brain experienced in aging humans.", "An non-aged animal model that also mimics loss of cholinergic neurons in, for example, AD, is transection of the fornix pathway connecting the septum from the hippocampus, which causes spontaneous degeneration of the same neurons which degenerate through aging (see, e.g., Example II).", "In rats and primates, such transections cause retrograde degeneration of cholinergic and non-cholinergic cell bodies in the septal nucleus and nucleus basalis (Ch4 region) of the brain.", "These animals are tractable to treatment with neurotrophins, and model clinical responsiveness to such treatment comparable to aged humans (especially the non-human primates, whose brains are most similar in size and structure to humans).", "Data demonstrating the use and efficacy of the methods of the invention in these animal models are provided in the Examples.", "Clinical evaluation and monitoring of treatment can be performed using the in vivo imaging techniques described above as well as through biopsy and histological analysis of treated tissue.", "In the latter respect, basal forebrain cholinergic neuronal numbers can be quantified in a tissue sample using, for example, anti-neurotrophin antibody (for immunoassay of secreted neurotrophin) or NGF-receptor (p75) and choline acetyltransferase (ChAT) for labeling of neurons.", "A sample protocol for in vitro histological analysis of treated and control tissue samples is described in the Examples.", "The invention having been fully described, examples illustrating its practice are set forth below.", "These examples should not, however, be considered to limit the scope of the invention, which is defined by the appended claims.", "Those of ordinary skill in the art will appreciate that while the Examples illustrate an ex vivo application of the invention, the results achieved will be accessible through in vivo delivery of the nervous system growth factor encoding transgenes described, as taught herein, with in vivo gene delivery sites and direct delivery means substituted for the grafting sites and grafting methods discussed in the Examples.", "In the examples, the abbreviation “min.” refers to minutes, “hrs” and “h” refer to hours, and measurement units (such as “ml”) are referred to by standard abbreviations.", "All printed materials cited are incorporated herein by reference.", "EXAMPLE I Adeno-Associated Virus Vector Construction and Viral Particle Production For adeno-associated viral vector construction, an expression cassette was cloned containing the following elements: 1) cytomegalovirus promoter (CMVie); 2) a multiple cloning site; 3) an internal ribosome entry site followed by the coding sequence for the active, β-sequence of human nerve growth factor (NGF) or the enhanced form of green fluorescent protein (EGFP); and, 4) a SV40 polyadenylation sequence.", "The complete cassette was cloned into the vector psub201 (American Type Culture Collection) after XbaI digestion to remove the AAV coding sequences.", "For NGF expression the coding sequence for human NGF (see, GENBANK Accession No.", "X52599) was inserted into the multiple cloning site of psub-CXIE resulting in the vector psub-CXIE-NGF.", "This vector, termed psub-CXIE, was used to prepare control GFP expressing virus particles.", "Thus, this vector was used for the production of particles coding for NGF and GFP.", "Recombinant adeno-associated virus was produced by co-transfection of 18 μg expression plasmid psub-CXIE or psub-CXIE-NGF, 18 μg pXX2 and 54 μg pXX6 per 150 mm plate of subconfluent 293 cells.", "Transfected cells were harvested 48 h later and adeno-associated virus was purified by Iodixanol density gradient centrifugation and Heparin affinity chromatography.", "For virus concentration and buffer exchanges Biomax™ 100K filters were used.", "Aliquots of virus were stored at −80C.", "The number of viral particles was determined using Southern dot blotting.", "EXAMPLE II In vivo Gene Transfer in an Animal Model of Cholineargic Cell Death Animals received injections of adeno-associated viral (AAV) vector into an in vivo rat model of cholinergic cell death, to determine the extent and parameters of AAV-NGF vector delivery to prevent neuronal degeneration using in vivo gene delivery.", "To prepare the animal model, adult Fischer 344 rats underwent fornix transections to induce basal forebrain cholinergic neuronal death.", "NGF-AAV vector (CXIE-NGF) or control, EGFP-AAV vector (CXIE) was injected into the cholinergic basal forebrain at a range of 2.5 to 10 μl of stock vector solution containing from 1010-1012 particles per ml (neurotrophic composition).", "Particles were injected over a time period of 3-5 min.", "into the right hemisphere at the following coordinates: AP −0.3; ML −0.5; DV −6 from brain surface.", "The skin was closed and animals were allowed to survive for 2-4 weeks.", "AAV vector delivery induced increasing zones of transfection with increasing concentration and volume of vector particles.", "Maximal levels of in vivo gene expression were achieved at the highest concentration of vector and highest volume of injection.", "Over the two week time period of this experiment, persistent in vivo gene expression was demonstrated.", "Gene expression was primarily manifested in neurons (>90%) as opposed to glia.", "No adverse effects of the injections were evident.", "Thus, vector doses of 2.5 to 10 μl vector stock at a range of 1010-1012 particles per ml were well-tolerated, resulted in optimal vector delivery to the host cholinergic neuronal system, and did not result in adverse events or undesired vector spread beyond the target neuronal nucleus.", "NGF and enhanced GFP expression were evident for at least two weeks in vivo.", "EXAMPLE III Model of Alzheimer's Disease Through Aging in Primates Twelve aged and four adult non-aged Macaca mulatta (rhesus) monkeys were experimental subjects.", "Non-aged animals (n=4, mean age=9.64±1.90 yrs) did not undergo surgical procedures and their intact brains were studied.", "Aged monkeys were divided into two experimental groups: NGF recipients (n=6, mean age=22.55±0.56 yrs) and control subjects (n=6, mean age=23.51±1.07 yrs).", "All procedures and animal care adhered strictly to NIH, AAALAC, USDA, Society for Neuroscience, and internal institutional guidelines (of the University of California, San Diego) for experimental animal health, safety and comfort.", "EXAMPLE IV Preparation of h-NGF Secreting Fibroblasts To demonstrate responsiveness to NGF, aged monkeys received intraparenchymal grafts of autologous fibroblasts genetically modified to produce and secrete human NGF, as previously described.", "Briefly, autologous fibroblasts obtained from skin biopsies were genetically modified in vitro to produce and secrete the active portion of human NGF.", "Transduction procedures were carried out using replication-incompetent retroviral vectors derived from Moloney murine leukemia virus (MLV).", "Transduced cells were selected by growth in the neomycin analog G418.Production of biologically active NGF was verified by induction of neurite outgrowth from PC12 cells as described; production of NGF mRNA was determined by Northern blot; and amounts of NGF produced from cells were assayed by NGF ELISA specific for human NGF and sensitive to 5 pg/ml.", "Optimal NGF-producing bulk clones were amplified to numbers sufficient for in vivo grafting by serial passaging.", "Cells were harvested by gentle trypsinization for in vivo grafting.", "EXAMPLE V Intraparenchymal Delivery into Primates of Fibroblasts Genetically Modified to Produce h-NGF Monkeys underwent pre-operative MRI scans (see, Tuszynski, et al., Gene Therapy, 3:305-314, 1996) to visualize basal forebrain target grafting regions (see, Mesulam et al., J. Comp.", "Neurol., 214:170-197, 1983).", "After generating stereotaxic grafting coordinates from MRI scans, each monkey received intraparenchymal grafts of autologous NGF-secreting fibroblasts.", "Stereotactic coordinates for surgery were generated from magnetic resonance images (MR) of the brain of each subject.", "The rostral and caudal boundaries of Ch4 were identified on each subject's MR scan, making reference to primate histological brain sections and to standard primate brain atlases.", "The total rostral-caudal distance of Ch4 was measured on the MR scan, and five graft injection sites were chosen that were equally distributed over this rostral-caudal distance.", "The sites for desired ventral-dorsal (VD) and medial-lateral (ML) injections were chosen such that cell grafts were deposited just dorsal to the desired target at each coordinate (within 500 um), and exactly centered in the mediolateral (ML) plane at the maximal density of cholinergic neuronal somata (estimated by review of histological sections at the corresponding AP level).", "Thus, five grafts were deposited on each side of the Ch4 region per subject, or ten total grafts per subject.", "Real-time coordinates for in vivo injections were calculated from calibration scales on the MR image.", "Subjects underwent surgical grafting in the same stereotaxic apparatus that MR scans were performed in.", "To place the grafts, animals were placed into a primate stereotaxic apparatus and a midline scalp incision was used to expose the skull.", "The AP and ML stereotaxic coordinates for the BFC system were used to define the margins of the craniotomy site.", "Following craniotomy, a ML zero reference point was obtained by measuring the midpoint of the superior sagittal sinus.", "The dura was incised and reflected to expose the pial surface.", "The pial surface at each injection site was used as a VD zero reference point for that injection site.", "Using the zero reference points obtained in the AP, ML, and VD planes and the stereotaxic injection coordinates calculated from that animal's MR scan, 5 ul of cells were injected into each of 5 sites over the rostral-caudal extent of the Ch4 targeted region bilaterally (10 grafts total per animal) using 25-gauge Hamilton syringe.", "Grafts were generally targeted to a position slightly dorsal to but within 500 um of Ch4 nuclei.", "The injection rate was controlled at 5 ul/min.", "Cells were injected at a concentration of 1.0×105 cells/ul (for a total of 10 million grafted cells per animal), a concentration that optimally maintains cells in suspension without clumping but sufficiently concentrated to maximize number of surviving cells in vivo.", "Monkeys survived for three months before sacrifice.", "Some control aged subjects received intraparenchymal grafts as noted above.", "These grafted cells consisted either of autologous fibroblasts transduced to express the reporter gene beta-galactosidase (n=6 monkeys).", "Beta-gal production was assessed in vitro using a specific anti-beta-gal antibody.", "Cells were grafted into intraparenchymal sites in numbers identical to those described above for NGF graft recipients.", "For all surgical procedures, primates were preanesthetized with 25 mg/kg ketamine IM.", "They were then anesthetized with isoflurane administered by endotracheal intubation.", "Post-operatively animals were closely monitored, and received supportive care and appropriate analgesics when indicated.", "Animals were placed in the same primate stereotaxic apparatus (Crist Instruments) that was used to perform MRI scans.", "A midline scalp incision exposed the skull.", "A 2.5×5 cm sagittally oriented craniotomy was performed on each side of the hemicranium, and the dura was incised and reflected to expose sites for stereotaxically guided cell injections.", "Ten ul of cells were injected into each site through a 25 ga. Hamilton syringe at a rate of 1 ul/minute.", "Postoperatively, all experimental subjects were observed closely for signs of discomfort or toxicity.", "After a three-month survival period, animals were perfused transcardially for one hour with a 4% solution of paraformaldehyde in 0.1M phosphate buffer followed by 5% sucrose solution in the same buffer for 20 minutes.", "The brain was stereotaxically blocked in the coronal plane.", "EXAMPLE VI Reversal of Age-Related p75 Expression Loss In AD brains, NGF accumulates in regions of basal forebrain cholinergic neurons and is decreased in the basal forebrain, leading to the hypothesis that insufficient retrograde transport of NGF promotes the degeneration of basal forebrain cholinergic neurons observed in AD.", "In humans, basal forebrain cholinergic neuron dysfunction has been closely linked with age-related cognitive and memory impairment.", "In the mammalian brain, it is believed that the p75 receptor collaborates with the TrkA receptor to form high-affinity binding sites for NGF.", "Although activation of TrkA is sufficient for NGF to rescue axotomized cholinergic neurons, disruption of NGF binding to p75 reduces NGF binding to TrkA.", "Hence, co-expression of the two receptors can lead to greater responsiveness to NGF.", "Conversely, loss of expression may lead to decreased responsiveness to NGF.", "Expression of both p75 and TrkA is regulated by NGF, so that a loss of NGF signalling further reduces the amount of both p75 and TrkA.", "Combined with a loss of expression of TrkA in AD brains, leading to reduced amounts of TrkA protein in both the basal forebrain and the cortex, decreased p75 expression may contribute to a decline in retrograde NGF signalling.", "Thus, p75 expression is a marker for NGF binding, basal forebrain cholinergic neuron dysfunction and cognitive impairment.", "To determine the effect of the method of the invention on p75 expression in treated primate brains, monkeys were treated as described in Example III.", "Each subject was then deeply anesthetized with ketamine and nembutal and perfused transcardially for 1 hour with a 4% solution of parafornaldehyde in 0.1M phosphate buffer, followed by 5% sucrose solution in the same buffer for 20 min.", "The brains were then stereotaxically blocked in the coronal plane to obtain a single block containing the full AP extent of Ch4.Coronal sections were cut on a freezing microtome set at 40 um.", "Every sixth section was processed for p75 immunoreactivity.", "Briefly, sections were washed thoroughly in Tris-buffered saline (TBS) and endogenous peroxidases were quenched by incubating in a 0.6% hydrogen peroxide solution.", "Sections were rinsed in TBS and then blocked using 5% donkey serum with 0.5% Triton X-100 in TBS (TBS++).", "Incubation in primary antibody (monoclonal diluted 1:100 in TBS++) occurred for 24 hours at room temperature.", "Sections were rinsed in TBS++, incubated in secondary antibody (biotinylated donkey-anti-mouse diluted 1:500 in TBS++) for 1 hour, rinsed again in TBS++, and then incubated for 90 minutes using a Vector ABC kit.", "p75-labeled neurons were then visualized using diaminobenzidine (DAB) as a chromogen.", "Sections were then mounted and coverslipped.", "p75-labeled neurons were quantified in Ch4i neurons using stereological procedures.", "Ch4i was targeted in this study since this region is the principal site of origin of cholinergic projections to cortical regions that modulate memory.", "Ch4 can be divided topographically into three subdivisions, the anterior (Ch4a), intermediate (Ch4i), and posterior (Ch4p).", "The anterior subdivision is further divided into medial (Ch4am) and lateral (Ch4al) sectors, which are divided by a vascular structure or rarefication in the density of neurons.", "However, as Ch4a travels in the posterior direction toward Ch4i, the division between Ch4am and Ch4al becomes less distinct and in some disappears.", "In this region the ansa peduncularis, the characteristic structure of Ch4i, begins to make its appearance.", "The ansa peduncularis divides Ch4i into ventral (Ch4iv) and dorsal (Ch4id) components.", "There is typically also a portion of the anterior commissure present over the lateral portion of Ch4id at this level that serves as the anterior boundary of Ch4i.", "At the posterior boundary of Ch4i, Ch4iv and Ch4id merge into a single nucleus embedded in the intersection of the globus pallidus, putamen, and optic tract.", "Stereological counts were performed on every sixth section through the entire extent of Ch4i.", "The NeuroZoom™ stereology computer program running on an Apple Macintosh PowerPC™ and connected to a Javelin™ video camera mounted on an Olympus Vanox™ HBT-3 microscope was used to conduct stereology by the well-known West optical dissector method.", "Briefly, the region of interest (Ch4i) was outlined in NeuroZoom using a 1× objective.", "Specific stereology parameters were then set in NeuroZoom as follows: Fraction (percent of area): 5% Counting frame size: x=66.46 um, y=53.73 um Section thickness: 40 um These parameters were adjusted to minimize the coefficient of error of the estimate (CE(P)) while maximizing the efficiency of sampling.", "The NeuroZoom program controlled movement from one counting frame to the next by moving a Lud1 motorized stage mounted on the microscope.", "Ch4i neurons were counted using a 60× high numerical aperture (1.40) oil objective.", "Cells were marked to be included in the count if they met the following criteria: 1) they were p75-labeled; 2) the soma was within the counting frame (or touching the inclusion boundary) but did not touch the exclusion boundary; 3) a clearly visible nucleus was present; and 4) the nucleus was best in focus within the inclusion volume (i.e., the top 12.5% and bottom 12.5% were excluded, and the nucleus was not in focus in either of these exclusion volumes).", "Multiple group comparisons were made by analysis of variance (ANOVA) with post-hoc analysis using Fisher's least squares difference.", "The number of p75-labeled Ch4i neurons was compared between four groups of rhesus monkeys, two of which were unoperated and two of which received intraparenchymal grafts of genetically-modified fibroblasts.", "Young monkeys (mean age=9.375±1.058) constituted one of the unoperated groups, while aged monkeys (mean age=25.139±2.455) comprised the other unoperated group.", "Of the two aged groups which received grafts to the basal forebrain, one (mean age=22.639±0.463) received grafts of cells modified to produce and secrete NGF, and the other (mean age=23.321±0.927) received grafts of cells modified to produce and secrete beta-gal.", "There were significantly fewer p75-labeled neurons in Ch4i from unoperated aged monkeys than from unoperated young monkeys (p<0.01).", "The mean number of p75-labeled Ch4i neurons from NGF-grafted aged monkeys was significantly greater than from control-grafted aged monkeys (p<0.04).", "Further, there number of p75-labeled Ch4i neurons in NGF-grafted aged monkeys did not differ from numbers in unoperated young monkey brains (p=0.1288).", "These results demonstrate that there is spontaneous loss of expression of the low-affinity neurotrophin receptor (p75) in cholinergic neurons in the basal forebrain, and that re-expression of p75 can be induced by intraparenchmal delivery of NGF.", "EXAMPLE VII Histology Confirming in vivo Uptake of Transgene Expression of NGF and Lack of Beta-Amyloid Induction Sections of brain tissue after humane sacrifice of the test animals were cut at 40 um intervals on a freezing microtome.", "Every sixth section was processed for Nissl stain or hematoxylin and eosin.", "Immunocytochemical labeling against amyloid was performed using an amyloid-specific monoclonal antibody (anti-A4).", "Sections lacking primary antibody were processed to verify specificity of labeling.", "A representative section per subject was quantified from each of the following regions: temporal, frontal, cingulate, insular, parietal and occipital cortices; amygdala and hippocampus; and the intermediate division of the Ch4 region (Nucleus Basalis of Meynert).", "Sampled sections from each subject were closely matched in region and size.", "The total number of amyloid plaques per region was quantified and recorded.", "Observers were blinded to the identity of the tissue being quantified.", "All grafted subjects showed surviving cell grafts within 500 um of each grafting site.", "There was no qualitative difference in fibroblast morphology and overall graft size between NGF- and control-graft recipients.", "Grafts were most frequently located adjacent to the intermediate division of the Ch4 region of the basal forebrain, but in all cases included at least one graft located within the anterior and posterior divisions of the Ch4 region.", "No amyloid plaques at all were detected in adult, non-aged primate tissue.", "In contrast, control aged monkeys showed a significant increase in amyloid immunolabeling in the frontal, temporal insular and cingulate cortices and amygdala, and extremely small increases in the parietal cortex and hippocampus relative to non-aged monkeys.", "No plaques at all were present in the cholinergic basal forebrain in any group.", "In aged control animals, plaques typically showed a dense central core and a less dense surrounding halo of immunreactive deposition product, an appearance typical of “mature” plaques observed in AD.", "This immunolabeling pattern is consistent with previous reports in aged primate brain.", "However, no increase in amyloid labeling was observed in the aged, NGF-grafted brains, indicating that three months of intraparenchymal NGF delivery does not increase beta-amyloid plaque deposition in the aged primate brain.", "Thus, the benefits of NGF grafting in the brains of primates exhibiting AD symptoms can be acheived without risk of stimulating amyloid deposition in response to the graft trauma.", "Initially, group differences were statistically determined by analysis of variance, with post-hoc analysis utilizing Fisher's least square difference.", "However, since non-aged adult monkeys showed no amyloid plaques, comparisons between NGF-treated and control aged monkeys were made using unpaired two-way student's t-test." ] ]

[ [ "Early diagnosis of conformational diseases", "A method for the diagnosis or detection of conformational diseases by assaying for a marker (the pathogenic conformer) of such diseases in a sample is described, which method comprises a cyclic amplification system to increase the levels of the pathogenic conformer which causes such diseases.", "In particular, such transmissible conformational diseases may be prion encephalopathies.", "Assays, diagnostic kits and apparatus based on such methods are also disclosed." ], [ "1.A method for the diagnosis or detection of a conformational disease which is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, by assaying a marker of said disease within a sample, which method comprises: (i) contacting said sample with an amount of the non-pathogenic conformer; (ii) disaggregating any aggregates eventually formed during step (i); and (iii) determining the presence and/or amount of said pathogenic conformer within the sample, the pathogenic conformer being a marker for the presence of said disease.", "2.The method of claim 1, wherein step (i) comprises step (ia) incubating said sample/non-pathogenic conformer.", "3.The method of claim 2, wherein steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).", "4.The method of claim 3, wherein the cycle is repeated from 5 to 40 times before carrying out step (iii).", "5.The method of any one of the preceding claims, wherein step (i) is carried out under physiological conditions.", "6.The method of any one of the preceding claims wherein the amount of the non-pathogenic conformer in step (i) is an excess amount.", "7.The method of any one of the preceding claims, wherein the conformational disease is a transmissible conformational disease.", "8.The method of any one of the preceding claims, wherein the sample to be analysed is subjected to a pre-treatment for selectively concentrating the pathogenic conformer in the sample.", "9.The method of claim 8, wherein the pathogenic conformer is PrPSc and the pre-treatment is the extraction from the sample of a fraction which is insoluble in mild detergents.", "10.An assay for a marker of a conformational disease which is characterized by a conformation transition of an underlying protein between a non-pathogenic and a pathogenic conformer, within a sample, which assay comprises the following steps: (i) contacting said sample with an amount of the non-pathogenic conformer; (ii) disaggregating any aggregates eventually formed during step (i); and (iii) determining the presence and/or amount of said pathogenic conformer within the sample, the pathogenic conformer being a marker for the presence of said disease.", "11.The assay of claim 10, wherein step (i) comprises step (ia) incubating said sample/non-pathogenic conformer.", "12.The assay according to claim 11, wherein steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).", "13.A diagnostic kit for use in the assay of any one of claims 10 to 12 which comprises a known amount of the non-pathogenic conformer, a multi-well microtitre plate and a multi-well sonicator.", "14.A method for identifying a compound which modulates the conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, comprising: (i) contacting an amount of the non-pathogenic conformer with an amount of the pathogenic conformer (a) in the presence of said compound and (b) in the absence of said compound; (ii) disaggregating any aggregates eventually formed during step (i); and (iii) determining the amount of the pathogenic conformer (a) in the presence of said compound and (b) in the absence of said compound.", "15.The method of any one of claims 1 to 9 or 14 or the assay of any one of claims 10 to 12, wherein the pathogenic conformer is PrPSc, the non-pathogenic conformer is PrPC and the underlying protein is the Prion Protein.", "16.A method for detecting the presence of a pathogenic form of prion protein within a sample, comprising: (i) contacting the sample with an amount of non-pathogenic prion protein; (ia) incubating the sample/non-pathogenic prion protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic prion protein within the sample.", "17.A method for diagnosing CJD within a patient, comprising: taking a sample from the patient; (i) contacting the sample with an amount of PrPC protein; (ia) incubating the sample/PrPC protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of PrPSc within the sample.", "18.A method for detecting the presence of a pathogenic form of β-amyloid protein within a sample, comprising: (i) contacting the sample with an amount of non-pathogenic β-amyloid protein; (ia) incubating the sample/non-pathogenic β-amyloid protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic β-amyloid protein within the sample.", "19.A method for diagnosing Alzheimer's disease in a patient, comprising: taking a sample from the patient; (i) contacting the sample with an amount of non-pathogenic β-amyloid protein; (ia) incubating the sample/non-pathogenic β-amyloid protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic β-amyloid protein within the sample.", "20.Apparatus for use in the method of any one of claims 1 to 9 or 14 or the assay of any one of claims 10 to 12, comprising a microtitre plate, multi-well sonicator and an amount of a non-pathogenic conformer." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Conformational diseases are a group of disorders apparently unrelated to each other, but sharing a striking similarity in clinical presentations that reflect their shared molecular mechanisms of initiation and self-association, with consequent tissue deposition and damage.", "The structural interest is due to the fact that these varied diseases each arise from an aberrant conformational transition in an underling protein, characteristically leading to protein aggregation and tissue deposition.", "Medically, the presentation of these conformational diseases reflects this molecular mechanism, with typically a slow and insidious onset when the transition is occurring in a normal protein, but a more sudden onset when it occurs in an unstable variant of the protein.", "Two examples of special significance of such conformational diseases are the Transmissible Spongiform Encephalopathies and Alzheimer dementia, a disease that threatens to overwhelm health care systems in the developed world (for a review see Carrell et al., 1997).", "Transmissible spongiform encephalopathies (TSE) also known as prion diseases are a group of neurodegenerative diseases that affect humans and animals.", "Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheiker disease (GSS) and fatal familial insomnia (FFI) in humans as well as scrapie and bovine spongiform encephalopathy (BSE) in animals are some of the TSE diseases (Prusiner, 1991).", "Although these diseases are relatively rare in humans, the risk for the transmissibility of BSE to humans through the chain food has taken the attention of the public health authorities and the scientific community (Cousens et al., 1997, Bruce et al., 1997).", "These diseases are characterized by an extremely long incubation period, followed by a brief and invariably fatal clinical disease (Roos et al., 1973).", "To date no therapy is available.", "The key characteristic of the disease is the formation of an abnormally shaped protein named PrP Sc , which is a post-translationally modified version of a normal protein, termed PrP C (Cohen and Prusiner, 1998).", "Chemical differences have not been detected to distinguish between PrP isoforms (Stahl et al., 1993) and the conversion seems to involve a conformational change whereby the α-helical content of the normal protein diminishes and the amount of β-sheet increases (Pan et al., 1993).", "The structural changes are followed by alterations in the biochemical properties: PrP C is soluble in non-denaturing detergents, PrP Sc is insoluble; PrP C is readily digested by proteases, while PrP Sc is partially resistant, resulting in the formation of a N-terminally truncated fragment known as “PrPres” (Baldwin et al., 1995; Cohen and Prusiner, 1998), “PrP 27-30” (27-30 kDa) or “PK-resistant” (proteinase K resistant) form.", "At present there is not an accurate diagnosis for TSE (WHO Report, 1998, Budka et al., 1995, Weber et al., 1997).", "Attempts to develop a diagnostic test for prion diseases are hampered by the apparent lack of an immune response to PrP Sc .", "The clinical diagnosis of CJD is based upon the combination of subacute progressive dementia (less than 2 years), myoclonus, and multifocal neurological dysfunction, associated with a characteristic periodic electroencephalogram (EEG) (WHO Report, 1998, Weber et al., 1997).", "However, variant CJD (vCJD), most of the iatrogenic forms of CJD and up to 40% of the sporadic cases do not have the EEG abnormalities (Steinhoff et al., 1996).", "On average the accuracy of clinical diagnosis is around 60% for CJD and highly variable for other prion-related diseases.", "The clinical diagnosis is more accurate only at the late-stage of the disease when clear symptoms have developed (Weber et al., 1997).", "Genetic analysis is useful for the diagnosis of inherited prion diseases, but these represent only 15% of the cases.", "Neuroimaging is useful only to exclude other conditions of rapidly progressive dementia due to structural lesions of the brain (Weber et al., 1997).", "The findings obtained by imaging of the brain by computed tomography (CT) and magnetic resonance imaging (MRI) depend mainly on the stage of the disease.", "CT is much less sensitive and in early phase no atrophy is detected in 80% of the cases (Galvez and Cartier, 1983).", "MRI hyperintense signals have been detected in the basal ganglia besides atrophy (Onofrji et al., 1993).", "Like the changes observed by CT, these alterations are by no means specific.", "Recent data have identified several neuronal, astrocytic and glial proteins that are elevated in CJD (Jimi et al., 1992).", "The protein S-100, neuron specific isoenzyme and ubiquitin are significantly increased in the cerebrospinal fluid (CSF) in the early phase of disease with decreasing concentrations over the course of the illness (Jimi et al., 1992).", "A marker of neuronal death, the 14-3-3 protein, has been proposed as a specific and sensitive test for sporadic CJD (Hsich et al., 1996).", "However, it is not useful for the diagnosis of vCJD, and much less specific in the genetic forms.", "As the 14-3-3 protein may be present in the CSF of patients with other conditions, the test is not recommended by WHO as a general screening for CJD and is reserved to confirm the clinical diagnosis (WHO Report, 1998).", "By combining clinical data with the biochemical markers a higher success in the diagnosis is achieved.", "However, according to the operational diagnosis currently in use in the European Surveillance of CJD, definitive diagnosis is established only by neuropathological examination and detection of PrP Sc either by immunohistochemistry, histoblot or western blot (Weber et al., 1997, Budka et al., 1995).", "Formation of PrP Sc is not only the most likely cause of the disease, but it is also the best known marker.", "Detection of PrP Sc in tissues and cells correlates widely with the disease and with the presence of TSE infectivity, and treatments that inactivate or eliminate TSE infectivity also eliminate PrP Sc (Prusiner, 1991).", "The identification of PrP Sc in human or animal tissues is considered key for TSE diagnosis (WHO Report, 1998).", "One important limitation to this approach is the sensitivity, since the amounts of PrP Sc are high (enough for detection with conventional methods) only in the CNS at the late stages of the disease.", "However, it has been demonstrated that at earlier stages of the disease there is a generalized distribution of PrP Sc (in low amounts), especially in the lymphoreticular system (Aguzzi, 1997).", "Indeed, the presence of PrP Sc has been reported in palatine tonsillar tissue and appendix obtained from patients with vCJD (Hill et al., 1997).", "Although it is not known how early in the disease course tonsillar or appendix biopsy could be used in vCJD diagnosis, it has been shown that in sheep genetically susceptible to scrapie, PrP Sc could be detected in tonsillar tissue presymptomatically and early in the incubation period.", "However, PrP Sc has not been detected in these tissues so far in any cases of sporadic CJD or GSS (Kawashima et al., 1997).", "The normal protein is expressed in white blood cells and platelets and therefore it is possible that some blood cells may contain PrP Sc in affected individuals (Aguzzi, 1997).", "This raises the possibility of a blood test for CJD, but this would require an assay with a much greater degree of sensitivity than those currently available.", "Prion replication is hypothesized to occur when PrP Sc in the infecting inoculum interacts specifically with host PrP C , catalyzing its conversion to the pathogenic form of the protein (Cohen et al., 1994).", "This process takes from many months to years to reach a concentration of PrP Sc enough to trigger the clinical symptoms.", "The infective unit of PrP Sc seems to be a β-sheet rich oligomeric structure, which converts the normal protein by integrating it into the growing aggregate ( FIG.", "1 ).", "The conversion has been mimicked in vitro by mixing purified PrP C with a 50-fold molar excess of previously denatured PrP Sc (Kocisko et al., 1994).", "The in vitro conversion systems described so far have low efficiency, since they require an excess of PrP Sc and therefore are not useful for diagnostic purposes because they cannot monitor undetectable amounts of the marker.", "The reason for the low efficiency is that the number of PrP Sc oligomers (converting units) remains fixed throughout the course of the assay.", "The converting units grow sequentially by the ends and as a result they become larger, but do not increase in number ( FIG.", "1 )." ], [ "FIELD OF THE INVENTION The present invention relates to a method for the diagnosis or detection of conformational diseases by assaying for a marker (i.e.", "the pathogenic conformer) of such diseases within a sample, which method comprises a cyclic amplification system to increase the levels of the pathogenic conformer.", "In particular, such conformational diseases may be prion encephalopathies.", "BACKGROUND OF THE INVENTION Conformational diseases are a group of disorders apparently unrelated to each other, but sharing a striking similarity in clinical presentations that reflect their shared molecular mechanisms of initiation and self-association, with consequent tissue deposition and damage.", "The structural interest is due to the fact that these varied diseases each arise from an aberrant conformational transition in an underling protein, characteristically leading to protein aggregation and tissue deposition.", "Medically, the presentation of these conformational diseases reflects this molecular mechanism, with typically a slow and insidious onset when the transition is occurring in a normal protein, but a more sudden onset when it occurs in an unstable variant of the protein.", "Two examples of special significance of such conformational diseases are the Transmissible Spongiform Encephalopathies and Alzheimer dementia, a disease that threatens to overwhelm health care systems in the developed world (for a review see Carrell et al., 1997).", "Transmissible spongiform encephalopathies (TSE) also known as prion diseases are a group of neurodegenerative diseases that affect humans and animals.", "Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheiker disease (GSS) and fatal familial insomnia (FFI) in humans as well as scrapie and bovine spongiform encephalopathy (BSE) in animals are some of the TSE diseases (Prusiner, 1991).", "Although these diseases are relatively rare in humans, the risk for the transmissibility of BSE to humans through the chain food has taken the attention of the public health authorities and the scientific community (Cousens et al., 1997, Bruce et al., 1997).", "These diseases are characterized by an extremely long incubation period, followed by a brief and invariably fatal clinical disease (Roos et al., 1973).", "To date no therapy is available.", "The key characteristic of the disease is the formation of an abnormally shaped protein named PrPSc, which is a post-translationally modified version of a normal protein, termed PrPC (Cohen and Prusiner, 1998).", "Chemical differences have not been detected to distinguish between PrP isoforms (Stahl et al., 1993) and the conversion seems to involve a conformational change whereby the α-helical content of the normal protein diminishes and the amount of β-sheet increases (Pan et al., 1993).", "The structural changes are followed by alterations in the biochemical properties: PrPC is soluble in non-denaturing detergents, PrPSc is insoluble; PrPC is readily digested by proteases, while PrPSc is partially resistant, resulting in the formation of a N-terminally truncated fragment known as “PrPres” (Baldwin et al., 1995; Cohen and Prusiner, 1998), “PrP 27-30” (27-30 kDa) or “PK-resistant” (proteinase K resistant) form.", "At present there is not an accurate diagnosis for TSE (WHO Report, 1998, Budka et al., 1995, Weber et al., 1997).", "Attempts to develop a diagnostic test for prion diseases are hampered by the apparent lack of an immune response to PrPSc.", "The clinical diagnosis of CJD is based upon the combination of subacute progressive dementia (less than 2 years), myoclonus, and multifocal neurological dysfunction, associated with a characteristic periodic electroencephalogram (EEG) (WHO Report, 1998, Weber et al., 1997).", "However, variant CJD (vCJD), most of the iatrogenic forms of CJD and up to 40% of the sporadic cases do not have the EEG abnormalities (Steinhoff et al., 1996).", "On average the accuracy of clinical diagnosis is around 60% for CJD and highly variable for other prion-related diseases.", "The clinical diagnosis is more accurate only at the late-stage of the disease when clear symptoms have developed (Weber et al., 1997).", "Genetic analysis is useful for the diagnosis of inherited prion diseases, but these represent only 15% of the cases.", "Neuroimaging is useful only to exclude other conditions of rapidly progressive dementia due to structural lesions of the brain (Weber et al., 1997).", "The findings obtained by imaging of the brain by computed tomography (CT) and magnetic resonance imaging (MRI) depend mainly on the stage of the disease.", "CT is much less sensitive and in early phase no atrophy is detected in 80% of the cases (Galvez and Cartier, 1983).", "MRI hyperintense signals have been detected in the basal ganglia besides atrophy (Onofrji et al., 1993).", "Like the changes observed by CT, these alterations are by no means specific.", "Recent data have identified several neuronal, astrocytic and glial proteins that are elevated in CJD (Jimi et al., 1992).", "The protein S-100, neuron specific isoenzyme and ubiquitin are significantly increased in the cerebrospinal fluid (CSF) in the early phase of disease with decreasing concentrations over the course of the illness (Jimi et al., 1992).", "A marker of neuronal death, the 14-3-3 protein, has been proposed as a specific and sensitive test for sporadic CJD (Hsich et al., 1996).", "However, it is not useful for the diagnosis of vCJD, and much less specific in the genetic forms.", "As the 14-3-3 protein may be present in the CSF of patients with other conditions, the test is not recommended by WHO as a general screening for CJD and is reserved to confirm the clinical diagnosis (WHO Report, 1998).", "By combining clinical data with the biochemical markers a higher success in the diagnosis is achieved.", "However, according to the operational diagnosis currently in use in the European Surveillance of CJD, definitive diagnosis is established only by neuropathological examination and detection of PrPSc either by immunohistochemistry, histoblot or western blot (Weber et al., 1997, Budka et al., 1995).", "Formation of PrPSc is not only the most likely cause of the disease, but it is also the best known marker.", "Detection of PrPSc in tissues and cells correlates widely with the disease and with the presence of TSE infectivity, and treatments that inactivate or eliminate TSE infectivity also eliminate PrPSc (Prusiner, 1991).", "The identification of PrPSc in human or animal tissues is considered key for TSE diagnosis (WHO Report, 1998).", "One important limitation to this approach is the sensitivity, since the amounts of PrPSc are high (enough for detection with conventional methods) only in the CNS at the late stages of the disease.", "However, it has been demonstrated that at earlier stages of the disease there is a generalized distribution of PrPSc (in low amounts), especially in the lymphoreticular system (Aguzzi, 1997).", "Indeed, the presence of PrPSc has been reported in palatine tonsillar tissue and appendix obtained from patients with vCJD (Hill et al., 1997).", "Although it is not known how early in the disease course tonsillar or appendix biopsy could be used in vCJD diagnosis, it has been shown that in sheep genetically susceptible to scrapie, PrPSc could be detected in tonsillar tissue presymptomatically and early in the incubation period.", "However, PrPSc has not been detected in these tissues so far in any cases of sporadic CJD or GSS (Kawashima et al., 1997).", "The normal protein is expressed in white blood cells and platelets and therefore it is possible that some blood cells may contain PrPSc in affected individuals (Aguzzi, 1997).", "This raises the possibility of a blood test for CJD, but this would require an assay with a much greater degree of sensitivity than those currently available.", "Prion replication is hypothesized to occur when PrPSc in the infecting inoculum interacts specifically with host PrPC, catalyzing its conversion to the pathogenic form of the protein (Cohen et al., 1994).", "This process takes from many months to years to reach a concentration of PrPSc enough to trigger the clinical symptoms.", "The infective unit of PrPSc seems to be a β-sheet rich oligomeric structure, which converts the normal protein by integrating it into the growing aggregate (FIG.", "1).", "The conversion has been mimicked in vitro by mixing purified PrPC with a 50-fold molar excess of previously denatured PrPSc (Kocisko et al., 1994).", "The in vitro conversion systems described so far have low efficiency, since they require an excess of PrPSc and therefore are not useful for diagnostic purposes because they cannot monitor undetectable amounts of the marker.", "The reason for the low efficiency is that the number of PrPSc oligomers (converting units) remains fixed throughout the course of the assay.", "The converting units grow sequentially by the ends and as a result they become larger, but do not increase in number (FIG.", "1).", "DETAILED DESCRIPTION OF THE INVENTION We have now found a method for the diagnosis or detection of a conformational disease, wherein the disease is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, by assaying a marker of said disease within a sample, which method comprises: (i) contacting said sample with an amount of the non-pathogenic conformer; (ii) disaggregating any aggregates eventually formed during step (i); and (iii) determining the presence and/or amount of said pathogenic conformer within the sample.", "Generally, the pathogenic conformer will be the marker for the presence of the said disease.", "Preferably, step (i) comprises step (ia) incubating said sample/non-pathogenic conformer.", "According to a preferred embodiment of the invention, steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).", "More preferably, the cycles are repeated from 5 to 40 times, and most preferably 5-20 times.", "The conformational diseases to be detected or diagnosed are those that are characterised by a conformational transition of an underlying protein.", "This “underlying protein” is a protein which is capable of adopting a non-pathogenic conformation and a pathogenic conformation.", "One example of such a protein is the prion protein, PrP.", "A further example of such a protein is the protein involved in Alzheimer's disease, i.e.", "the β-amyloid protein.", "The conformational diseases to be diagnosed or detected are preferably transmissible conformational diseases, such as TSE (as defined in the Background section).", "In the case of diagnosis of TSE and according to a preferred embodiment of the invention, the marker of the disease as well as the pathogenic conformer is PrPSc, whereas the non-pathogenic conformer of the protein of interest is PrPC.", "The amount of the non-pathogenic conformer that is used in step (i) (and optionally in step (ib)) will generally be a known amount, although this need not be the case if one merely wishes to establish the presence or absence of the pathogenic conformer.", "Preferably, the amount of non-pathogenic conformer that is used in step (i) (and optionally in step (ib)) will be an excess amount.", "Generally, the initial ratio of non-pathogenic conformer to pathogenic conformer (if present in the sample) will be greater than 100:1, preferably greater than 1000:1 and most preferably greater than 1000000:1.In a further preferred embodiment of the invention, the non-pathogenic conformer in step (i) is present in a brain homogenate of a healthy subject and/or may be added to it, before carrying out step (i); in this case, therefore, the brain homogenate containing a (preferably known) excess of the non-pathogenic conformer is added during step (i).", "Preferably, the brain homogenate of the healthy subject comes from the same species from which the sample to be analyzed comes (e.g.", "human brain homogenate for human sample to be analyzed, rat brain homogenate from rat sample to be analyzed).", "More preferably, the non-pathogenic conformer is present in a specific fraction of the brain homogenate, for example in the lipid-rafts from brain homogenate.", "The preparation of such fractions can be carried out for example as described in Sargiacomo M et al., 1993.Thus the invention further relates to a method or assay as described herein wherein a tissue or tissue fraction is added to the non-pathogenic conformer in step (i).", "Preferably, the tissue is brain tissue, or a homogenate or fraction derived therefrom, from a healthy subject (i.e.", "one where the pathogenic conformer is not present).", "It has been reported (Kocisko et al., 1994) that less glycosylated forms of PrPC are preferentially converted to the PrPSc form.", "In particular, PrPC which was treated with phosphatidylinositol specific phospholipase C was routinely more efficiently converted to the pathogenic form than the complete, more heavily glycosylated PrPC.", "A further embodiment of the invention therefore relates to a method or assay as herein described wherein the non-pathogenic conformer is PrPC which has a reduced level of glycosylation (in particular N-linked glycosylation) in comparison with the wild-type PrPC.", "Preferably, the PrPC has been treated to remove some, all or a significant amount of the glycosylation prior to its use as the non-pathogenic conformer in the methods and assays described herein; and more preferably, the non-pathogenic conformer is PrPC which is essentially unglycosylated.", "In the case of diagnosis of TSE, if aggregates of the pathogenic form are present within the sample, during step (i) they will induce the PrPC→PrPSc transition and during step (ii) such aggregates will be broken down into smaller still infective units, each of which is still capable of inducing the conversion of other PrPC.", "This kind of method is herein called “cyclic amplification” and is represented in FIG.", "2.This system results in an exponential increase in the amount of PrPSc eventually present in the sample that can easily be detected.", "According to a further preferred embodiment of the invention, it is therefore possible to calculate the amount of PrPSc initially present in the sample starting from the known amount of PrPC, determining the amount of PrPSc present within the sample at the end of the assay and considering the number of cycles performed.", "If, on the contrary, no PrPSc (either as such or in the form of aggregates) is present in the sample, no PrPC molecule will be converted into PrPSc and at the end of the assay the marker will be completely absent (no pathogenic conformer detected in the sample).", "It has been shown that the infective unit of PrPSc is a β-sheet rich oligomer, which can convert the normal protein by integrating it into the growing aggregate, where it acquires the properties associated with the abnormal form (protease resistance and insolubility) (Jarrett and Lansbury, Jr., 1993, Caughey et al., 1997).", "After incubation of the two forms of PrP, the oligomeric species increases its size by recruiting and transforming PrPC molecules.", "This process has low efficiency, since it depends on a fixed number of oligomers growing by the ends.", "The number of converting units is not increased in the course of the reaction when they only become larger.", "It is assumed that this process is what happens in the animal or human body after infection; a process known to take months or even several years.", "In this invention we describe a procedure to break down the oligomers to a smaller ones, each of which is then capable of converting PrPC.", "Therefore, the system has direct applications to the diagnosis of conformational diseases, and in particular transmissible conformational diseases, such as TSE by amplifying otherwise undetectable amounts of PrPSc in different tissues or biological fluids.", "The system may allow the early identification of people at risk of developing TSE and could also be very useful to follow biochemically the efficacy of TSE therapeutic compounds during clinical trials.", "According to a preferred embodiment of the invention the sample to be analysed is subjected to a “pre-treatment” step, which has the purpose of “selectively concentrating” in the sample the pathogenic conformer that is to be detected.", "In the case of TSE both PrPC and PrPSc have been reported to be located in a special region of the plasma membrane which is resistant to mild detergent treatment (such as ice-cold Triton X-100) due to the relatively high content of cholesterol and glycosphingolipids (M. Vey et al., 1996).", "These membrane domains are named lipid-rafts or detergent-resistant membranes (DRM) or caveolae-like domains (CLDs) and are rich in signaling proteins, receptors and GPI-anchored proteins.", "We have confirmed that 100% of PrPC in brain is attached to this fraction, which contains <2% of the total proteins (see Example 6 and FIG.", "7).", "Thus, the simple step of lipid-raft isolation from the sample allows a dramatic enrichment in PrPC.", "Similar results were obtained by the Applicant in the isolation of lipid-rafts from scrapie brain homogenate, in which PrPSc was recovered in the rafts.", "Thus one embodiment of the invention includes a step wherein the sample to be analysed is subjected to a pre-treatment step for selectively concentrating the pathogenic conformer in the sample.", "Preferably, the pathogenic conformer is PrPSc and the pretreatment is the extraction from the sample of a fraction which is insoluble in mild detergents.", "Steps (i) and (ia) are preferably performed under physiological conditions (pH, temperature and ionic strength) and, more preferably, protease inhibitors and detergents are also added to the solution.", "The conditions will be chosen so as to allow any pathogenic conformer, if present in the sample, to convert the non-pathogenic conformer into pathogenic conformer thus forming an aggregate or oligomer of pathogenic conformers.", "Appropriate physiological conditions will readily be apparent to those skilled in the art.", "The length of the incubation will be for a time which will allow some, all or a significant portion of the non-pathogenic conformer to be converted to pathogenic conformer, assuming that the sample contains some pathogenic conformer.", "The time will readily be determinable by those skilled in the art.", "Preferably, each incubation will be between 1 minute to 4 hours, most preferably 30 minutes to 1 hour, and particularly preferably approximately 60 minutes.", "Incubation step (ia) may also comprise the further step (ib) which comprises the addition of a further amount of non-pathogenic conformer.", "Various methods can be used for disaggregating the aggregates during step (ii) of the method of the present invention.", "They include: treatment with solvents (such as sodium dodecyl sulfate, dimethylsulfoxide, acetonitrile, guanidine, urea, trifluoroethanol, diluted trifluroacetic acid, diluted formic acid, etc.", "), modification of the chemical-physical characteristics of the solution such as pH, temperature, ionic strength, dielectric constant, and physical methods, such as sonication, laser irradiation, freezing/thawing, French press, autoclave incubation, high pressure, stirring, mild homogenization, other kinds of irradiation, etc.", "Sonication is the preferred method according to the invention Disaggregation may be carried out for a time which disaggregates some, all or a significant portion of the aggregates which have formed during step (ii).", "It is not necessary for all of the aggregates to be disaggregated in any one disaggregation step.", "In this way, the number of converting units is increased in each disaggregation step.", "The disaggregation time will readily be determinable by those skilled in the art and it may depend on the method of disaggregation used.", "Preferably, disaggregation is carried out for 1 second to 60 minutes, most preferably 5 seconds to 30 minutes and particularly preferably, 5 seconds to 10 minutes.", "If disaggregation is carried out by sonication, sonication is preferably for 5 seconds to 5 minutes, and most preferably for 5 to 30 seconds.", "Sonication has been used in the past as part of several methods to purify PrP with the goal of increasing solubility of large aggregates, but it has never been described to amplify in vitro conversion of PrP.", "The use of a traditional single-probe sonicator imposes a problem for handling many samples simultaneously, such as a diagnostic test will require.", "There are on the market some 96-well format microplate sonicators, which provide sonication to all the wells at the same time and can be programmed for automatic operation.", "These sonicators can be easily adapted to be used in the diagnostic method of the present invention.", "Thus one embodiment of the invention relates to the use, in step (ii), of a multi-well sonicator.", "The detection of the newly converted pathogenic conformer, e.g.", "PrPSc, (iii) after the cyclic amplification procedure described in steps (i) to (ii) could be carried out according to any of the known methods.", "Specific detection of PrPSc is usually (but not always, see below) done by a first step of separation of the two PrP isoforms (normal protein and pathogenic protein).", "Separation is done on the basis of the peculiar biochemical properties of PrPSc that distinguish it from most of the normal proteins of the body, namely: PrPSc is partially resistant to protease treatment and is insoluble even in the presence of non-denaturant detergents.", "Therefore the first step after the amplification procedure is usually the removal or separation of PrPC in the sample, either by treatment with proteases or by centrifugation to separate the soluble (PrPC) from the insoluble (PrPSc) protein.", "Thereafter, detection of PrPSc can be done by any of the following methods, inter alia: A) Immunobloting after SDS-PAGE.", "This is done through a routine procedure well known for those with skill in the art and using some of the many commercially available anti-PrP antibodies.", "B) Elisa assay.", "Solid phase detection can be done by either a simple assay in which the sample is loaded on the plate and the amount of PrPSc detected afterwards by using anti-PrP antibodies or more preferably by using sandwich Elisa in which the plate is first coated with an anti-PrP antibody that captures specifically PrP from the sample, which is finally detected by using a second anti-PrP antibody.", "Both forms of Elisa can also be used with labelled (radioactivity, fluorescence, biotin, etc) anti-PrP antibodies to further increase the sensitivity of the detection.", "C) Radioactivity assays.", "Normal PrPC used as a substrate for the amplification procedure can be radioactively labelled (3H, 14C, 35S, 125I, etc) before starting the procedure and after the removal of the non-converted PrPC, radioactivity of newly converted PrPSc could be quantitated.", "This procedure is more quantitative and does not rely on the use antibodies.", "D) Fluorescence assays.", "Normal PrPC used as a substrate for the amplification procedure can be labelled with fluorescent probes before starting the procedure and after the removal of non-converted PrPC, fluorescence of the newly converted PrPSc could be quantitated.", "It is possible that the fluorescence assay might not require the removal of non-converted PrPC, because the fluorescence properties of PrPC and PrPSc might be different due to the distinct conformation of the two isoforms.", "E) Aggregation assays.", "It is well known that PrPSc (and not PrPC) is able to aggregate forming amyloid fibrils or rod-type structures.", "Therefore detection of PrPSc could be done by using the methods used to quantify the formation of these type of aggregates, including electron microscopy, staining with specific dyes (Congo red, Thioflavin S and T, etc), and turbidimetric assays.", "Aggregation assays do not require the step of separation of the two isoforms, because is known that normal PrPC does not aggregate.", "F) Structural assays.", "The most important difference between the normal and the pathogenic PrP is their secondary and tertiary structures.", "Therefore, methods that allow the structural evaluation of proteins can be used, including NMR, Circular dichroism, Fourier-transformed infrared spectroscopy, Raman spectroscopy, intrinsic fluorescence, UV absorption, etc.", "The most widely used PrP monoclonal antibody is “3F4” (Kascsak et al., 1987), which is a monoclonal antibody derived from a mouse immunized with hamster 263K PrPres (the protease-resistant conformer).", "This antibody is also able to recognize the non-pathogenic conformer from hamsters and humans, but not from bovine, mouse, rat, sheep or rabbit brains; it is also able to bind the human pathogenic conformer, but only after denaturation of the protein.", "Such antibodies may be labeled to allow easy detection of the marker.", "For example time-resolved fluorescence measurements with europium-labeled 3F4 antibody has been used by some scientists (Safar et al., 1998).", "The above-described methods of detection may be used for the detection of other pathogenic conformers, for example the pathogenic form of β-amyloid protein, mutatis mutandis.", "In an alternative embodiment the non-pathogenic conformer added in excess may be labeled and detectable so that the amount of the non-aggregated conformer at the end of the assay will allow a determination of the amount of pathogenic conformer initially present in the sample.", "According to a further alternative embodiment, the pathogenic conformer (the marker) could be directly detected with an antibody directed against it.", "In broader terms a label or labeling moiety may be added to the pathogenic conformer, to the non-pathogenic conformer or to an antibody against one of the conformers depending on the kind of assay that is performed.", "Another object of the invention is an assay for a marker of a conformational disease which is characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, within a sample, which assay comprises the following steps: (i) contacting said sample with an amount of the non-pathogenic conformer, (ii) disaggregating any aggregates eventually formed during step (i) and (iii) determining the presence and/or amount of said pathogenic conformer within the sample.", "In general, the pathogenic conformer will be the marker for the presence of said disease.", "Preferably, step (i) comprises step (ia) incubating said sample/non-pathogenic conformer.", "According to a preferred embodiment of the invention, steps (ia) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).", "More preferably, the cycles are repeated from 5 to 40 times, and most preferably 5 to 20 times.", "A further object of the present invention is a diagnostic kit for use in the assay specified, which comprises an amount of the non-pathogenic conformer, and optionally additionally a micro-titre plate and a multi-well sonicator.", "Using the method of the invention, it is possible to detect 1 to 10 fg of pathogenic conformer initially present in a sample, which is equivalent to 3 to 30×10−20 moles.", "The sample will generally be a biological sample or tissue, and any such biological sample or tissue can be assayed with the method of the present invention.", "In the case of a tissue, the assay and method of the present invention may be carried out on homogenates or direct on ex vivo samples.", "The methods and assays will generally be carried out on ex vivo or in vitro samples.", "Preferably, the sample is a biological fluid, such as blood, lymph, urine or milk; brain tissue, spinal cord, tonsillar tissue or appendix tissue; a sample derived from blood such as blood cell ghosts or buffy coat preparations; or a plasma membrane preparation such as lipid-rafts, detergent resistant membranes or caveolae-like domains.", "The sample might alternatively be a composition comprising a compound (particularly a protein) derived from a human or animal source, such as growth hormone, or a tissue extract, such as pituitary extract.", "Such a sample composition might be contaminated with a pathogenic conformer.", "The sample might also comprise a food product or drink, or a portion of a food product or drink (either destined for human consumption or animal consumption) in order to establish the presence or absence of pathogenic conformer in that product or drink.", "Preferably, the non-pathogenic conformer added in step (i) will be from the same species as the sample.", "It may, for example, be derived from a healthy (i.e.", "non-pathogenic) form (e.g.", "tissue) of the biological sample to be tested.", "Alternatively, the non-pathogenic conformer may be produced synthetically or recombinantly, using means known in the art.", "It will be understood, however, that the non-pathogenic conformer need not be in pure or even substantially pure form.", "In most cases, the non-pathogenic conformer will be in the form of a tissue homogenate or a fraction thereof which contains the relevant non-pathogenic conformer.", "Preferred examples include brain homogenates and fractions derived therefrom, e.g.", "lipid rafts.", "Preferably, the sample and/or the non-pathogenic conformer will be of human origin or from a domestic animal, e.g.", "a cow, sheep, goat or cat.", "Another object of the present invention is to provide a method for identifying a compound which modulates the conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, comprising: (i) contacting an amount of the non-pathogenic conformer with an amount of the pathogenic conformer in the presence and in the absence of said compound, (ii) disaggregating any aggregates eventually formed during step (i), (iii) determining the amount of the pathogenic conformer in the presence and in the absence of said compound.", "If desired, step (i) may comprise step (ia) incubating said sample/non-pathogenic conformer, and a cycle carried out between steps (ia) and (ii) as described above for the methods and assays of the invention, mutatis mutandis.", "If the amount of pathogenic conformer measured in the presence of the compound is higher than that measured in the absence, it means that the compound is a factor which “catalyzes” the conformational transition; if such amount is lower, it means that the compound is a factor which inhibits such transition.", "According to the above method, “identifying” should also be interpreted to mean “screening” of a series of compounds.", "A “label” or “labelling moiety” may be any compound employed as a means for detecting a protein.", "The label or labelling moiety may be attached to the protein via ionic or covalent interactions, hydrogen bonding, electrostatic interactions or intercalation.", "Examples of labels and labelling moieties include, but are not limited to fluorescent dye conjugates, biotin, digoxigenin, radionucleotides, chemiluminescent substances, enzymes and receptors, such that detection of the labelled protein is by fluorescence, conjugation to streptaviden and/or avidin, quantitation of radioactivity or chemiluminescence, catalytic and/or ligand-receptor interactions.", "Preferably it is a fluorescent or a phosphorescent label.", "The term “conformational diseases” refers to that group of disorders arising from a propagation of an aberrant conformational transition of an underlying protein, leading to protein aggregation and tissue deposition.", "Such diseases can also be transmitted by an induced conformational change, propagated from a pathogenic conformer to its normal or non-pathogenic conformer and in this case they are called herein “transmissible conformational diseases”.", "Examples of such kinds of diseases are the prion encephalopathies, including the bovine spongiform encephalopathy (BSE) and its human equivalent Creutzfeld-Jakob (CJD) disease, in which the underlying protein is the PrP.", "The term “sporadic CJD” abbreviated as “sCJD” refers to the most common manifestation of Creutzfeldt-Jakob Disease (CJD).", "This disease occurs spontaneously in individuals with a mean age of approximately 60 at a rate of 1 per million per year individuals across the earth.", "The term “Iaterogenic CJD” abbreviated as “iCJD” refers to disease resulting from accidental infection of people with human prions.", "The most noted example of such is the accidental infection of children with human prions from contaminated preparations of human growth hormone.", "The term “Familial CJD” refers to a form of CJD, which occurs rarely in families and is inevitably caused by mutations of the human prion protein gene.", "The disease results from an autosomal dominant disorder.", "Family members who inherit the mutations succumb to CJD.", "The term “Gerstmann-Strassler-Scheinker Disease” abbreviated as “GSS” refers to a form of inherited human prion disease.", "The disease occurs from an autosomal dominant disorder.", "Family members who inherit the mutant gene succumb to GSS.", "The term “prion” shall mean a transmissible particle known to cause a group of such transmissible conformational diseases (spongiform encephalopathies) in humans and animals.", "The term “prion” is a contraction of the words “protein” and “infection” and the particles are comprised largely if not exclusively of PrPSc molecules.", "Prions are distinct from bacteria, viruses and viroids.", "Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats.", "Four prion diseases known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI).", "As used herein prion includes all forms of prions causing all or any of these diseases or others in any animals used and in particular in humans and in domesticated farm animals.", "The terms “PrP gene” and “prion protein gene” are used interchangeably herein to describe genetic material which expresses the prion proteins and polymorphisms and mutations such as those listed herein under the subheading “Pathogenic Mutations and Polymorphisms.” The PrP gene can be from any animal including the “host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.", "The term “PrP gene” refers generally to any gene of any species which encodes any form of a PrP amino acid sequences including any prion protein.", "Some commonly known PrP sequences are described in Gabriel et al., 1992, which is incorporated herein by reference to disclose and describe such sequences.", "Abbreviations used herein include: CNS for central nervous system; BSE for bovine spongiform encephalopathy; CJD for Creutzfeldt-Jakob Disease; FFI for fatal familial insomnia; GSS for Gerstmann-Strassler-Scheinker Disease; PrP for prion protein; PrPC for the normal, non-pathogenic conformer of PrP; PrPSc for the pathogenic or “scrapie” isoform of PrP (which is also the marker for prion diseases).", "Pathogenic Mutations and Polymorphisms There are a number of known pathogenic mutations in the human PrP gene.", "Further, there are known polymorphisms in the human, sheep and bovine PrP genes.", "The following is a non-limiting list of such mutations and polymorphisms: MUTATION TABLE Pathogenic human Human Sheep Bovine mutations polymorphisms polymorphisms polymorphisms 2 octarepeat Codon 129 Codon 171 5 or 6 insert Met/Val Arg/Glu octarepeats 4 octarepeat Codon 219 Codon 136 insert Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro-Len Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178 Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala The normal amino acid sequence, which occurs in the vast majority of individuals, is referred to as the wild-type PrP sequence.", "This wild-type sequence is subject to certain characteristic polymorphic variations.", "In the case of human PrP, two polymorphic amino acids occur at residues 129 (Met/Val) and 219 (Glu/Lys).", "Sheep PrP has two amino acid polymorphisms at residues 171 and 136, while bovine PrP has either five or six repeats of an eight amino acid motif sequence in the amino terminal region of the mature prion protein.", "While none of these polymorphisms are of themselves pathogenic, they appear to influence prion diseases.", "Distinct from these normal variations of the wild-type prion proteins, certain mutations of the human PrP gene which alter either specific amino acid residues of PrP or the number of octarepeats have been identified which segregate with inherited human prion diseases.", "In order to provide further meaning to the above chart demonstrating the mutations and polymorphisms, one can refer to the published sequences of PrP genes.", "For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., 1992.The sequence for the Syrian hamster is published in Baslet et al 1986.The PrP gene of sheep is published by Goldmann et al., 1990.The PrP gene sequence for bovine is published in Goldmann et al., 1991.The sequence for chicken PrP gene is published in Harris et al., 1991.The PrP gene sequence for mink is published in Kretzschmar et al., 1992.The human PrP gene sequence is published in Kretzschmar et al., 1986.The PrP gene sequence for mouse is published in Locht et al., 1986.The PrP gene sequence for sheep is published in Westaway et al., 1994.These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequence.", "The invention also provides a method for detecting the presence of a pathogenic form of prion protein within a sample (preferably a blood or brain sample) comprising: (i) contacting the sample with an amount of non-pathogenic prion protein; (ia) incubating the sample/non-pathogenic prion protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic prion protein within the sample.", "A further embodiment of the invention provides a method for diagnosing CJD within a patient, comprising: taking a sample from the patient (preferably a blood or brain sample); (i) contacting the sample with an amount of PrPC protein; (ia) incubating the sample/PrPC protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of PrPSc within the sample.", "The invention also provides a method for detecting the presence of a pathogenic form of β-amyloid protein within a sample (preferably a blood or brain sample), comprising: (i) contacting the sample with an amount of non-pathogenic β-amyloid protein; (ia) incubating the sample/non-pathogenic β-amyloid protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic β-amyloid protein within the sample.", "A further embodiment of the invention provides a method for diagnosing Alzheimer's disease in a patient, comprising: taking a sample (preferably a blood or brain sample) from the patient; (i) contacting the sample with an amount of non-pathogenic β-amyloid protein; (ia) incubating the sample/non-pathogenic β-amyloid protein; (ii) disaggregating any aggregates formed during step (ia); repeating steps (ia)-(ii) two or more times; and then (iii) determining the presence and/or amount of pathogenic β-amyloid protein within the sample.", "The invention furthermore provides apparatus for use in the methods described above, particularly apparatus comprising a microtitre plate, multi-well sonicator and an amount of a non-pathogenic conformer.", "A further embodiment of the invention provides a method for the diagnostic detection of a conformational disease, characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, by assaying a marker of said disease within a sample, which method comprises (i) contacting said sample with a known amount of the non-pathogenic conformer, (ii) disaggregating the aggregates eventually formed during step (i) and (iii) determining the presence and/or amount of said pathogenic conformer within the sample.", "Preferably, steps (i) and (ii) form a cycle which is repeated at least twice before carrying out step (iii), most preferably steps (i) and (ii) form a cycle, which is repeated from 5 to 40 times before carrying out step (iii).", "The invention also provides an assay for a marker of a conformational disease, characterized by a conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, within a sample, which assay comprises the following steps: (i) contacting said sample with a known amount of the non-pathogenic conformer, (ii) disaggregating the aggregates eventually formed during step (i) and (iii) determining the presence and/or amount of said pathogenic conformer within the sample.", "Preferably, the steps (i) and (ii) form a cycle which is repeated at least twice before carrying out step (iii).", "The invention further provides a method for identifying a compound which modulates the conformational transition of an underlying protein between a non-pathogenic and a pathogenic conformer, comprising: (i) contacting a known amount of the non-pathogenic conformer with a known amount of the pathogenic conformer in the presence and in the absence of said compound, (ii) disaggregating the aggregates eventually formed during step (i), (iii) determining the amount of the pathogenic conformer in the presence and in the absence of said compound.", "The present invention has been described with reference to the specific embodiments, but the content of the description comprises all modifications and substitutions, which can be brought by a person skilled in the art without extending beyond the meaning and purpose of the claims.", "The invention will now be described by means of the following Examples, which should not be construed as in any way limiting the present invention.", "The Examples will refer to the Figures specified here below.", "DESCRIPTION OF THE DRAWINGS FIG.", "1.Schematic representation of the conversion PrPC→PrPSc.", "The infective unit of PrPSc is a β-sheet rich oligomer, which converts PrPC by integrating it into the growing aggregate, where it acquires the properties associated with PrPSc.", "FIG.", "2.Diagrammatic representation of the cyclic amplification procedure.", "The system is based on cycles of incubation of PrPSc in the presence of excess of PrPC followed by cycles of sonication.", "During the incubation periods, oligomeric PrPSc is enlarged by incorporating PrPC into the growing aggregate, while during sonication the aggregates are broken down with the aim of multiplying the converting units.", "In the figure, two cycles of sonication/incubation are shown.", "FIG.", "3.Amplification of PrPSc by sonication cycles.", "A small amount of scrapie brain homogenate containing PrPSc was incubated with healthy rat brain homogenate (lane 1, control experiment) or with healthy hamster brain homogenate (lane 2 and 3).", "The latter sample was divided in two groups one of which was subjected to five cycles of incubation/sonication (lane 3).", "Half of the above samples were loaded directly in a gel and stained for total protein with Coomasie (panel A).", "The other half were treated with PK and immunoblotted using the anti-PrP antibody 3F4 (panel B).", "Panel C shows some controls in which healthy brain homogenate was incubated alone (lanes 1 and 2) or in the presence of diluted scrapie brain homogenate (lanes 3 and 4).", "Half of the samples (lanes 2 and 4) were subjected to 5 cycles of sonication/incubation.", "Lanes 2, 3 and 4 were treated with proteinase K. FIG.", "4.Sensitivity of the cyclic amplification system.", "The minimum concentration of PrPSc that can be used for detection after amplification was studied by serially diluting the scrapie brain homogenate and incubating with healthy hamster brain homogenate with or without sonication cycles.", "Panel A shows the control experiment in which scrapie hamster brain was diluted serially in rat brain homogenate.", "Panel B corresponds to the experiment in which the serial dilutions of scrapie hamster brain were incubated with healthy hamster brain and subjected to 5 cycles of incubation/sonication.", "Densitometric evaluation of the immunoblots in A and B is shown in panel C. The dilutions were done considering as starting material the brain and were the following: 100 (lane 1), 200 (lane 2), 400 (lane 3), 800 (lane 4), 1600 (lane 5) and 3200 (lane 6).", "FIG.", "5.Relationship between the PrPres signal and the number of amplification cycles.", "Diluted scrapie brain homogenate was incubated with an excess of healthy hamster brain homogenate.", "Samples were subjected to 0, 5, 10, 20 or 40 cycles and the PrPres signal evaluated by immunoblot.", "FIG.", "6.Amplification of PrPSc in blood samples.", "Heparinized rat blood was spiked with Scrapie hamster brain homogenate to reach a final dilution of 10:1.This mixture was incubated for 15 min at RT.", "10 fold serial dilutions were made of this material using heparinized rat blood.", "Samples were subjected to 11 cycles of incubation-sonication and the PrPres signal evaluated by immunoblot.", "FIG.", "7: Prion protein is present in lipid-rafts.", "Lipid-rafts (also called detergent-resistant membrane fraction or DRM) were isolated using a modification of previouly described protocols.", "One-hundred mg of brain tissue was homogenized in 1 ml of PBS containing 1% triton X-100 and 1× complete cocktail of protease inhibitors (Boehringer).", "Tissue was homogenized with 10 passages through 22 G syringe needle and incubated for 30 minutes at 4° C. on a rotary shaker.", "The sample was diluted 1:2 in sucrose 60% and placed in the bottom of a centrifuge tube.", "7 ml of sucrose 35% were place carefully over the sample.", "1.5 ml of sucrose 15% was layered in the top of the gradient.", "The tube was centrifuged at 150,000 g for 18 hrs at 4° C. The lipid rafts float to the 15%-35% sucrose interface (panel A).", "Different fractions were collected and analyzed by total protein staining with silver nitrate (panel B) and immunoblot to detect PrP (panel C).", "To remove sucrose from the sample, lipid raft fraction was recovered washed in PBS and centrifuged at 28,000 rpm during 1 hr at 4° C. The pellet was washed and resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.", "All PrPC was located in this fraction (panel D).", "FIG.", "8: The factors needed for amplification are present in lipid-rafts.", "Lipid-rafts were isolated from healthy hamster brain as describe in FIG.", "2 and mixed with 700-fold diluted PrPSc highly purified from scrapie hamster brain.", "Samples were either frozen (line 3) or amplified for 20 h (line 4).", "Lines 1 and 2 represent the same procedures but using total brain homogenate for amplification.", "FIG.", "9: Presymptomatic detection of PrPSc in hamster brain.", "Hamsters were inoculated intra-cerebrally (i.c.)", "with saline (control group) or with 100-fold diluted scrapie brain homogenate.", "Every week 4 hamsters per group were sacrificed and brains were extracted and homogenized.", "Half of the samples were frozen immediately (white bars) and the other half subjected to 20 cycles of incubation/sonication (black bars).", "All samples were treated with PK and immunoblotted.", "The intensity of the bands was evaluated by densitometry.", "Each bar represent the average of samples from 4 animals.", "No detection was observed in any of the control brains either without or with amplification and these results are not shown in the Figure.", "FIG.", "10: Amplification of human PrPSc.", "The studies were done using brain samples of 11 different confirmed cases of sporadic CJD, as well as 5 from familial CJD and 4 age-matched controls, which included patients affected by other neurological disorders.", "Brain was homogenized and subjected to 20 amplification samples.", "Representative results of a control (A) and three different sporadic CJD (B) cases (1, 2, 3) are shown in the Figure.", "FIG.", "11: Detection of PrPSc in blood after preparation of blood cells ghosts.", "Cell ghosts from 0.5 ml of heparinized blood coming from healthy (C) and scrapie-affected hamsters (Sc) were prepared as described in the text.", "Half of the samples were not subjected to amplification and the other half were mixed with normal hamster brain homogenate and subjected to 20 amplification cycles.", "All samples were then treated with PK and analyzed by immunoblots.", "One representative experiment is shown in the Figure.", "FIG.", "12: Detection of PrPSc in blood after sarkosyl extraction.", "0.5 ml of heparinized blood coming from healthy (C) and scrapie-affected hamsters (Sc) was subjected to sarkosyl extraction as described in the text.", "Half of the samples were not subjected to amplification and the other half were mixed with normal hamster brain homogenate and subjected to 20 amplification cycles.", "All samples were then treated with PK and analyzed by immunoblots.", "One representative sample of control animals and two for scrapie-affected animals is shown in the Figure.", "FIG.", "13: Detection of PrPSc in blood after lipid rafts purification.", "Lipid-rafts were extracted as described in the text from 0.5 ml of heparinized blood coming from healthy (C) and scrapie-affected hamsters (Sc).", "Half of the samples were not subjected to amplification and the other half were mixed with normal hamster brain homogenate and subjected to 20 amplification cycles.", "All samples were then treated with PK and analyzed by immunoblots.", "One representative sample of control animals and two for scrapie-affected animals is shown in the Figure.", "FIG.", "14: Detection of PrPSc in blood after preparation of buffy coats.", "The buffy coat fraction of blood was separated by centrifugation from 0.5 ml of heparinized blood coming from healthy (C) and scrapie-affected hamsters (Sc).", "Half of the samples were not subjected to amplification and the other half were mixed with normal hamster brain homogenate and subjected to 20 amplification cycles.", "All samples were then treated with PK and analyzed by immunoblots.", "One representative experiment is shown in the Figure.", "EXAMPLES Example 1 Amplification of PK Resistant PrP by Cyclic in Vitro Conversion Hamster brain homogenate extracted from scrapie affected animals was diluted until the signal of PrPSc was barely detected by immunoblot after treatment with proteinase K (PK) FIG.", "3B, lane 1).", "PK treatment is done routinely in the field to distinguish between the normal and abnormal forms of PrP, which differ in their sensitivity to protease degradation (PrPSc is partially resistant and PrPC is degraded) (Prusiner, 1991).", "The form of PrP that is resistant to PK treatment will be named from now on PrPres.", "Incubation of a sample of diluted scrapie brain homogenate with a healthy hamster brain homogenate containing an excess of PrPC, resulted in the increase in PrPres signal (FIG.", "3B, lane 2).", "This suggests that the incubation of the two brain homogenates resulted in the conversion of PrPC to PrPSc.", "When the samples were incubated under the same conditions but subjected to five cycles of incubation/sonication, the amount of PrPres was dramatically increased (FIG.", "3B, lane 3).", "Densitometric analysis of the immunoblot indicates that the PrPres signal was increased 84-fold by cyclic amplification in comparison with the PrPres signal presented in the diluted scrapie brain homogenate (lane 1).", "The conversion is dependent of the presence of PrPSc since no PrPres was observed when the normal hamster brain homogenate was incubated alone under the same conditions either with or without sonication (FIG.", "3C, lane 2).", "To rule out artifacts of the transfer, the total protein loaded in the gel was maintained constant (FIG.", "3A) by adding rat brain homogenate to the diluted scrapie sample, taking advantage of the fact that rat PrP is not detected by the antibody used for the immunoblot.", "Example 2 Sensitivity of Detection by Cyclic Amplification To evaluate the minimum concentration of PrPSc that can be used for detection after amplification, the scrapie brain homogenate was serially diluted directly in healthy hamster brain homogenate.", "Without incubation, the signal of PrPres diminishes progressively until it was completely undetectable at 800-fold dilution (FIGS.", "4A, C).", "In contrast when the same dilution was incubated with healthy hamster brain homogenate and subjected to 5 cycles of incubation/sonication, the limit of PrPres detection was decreased dramatically.", "Indeed, clear signal was easily detected even at a 3200-fold dilution (FIGS.", "4B, C).", "Example 3 Exponential Increase in PrPres with Number of Cycles To study whether the intensity of the PrPres signal after cyclic amplification depends on the number of cycles of incubation/sonication performed, diluted scrapie brain homogenate was incubated with an excess of healthy hamster brain homogenate.", "Samples were subjected to 0, 5, 10, 20 or 40 cycles and the PrPres signal evaluated by immunoblot.", "The levels of PrPres increased exponentially with the number of incubation/sonication cycles (FIG.", "5).", "This result suggests that increasing the number of cycles could further diminish detection limits.", "Example 4 Sonication Experiments in Blood Samples by Spiking with PrPSc Heparinized rat blood was spiked with Scrapie hamster brain homogenate to reach a final dilution of 10:1.This mixture was incubated for 15 min at RT.", "10 fold serial dilutions were made of this material using heparinized rat blood.", "50 μl of each dilution were centrifuged at 3,000 rpm for 10 min.", "Plasma was separated from the pellet.", "10 μl of plasma were mixed in 50 μl of healthy hamster brain homogenate containing the PrPC substrate for the conversion reaction.", "Samples were subjected to 11 cycles of incubation-sonication.", "As a control same samples were mixed in 50 μl of healthy hamster brain homogenate and kept at −20° C. until needed.", "15 μl of sonicated and control samples were digested with proteinase K, separated by SDS-PAGE and analyzed by western blotting and PrPSc was detected as disclosed in the “Methods” section.", "The results are reported in FIG.", "6.These results show a clear increase in the detection of the protein after the amplification procedure, which is especially evident at the lower concentration of PrPSc (for example at the 1280 dilution).", "If we compare such results with those obtained on infected brain tissues, we have the confirmation that the amplification process works similarly in blood.", "Example 5 High Throughput Cyclic Amplification The use of a single-probe traditional sonicator imposes a problem for handling many samples simultaneously, as a diagnostic test will require.", "We have adapted the cyclic amplification system to a 96-well format microplate sonicator (Misonix 431MP—20 kHz), which provides sonication to all of the wells at the same time and can be programmed for automatic operation.", "This improvement not only decreases processing time, but also prevents loss of material when compared to using a single probe.", "Cross contamination is eliminated since there is no direct probe intrusion into the sample.", "The latter is essential to handle infectious samples and minimize false positive results.", "Twenty cycles of 1 h incubation followed by sonication pulses of 15 sec or 30 sec gave a significant amplification of PrPres signal, similar to that previously observed using a traditional sonicator.", "Example 6 The Factors Necessary for Amplification Are in a Detergent-Resistant Membrane Fraction The subcellular location where the PrP conversion occurs during the disease pathogenesis is not yet ascertained.", "However, both PrPC and PrPSc have been reported to be located in a special region of the plasma membrane which is resistant to mild detergent treatment due to the relatively high content of cholesterol and glycosphingolipids (Vey et al., 1996; Harmey et al., 1995).", "These membrane domains are named lipid-rafts or detergent-resistant membranes (DRM) and are rich in signaling proteins, receptors and GPI-anchored proteins.", "We have confirmed that 100% of PrPC in brain is attached to this fraction, which contains <2% of the total proteins (FIG.", "7).", "Thus, the simple step of lipid-raft isolation allows a dramatic enrichment in PrPC.", "Similar results were obtained in the isolation of lipid-rafts from scrapie brain homogenate, in which PrPSc was recovered in the rafts.", "To evaluate whether the factors needed to amplify PrP are contained in lipid-rafts, we purified them from the brain of healthy animals and added minute quantities of highly pure PrPSc extracted from the brain of sick animals.", "Amplification in lipid-rafts was equivalent to that obtained with total brain extract (FIG.", "8), since the amount of PrPres produced after amplification was similar in both conditions.", "This result indicates that all elements required for PrP conversion and amplification (including the so-called “Factor X”; (Telling et al., 1995)) are contained in this specialized membrane domain.", "Therefore, identification and isolation of the factors needed for PrP conversion should be possible by further separation of proteins from the lipid-rafts and monitoring their activity by cyclic amplification.", "In addition, lipid-rafts constitute a possible replacement for the use of total brain homogenate in the cyclic amplification procedure as a source of PrPC substrate and other endogenous factors implicated in the conversion.", "Example 7 Pre-Symptomatic Diagnosis in Experimental Animals To study the pre-symptomatic diagnosis of hamsters experimentally infected with scrapie, we screened 88 brain samples at different stages during the preclinical phase, half of which were non-infected controls.", "Brain was taken every week (4 per each group) and subjected to 20 cycles of amplification.", "The results showed that the method is able to detect the abnormal protein in the brain even at the second week after inoculation, far before the animals develop any symptoms (FIG.", "9).", "Without cyclic amplification, PrPSc was detected in the brain at week six post-infection, only 4 weeks before the appearance of the clinical disease.", "No amplification was detected in any of the control animals that were not infected with scrapie.", "Example 8 Application of Cyclic Amplification to Human Brain Samples To analyze the application of the cyclic amplification procedure to human samples from brain of people (cadavers) affected by Creutzfeldt-Jakob disease (CJD), we incubated brain homogenates of several CJD patients (or normal controls) with healthy human brain homogenate and carried out the cyclic amplification procedure.", "The results show that there was significant amplification in samples of sporadic CJD brain analyzed and in none of the 4 control samples (FIG.", "10).", "Interestingly, amplification was obtained only in the samples that had shown to be infectious and thus able to convert non-mutated PrPC, while it did not work when the mutant protein is not capable to convert the wild type protein.", "These data support the conclusion that the method works in human samples similarly as shown before for animal samples.", "Example 9 Diagnosis in Blood by Cyclic Amplification Infectivity studies suggested that at least in experimental animals PrPSc is present in blood in late-stage animals (Brown et al., 2001).", "In order to perform the blood detection of PrPSc by cyclic amplification, we preferred first to selectively concentrate the sample in the protein to be detected and to eliminate the bulk of very abundant blood proteins, such as albumin or hemoglobin.", "The following four different protocols have been shown effective for this purpose.", "1.Preparation of Blood Cells Ghosts Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. The plasma and cellular fraction were separated and frozen at −80° C. until needed.", "0.5 ml of blood cell package was washed 3 times in 12-15 vol of fresh cold PBS, pH 7.6.The cells were resuspended in 12-15 vol of 20 mOsM sodium phosphate buffer pH 7.6 and stirred gently for 20 min on ice, then centrifuged at 30,000 rpm for 10 min at 4° C. The supernatant was discarded, the pellet was washed 3 times in 20 mOsM sodium phosphate buffer.", "The final pellet was resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.", "15 μl of this suspension was mixed v/v with 10% healthy hamster brain homogenate and subjected to 20 cycles of incubation-sonication.", "20 μl of sonicated and control samples were digested with proteinase K, separated by SDS-PAGE and analyzed by western blotting and PrPSc was detected as disclosed in the “Methods” section.", "The results show the detection of the PrPSc after the amplification procedure in the blood samples from infected animals (FIG.", "11).", "In the blood samples from non-infected animals there is no signal after amplification.", "Without amplification is not possible to detect the presence of PrPSc (FIG.", "11).", "2.Sarkosyl Extraction Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. 0.5 ml of blood cell package was diluted (v/v) in 20% sarkosyl and incubated for 30 minutes.", "The sample was centrifuged in Beckman TL100 ultracentrifuged at 85,000 rpm for 2 hrs at 4° C. The pellet was washed and resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.", "15 μl of this suspension was mixed v/v with 10% healthy hamster brain homogenate and subjected to 20 cycles of incubation-sonication.", "20 μl of sonicated and control samples were digested with proteinase K, separated by SDS-PAGE and analyzed by western blotting and PrPSc was detected as disclosed in the “Methods” section.", "The results show the detection of the PrPSc after the amplification procedure in the blood samples from infected animals (FIG.", "12).", "In the blood samples from non-infected animals there is no signal after amplification.", "Without amplification is not possible to detect the presence of PrPSc (FIG.", "12).", "3.Lipid Raft Extraction Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. 0.5 ml of blood cell package was diluted (v/v) in PBS with 1% Triton X-100 and incubated for 30 minutes at 4° C. The sample was diluted 1:2 in sucrose 60% and placed in the bottom of a centrifuge tube.", "7 ml of sucrose 35% were placed carefully over the sample.", "1.5 ml of sucrose 15% was layered in the top of the gradient.", "The tube was centrifuged at 150,000 rpm for 18 hrs at 4° C. The lipid rafts were recovered washed in PBS and centrifuged at 28,000 rpm during 1 hr at 4° C. The pellet was washed and resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.", "15 μl of this suspension was mixed v/v with 10% healthy hamster brain homogenate and subjected to 20 cycles of incubation-sonication.", "20 μl of sonicated and control samples were digested with proteinase K, separated by SDS-PAGE and analyzed by western blotting and PrPSc was detected as disclosed in the “Methods” section.", "The results show the detection of the PrPSc after the amplification procedure in the blood samples from infected animals (FIG.", "13).", "In the blood samples from non-infected animals there is no signal after amplification.", "Without amplification is not possible to detect the presence of PrPSc (FIG.", "13).", "4.Buffy Coat Preparation.", "Heparinized hamster blood was centrifuged at 1,500 rpm at 4° C. for 10 min.", "The buffy coat was carefully recovered using standard procedures and kept at −80° C. until needed.", "The frozen buffy coat was resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.", "15 μl of this suspension was mixed v/v with 10% healthy hamster brain homogenate and subjected to 20 cycles of incubation-sonication.", "20 μl of sonicated and control samples were digested with proteinase K, separated by SDS-PAGE and analyzed by western blotting and PrPSc was detected as disclosed in the “Methods” section.", "The results show the detection of the PrPSc after the amplification procedure in the blood samples from infected animals (FIG.", "14).", "In the blood samples from non-infected animals there is no signal after amplification.", "Without amplification is not possible to detect the presence of PrPSc (FIG.", "14).", "Methods Preparation of Brain Homogenates.", "Brains from Syrian golden hamsters healthy or infected with the adapted scrapie strain 263 K were obtained after decapitation and immediately frozen in dry ice and kept at −80° C. until used.", "Brains were homogenized in PBS and protease inhibitors (w/v) 10%.", "Detergents (0.5% Triton X-100, 0.05% SDS) were added and clarified with low speed centrifugation (10,000 rpm) for 1 min.", "Preparation of the Samples and Cyclic Amplification.", "Serial dilutions of the scrapie brain homogenate were made directly in the healthy brain homogenate.", "30 μl of these dilutions were incubated at 37° C. with agitation.", "Each hour a cycle of sonication (5 pulses of 1 sec each) was done using a microsonicator with the needle immersed in the sample.", "These cycles were repeated several times (5-20).", "PrPSc Detection.", "The samples were digested with PK 100 μg/mL for 90 min at 37° C. 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[ [ "Method, apparatus, and system for building context dependent models for a large vocabulary continuous speech recognition (lvcsr) system", "According to one aspect of the invention, a method is provided in which a set of multiple mixture monophone models is created and trained to generate a set of multiple mixture context dependent models.", "A set of single mixture triphone models is created and trained to generate a set of context dependent models.", "Corresponding states of the triphone models are clustered to obtain a set of tied states based on a decision tree clustering process.", "Parameters of the context dependent models are estimated using a data dependent maximum a posteriori (MAP) adaptation method in which parameters of the tied states of the context dependent models are derived by adapting corresponding parameters of the context independent models using the training data associated with the respective tied states." ], [ "1.A method comprising: training a set of multiple mixture monophone models using a set of monophone transcripts to generate a set of multiple mixture context independent models; training a set of single mixture triphone models using set of triphone transcripts to generate a set of context dependent models; and estimating parameters of the context dependent models using a data dependent maximum a posteriori (MAP) adaptation method wherein parameters of tied states of the context dependent models are derived by adapting corresponding parameters of the context independent models through MAP adaptation method using training data associated with the corresponding tied states.", "2.The method of claim 1 wherein training the set of multiple mixture monophone models includes: constructing single mixture monophone models based upon the set of monophone transcripts; and estimating parameters of the single mixture monophone models using the Baum-Welch method.", "3.The method of claim 2 wherein the single mixture monophone models are split up and the corresponding parameters are re-estimated iteratively for a predetermined number of iterations to obtain the multiple-mixture context independent models.", "4.The method of claim 1 wherein training the set of triphone models further including: constructing a decision tree for each state of each center phone of the single mixture triphone models; and clustering context dependent states of each respective center phone into corresponding groups using a decision tree algorithm to obtain a set of corresponding tied states, each tied state having a corresponding distribution of training data.", "5.The method of claim 4 wherein estimating parameters including: deriving parameters of the tied states of the context dependent models based upon the parameters associated with the context independent models and the training data associated with the tied states of the context dependent models; 6.The method of claim 5 wherein deriving the parameters of the tied states of the context dependent models includes; adapting the parameters of the context independent models by the maximum a posteriori (MAP) adaptation method using training data associated with the tied states of the context dependent models.", "7.The method of claim 6 wherein adapting the parameters includes: computing new sufficient statistic estimates associated with the parameters of the context independent models; and combining the new sufficient statistic estimates with old sufficient statistic estimates from the parameters of the context independent models using a data-dependent mixing coefficient.", "8.The method of claim 7 wherein, based upon the data-dependent mixing coefficient, mixtures of high counts of data from the tied states of the context dependent models rely more on the new sufficient statistic estimates for final parameter estimation.", "9.The method of claim 7 wherein, based upon the data-dependent mixing coefficient, mixtures of low counts of data from the tied states of the context dependent models rely more on the old sufficient statistic estimates for final parameter estimation.", "10.An acoustic model comprising: a set of context dependent models having a set of tied states whose parameters are estimated using a data dependent maximum a posteriori (MAP) adaptation method wherein parameters of the tied states are derived based on corresponding parameters of a set of multiple mixture context independent models using training data associated with the respective tied states.", "11.The acoustic model of claim 10 wherein a set of multiple mixture monophone models is trained using a set of monophone transcripts to generate the corresponding context independent models.", "12.The acoustic model of claim 11 wherein single mixture monophone models are split up and re-estimated iteratively for a predetermined number of iterations to obtain the set of multiple mixture monophone models.", "13.The acoustic model of claim 10 wherein a set of single mixture triphone models is trained using a set of triphone transcripts to generate the context dependent models.", "14.The acoustic model of claim 13 wherein a decision tree is constructed for each state of each center phone of the single mixture monophone models and context dependent states of each respective center phone are clustered into corresponding groups using a decision tree algorithm to obtain the set of tied states, each tied having a corresponding distribution of training data.", "15.The acoustic model of claim 14 wherein the parameters of the tied states are derived by adapting the parameters of the context independent models by the MAP adaptation method using the training data associated with the tied states of the context dependent models.", "16.The acoustic model of claim 15 wherein new sufficient statistic estimates associated with the parameters of the context independent models are computed and combined with old sufficient statistic estimates from the parameters of the context independent models using a data dependent mixing coefficient.", "17.The acoustic model of claim 16 wherein, based upon the data dependent mixing coefficient, mixtures with high counts of data from the tied states of the context dependent models rely more on the new sufficient statistics for final parameter estimation.", "18.The acoustic model of claim 16 wherein, based upon the data dependent mixing coefficient, mixtures with low counts of data from the tied states of the context dependent models rely more on the old sufficient statistics for final parameter estimation.", "19.A system comprising: a feature extraction unit to convert an input signal representing an input speech into a set of feature vectors, each feature vector representing a corresponding frame of the input signal; an acoustic model including a set of context dependent models having a set of tied states whose parameters are estimated using a data dependent maximum a posteriori (MAP) adaptation method wherein parameters of the tied states are derived based on corresponding parameters of a set of multiple mixture context independent models using training data associated with the respective tied states; and a decoder coupled to the feature extraction unit and the acoustic model, the decoder to recognize the input speech based, at least in part, upon the feature vectors and the acoustic model.", "20.The system of claim 19 wherein a set of multiple mixture monophone models is trained using a set of monophone transcripts to generate the corresponding context independent models and wherein single mixture monophone models are split up and re-estimated iteratively for a predetermined number of iterations to obtain the set of multiple mixture monophone models.", "21.The system of claim 19 wherein a set of single mixture triphone models is trained using a set of triphone transcripts to generate the context dependent models and wherein a decision tree is constructed for each state of each center phone of the single mixture monophone models and context dependent states of each respective center phone are clustered into corresponding groups using a decision tree algorithm to obtain the set of tied states, each tied having a corresponding distribution of training data.", "22.The system of claim 21 wherein the parameters of the tied states are derived by adapting the parameters of the context independent models by the MAP adaptation method using the training data associated with the tied states of the context dependent models.", "23.The system of claim 22 wherein new sufficient statistic estimates associated with the parameters of the context independent models are computed and combined with old sufficient statistic estimates from the parameters of the context independent models using a data dependent mixing coefficient.", "24.The system of claim 23 wherein, based upon the data dependent mixing coefficient, mixtures with high counts of data from the tied states of the context dependent models rely more on the new sufficient statistics for final parameter estimation.", "25.The system of claim 24 wherein, based upon the data dependent mixing coefficient, mixtures with low counts of data from the tied states of the context dependent models rely more on the old sufficient statistics for final parameter estimation.", "26.A machine-readable medium comprising instructions which, when executed by a machine, cause the machine to perform operations including: training a set of multiple mixture monophone models using a set of monophone transcripts to generate a set of multiple mixture context independent models; training a set of single mixture triphone models using set of triphone transcripts to generate a set of context dependent models; and estimating parameters of the context dependent models using a data dependent maximum a posteriori (MAP) adaptation method wherein parameters of tied states of the context dependent models are derived by adapting corresponding parameters of the context independent models through MAP adaptation method using training data associated with the corresponding tied states.", "27.The machine-readable medium of claim 26 wherein training the set of triphone models further including; constructing a decision tree for each state of each center phone of the single mixture triphone models: and clustering context dependent states of each respective center phone into corresponding groups using a decision tree algorithm to obtain a set of corresponding tied states, each tied state having a corresponding distribution of training data.", "28.The machine-readable medium of claim 27 wherein estimating parameters including: deriving parameters of the tied states of the context dependent models based upon the parameters associated with the context independent models and the training data associated with the tied states of the context dependent models; 29.The machine-readable medium of claim 28 wherein deriving the parameters of the tied states of the context dependent models includes: adapting the parameters of the context independent models by the maximum a posteriori (MAP) adaptation method using training data associated with the tied states of the context dependent models, including: computing new sufficient statistic estimates associated with the parameters of the context independent models; and combining the new sufficient statistic estimates with old sufficient statistic estimates from the parameters of the context independent models using a data-dependent mixing coefficient.", "30.The machine-readable medium of claim 29 wherein, based upon the data-dependent mixing coefficient, mixtures of high counts of data from the tied states of the context dependent models rely more on the new sufficient statistic estimates for final parameter estimation and mixtures of low counts of data from the tied states of the context dependent models rely more on the old sufficient statistic estimates for final parameter estimation." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words).to determine the most probable sentence or word sequence given the input sequence of observations.", "In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.", "In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).", "In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMMs.", "In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.", "In practice, contextual effects can cause significant variations with respect to the way different sounds are produced.", "Contextual variations of sounds can be more accurately modeled using context dependent models.", "In other words, to achieve good phonetic discrimination, different CDHMMs have to be trained for each different context.", "In general, triphone models have been used as context dependent models in which every phone has a distinct HMM model for every unique pair of left and right neighbors.", "The use of Gaussian mixture output distribution allows each state distribution to be modeled accurately.", "However, when context dependent models (e.g., triphones) are used, there is a very large number of parameters to train with little or insufficient training data.", "One of the early approaches to deal with this problem is to tie all Gaussian components together to form a pool which is shared among HMM states.", "This approach is called tied-mixture approach.", "In a tied-mixture system, only the mixture component weights are state-specific and they can smoothed by interpolating with context dependent models.", "Recently, another approach called decision tree state tying has been used to improve the trainability of speech recognition systems and to strike a better balance between the level of detail of the phonetic models (e.g., the number of parameters in the system) and the ability to accurately estimate those parameters from the available training data.", "Context modeling based on decision tree state tying approach has become increasingly popular for modeling speech variations in LVCSR systems.", "In the conventional framework, the stochastic classifier for each tied state is trained using the Baum-Welch algorithm with the training data corresponding to the specific tied state.", "However, the context dependent classifiers trained using this conventional method are not very reliable because the training data corresponding to each tied state is still limited and model parameters can be easily affected by undesired sources of information such speaker and channel information contained in the training data." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The features of the present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.", "1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.", "2 is a diagram illustrating an HMM-based phone model; FIG.", "3 is a diagram illustrating a plurality of triphones in which similar states are tied together; FIG.", "4 illustrates an example of a decision tree that is used for state clustering or state tying; FIG.", "5 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; and FIG.", "6 shows a flow diagram of one embodiment of a method according to the teachings of the present invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to the field of speech recognition.", "More specifically, the present invention relates to a method, apparatus, and system for building context dependent models for a large vocabulary continuous speech recognition (LVCSR) system using a context adaptation approach.", "BACKGROUND OF THE INVENTION Modern speech recognition systems are based on principles of statistical pattern recognition and typically employ an acoustic model and a language model to decode an input sequence of observations (also referred to as acoustic events or acoustic signals) representing an input speech (e.g., a sentence or string of words).to determine the most probable sentence or word sequence given the input sequence of observations.", "In other words, the function of a modern speech recognizer is to search through a vast space of potential or candidate sentences and to choose the sentence or word sequence that has the highest probability of generating the input sequence of observations or acoustic events.", "In general, most modern speech recognition systems employ acoustic models that are based on continuous density hidden Markov models (CDHMMs).", "In particular, CDHMMs have been widely used in speaker-independent LVCSR because they outperform discrete HMMs and semi-continuous HMMs.", "In CDHMMs, the probability function of observations or state observation distribution is modeled by multivariate mixture Gaussians (also referred to herein as Gaussian mixtures) which can approximate the speech feature distribution more accurately.", "In practice, contextual effects can cause significant variations with respect to the way different sounds are produced.", "Contextual variations of sounds can be more accurately modeled using context dependent models.", "In other words, to achieve good phonetic discrimination, different CDHMMs have to be trained for each different context.", "In general, triphone models have been used as context dependent models in which every phone has a distinct HMM model for every unique pair of left and right neighbors.", "The use of Gaussian mixture output distribution allows each state distribution to be modeled accurately.", "However, when context dependent models (e.g., triphones) are used, there is a very large number of parameters to train with little or insufficient training data.", "One of the early approaches to deal with this problem is to tie all Gaussian components together to form a pool which is shared among HMM states.", "This approach is called tied-mixture approach.", "In a tied-mixture system, only the mixture component weights are state-specific and they can smoothed by interpolating with context dependent models.", "Recently, another approach called decision tree state tying has been used to improve the trainability of speech recognition systems and to strike a better balance between the level of detail of the phonetic models (e.g., the number of parameters in the system) and the ability to accurately estimate those parameters from the available training data.", "Context modeling based on decision tree state tying approach has become increasingly popular for modeling speech variations in LVCSR systems.", "In the conventional framework, the stochastic classifier for each tied state is trained using the Baum-Welch algorithm with the training data corresponding to the specific tied state.", "However, the context dependent classifiers trained using this conventional method are not very reliable because the training data corresponding to each tied state is still limited and model parameters can be easily affected by undesired sources of information such speaker and channel information contained in the training data.", "BRIEF DESCRIPTION OF THE DRAWINGS The features of the present invention will be more fully understood by reference to the accompanying drawings, in which: FIG.", "1 is a block diagram of one embodiment of a speech recognition system according to the teachings of the present invention; FIG.", "2 is a diagram illustrating an HMM-based phone model; FIG.", "3 is a diagram illustrating a plurality of triphones in which similar states are tied together; FIG.", "4 illustrates an example of a decision tree that is used for state clustering or state tying; FIG.", "5 shows a flow diagram of one embodiment of a method according to the teachings of the present invention; and FIG.", "6 shows a flow diagram of one embodiment of a method according to the teachings of the present invention.", "DETAILED DESCRIPTION In the following detailed description numerous specific details are set forth in order to provide a thorough understanding of the present invention.", "However, it will be appreciated by one skilled in the art that the present invention may be understood and practiced without these specific details.", "In the discussion below, the teachings of the present invention are utilized to implement a method, apparatus, system, and machine-readable medium for building context dependent models for a speech recognition system using a context adaptation approach.", "In one embodiment, a set of multiple mixture monophone models are created and trained to generate multiple mixture context independent models.", "A set of single mixture triphone models are also created and trained to generate context dependent models having a set of tied states.", "The parameters of the context dependent models are then estimated using a data dependent maximum a posteriori (MAP) adaptation method in which the parameters of the tied states of the context dependent models are derived by adapting corresponding parameters of the context independent models through MAP adaptation method using the corresponding training data associated with the respective tied states.", "In one embodiment, a set of single mixture monophone models is used to generate the multiple mixture monophone models.", "The single mixture models are split up and the corresponding model parameters are re-estimated to increase the number of mixtures.", "The split up and re-estimating process is repeated several times to obtain the multiple mixture context independent models.", "In one embodiment, after the single mixture triphone models have been created and trained, a decision tree is constructed for each state of each center phone and the corresponding context dependent states of each respective center phone are clustered into corresponding groups using a decision tree algorithm to obtain a set of tied states.", "The parameters of the tied states are then derived based upon the parameters associated with the context independent models and the training data associated with the specific tied states of the context dependent models.", "In one embodiment, the parameters of the tied states are derived by adapting the parameters of the context independent models by MAP adaptation method using the training data associated with the respective tied states.", "In one embodiment, new sufficient statistic estimates associated with the parameters of the context independent models are computed first and then combined with old sufficient statistic estimates of the corresponding parameters using a data-dependent mixing coefficient.", "In one embodiment, the data dependent mixing coefficient is designed so that mixtures with high counts of data from the context dependent tied states rely more on the new sufficient statistic estimates for final parameter estimation and the mixtures with low counts of data from the context dependent tied states rely more on the old sufficient statistic estimates for final parameter estimation.", "The teachings of the present invention are applicable to any method, apparatus, and system for speech recognition that employs state tying in building context dependent models.", "However, the present invention is not limited to state tying context modeling and can be applied to other types of acoustic modeling in speech recognition systems.", "In addition, the present invention can also be applied to data modeling in other fields or disciplines including, but not limited to, image processing, signal processing, geometric modeling, computer-aided-design (CAD), computer-aided-manufacturing (CAM), etc.", "FIG.", "1 illustrates a block diagram of one embodiment of a speech recognition system 100 according to the teachings of the present invention.", "The system 100, as shown in FIG.", "1, includes an analog to digital converter (A/D) 110, a feature extractor or spectral analysis unit 120, a decoder 130, an acoustic model 140, and a language model 150.An input signal 105 representing the input speech is first digitized using the A/D 110.The digital signal is then sliced up into frames typically of 10, 15, or 20 ms. Each frame of the signal is then converted into a corresponding feature vector which is used to characterize the spectral properties of the input signal.", "In the present embodiment, the feature vector is multi-dimensional and has a plurality of feature components.", "In one embodiment, the feature components include 12 MFCC components, 12 delta MFCCs, and 12 delta-delta MFCCs.", "As such the feature vectors have 36 dimensions.", "The feature vectors generated by the feature extractor unit 120 are then inputted into the decoder 130 which determines the sentence or sequence of words that has the highest probability given the acoustic events characterized by the feature vectors, based upon the acoustic model 140 and the language model 150.The acoustic model 140, in one embodiment, includes a set of context dependent models having a set of tied states clustered based on a decision tree clustering algorithm.", "In one embodiment, the parameters of the context dependent models are estimated using a data dependent MAP adaptation method in which the tied states of the context dependent models are derived by adapting the parameters of previously trained context independent models through MAP adaptation method using the training data associated with the respective tied states.", "The acoustic model 140 and the construction of the context dependent models are described in greater detail below.", "FIG.", "2 illustrates a diagram of one embodiment of an HMM-based phone model structure used in the acoustic model 140 to model a phonetic unit (e.g., a phoneme or a subword unit, etc.).", "Each individual phonetic unit is represented or modeled by a corresponding HMM.", "As shown in FIG.", "2, an HMM has a set of sequence of states (1-5) that are connected by a set of transition probabilities (a12,a23,a34,a45), and a set of observation probabilities or likelihoods (b2(o1,b2(o2),b3(o3),b4(o4),b4(o5),b4(o6)).", "Each transition probability aij represents the probability of transitioning from a state i to a state j.", "Each observation probability or distribution bi(oj)represents the probability of an observation vector oj being generated from a state i.", "Thus, the transition probabilities model the durational variability in speech and the output probabilities model the spectral variability.", "Accordingly, the set of states, the set of transition probabilities and the set of output probabilities are the parameters that are used to define an HMM.", "The HMM shown in FIG.", "2 has a left-right topology.", "In many modern speech recognition systems, each state output distribution or observation probability function is modeled by a multivariate mixture Gaussian as follows: bj(oi)=k=1McjkN(oi,mjk,Vjk) where cjk is the weight of mixture component k in state j and N(oi,mjk,Vjk) denotes a multivariate Gaussian of mean mjk and covariance Vjk for the kth mixture component in state j.", "As discussed above, the use of continuous density HMMs for the acoustic models results in a very large number of model parameters to train with insufficient training data.", "Thus, various approaches, in particular the decision tree based state tying method, have been developed in recent years to strike a good balance between model complexity and available training data.", "FIG.", "3 shows an example of a set of context dependent models (e.g., triphones) with similar states being tied or clustered based on decision trees.", "This allows the data associated with each individual state to be pooled in order to give more robust estimates for the parameters of the respective tied state.", "As illustrated in FIG.", "4, each triphone (e.g., TR1-TR4) has its own output distribution before state tying.", "After state tying, several states share the same output distributions.", "FIG.", "4 illustrates an example of a decision tree clustering process in which all corresponding states of all allophonic variants of each basic phone (e.g.", "also called monophone or center phone) are clustered based on a decision tree constructed for each state of each basic phone.", "Each tree has a yes/no phonetic question (e.g., “Is the right context a nasal”, etc.)", "at each node.", "Initially, all corresponding states of all allophonic variants of each basic phone are tied to form a single pool.", "Phonetic questions are then used to cluster the pool into corresponding subsets.", "All states in the same leaf node are then tied.", "For example, as shown in FIG.", "4, a decision tree is used to tie the center states of all triphones that are derived from the monophone or basic phone “aw”.", "All of the corresponding states traverse down the respective tree and end up at one of the leaf nodes depending on the answer to each of the phonetic questions.", "As discussed above, in the conventional framework of context dependent model training, the stochastic classifier for each tied state is trained using Baum-Welch algorithm with the training data corresponding to the specific tied state.", "However, the context dependent classifiers trained using this conventional method are not very reliable because the training data corresponding to each tied state is limited and model parameters are easily affected by undesired sources of information such as speaker and channel information contained in the training data.", "As described herein, the present invention provides a new framework and method for building context dependent models that are more robust and more reliable than those that are trained by the conventional method.", "In one embodiment, according to the teachings of the present invention, all states of each center phone of context dependent models are clustered into groups by a decision tree algorithm.", "The tied states of context dependent models are then derived by adapting the parameters of previously trained multiple-mixture context independent models using the training vectors corresponding to the tied state and maximum a posteriori probability (MAP) method.", "In one embodiment, a set of multiple mixture monophone models are trained to generate the multiple mixture context independent models.", "A set of single mixture triphone models are also crated and trained to generate an initial set of untied context dependent models.", "Context dependent states of the context dependent models are then clustered into groups using a decision tree clustering algorithm to generate a set of tied states for the context dependent models.", "After state tying of the context dependent models, the parameters of the tied states are then estimated by adapting the parameters of the context independent models through MAP method using training data corresponding to the tied states.", "In this new framework according to the teachings of the present invention, the multiple mixture context independent models cover the space of broader speaker and environment classes of speech signal.", "The adaptation using MAP method is considered as the context dependent tuning of those speaker and environment classes observed in different contexts of training speech.", "Mixture parameters for those speaker and environment classes not observed in the training speech of a specific tied state can be copied from the context independent model.", "The construction and training of context dependent models according to the teachings of the present invention are described in more details below.", "In one embodiment, the construction and training process of context dependent models according to the present invention includes the following phases: a context independent model training phase, a triphone model training and decision tree based clustering phase, and a context dependent model training and adaptation phase.", "Each phase of the process is described in detail below.", "Context Independent training: In this phase, multiple-mixture monophone models are trained as the context independent models.", "The mixture splitting up process is the same as a baseline system.", "It is well known that the speech signal contains information about various sources.", "Speech recognition systems need to focus on linguistic information while ignoring other undesired sources of information such as speaker and channel information.", "One way to achieve this is by training stochastic classifiers with data that contain the various sources of information.", "For example speaker independence can be achieved in a speech recognition system by training the system using speech data collected from multiple speakers.", "Multiple mixture context independent models are constructed to cover the space of broader speaker and environment classes of speech signal.", "High prediction and classification power can be obtained with the context dependent models adapted from these multiple mixture context independent models as described below.", "In one embodiment, the context independent models (e.g., monophone models) are built using Baum-Welch algorithm as in the conventional framework except that multiple mixture monophone models are built instead of single mixture models in the convention framework.", "To construct multiple mixture context independent models as background models for context adaptation, single mixture models are split up and the model parameters are re-estimated.", "After several splitting and re-estimating iterations, multiple mixture context independent models are obtained.", "In various experiments according to teachings of the present invention various numbers of mixtures per state were tried (e.g., 24, 32, 48, 56 mixtures per state, etc.).", "Triphone Model Training and Decision Tree Based State Clustering: Before decision tree clustering, a set of single mixture triphone models are constructed and trained.", "In one embodiment, the single mixture triphone models are built as in the conventional framework.", "After the initial triphone models are created and trained, one decision tree is then constructed for each state of each center phone and all the context dependent states of the respective phone are clustered into groups by the decision tree algorithm.", "The node splitting and model selection of decision tree based state tying is a data-driven procedure.", "It integrates both a priori phonetic knowledge and acoustics similarities derived from training data.", "However, because linguistic information and other unwanted information are bound together in an unknown way in the current spectral-based features, training data corresponding to each tied sate may not only contain similar acoustic information but also contain similar speaker and channel information.", "In the conventional framework, while the parameters of the context dependent model are estimated only with these biased data using Baum-Welch algorithm, the context dependent models will not be reliable and robust.", "Context Dependent Model Training Using Data-Dependent Adaptation Coefficient In one embodiment, to make the context dependent models more robust and reliable, the tied states of context dependent models are derived by adapting the parameters of the context independent model through MAP method using the training data corresponding to the tied states.", "In one embodiment, the adaptation process is a two-part estimation process.", "In the first part, sufficient statistic estimates for the weight, mean, and covariance of the Gaussian mixtures are computed.", "This part is identical to the first step of Baum-Welch algorithm.", "In the second part, the new sufficient statistic estimates are then combined with the old sufficient statistics from the context independent parameters using a data-dependent mixing coefficient.", "In one embodiment, the data-dependent mixing coefficient is designed so that mixtures with high counts of data from the context dependent clustered state rely more on the new sufficient statistics for final parameter estimation and mixtures with low counts of data from context dependent clustered state rely more on the old sufficient statistics for final parameter estimation.", "For a given context dependent model and the corresponding training vectors, X={x1, .", ".", ".", ", xτ}, the probabilistic alignment of the training vectors into the mixture components is determined.", "That is, for a mixture i in this model, the probabilistic alignment is computed as follow: Pr(i|xi)=wipi(xi)/j=1Mwjpj(xi) where wi is the weight of ith mixture and pi(x) is the Gaussian densities corresponding to the ith mixture.", "Pr(i|xi) and xi are then used to compute the sufficient statistics for the weight, mean, and variance parameters as shown below: ni=i=1TPr(i|xi); Ei(x)=1/ni i=1TPr(i|xi)xi; Ei(x2)=1/ni i=1TPr(i|xi)xi2; For each mixture i of the context dependent cluster state j, the adaptation of sufficient statistics from the training data can be written as follows: {overscore (wi)}=[αiwni/T+(1−α)wi]γ (1) {overscore (μi)}=αimEi(x)+(1−αim)μi (2) {overscore (σi2)}=αivEi(x2)+(1−αiv)(σi2+μi2)−{overscore (μi2)} (3) The adaptation coefficients controlling the balance between old and new estimates are {αiv, αim, αiv} for the weights, means and variances, respectively.", "The scale factor, γ, is computed over all adapted mixture weights to ensure they sum to unity.", "For each mixture i of the context dependent cluster state j and each parameter, a data-dependent adaptation coefficient αip,p└ {w,m,v}, is used in the above equations.", "This is defined as follows: αip=ni/ni+rp (4) where rp is a fixed relevance factor for parameter ρ.", "Using a data-dependent adaptation coefficient allows a mixture-dependent adaptation of parameters.", "If a mixture component has a low probabilistic count, ni, of new data, then αip♦0 causing the deemphasis of the new (potentially under trained) parameters and the emphasis of the old (better trained) parameters.", "For mixture components with high probabilistic counts, αip♦1, causing the use of the new context-dependent parameters.", "In one embodiment, the relevance factor is a way of controlling how much new data should be observed in a mixture before the new parameters are used to replace the old parameters.", "This approach should thus be more robust given limited training data.", "The use of parameter-dependent relevance factors (and hence parameter-dependent adaptation coefficients αip) can further allow for the tuning of different adaptation rates for the weights, means, and variances.", "In one embodiment, a single adaptation coefficient is used for all parameters (αiw=αim=αiv=ni/(ni+r)) with the same relevance factor.", "In this case, for mixture component with probabilistic counts larger than this relevance factor, the new parameters are emphasized, otherwise the old parameters are emphasized.", "Since the adaptation is data dependent, not all Gaussians in the context independent models are adapted during the context dependent model training phase.", "In various experiments according to the teachings of the present invention, there are usually about 1/10 of the Gaussian mixtures of the final context dependent models that are not updated from the context independent models and the parameters of these mixtures are merely copied from the context independent models.", "This factor can be used to reduce model storage requirements.", "In this new framework of building context dependent models, each tied state can be considered as a context class.", "Accordingly, every context class contains its own context information.", "To improve the prediction power of the context dependent models, the distribution parameters of each classifier are estimated not only from its own training data but also from the context independent models.", "Thus the context adaptation method described herein can be considered as a smooth technique.", "FIG.", "5 illustrates a flow diagram of one embodiment of a method according to the teachings of the present invention.", "At block 510, single mixture monophone models are built.", "At block 520, multiple mixture monophone models (context independent models) are built based on the single mixture monophone models.", "At block 525, single mixture triphone models are built as the initial context dependent models.", "At block 530, decision tree based state tying is performed to create tied states for the context dependent models.", "At block 540, parameters of the context dependent models are estimated using a data dependent MAP adaptation method.", "FIG.", "6 shows a flow diagram of a method according to the teachings of the present invention.", "At block 610, a set of multiple mixture monophone models is trained based on a set of monophone transcripts to generate a set of multiple mixture context independent models.", "At block 620, a set of single mixture monophone models is trained based on a set of triphone transcripts to generate a set of context dependent models.", "At block 630, parameters of the context dependent models are estimated using a data dependent MAP adaptation method in which parameters of the tied states of the context dependent models are derived by adapting the corresponding parameters of the context independent models through MAP adaptation method using the training data corresponding to the respective tied states.", "The invention has been described in conjunction with the preferred embodiment.", "It is evident that numerous alternatives, modifications, variations and uses will be apparent to those skilled in the art in light of the foregoing description." ] ]

[ [ "Dna sequences coding for a polyol carrier and use thereof, in particular for preparing transgenic plants", "The invention concerns the use of a DNA sequence coding for a polyol carrier, in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, more preferably 6 carbon atoms, the polyols being advantageously selected among mannitol, sorbitol, dulcitol, galactitol, inositol, myo-inositol, ribitol and xylitol, and being preferably mannitol, for preparing transgenic plants." ], [ "1.Use of a DNA sequence coding for a linear polyol carrier, in plants and fungi, such as polyols having main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, myo-inositol, ribitol and xylitol, and being in particular mannitol, for the preparation of transgenic plants.", "2.Use according to claim 1, in which the DNA sequence is chosen from one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.3.Protein characterized in that it comprises or is constituted by: sequence SEQ ID NO: 2, or any sequence derived from SEQ ID NO: 2, in particular by substitution, suppression or addition of one or more amino acids, having the property of transporting linear polyols in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, ribitol and xylitol, and being in particular mannitol, any homologous sequence of SEQ ID NO: 2, preferably having a homology of at least approximately 50% with sequence SEQ ID NO: 2 and possessing the property of transporting, in plants and fungi, polyols as defined above, or any fragment of one of the sequences defined above, on the condition that it possesses the property of transporting, in plants and fungi, polyols as defined above, in particular any fragment being constituted by at least approximately 10 amino acids adjacent in the sequence SEQ ID NO: 2.4.Nucleotide sequence coding for a protein as defined in claim 3.5.DNA sequence which comprises or is constituted by: nucleotide sequence SEQ ID NO: 1, or any nucleotide sequence derived, by degeneration of the genetic code, from the sequence SEQ ID NO: 1 coding for a protein represented by SEQ ID NO: 2, or any nucleotide sequence derived, in particular by substitution, suppression or addition of one or more nucleotides, from the sequence SEQ ID NO: 1 coding for a protein derived from SEQ ID NO: 2, as defined in claim 3, or any homologous nucleotide sequence of SEQ ID NO: 1, preferably having a homology of at least approximately 35% with the sequence SEQ ID NO: 1 coding for a homologous protein of SEQ ID NO: 2, as defined in claim 3, or any fragment of the nucleotide sequence SEQ ID NO: 1 or of the nucleotide sequences defined above, said fragment being preferably constituted of at least approximately 30 nucleotides adjacent in said sequence, or any complementary nucleotide sequence of the above-mentioned sequences or fragments, or any nucleotide sequence capable of hybridizing in stringent conditions with the complementary sequence of one of the abovementioned sequences fragments.", "6.Recombinant vector, in particular plasmid, cosmid, phage or virus DNA, containing a nucleotide sequence according to any one of claims 4 or 5.7.Recombinant vector according to claim 6, containing the elements necessary for the expression in a host cell of polypeptides coded by the nucleic acids according to one of claims 4 or 5, inserted into said vector.", "8.Host cell, chosen in particular from bacteria, viruses, yeasts, fingi, plants or mammal cells, said host cell being transformed, in particular using a recombinant vector according to any one of claims 6 or 7.9.Antisense oligonucleotides or antisense messenger RNA derivatives of the nucleotide sequences according to one of claims 4 or 5.10.Plant cells containing in their genome a nucleotide sequence according to one of claims 4 or 5.11.Transgenic plants, parts of plants, plant seeds or plant propagation material containing cells according to claim 10.12.Transgenic plant according to claim 11, which, in its native state, does not contain or express the gene of the mannitol carrier, into the genome of which is introduced a nucleotide sequence according to one of claims 4 or 5.13.Transgenic plant according to claim 11, which, in its native state, contains or expresses the gene of the mannitol carrier, into the genome of which is introduced a nucleotide sequence according to one of claims 4 or 5.14.Process of screening genetically modified plants with at least one nucleotide sequence of interest which comprises the following stages: the transformation of plant cells with a vector containing an insertion sequence, said insertion sequence comprising the nucleotide sequence of interest and a nucleotide sequence coding for a polyol carrier as defined in one of claims 1, 2, 4 and 5, the culture of the cells thus transformed on a medium containing said polyol as the only source of carbon, to obtain transgenic plants or fragments of transgenic plants containing said insertion sequence.", "15.Process for obtaining transgenic plants resistant to pathogens, which comprises the following stages: the transformation of plant cells with a nucleotide sequence coding for a polyol carrier as defined in one of claims 1, 2, 4 and 5, the culture of the cells thus transformed to obtain transgenic plants or fragments of transgenic plants.", "16.Process for obtaining transgenic plants resistant to saline stress, which comprises the following stages: the transformation of plant cells with a nucleotide sequence coding for a polyol carrier as defined in one of claims 1, 2, 4 and 5, the culture of the cells thus transformed to obtain transgenic plants or fragments of transgenic plants." ], [ "The invention relates to DNA sequences coding for a polyol carrier and their use, in particular for the preparation of transgenic plants.", "The plants are capable of synthesizing, via photosynthesis, primary compounds such as glucides by using light energy.", "Only certain organs of the plant, mainly the adult leaves, are capable of manufacturing and exporting the glucides towards the storage organs, such as the tubers, the seeds and the fruits, used in human and animal foodstuffs.", "In the majority of plants, the main glucide transported is saccharose, but in a large number of plants, other compounds are also transported such as polyols of which mannitol is an example.", "Polyols are, like saccharose, primary products of photosynthesis.", "It has furthermore been estimated that approximately 30% of the global production of primary carbon was used for the synthesis of polyols.", "Polyols, cyclic or non-cyclic, are very widespread in plants; they are low-molecular weight, very soluble and non-reducing compounds.", "The three non-cyclic polyols (alditols) which are most widespread amongst the Angiosperms are galactitol, sorbitol and mannitol.", "Sorbitol is the main photosynthetic product in several species of Rosaceae such as the apple, the pear, the peach and the plum.", "Mannitol, the most widespread of the alditols, is present in more than 100 species of higher plants, in particular in the Rubiaceae (coffee), the Oleaceae (privet, ash, olive) and the Apiaceae (celery, carrot, parsley) (Lewis, 1984).", "It is produced in the mesophyll cells (cells containing chlorophyll).", "To circulate, it must re-enter the sieve tubes (veins).", "However, there is no continuity between the mesophyll cells and the sieve tubes: a mannitol carrier is therefore needed.", "In this way, the mannitol leaves the mesophyll cells and uses the carrier to enter the sieve tubes.", "The compounds synthesized in the adult leaves are transported towards the storage organs and cross a certain number of membranes using the specialized proteins that are the carriers.", "These carriers play a considerable role in the plant as they are essential for its growth.", "The existence of a mannitol carrier in a plant such as celery has been shown by different biochemical experiments (Salmon et al., 1995).", "This publication has shown that there was a mannitol carrier in celery and that the expression of this carrier was very sizeable in the tissues of the phloem.", "However, nothing is said as to the identification of the mannitol carrier.", "If numerous carriers of sugars, such as saccharose and the hexoses have been cloned during the course of the last few years, none of them is capable of transporting polyol.", "At present, no carrier of linear polyol has been identified in a living organism.", "In bacteria, a multienzymatic system capable of both transporting and phosphorylating mannitol has been described (Boer et al., 1994).", "However, such systems have never been described in the higher organisms.", "A subject of the invention is carriers of polyols in plants and fungi, and their DNA sequences.", "A subject of the invention is also the use of DNA sequences of a polyol carrier for obtaining transgenic plants.", "A subject of the invention is also the use of DNA sequences of a polyol carrier, in particular within the scope of obtaining plants resistant to pathogens or plants resistant to saline stress.", "A subject of the invention is also the use of DNA sequences of a polyol carrier within the scope of a method of screening genetically modified plants.", "The invention relates to the use of a DNA sequence coding for a linear polyol carrier, in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, ribitol and xylitol, and being in particular mannitol, for the preparation of transgenic plants.", "In the expression “plants and fungi”, are included algae, mosses (Bryophytes), ferns (Pteridophytes), higher plants (Gymnosperms and Angiosperms) and fungi.", "It can be recalled that, by definition, a polyol is a “polyalcohol” containing as many alcohol functions as carbon atoms.", "It can also be specified that the terms polyol, polyalcohol and alcohol sugar are equivalents.", "According to an advantageous embodiment, the invention relates to the use, for the preparation of transgenic plants, of a DNA sequence chosen from one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.SEQ ID NO: 1 is a new nucleic acid sequence identified in celery (Apium graveolens L.), coding for a mannitol carrier.", "SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 are sequences of nucleic acids coding for proteins, the functions of which were unknown until now.", "SEQ ID NO: 3 (Beet 1) and SEQ ID NO: 4 (Beet 2) originate from the Beetroot (Beta vulgaris).", "SEQ ID NO: 5 (Pst 1), SEQ ID NO: 6 (Pst 2), SEQ ID NO: 7 (Pst 3), SEQ ID NO: 8 (Pst 4) and SEQ ID NO: 9 (Pst 5) originate from Arabidopsis thaliana.", "SEQ ID NO: 10 (Bs) originates from Bacillus subtilis.", "The invention also relates to a new protein, characterized by the fact that it comprises or is constituted by: sequence SEQ ID NO: 2, or any sequence derived from SEQ ID NO: 2, in particular by substitution, suppression or addition of one or more amino acids, having the property of transporting linear polyols in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, in particular 6 carbon atoms, these polyols being advantageously chosen from mannitol, sorbitol, dulcitol, galactitol, inositol, ribitol and xylitol, and being in particular mannitol, any homologous sequence of SEQ ID NO: 2, preferably having a homology of at least approximately 50% with sequence SEQ ID NO: 2 and possessing the property of transporting, in plants and fungi, polyols as defined above, or any fragment of one of the sequences defined above, on the condition that it has the property of transporting, in plants and fungi, polyols as defined above, in particular any fragment being constituted of at least approximately 10 amino acids adjacent in the sequence SEQ ID NO: 2.The property of transporting polyols presented by a polyol carrier can be verified by one or other of the following tests: the use of S. cerevisiae yeast or the use of purified plasmic membrane of phloem vesicles.", "The use of the yeast Saccharomyces cerevisiae (Noiraud et al., 2000) comprises the transformation of yeasts with the nucleotide sequence to be tested, these yeasts are capable of growing on said polyol.", "To verify that the polyol is transported in these yeasts, radioactively labelled polyol can be used.", "For each experiment, a control is perfected with a strain of yeast incapable of growing on the polyol, and which does not transport said polyol.", "The test using a purified plasmic membrane from phloem vesicles is that described by Salmon et al.", "(1995).", "According to an advantageous embodiment of the invention, the protein of the invention, as defined above, is characterized in that it is constituted by the sequence SEQ ID NO: 2.The invention also relates to the protein fragments as defined above, chosen from the following sequences: Ala Cys Ala Leu Leu Ala Ser Met Asn Ser Ile Leu Leu Gly Tyr Asp Thr Gly Val Leu Ser Gly Ala Ser Ile (SEQ ID NO: 11) delimited from the amino acid in position (26) to the amino acid in position (50) of the sequence SEQ ID NO: 2, Gln Ile Glu Ile Ile Ile Gly Ile Ile Asn Ile Tyr Ser Leu Leu Gly Ser Ala Ile Ala Gly (SEQ ID NO: 12) delimited from the amino acid in position (62) to the amino acid in position (82) of the sequence SEQ ID NO: 2, Tyr Thr Met Val Leu Ala Gly Ile Ile Phe Phe Leu Gly Ala Ile Phe Met Gly Leu Ala (SEQ ID NO: 13) delimited from the amino acid in position (92) to the amino acid in position (111) of the sequence SEQ ID NO: 2, Phe Leu Met Phe Gly Arg Phe Val Ala Gly Ile Gly Val Gly Tyr Ala Met Met Ile Ala Pro Val Tyr Thr Ala (SEQ ID NO: 14) delimited from the amino acid in position (116) to the amino acid in position (140) of the sequence SEQ ID NO: 2, Phe Leu Thr Ser Phe Pro Glu Val Phe Ile Asn Ser Gly Val Leu Leu Gly Tyr Val Ser Asn Phe Ala Phe Ala (SEQ ID NO: 15) delimited from the amino acid in position (150) to the amino acid in position (174) of the sequence SEQ ID NO: 2, Ile Met Leu Gly Ile Gly Ala Phe Pro Ser Val Ala Leu Ala Ile Ile Val Leu Tyr Met (SEQ ID NO: 16) delimited from the amino acid in position (184) to the amino acid in position (203) of the sequence SEQ ID NO: 2, Ala Ala Ile Thr Gly Ile Gly Ile His Phe Phe Gln Gln Ala Cys Gly Ile Asp Ala Val Val Leu (SEQ ID NO: 17) delimited from the amino acid in position (281) to the amino acid in position (302) of the sequence SEQ ID NO: 2, Leu Leu Ala Thr Ile Ala Val Gly Val Cys Lys Thr Val Phe Ile Leu Ile Ser Thr Phe (SEQ ID NO: 18) delimited from the amino acid in position (320) to the amino acid in position (339) of the sequence SEQ ID NO: 2, Leu Met Leu Thr Ser Met Gly Gly Met Val Ile Ala Leu Phe Val Leu Ala Gly Ser Leu Thr Val (SEQ ID NO: 19) delimited from the amino acid in position (349) to the amino acid in position (370) of the sequence SEQ ID NO: 2, Gly Gly Leu Ala Ile Phe Thr Val Tyr Ala Phe Val Ser Ile Phe Ser Ser Gly Met Gly Pro Ile Ala Trp Val Tyr (SEQ ID NO: 20) delimited from the amino acid in position (382) to the amino acid in position (407) of the sequence SEQ ID NO: 2, Cys Ser Ile Gly Val Ala Val Asn Arg Gly Met Ser Gly Ile Ile Gly Met Thr Phe Ile Ser (SEQ ID NO: 21) delimited from the amino acid in position (421) to the amino acid in position (441) of the sequence SEQ ID NO: 2, and Ala Phe Leu Leu Phe Ala Val Val Ala Ser Ile Gly Trp Val Phe Met Tyr Thr Met Phe (SEQ ID NO: 22) delimited from the amino acid in position (451) to the amino acid in position (470) of the sequence SEQ ID NO: 2.The invention also relates to a nucleotide sequence coding for a protein as defined above.", "An advantageous DNA sequence of the invention comprises or is constituted by: the nucleotide sequence SEQ ID NO: 1, or any nucleotide sequence derived by degeneration of the genetic code, of the sequence SEQ ID NO: 1 coding for a protein represented by SEQ ID NO: 2, or any nucleotide sequence derived, in particular by substitution, suppression or addition of one or more nucleotides, of the sequence SEQ ID NO: 1 coding for a protein derived from SEQ ID NO: 2, as defined above, or any homologous nucleotide sequence of SEQ ID NO: 1, preferably having a homology of at least approximately 35% with the sequence SEQ ID NO: 1 coding for a homologous protein of SEQ ID NO: 2, as defined above, or any fragment of the nucleotide sequence SEQ ID NO: 1 or of the nucleotide sequences defined above, said fragment being preferably constituted of at least approximately 30 nucleotides adjacent in said sequence, or any complementary nucleotide sequence of the above-mentioned sequences or fragments, or any nucleotide sequence capable of hybridizing in stringent conditions with the complementary sequence of one of the abovementioned sequences or fragments.", "By stringent conditions of hybridization is understood: temperature of hybridization: 65° C., hybridization medium: sodium phosphate buffer 250 mM, pH 7.2; 6.6% (w/v) of SDS; 1 mM EDTA; 1% (w/v) of bovine serum albumin, washing temperature: 65° C., successive rinsing media: 2×SSC (1.75% NaCl; 0.88% sodium citrate), SDS 0.1% 1×SSC (0.875% NaCl; 0.44% sodium citrate), SDS 0.1% 0.5×SSC (0.44% NaCl; 0.22% sodium citrate), SDS 0.1%.", "The invention also relates to the fragments of nucleotide sequences as defined above, chosen from the following sequences: GCT TGT GCT CTT TTA GCT TCC ATG AAT TCC ATC TTA CTC GGC TAT GAC ACC GGA GTG TTG AGT GGA GCA TCA ATA (SEQ ID NO: 23) delimited from the nucleotide in position (92) to the nucleotide in position (166) of the sequence SEQ ID NO: 1, CAA ATC GAA ATA ATC ATC GGA ATC ATC AAC ATC TAC TCT CTT CTT GGT TCG GCC ATA GCC GGA (SEQ ID NO: 24) delimited from the nucleotide in position (200) to the nucleotide in position (262) of the sequence SEQ ID NO: 1, TAC ACC ATG GTA CTA GCT GGT ATC ATA TTT TTT CTA GGA GCC ATT TTC ATG GGG CTT GCT (SEQ ID NO: 25) delimited from the nucleotide in position (290) to the nucleotide in position (349) of the sequence SEQ ID NO: 1, TTT CTC ATG TTT GGT CGC TTT GTT GCT GGA ATT GGT GTC GGT TAT GCC ATG ATG ATC GCT CCC GTC TAC ACT GCC (SEQ ID NO: 26) delimited from the nucleotide in position (362) to the nucleotide in position (436) of the sequence SEQ ID NO: 1, TTC CTC ACT TCT TTT CCT GAG GTT TTC ATT AAT TCT GGT GTG TTG CTC GGG TAT GTA TCC AAC TTT GCA TTT GCC (SEQ ID NO: 27) delimited from the nucleotide in position (464) to the nucleotide in position (538) of the sequence SEQ ID NO: 1, ATT ATG CTG GGA ATT GGA GCA TTT CCT TCA GTT GCC TTG GCC ATA ATT GTG TTA TAT ATG (SEQ ID NO: 28) delimited from the nucleotide in position (566) to the nucleotide in position (625) of the sequence SEQ ID NO: 1, GCT GCA ATT ACG GGT ATT GGT ATT CAT TTC TTC CAA CAG GCT TGT GGT ATT GAT GCT GTT GTT TTA (SEQ ID NO: 29) delimited from the nucleotide in position (857) to the nucleotide in position (922) of the sequence SEQ ID NO: 1, CTC CTT GCG ACA ATT GCT GTT GGA GTC TGC AAA ACA GTC TTT ATT CTG ATA TCA ACG TTT (SEQ ID NO: 30) delimited from the nucleotide in position (974) to the nucleotide in position (1033) of the sequence SEQ ID NO: 1, CTG ATG CTA ACA AGT ATG GGG GGT ATG GTT ATT GCT CTA TTT GTA CTG GCA GGC TCA TTG ACG GTT (SEQ ID NO: 31) delimited from the nucleotide in position (1061) to the nucleotide in position (1126) of the sequence SEQ ID NO: 1, GGT GGT TTG GCA ATA TTT ACA GTG TAT GCT TTT GTG TCG ATA TTT TCA AGT GGC ATG GGT CCA ATT GCT TGG GTC TAT (SEQ ID NO: 32) delimited from the nucleotide in position (1160) to the nucleotide in position (1237) of the sequence SEQ ID NO: 1, TGT AGT ATC GGA GGT GCA GTT AAC CGT GGC ATG AGT GGC ATA ATT GGA ATG ACA TTT ATA TCG (SEQ ID NO: 33) delimited from the nucleotide in position (1277) to the nucleotide in position (1339) of the sequence SEQ ID NO: 1, GCA TTC CTT TTA TTT GCT GTG GTT GCA TCT ATC GGA TGG GTC TTT ATG TAC ACA ATG TTC (SEQ ID NO: 34) delimited from the nucleotide in position (1367) to the nucleotide in position (1426) of the sequence SEQ ID NO: 1, The nucleic acid sequence SEQ ID NO: 23 codes for the protein fragment SEQ ID NO: 11.The nucleic acid sequence SEQ ID NO: 24 codes for the protein fragment SEQ ID NO: 12.The nucleic acid sequence SEQ ID NO: 25 codes for the protein fragment SEQ ID NO: 13.The nucleic acid sequence SEQ ID NO: 26 codes for the protein fragment SEQ ID NO: 14.The nucleic acid sequence SEQ ID NO: 27 codes for the protein fragment SEQ ID NO: 15.The nucleic acid sequence SEQ ID NO: 28 codes for the protein fragment SEQ ID NO: 16.The nucleic acid sequence SEQ ID NO: 29 codes for the protein fragment SEQ ID NO: 17.The nucleic acid sequence SEQ ID NO: 30 codes for the protein fragment SEQ ID NO: 18.The nucleic acid sequence SEQ ID NO: 31 codes for the protein fragment SEQ ID NO: 19.The nucleic acid sequence SEQ ID NO: 32 codes for the protein fragment SEQ ID NO: 20.The nucleic acid sequence SEQ ID NO: 33 codes for the protein fragment SEQ ID NO: 21.The nucleic acid sequence SEQ ID NO: 34 codes for the protein fragment SEQ ID NO: 22.The invention also relates to a recombinant vector, in particular plasmid, cosmid, phage or virus DNA, containing a nucleotide sequence as mentioned above.", "The invention also relates to a recombinant vector as defined above, containing the elements necessary for expression in a host cell of polypeptides coded by the nucleic acids as defined above, inserted into said vector.", "According to an advantageous embodiment of the invention, the recombinant vector defined above contains in particular a promoter recognized by the RNA polymerase of the host cell, in particular an inducible promoter and optionally a transcription or termination sequence, and optionally a signal and/or anchoring sequence.", "According to another advantageous embodiment of the invention, the recombinant vector, such as defined above, contains the elements which allow the expression of a nucleotide sequence, as defined above, as a mature protein or fusion protein.", "The invention also relates to a host cell, chosen in particular from bacteria, viruses, yeasts, fungi, plants or the cells of mammals, said host cell being transformed, in particular using a recombinant vector as defined above.", "According to an advantageous embodiment of the invention, the host cell, as defined above, contains the regulation elements allowing the expression of the nucleotide sequence as defined above.", "The invention also relates to the product of the expression of a nucleic acid expressed by a host cell transformed as defined above.", "The invention also relates to an antibody characterized in that it is directed in a specific manner against a protein of the invention.", "The invention is not limited to polyclonal antibodies; the invention also relates to any monoclonal antibody produced by any hybridoma capable of being formed according to standard methods starting from, on the one hand, animal, in particular mouse or rat, spleen cells, the cells of the animal being immunized against the protein of the invention, and on the other hand cells of a cell line of myeloma, said hybridoma being capable of being chosen according to the capacity of the cell line to produce monoclonal antibodies recognizing the protein used beforehand for the immunization of the animals.", "The invention also relates to a nucleotide probe capable of hybridizing with any one of the nucleic sequences of the invention.", "The invention also relates to the antisense oligonucleotides or antisense messenger RNA, derived from the nucleotide sequences as defined above.", "By modification of the expression of the mannitol carrier, using antisense oligonucleotides, it can then be determined if a reduction of the expression of the mannitol carrier has the result of reducing tolerance to saline stress.", "The invention also relates to plant cells containing in their genome a nucleotide sequence as defined above.", "The invention also relates to the transgenic plants, parts of plants, plant seeds or plant propagation material containing cells such as defined above.", "The invention relates in particular to transgenic plants which, in their native state, do not contain or express the gene of the mannitol carrier, in the genome of which said nucleotide sequence is introduced.", "The invention relates in particular to the transgenic plants which, in their native state, contain or express the gene of the mannitol carrier, in the genome of which said nucleotide sequence is introduced.", "The invention also relates to a process for the preparation of a recombinant protein as defined above, comprising the following stages: Culture in an appropriate medium of a host cell which has been transformed beforehand by an appropriate vector containing a nucleic acid of the invention, and Recovery of the protein produced by the abovementioned host cell transformed from the abovementioned culture medium or from the host cell.", "For example, a process for the preparation of a transgenic celery as defined above, comprises the following stages: inoculation of the celery tissues, coculture of the celery segments and of A. tumefaciens bacteria, elimination of the A. tumefaciens bacteria, regeneration of the transformed celery plants (Nadel et.", "al., 1989).", "The nucleotide sequences of the invention can be introduced into plasmids and be combined with regulation elements for expression in eukaryotic cells.", "These regulation elements are on the one hand transcription promoters and on the other hand transcription terminators.", "With the nucleotide sequences of the invention contained in the plasmids, the eukaryotic cells can be transformed with the intention of expressing a translatable mRNA which makes the synthesis of a polyol carrier in the cells possible or with the intention of expressing a non-translatable mRNA, which prevents the synthesis of a polyol carrier endogenous in the cells.", "The processes of genetic modification of dicotyledons and monocotyledons are already known (Gasser et al., 1989).", "For expression in plants, the nucleotide sequences of the invention must be conjugated with transcription regulation elements.", "Such elements, called promoters, are already known (EP 375091).", "In addition, coding regions with the termination signals of the transcription with which they can be correctly transcribed must be provided.", "Such elements are also described (Gielen et al., 1989).", "The initiation region of the transcription can be native and/or homologous as well as foreign and/or heterologous to the plant host.", "If desired, the termination regions are interchangeable amongst themselves.", "The DNA sequence of the initiation and termination regions of the transcription can be prepared synthetically or obtained naturally, or obtained from a mixture of natural or synthetic DNA constituents.", "To introduce foreign genes in higher plants, a large number of cloning vectors are available which include a replication signal for E. coli and a marker which allows selection of the transformed cells.", "For the introduction of the nucleotide sequences of the invention into a plant host cell, in addition to transformation using Agrobacteria, there are many other techniques.", "These techniques include the fusion of protoplasts, the microinjection of DNA and electroporation, as well as ballistic methods and viral infection.", "Starting from the transformed plant material, whole plants can be regenerated in a suitable medium, containing antibiotics or biocides for the selection.", "The resulting plants can then be tested for the presence of the DNA introduced.", "There is no particular requirement for the plasmids regarding the injection and the electroporation.", "Single plasmids can be used such as the pUC derivatives.", "The presence of a marker gene is necessary for the regeneration of whole plants from such transformed cells.", "The transformed cells develop in the plants in the usual manner (McCormick et al., 1986).", "These plants can develop normally and be crossed with plants which possess the same transformed genes or different genes.", "The resulting hybrids have the corresponding phenotypic properties.", "The DNA sequences of the invention can also be introduced into plasmids and be combined with regulation elements for an expression in prokaryotic cells.", "The DNA sequences of the invention can also be introduced into plasmids which allow a mutagenesis or a sequence modification by means of a recombination of DNA sequences in prokaryotic or eukaryotic systems.", "The transgenic plants of the invention are in particular characterized by an increase of the capacity to transport a polyol of the invention and to accumulate it in the organs from which it is extracted.", "They can be used to direct the flow of said polyol with the aid of said carrier towards the organs which accumulate little salt, thus facilitating extraction.", "The invention also relates to a process of screening genetically modified plants with at least one nucleotide sequence of interest which comprises the following stages: the transformation of plant cells with a vector containing an insertion sequence, said insertion sequence comprising the nucleotide sequence of interest and a nucleotide sequence coding for a polyol carrier as defined above, the culture of the cells thus transformed on a medium containing said polyol as an only source of carbon, to obtain transgenic plants or fragments of transgenic plants containing said insertion sequence.", "This process relates to plants not synthesizing polyol or plants which synthesize it.", "It concerns, more particularly, the transformation of fragments or of plant cells with a nucleotide sequence coding for a polyol carrier, in particular mannitol.", "The screening is then carried out on a medium containing said polyol as the only source of carbon.", "The plants expressing the polyol carrier thus have an advantage in growth over the non-transformed plants.", "At this stage, it can be supposed that any plant is capable of using said polyol as a source of carbon.", "However, it can prove necessary to do a co-transformation with a gene coding a protein capable of degrading said polyol.", "The use of an active promoter only in the initial phases of regeneration or inducible by a simple compound makes it possible to restrict the expression of the polyol carrier to the selection phases.", "The invention therefore concerns a simple selection system based on a plant gene which is no longer necessary once the selection is finished and on the use of a natural product as a selection agent.", "This system avoids having to resort to the use of products likely to be toxic, such as antibiotics.", "The invention also relates to a process for obtaining transgenic plants resistant to pathogens, which comprises the following stages: transformation of plant cells with a nucleotide sequence coding for a polyol carrier as defined above, culture of the thus transformed cells to obtain transgenic plants or fragments of transgenic plants.", "This process relates to the transformation of plants not synthesizing polyol or plants which synthesize it, with a nucleotide sequence of a polyol carrier, in particular mannitol, placed either under the control of a ubiquist promoter (type CaMV 35S) or under the control of an inducible promoter in response to the attack of the pathogen.", "The usefulness resides in the fact that the plant, in transporting more polyol, produced by the pathogen, towards its own cells, suppresses one of the means of defence put in place by the pathogen to fight against the activated oxygen released by the plant in response to this attack.", "In order to increase the effectiveness of the process, the expression of the polyol carrier can be conjugated with an enzyme degrading said polyol.", "The invention relates to a process for obtaining transgenic plants resistant to saline stress, which comprises the following stages: transformation of plant cells with a nucleotide sequence coding for a polyol carrier as defined above, culture of the cells thus transformed to obtain transgenic plants or fragments of transgenic plants.", "This process relates to the transformation of plants not synthesizing polyol or plants which synthesize it with a nucleotide sequence coding for a polyol carrier placed under the control of a phloem-specific promoter (or of the promoter of the polyol carrier).", "If the plant synthesizes said polyol, the increase of the transport of said polyol could lead to an accumulated tolerance to saline stress.", "In the opposite case, it is also advisable to introduce genes allowing the synthesis of said polyol, but limiting this synthesis to the leaves in order to avoid harmful effects on the growth of the plant.", "DESCRIPTION OF THE FIGURES FIG.", "1 represents the growth test of the yeast MaDH4 expressing the proteic sequence AgMaT1.The cDNA of AgMaT1, under the control of the promoter ADH1, was introduced in the cells of the MaDH4 strain, and the growth of the transformed cells on mannitol was studied.", "The transformed cells were grown on the SC (synthetic complete) liquid medium without tryptophan containing either 2% glucose (SC-glu) or 2% mannitol (SC-mann).", "MaDH4-YEP112A1XE: MaDH4 containing the empty plasmid; MaDH4-AgMaT1: MaDH4 containing the plasmid with the nucleic acid of AgMaT1.The white squares correspond to MaDH4 yeasts transformed with the empty plasmid YEP112A1XE (defined hereafter) and grown on SC-glucose medium.", "The black squares correspond to MaDH4 yeasts transformed with the empty plasmid YEP 112A1XE (defined hereafter) and grown on SC-mannitol medium.", "The white circles correspond to MaDH4 yeasts transformed with AgMaT1/YEP112A1XE (plasmid YEP112A1XE containing the nucleic acid of AgMaT1) and grown on SC-glucose medium.", "The black circles correspond to MaDH4 yeasts transformed with AgMaT1/YEP112A1XE (plasmid YEP112A1XE containing the nucleic acid of AgMaT1) and grown on SC-mannitol medium.", "The curves represent the evolution according to the absorbance time (at 600 nm) of the yeast cultures.", "This increase of absorbance corresponds in fact to an increase of the number of yeasts in the culture medium and is representative of the growth rate of the yeasts.", "Therefore the yeasts transformed with the plasmids YEP112A1XE and AgMaT1/YEP112A1XE grow on glucose but only the yeasts transformed with the AgMaT1/YEP112A1XE plasmid are capable of growing on mannitol.", "It is therefore proof that AgMaT1 codes for a mannitol carrier.", "FIG.", "2 represents the absorption of mannitol in cells of S. cerevisiae.", "The external concentration of mannitol 3H is 500 μM and the pH is 4.5.The squares represent the absorption in transformed cells with the nucleic acid of AgMaT1 whilst the circles represent the absorption in control cells transformed with the empty YEP112A1XE plasmid.", "Only the transformed cells with the AgMaT1/YEP112A1XE plasmid are capable of absorbing the mannitol 3H placed in the external medium.", "Material and Methods Plant Material Celery plants (Apium graveolens L. dulce variety, Vert d'Elne cultivar) were grown in greenhouses according to the conditions described by Davis et al.", "(1988).", "The phloemian bundles were isolated from adult petioles according to the technique described by Daie (1987).", "Bacterial Strains and Yeasts.", "The following strains were used in this study: Escherichia coli strains DH5α (supE44, ΔlacU169 (φ80, lacZM15), hsdR17, recA, endA1, gyrA96, thi-1, relA1) (strains commercially available from Clontech).", "XL1Blue MRF′ (Stratagene) and SOLR (Stratagene) were cultured according to standard techniques (Sambrook et al., 1989).", "The Saccharomyces cerevisiae MaDH4 strain (ura3, trp1LEU2, gap1-1, put4-1, uga4-1), the preparation of which is indicated hereafter, expresses the mannitol dehydrogenase gene of yeast and has been used for the functional characterization of the cDNA of AgMaT1.The 2a strain was obtained by crossing between the Δα (MATα, ura3, trp1, leu2) (Marcireau et al., 1992) and Σ22574d (MATα, ura3-1, gap1-1, put4-1, uga4-1) (Jauniaux et al., 1987) strains.", "Expression Vector in Yeasts The plasmid YIP 128A1, described in Riesmeier et al.", "(1992) is used.", "The mannitol dehydrogenase gene of yeast (YEL070) was amplified by PCR (polymerization chain reaction) using the oligonucleotides MDHPST5 (5-GACTCGA-GATGACAAAATCAGACGAAACAAC-3) and MDHBGL3 (5-GAAGATCTTCACACTTGGTCTAAAATTTCC-3) on the genomic DNA of the Saccharomyces Δα strain.", "The PCR product was cloned in the pBluescript SK vector digested beforehand by Pst1 and BamH1.After sequencing to confirm the sequence of the amplified gene, the PCR product was digested by Pst1 and Xba1 and cloned in the Pst1/Xba1 sites of YIP128A1.The construction was integrated into the genome of S. cerevisiae by the EcoV site in the leu2 gene in order to obtain the MaDH4 strain.", "5′RACE-PCR (Rapid Amplification of the cDNA Ends by PCR), The total RNA of celery leaves was isolated according to the method of Kay et al.", "(1987).", "The first cDNA strand was reverse transcribed from the total RNA with the degenerated primer (5′-CCNACNCC(G/A)AANGGNA(G/A)NA(G/A)-3) derived from the sequence LLGFGVG using reverse transcriptase SuperScript™ II (Stratagene).", "After degradation of the RNA matrix by RNaseH (Eurogentec), an anchoring primer (dC)16 was created at the 3′ end of the single-stranded cDNA by a deoxynucleotydil transferase (GibcoBRL).", "A PCR amplification was carried out using the (dG)16 and LLGFGVG primers under the following conditions: 2 minutes at 95° C. then 30 cycles comprising denaturation for 2 minutes at 95° C., fixation for 2 minutes at 55° C. and extension for 2 minutes at 72° C. The PCR products were analyzed by agarose gel electrophoresis then cloned in the pGEM-T Easy plasmid (Promega).", "Construction and Screening of a cDNA Bank of Celery Phloem The total RNA of the phloem bundles was isolated according to the method described by Kay et al.", "(1987).", "The polyA+ RNA was purified with the PolyATtract mRNA isolation system (Promega).", "A unidirectional EcOR1/XhoI bank was constructed in the Uni-ZapXR phage (Stratagene).", "The recombinant phages (900,000) were screened with the radioactively labelled product of 5′RACE-PCR as probe, in accordance with the manufacturer's protocol (Stratagene).", "The Hybond TM-N nylon filters (Amersham) were hybridized overnight at 42° C. according to standard conditions (Stratagene).", "The filters were then rinsed for 15 minutes at 42° C. in SSC 2×(SSC 1×=0.15 M NaCl; 0.015 M sodium citrate) with 0.1% SDS, then for 15 minutes in the same medium but at 50° C. and 30 minutes at 50° C. in SSC 1× and 0.1% SDS.", "The excision in vivo was carried out on the 24 clones which produced a positive signal during the 3 successive screening turns.", "The identified cDNAs were partially sequenced.", "The sequence comparisons were carried out on the National Center for Biotechnology Information site.", "The transmembrane regions were predicted with the Tmpred program (Hofinann and Stoffel, 1993).", "Expression of AgMaT1 in Saccharomyces Cerevisiae The cDNA of AgMaT1 was ligated in the Pst1-XhoI sites of the yeast vector YEP112A1XE (Riesmeier et al., 1992).", "This vector allows the expression of the cDNA under the control of the yeast promoter ADH1.The MaDH4 yeast cells were rendered competent and transformed according to the protocol described by Dohmen et al.", "(1991).", "Determination of the Growth Rate The yeast cultures were grown on SC medium comprising either 2% glucose, or 2% mannitol.", "Aliquot fractions were taken regularly from the cultures and their absorbance was measured at 600 nm.", "Determination of the Mannitol Dehydrogenase Activity The cells were cultured until in logarithmic growth phase, rinsed in distilled water and resuspended at 80% (weight/volume) in extraction buffer (50 mM potassium phosphate pH 7.5, 1 mM DTT and 0.5% Triton X100).", "The cells were broken apart by vortex with glass beads.", "The cellular debris was eliminated by centrifuging and the crude extract used for the enzymatic assay.", "The mannitol dehydrogenase activity was measured at 30° C. according to Quain and Boulton (1987).", "Measurement of the Transport of Radiolabelled Mannitol The cells were cultured until the start of the logarithmic phase (corresponding to an absorbance of 0.6 to 600 nm), washed in distilled water and resuspended at 1% (weight/volume) in SC medium buffered to pH 4.5 with 25 mM MES.", "A 100 μl aliquot fraction of the cell suspension was incubated for 60, 120, 180 and 300 seconds in 100 μl of a solution containing of 500 μM [3H]-mannitol.", "The reaction was stopped by adding 8 ml of water at 4° C. and by filtration through glass fibre filters (Sartorius).", "The radioactivity incorporated in the yeast cells was determined by counting using liquid scintillation (Packard).", "For the experiments with inhibitors or competitors, the product was added 30 seconds before the radioactive mannitol.", "Study of the Expression of AgMaT1 by RT-PCR (Reverse Transcription Followed by Polymerase Chain Amplification) The total RNA of celery phloem was isolated according to the method of Kay et al.", "(1987).", "The first strand of cDNA was reverse transcribed from the total RNA with the oligo dT primer by using the reverse transcriptase SuperScript™ II (Stratagene).", "After degradation of the RNA matrix by RNaseH (Eurogentec), PCR amplification was carried out using the primers 5′ (ATTCTGGTGTGTTGCTCG) and 3′ (CAATGAACAGTATGATGTG) which allow the amplification of a fragment of 661 nucleotides.", "The PCR conditions were as follows: 2 minutes at 95° C. then 30 cycles comprising denaturation for 30 seconds at 95° C., fixation for one minute at 47° C. and extension for 45 seconds at 72° C. The PCR products were analyzed by agarose gel electrophoresis and the intensity of the signal obtained was quantified using Photoshop 5.0 software (Adobe systems Inc.).", "The extension factor e1F4A(10) (Mandel et al., 1995) was used as control gene, the expression of which is invariable.", "Results Molecular Cloning of AgMaT1 A certain number of proteins which transport sugars or metabolites show similarities in their sequences.", "It has been suggested that these transport proteins have evolved from the duplication of an ancestral protein with 6 transmembrane regions (Maiden et al., 1987).", "Several preserved amino acid regions were identified such as the amino acid sequences at the ends of the 6th and 12th transmembrane domains, PESPR and PETKG respectively (Griffith et al., 1992).", "Comparison between the different glucose carriers (MST1, STP1, STP4, HUP1, HUP3, GLUT1), the D-xylose carrier of L. brevis, the arabinose carrier of E. coli (ARAE), the galactose carrier of E. coli (GALP) and the myo-inositol carriers of yeast (genes ITR1 and ITR2) indicated a preserved region LLGFGVG.", "This sequence was chosen as matrix for designing the degenerated 5′RACE primer for PCR.", "The first strand of cDNA was reverse transcribed from the entire RNA of mature celery leaves, primed with a degenerated primer LLGFGVG.", "After amplification, a band of 1 kb was observed on the agarose gel.", "All the fragments of this PCR reaction were cloned in a pGEM-T Easy vector (Promega), and several clones were obtained.", "In order to obtain an entire clone, a cDNA library was constructed originating from phloem bundles isolated from mature celery petioles and this library was screened with the 5′RACE-PCR clone.", "After having screened 900,000 transformants, 24 positive clones were identified.", "The positive transformants with inserts of approximately 1.8-2.0 kb were chosen and partially sequenced.", "One of these clones, called AgMaT1, was chosen for detailed analysis.", "It contained 1778 pb with an open reading frame which codes for a protein containing 513 amino acids with a molecular mass estimated at 56 kDa.", "Hydropathic analysis of the deduced sequence of amino acids indicates that AgMaT1 contains 12 transmembrane domains and a long hydrophilic central region of 77 amino acid residues.", "The amino acid sequence of AgMaT1 was compared with those of the databases and it was found that this sequence was related to the sugar carriers in numerous organisms.", "The percentage identity of the amino acids is approximately equal to 50%.", "However, a greater percentage of identity (65%) was found with two optional sugar carriers of Beta vulgaris (Beet 1 and Beet 2).", "An asparagine residue, which is part of an N-glycosylation consensus sequence (Asn372), is situated on the external side and therefore must be glycosylated.", "In addition, the consensus sequences, which are the common characteristics of the sub-group of sugar carriers of MFS, are present in AgMaT1.The sequences of PESPRXL and PETQGRXXXE were found respectively at the ends of the 6th and 12th transmembrane domains, or the (R/K)XGR(R/K) motif between the 2nd and the 3rd and also the 8th and 9th transmembrane helices (Griffith et al., 1992).", "Note: The main difficulty encountered during cloning was the total absence of characterisation of such a carrier in any living organism.", "In fact the only mannitol carrier is a bacteria mannitol-phospho-transferase (Boer et al., 1994) which carries out both the transport and phosphorylation of mannitol.", "This combined system is present in bacteria for numerous substrates but it does not exist in Eukaryotic organisms.", "However, according to a first strategy, a first screening of the cDNA bank was carried out with the part of the gene of mannitol-phospho-transferase corresponding to the transmembrane field.", "This screening did not allow a result to be obtained, which is justified a posteriori by the absence of significant homology between AgMaT1 and the mannitol-phospho-transferase.", "A second strategy, which turns out not to be operational, is inspired by that used for identifying the carrier of oligosaccharides in the plants (Patent EP 0,647,273).", "This consists of complementing the cells of Saccharomyces cerevisiae with a cDNA bank in an expression vector.", "The yeasts are in fact capable of using mannitol as a source of carbon, but they require a fairly long induction period on mannitol.", "As has already been specified, no mannitol carrier has been identified in yeast.", "The reasoning being that if a yeast expressed a plant mannitol carrier, this would confer on it a growth advantage and that therefore, it would grow quicker on a medium containing mannitol.", "The operation was carried out in this way but none of the cDNAs obtained showed any of the characteristics of membrane proteins and in fact resembled transcription factors.", "The selection system in fact allowed the cDNA which was involved in the expression of yeast genes to be identified and not the carriers.", "Faced with the above difficulties, the Inventors formulated an improbable a priori hypothesis according to which the mannitol carrier would be part of the super family of glucide carriers described by Marger and Saier (1993).", "To do this, a species, celery, was used in which the existence of a mannitol carrier had been demonstrated (Salmon et al., 1995) and to construct a cDNA bank from the tissue (the phloem) in which the carrier was more expressed.", "The second stage was the selection of the cDNA obtained according to their capacity to confer the possibility of transporting mannitol to the yeasts.", "In these experiments the control was the strain of yeast transformed with the empty expression plasmid.", "In this way the mannitol carrier function of the cDNA of AgMaT1 was demonstrated.", "During this experiment, other sequences were identified: in total 24 clones were obtained.", "Among all these clones, two were sequenced which showed the hydropathy profiles of carriers.", "The first, M22 (AgMaT1), conferred the ability to transport mannitol to the yeasts whilst the second, M7, did not confer it.", "Construction of a Strain of Yeast Capable of Metabolizing Intracellular Mannitol Initial studies were carried out in order to characterize the ability of a yeast to absorb and to metabolize mannitol (Quain and Boulton, 1987).", "Out of the 40 polyploid strains of S. cerevisiae screened, half of them have shown good growth on 5% mannitol after long-term adaptation (Quain and Boulton, 1987).", "As a result, it was decided to test different strains of yeasts for their ability to transport and metabolize the mannitol and 2 strains were retained.", "This was firstly carried out by analyzing the growth characteristics on a medium containing mannitol as the only carbon source.", "Σ22574d, generally deficient in a general carrier of amino acids and carrier of proline, is incapable of growth on a medium containing mannitol as the only carbon source.", "On the contrary, Δα was capable of growing on mannitol after long-term adaptation.", "After adaptation, the strain could be maintained successfully on a solid medium containing 5% mannitol.", "But maintenance of the adapted Δα strain on a solid medium only containing glucose leads to the total loss of the adapted growth.", "Such a growth adaptation on mannitol is probably due to the induction of the key degradation enzymes or the transport permeases.", "In accordance with the previous observations, NAD+ dependant D-mannitol dehydrogenase could be detected in the Δα yeasts (Table 1).", "TABLE 1 Activity of mannitol dehydrogenase in different yeast strains.", "The strains are developed in a liquid medium containing either 2% glucose or 2% mannitol.", "The results are the averages ± SD of the three independent experiments.", "ND, not detected.", "Activity (μmol of oxidized mannitol · (mg of protein)−1 · min−1) Strain glucose mannitol Δα 0.011 ± 0.003 0.240 ± 0.007 Σ22574d 0.006 ± 0.002 ND 2a 0.001 ± 0.001 ND MaDH4 0.410 ± 0.011 ND In order to obtain an auxotrophy to tryptophan, the Δα strain (Trp) was crossed with the Σ22574d strain (Trp+).", "Yeast 2a was chosen, which cannot grow on a medium containing mannitol, with an auxotrophy to tryptophan and to leucine.", "No mannitol dehydrogenase activity was detected in cells 2a (Table 1).", "It was necessary to introduce a limited mannitol hydrolysis activity inside the yeast.", "The cDNA of the gene of the yeast mannitol dehydrogenase was cloned in YIP128A1 under the control of the ADH1 promoter and it was integrated in a stable manner in the leu2 gene of 2a.", "Several transformants have shown a mannitol dehydrogenase activity.", "The strain with the most significant activity, called MaDH4, was used for the subsequent analyses (Table 1).", "Heterologous Expression of the AgMaT1 Protein For a subsequent characterization of the function of the AgMaT1 protein, it was necessary to express the carrier in a functional manner in a heterologous system such as yeast cells.", "The cDNA of AgMaT1 was sub-cloned in the PstI/XhoI sites of the YEP 112A1XE shuttle vector which has a promoter/terminator box of the gene of alcohol dehydrogenase ADH1 of S. cerevisiae (Riesmeier et al., 1992).", "The competent MaDH4 cells were transformed with this construction and YEP 112A1XE was used as control.", "All of the constructions were firstly tested for their ability to grow on mannitol as the only carbon source.", "As indicated in FIG.", "1, the MaDH4 strain, transformed with the empty plasmid YEP 112A1XE is not capable of growing on mannitol.", "The cells expressing AgMaT1 could grow very well on this polyol.", "In order to directly test the ability of the transformed cells to transport mannitol, the yeast cells were incubated in a medium containing [3H]-mannitol for a few seconds to several minutes, the cells were washed and the radioactivity absorbed was measured by electric scintillation counting.", "FIG.", "2 indicates that the transport of mannitol in the control cells of S. cerevisiae is negligible.", "However, the MaDH4 yeast strains, expressing AgMaT1, transport the [3H]-mannitol at high speeds when they grow on a medium containing mannitol.", "The same result is obtained with the cells of transformed yeast growing on glycerol (data not indicated).", "Other polyols such as dulcitol, sorbitol, xylitol, myo-inositol appear capable of inhibiting by half the absorption of mannitol.", "The oside form of mannitol, mannose, appears to be recognized by AgMaT1.Variation of the Expression of AgMaT1 During Saline Stress The expression of AgMaT1 was monitored in plants having been subjected to saline stress for 4 weeks (daily watering with 300 mM of NaCl, Noiraud et al., 2000).", "The phloem of these plants as well as of the corresponding control plants (watered with water not containing NaCl) was removed in order to extract the RNA which was used to carry out RT-PCR reactions.", "If the expression of AgMaT1 in the phloem of the control plants is taken as base 100, the expression of AgMaT1 in the phloem of plants treated with NaCl is 500%, which represents a very significant stimulation and is in accordance with the role of AgMaT1 in saline stress tolerance in celery.", "Transformation Protocol of Petioles or Leaves of Celery Celery plants (approximately 10 cm in height) regenerated from embryogenic cells are used as plant material for the transformation.", "Inoculation of the Celery Tissues Agrobacterium tumefaciens bacteria are cultured for 24 hours at 28° C. under agitation in LB medium (Liquid Broth: 1% tryptone, 0.5% autolytic extract of yeast, 0.5% NaCl) with the appropriate antibiotic.", "The petioles of celery plants are fragmented into sections of approximately 0.5 cm.", "For each fragment, a longitudinal section is produced.", "The celery segments are incubated in MS medium (Murashige & Skoog) 1×(normal concentration, i.e.", "no dilution) liquid containing {fraction (1/25)}th of the culture of Agrobacterium tumefaciens bacteria for 60 minutes at ambient temperature.", "Composition of the MS Medium Macro-elements CaCl2 2.99 mM KH2PO4 1.25 mM KNO3 18.79 mM MgSO4 1.50 mM NH4NO3 20.61 mM Vitamins Glycine 26.64 mM Myo-inositol 0.56 mM Nicotinic acid 4.06 μM Pyridoxine-HCl 2.43 μM Thiamine-HCl 0.30 μM Micro-elements CoCl2, 6 H2O 0.11 μM CuSO4, 5 H2O 0.10 μM FeNaEDTA 0.10 μM H3Bo3 0.10 μM KI 5.00 μM MnSO4, H2O 0.10 mM Na2MoO4, 2 H2O 1.03 μM ZnSO4, 7 H2O 29.91 μM The excess bacteria are then removed from the celery segments by arranging them on absorbent paper for 2-3 minutes.", "Coculture of the Celery Segments and the A. Tumefaciens Bacteria The cambial surface of the celery segments is left in contact with the gelosed regeneration medium RM.", "The Petri dishes are placed in a chamber air-conditioned at 25° C. for 48 hours and subjected to light/dark cycles of 16 hours/8 hours.", "Elimination of the A. Tumefaciens Bacteria After coculture for 48 hours, the celery segments are removed form the dishes of RM medium and transferred into MS 1 X liquid supplemented with cefotaxime at a final concentration of 250 μg/mL.", "After incubation for 60 minutes, the celery segments are dried on absorbent paper for 2-3 minutes.", "Regeneration of Transformed Celery Plants The cambial surface of the celery segments is left in contact with a gelosed callogenesis initiation medium CIM.", "The CIM Petri dishes are placed in a chamber air-conditioned at 25° C. and subjected to light/dark cycles of 16 hours/8 hours until the development of calluses (2-3 weeks).", "The celery segments are then transferred onto a gelosed organogenesis induction medium OIM (2-3 weeks).", "After the appearance of buds, these are removed and placed on gelosed rooting medium RM.", "A few weeks (3-4 weeks) are necessary for the development of young celery shoots.", "Composition of the Media Regeneration Medium RM MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L 6-Benzylaminopurine (BAP) 1.0 mg/L α-naphthylacetic acid (NAA) 0.1 mg/L Gibberellic acid (GA3) 0.1 mg/L Agar 0.8% Callogenesis Initiation Medium (CIM) MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L 6-Benzylaminopurine (BAP) 1.0 mg/L α-naphthylacetic acid (NAA) 0.1 mg/L Gibberellic acid (GA3) 0.1 mg/L Kanamycin 125.0 mg/L Cefotaxime 200.0 mg/L Agar 0.8% Organogenesis Induction Medium (OIM) MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L 6-Benzylaminopurine (BAP) 1.0 mg/L Gibberellic acid (GA3) 0.1 mg/L Kanamycin 75.0 mg/L Cefotaxime 200.0 mg/L Agar 0.8% Rooting Medium (RM) MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L α-indolyacetic acid (IAA) 0.1 mg/L Kanamycin 75.0 mg/L Cefotaxime 200.0 mg/L Agar 0.8% REFERENCES Lewis D H (1984) Physiology and metabolism of alditols.", "In D H Lewis eds, Storage carbohydrates in vascular plants, Cambridge University Press, Cambridge, pp 157-179, Boer et al.", "(1994) Journal of Biological Chemistry, 269: 17863-17871, Daie J (1987) Sucrose uptake in isolated phloem of celery is a single saturable transport system.", "Planta 171: 474-482, Davis et al.", "(1988) Biosynthesis of sucrose and mannitol as a function of leaf age in celery (Apium graveolens L.).", "Plant Physiol.", "86: 129-133, Dohmen et al.", "(1991) An efficient transformation procedure enabling long term storage of competent cells of various yeast genera.", "Yeast 7: 691-692, Gasser et al.", "(1989) Science, 244: 1293-1299, Gielen et al.", "(1989) EMBO J, 8: 23-29, Griffith et al.", "(1992) Membrane transport proteins: implications of sequence comparisons.", "Curr.", "Opin.", "Cell Biol.", "4: 684-695, Hofmann K and Stoffel W (1993) Tmbase—A database of membrane spanning protein segments.", "Biol.", "Chem.", "Hoppe-Seyler 347: 166, Jauniaux et al.", "(1987) Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae.", "Cloning of the PUT4 gene and study of PUT4 RNA levels in the wild-type and the mutant strains.", "Eur.", "J. Biochem.", "164: 601-606, Kay et al.", "(1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes.", "Sci.", "236: 1299-1302, Maiden et al.", "(1987) Mammalian and bacterial sugar porters are homologous.", "Nature 325: 641-643, Mandel et al.", "(1995) Plant Mol.", "Biol., 29: 995-1004, Marcireau et al.", "(1992) Construction and growth properties of a yeast strain defective in sterol 14-reductase.", "Curr Genet, 22: 267-272, Marger M D, Saier J M H (1993) A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport.", "Trends Biochem.", "Sci.", "18: 13-20, McCormick et al.", "(1986) Plant Cell Reports, 5: 81-84, Nadel et al.", "(1989) Plant Cell and Organ Culture, 18: 181-189, Noiraud et al.", "(2000) The sucrose transporter of celery.", "Identification and expression during salt stress.", "Plant Physiology, 122: 1447-1455, Quain D E, Boulton C A (1987) Growth and metabolism of mannitol by strains of Saccharomyces cerevisiae.", "J. Gen. Bacteriol.", "133: 1675-1684, Riesmeier et al.", "(1992) Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.", "EMBO J.", "11: 4705-4713, Salmon et al.", "(1995) Study of sucrose and mannitol transport in plasma-membrane vesicles from phloem and non-phloem tissues of celery (Apium graveolens L.) petioles.", "Planta 197: 76-83, Sambrook et al.", "(1989) Molecular cloning.", "Cold Spring Harbor Laboratory Press." ] ]

[ [ "Carrier-free 103pd brachytherapy seeds", "A brachytherapy seed comprising (a) carrier-free 103Pd isotope, (b) a substrate for the carrier-free 103Pd radioisotope, and (c) a casing for encapsulating the carrier-free 103Pd-laden substrate, is disclosed." ], [ "1.A brachytherapy seed which comprises: (a) a substrate having a surface suitable for the electroless deposition of palladium, (b) carrier-free 103Pd disposed on said surface of said substrate to provide a carrier-free 103Pd-laden substrate; and (c) a biocompatible casing for encapsulating the carrier-free 103Pd-laden substrate.", "2.The brachytherapy seed of claim 1, wherein the surface suitable for the electroless deposition of palladium is metallic.", "3.The brachytherapy seed of claim 2, wherein the metallic surface comprises nickel, copper, or a mixture thereof.", "4.The brachytherapy seed of claim 1, wherein the substrate further comprises a radiopaque metal.", "5.The brachytherapy seed of claim 4, wherein the substrate comprises a radiopaque metal core surrounded by a sheath of a radiotransparent metal.", "6.The brachytherapy seed of claim 5, wherein the radiopaque metal core comprises silver, gold, platinum, iridium, palladium, thallium, copper, iron or lead or a mixture thereof.", "7.The brachytherapy seed of claim 5, wherein the radiotransparent metal comprises aluminium.", "8.The brachytherapy seed of claim 7, wherein the radiopaque core comprises silver.", "9.The brachytherapy seed of claim 1, wherein the substrate further comprises a radiotransparent core.", "10.The brachytherapy seed of claim 9, wherein the radiotransparent core comprises graphite, a polymer, or a metal having a low atomic number.", "11.The brachytherapy seed of claim 10, wherein the metal having a low atomic number is aluminium.", "12.The brachytherapy seed of claim 1, wherein the biocompatible casing comprises stainless steel, titanium, a titanium alloy, tantalum, a nickel alloy, a copper alloy, or an aluminium alloy.", "13.A substrate in a form suitable for use in a brachytherapy seed, which has a surface coating suitable for the electroless deposition of palladium and further comprises a radiopaque metal core chosen from silver, platinum, iridium, palladium, thallium, copper, iron or lead or a mixture thereof, surrounded by a sheath of a radiotransparent metal.", "14.The substrate of claim 13, wherein the radiotransparent metal comprises aluminium.", "15.The substrate of claim 13, wherein the radiopaque metal core comprises silver, the sheath comprises aluminium, and the surface coating comprises nickel, or copper, or a mixture thereof.", "16.A substrate in a form suitable for use in a brachytherapy seed, which comprises a radiotransparent core with a surface coating, wherein the surface coating is suitable for the electroless deposition of palladium.", "17.The substrate of claim 16, wherein the radiotransparent core comprises graphite, a polymer, or a metal having a low atomic number.", "18.The substrate of claim 16, wherein the surface coating is metallic.", "19.The substrate of claim 18, wherein the metallic surface coating comprises nickel, or copper, or a mixture thereof.", "20.The substrate of claim 16, wherein the metal having a low atomic number is aluminium 21.A method of preparing a carrier-free 103Pd-laden substrate which comprises: immersing a substrate having a surface suitable for the electroless deposition of palladium into an electroless bath comprising carrier-free 103Pd for a sufficient time to deposit a pre-selected amount of carrier-free 103Pd onto the surface suitable for the electroless deposition of palladium of said substrate.", "22.The method of claim 21, wherein the electroless bath further comprises an acid, a reducing agent, an optional complexing agent, an optional buffer, or mixtures thereof.", "23.The method of claim 21, wherein the electroless bath further comprises a base, a reducing agent, an optional complexing agent, an optional buffer, and mixtures thereof.", "24.A method of treating a disease or condition responsive to brachytherapy, which comprises placement of one or more brachytherapy seeds of claim 1 at a target location within an individual, and allowing the seeds to remain at the target location for a sufficient time to deliver a therapeutically effective radiation dose.", "25.The method of claim 24, wherein the seeds are placed within the individual temporarily.", "26.The method of claim 24, wherein the seeds are placed within the individual permanently.", "27.The method of claim 24, wherein the disease or condition is selected from the group consisting of: head cancer, neck cancer, melanoma, brain cancer, non-small cell lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, liver cancer, proliferative disease, arthritis, urethral stricture, and fibroid uterine tumor." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Radiation therapy is the treatment of diseases and conditions, especially the treatment of tumors, including malignant tumors, with radiation.", "In radiation therapy, the ultimate aim is to destroy the malignant tissue without causing excessive radiation damage to nearby healthy, and possibly vital, tissue.", "This is difficult to accomplish because of the proximity of malignant tissue to healthy tissue.", "Medical personnel and investigators have developed methods for preferentially irradiating deep seated diseased tissue as opposed to healthy tissue.", "These methods include the use of high energy X-ray beams together with cross fire and rotational techniques which create a radiation pattern that is maximized at the site of the diseased tissue.", "Nonetheless, some absorption and damage inevitably occurs to healthy tissue in the path through which radiation passes to arrive at deep-seated diseased tissue.", "One method of limiting the zone of irradiation utilizes radioactive articles in the form of small, radioactive “seeds,” which are permanently or temporarily implanted at the zone to be irradiated.", "Such seeds contain a radioactive source disposed within a sealed capsule.", "The small size of the therapeutic seeds allows the seeds to be inserted or implanted within or near the tissue to be treated, for example, to totally surround the treated tissue.", "Radiation treatment can involve a temporary implantation of a radioactive source for a calculated period, followed by its removal.", "Alternatively, the radioactive source can be implanted in the patient permanently and left to decay to an inert state over a predictable time.", "The use of temporary or permanent implantation depends on the disease or condition being treated, the radioisotope selected, and the duration and intensity of required treatment.", "The advantages of interstitial implantation of a radiation-emitting article for localized tumor treatment have long been recognized.", "Interstitially implanted articles concentrate the radiation at a zone where radiation treatment is needed, e.g.", "near or within diseased tissue in order to directly affect diseased tissue, while exposing normal, healthy tissue to substantially less radiation than beaming radiation into the body from an external source.", "Implanting radioactive articles in proximity to or directly within diseased or damaged tissue within a body is a therapy referred to as brachytherapy (i.e.", "short-range therapy).", "Brachytherapy is a general term for a medical treatment involving placement of a radioactive source near diseased tissue.", "Brachytherapy has been proposed for use in the treatment of a variety of diseases and conditions, including arthritis and cancer, for example, breast, brain, liver, and ovarian cancer, and especially prostate cancer in men [see for example, J. C. Blasko et al., The Urological Clinics of North America, 23, 633-650 (1996), and H. Ragde et al., Cancer, 80, 442-453 (1977)].", "This form of therapy permits the application of larger doses of radiation directly to diseased or damaged tissue, like tumors.", "Permanent implants for prostate cancer treatment comprise radioisotopes with relatively short half-lives and lower energies relative to radioisotopes used in temporary implants.", "Examples of permanently implantable radioisotopes include iodine-125 and palladium-103.The radioisotope generally is disposed on a substrate, which is encapsulated in a metal casing, for example, a titanium casing, to form a “seed,” which is then implanted in the patient.", "Radioactive seeds are disclosed, for example, in Lawrence U.S. Pat.", "No.", "3,351,049 and Kubiatowicz U.S. Pat.", "No.", "4,323,055.U.S.", "Pat.", "No.", "3,351,049 discloses conventional brachytherapy seeds comprising titanium containers encapsulating ion exchange resin beads onto which a radioactive ion, e.g.", "125 I or 103 Pd has been adsorbed.", "U.S. Pat.", "No.", "3,351,049 also discloses that 103 Pd, preferably carrier-free, could be plated on a 3.5 mm long plastic rod.", "However, U.S. Pat.", "No.", "3,351,049 does not disclose any method of plating carrier-free 103 Pd onto the plastic rod.", "As discussed hereafter, the uniform distribution of carrier-free 103 Pd on a substrate has been an ongoing and unsolved problem.", "U.S. Pat.", "No.", "4,323,055 discloses brachytherapy seeds comprising a coating of radioactive silver iodide on a silver wire encapsulated inside a titanium container.", "WO 97/19706 discloses the immobilization of a radioactive powder within a polymeric matrix.", "The seeds disclosed in prior patents comprise a tiny sealed capsule having an elongate cavity containing the radioisotope, e.g., iodine-125 ( 125 I) or palladium-103 ( 103 Pd), adsorbed onto a substrate.", "Such seeds are suitable for use with radioisotopes that emit radiation capable of penetrating the capsule walls.", "Therefore, the seeds generally contain radioisotopes that emit γ-radiation or low-energy X-rays, as opposed to β-emitting radioisotopes.", "Because of the low energy X-rays emitted by 125 I and 103 Pd, and the short half-life of 125 I and 103 Pd, the seeds can remain implanted in the tissue of a patient indefinitely without excessive damage to surrounding healthy tissue or excessive exposure to other individuals near the patient.", "In order to function effectively, radiation emitting from the radioisotope within the seed should not be blocked or otherwise unduly attenuated.", "Seeds based on metal wire substrates have the disadvantage that a portion of the radioactivity is absorbed by the wire substrate itself, i.e.", "the radioactive emissions from the seed are attenuated by the wire.", "The amount of radioactivity absorbed by the wire increases as the atomic number (i.e.", "Z) of the metal wire substrate increases.", "The precise amount of attenuation is related to the identity and the dimensions of the wire substrate.", "For example, silver iodide-125 coated on an 0.5 mm diameter silver wire has up to about 40-50% of the radioactivity absorbed by the silver wire.", "Therefore, in the manufacture of a radioactive seed of a preselected activity, additional 125 I is loaded onto the wire to account for the absorption of radioactivity by the wire and also by the seed capsule.", "As the preselected radioactivity of the seed increases, the cost of the extra amount of radioisotope which is loaded onto the wire substrate also increases.", "Radiation emitted from the radioisotope also should be distributed uniformly from the seed in all directions, i.e.", "an isotropic radial distribution.", "Providing a uniform distribution of radiation from a seed has been difficult to accomplish.", "For example, present-day seeds have a radioisotope adsorbed onto a carrier substrate, which is placed into a metal casing that is welded at the ends.", "The most advantageous materials of construction for the casing which encapsulates the radioisotope-laden carrier are stainless steel, titanium, and other low atomic number metals, with titanium and titanium alloys being preferred.", "However, problems exist with respect to sealing casings made from these materials.", "In particular, metallic casings typically are sealed by welding, but welding of such small casings is difficult because welding can locally increase the casing wall thickness, or can introduce higher atomic number materials at the ends of the casing where the welds are located.", "The presence of such localized anomalies can significantly alter the geometrical configuration at the welded ends, resulting in undesirable shadow effects in the radiation pattern emanating from the seed.", "Such seeds also have the disadvantage of providing a non-homogeneous radiation dose to the target due to their construction, i.e.", "the relatively thick ends attenuate the emanating radiation more than the relatively thin body of the seed.", "Problems also have been encountered in homogeneously applying the radioisotope to the substrate.", "Brachytherapy seeds are small in size, and the amount of radioisotope present in each seed is extremely small, e.g.", "less than 1×10 −6 g of radioactive isotope per seed.", "The amount of radioisotope present in each seed necessarily decreases as the specific activity of the isotope increases.", "This presents severe handling and manufacturing problems with respect to homogeneously applying a small chemical amount of radioisotope onto the substrate, especially when the radioisotope is carrier-free.", "These problems, together with safety problems, increase in scope as the radioactivity of the isotope increases.", "Several patents are directed to implantable radioactive seeds for use in brachytherapy.", "Examples of such patents include Kubiatowicz U.S. Pat.", "No.", "4,323,055; Suthanthiran U.S. Pat.", "No.", "4,891,165; Russell, Jr. et al.", "U.S. Pat.", "Nos.", "4,784,116 and 4,702,228; Lawrence U.S. Pat.", "No.", "3,351,049; Good U.S. Pat.", "No.", "5,342,283; Carden, Jr. U.S. Pat.", "No.", "5,405,309; and Langton et al.", "U.S. Pat.", "No.", "5,460,592.U.S.", "Pat.", "No.", "5,405,309 addresses the previously mentioned problem of uniformly distributing carrier-free 103 Pd on a substrate.", "The specific problem addressed by U.S. Pat.", "No.", "5,405,309 is that carrier-free radioisotopes are present in a seed at vanishingly small amounts, and that use of an extremely dilute carrier-free radioisotope solution presents significant handling problems, in addition to safety problems associated with an intensely radioactive composition.", "U.S. Pat.", "No.", "5,405,309 teaches that 103 Pd can be applied more easily, evenly, and safely to a substrate by admixing non-radioactive palladium metal (i.e.", "carrier Pd) with carrier-free 103 Pd to increase the physical mass of the palladium and facilitate application of the palladium onto the substrate.", "Because 103 Pd is expensive to produce, it is important that application of the 103 Pd radioisotope onto the substrate is as efficient and reproducible as possible.", "The method of U.S. Pat.", "No.", "5,405,309 utilizes electroplating, i.e.", "a process involving passage of an electric current, to achieve a homogeneous distribution of 103 Pd on the substrate.", "However, the addition of non-radioactive palladium metal, i.e.", "carrier Pd, to facilitate electroplating of 103 Pd attenuates the low energy X-ray emissions of the 103 Pd adsorbed onto the substrate by providing an additional high Z material that attenuates radiation emanating from radioactive 103 Pd.", "The result is that additional 103 Pd must be applied to the substrate to attain at least a threshold radioactive dose.", "This adds to the cost of such 103 Pd seeds.", "It is a major disadvantage to prepare costly carrier-free 103 Pd (eg.", "using a high energy cyclotron), then to use a process that provides a product wherein a portion of the radioactive emissions are effectively lost by dilution of carrier-free 103 Pd with carrier Pd.", "Although the above patents illustrate improvements in seeds for use in brachytherapy, the art still suffers from the problem of providing a 103 Pd seed that, simultaneously, is easy to manufacture and has a uniform distribution of radioisotope on the substrate, while minimizing attenuation of radioactivity emanating from the seed.", "The present invention is directed to providing 103 Pd brachytherapy seeds having these attributes." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is directed to 103 Pd-containing brachytherapy seeds.", "More particularly, the present invention is directed to carrier-free 103 Pd brachytherapy seeds, wherein the carrier-free 103 Pd is homogeneously deposited on a substrate to provide a uniform radiation dose in the treatment of a disease, like cancer.", "Accordingly, one aspect of the present invention is to provide brachytherapy seeds comprising carrier-free 103 Pd adsorbed on the surface of a suitable substrate.", "Preferably, the seed has a total activity of about 10 to 75 MBq (ca.", "0.3 to 2 mCi), preferably 25 to 50 MBq (ca.", "0.7 to 1.4 mCi), and more preferably 30 to 45 MBq (ca.", "0.8 to 1.2 mCi).", "Another aspect of the present invention is to provide a method of manufacturing a brachytherapy seed comprising carrier-free 103 Pd adsorbed onto a substrate.", "The method comprises an electroless deposition of carrier-free 103 Pd onto a substrate.", "Yet another aspect of the present invention is to provide a substrate for a carrier-free 103 Pd brachytherapy seed, wherein, in one embodiment, the substrate comprises a core of a radiopaque metal (e.g.", "silver), a sheath of a radiotransparent metal (e.g.", "aluminium) surrounding the core, and a coating of a material suitable for an electroless deposition of 103 Pd (e.g.", "copper or nickel) applied over the radiotransparent metal sheath.", "In a preferred embodiment, the outer metal coating comprises nickel.", "In another embodiment, the core of the substrate comprises a radiotransparent core, such as a polymer, graphite, or a low atomic number (Z) metal, such as aluminium.", "The radiotransparent core is coated with a material suitable for an electroless deposition of 103 Pd.", "These and other aspects of the invention will become apparent from the following detailed description of the preferred embodiments, taken in conjunction with the Figures." ], [ "FIELD OF THE INVENTION The present invention relates to radiotherapy and to carrier-free palladium-103 (103Pd) brachytherapy seeds used in therapeutic medical treatments.", "In particular, the present invention relates to radioactive brachytherapy seeds comprising: (a) a carrier-free 103Pd radioisotope, (b) a substrate for the carrier-free 103Pd radioisotope, and (c) a casing for encapsulating the carrier-free 103Pd-laden substrate.", "BACKGROUND OF THE INVENTION Radiation therapy is the treatment of diseases and conditions, especially the treatment of tumors, including malignant tumors, with radiation.", "In radiation therapy, the ultimate aim is to destroy the malignant tissue without causing excessive radiation damage to nearby healthy, and possibly vital, tissue.", "This is difficult to accomplish because of the proximity of malignant tissue to healthy tissue.", "Medical personnel and investigators have developed methods for preferentially irradiating deep seated diseased tissue as opposed to healthy tissue.", "These methods include the use of high energy X-ray beams together with cross fire and rotational techniques which create a radiation pattern that is maximized at the site of the diseased tissue.", "Nonetheless, some absorption and damage inevitably occurs to healthy tissue in the path through which radiation passes to arrive at deep-seated diseased tissue.", "One method of limiting the zone of irradiation utilizes radioactive articles in the form of small, radioactive “seeds,” which are permanently or temporarily implanted at the zone to be irradiated.", "Such seeds contain a radioactive source disposed within a sealed capsule.", "The small size of the therapeutic seeds allows the seeds to be inserted or implanted within or near the tissue to be treated, for example, to totally surround the treated tissue.", "Radiation treatment can involve a temporary implantation of a radioactive source for a calculated period, followed by its removal.", "Alternatively, the radioactive source can be implanted in the patient permanently and left to decay to an inert state over a predictable time.", "The use of temporary or permanent implantation depends on the disease or condition being treated, the radioisotope selected, and the duration and intensity of required treatment.", "The advantages of interstitial implantation of a radiation-emitting article for localized tumor treatment have long been recognized.", "Interstitially implanted articles concentrate the radiation at a zone where radiation treatment is needed, e.g.", "near or within diseased tissue in order to directly affect diseased tissue, while exposing normal, healthy tissue to substantially less radiation than beaming radiation into the body from an external source.", "Implanting radioactive articles in proximity to or directly within diseased or damaged tissue within a body is a therapy referred to as brachytherapy (i.e.", "short-range therapy).", "Brachytherapy is a general term for a medical treatment involving placement of a radioactive source near diseased tissue.", "Brachytherapy has been proposed for use in the treatment of a variety of diseases and conditions, including arthritis and cancer, for example, breast, brain, liver, and ovarian cancer, and especially prostate cancer in men [see for example, J. C. Blasko et al., The Urological Clinics of North America, 23, 633-650 (1996), and H. Ragde et al., Cancer, 80, 442-453 (1977)].", "This form of therapy permits the application of larger doses of radiation directly to diseased or damaged tissue, like tumors.", "Permanent implants for prostate cancer treatment comprise radioisotopes with relatively short half-lives and lower energies relative to radioisotopes used in temporary implants.", "Examples of permanently implantable radioisotopes include iodine-125 and palladium-103.The radioisotope generally is disposed on a substrate, which is encapsulated in a metal casing, for example, a titanium casing, to form a “seed,” which is then implanted in the patient.", "Radioactive seeds are disclosed, for example, in Lawrence U.S. Pat.", "No.", "3,351,049 and Kubiatowicz U.S. Pat.", "No.", "4,323,055.U.S.", "Pat.", "No.", "3,351,049 discloses conventional brachytherapy seeds comprising titanium containers encapsulating ion exchange resin beads onto which a radioactive ion, e.g.", "125I or 103Pd has been adsorbed.", "U.S. Pat.", "No.", "3,351,049 also discloses that 103Pd, preferably carrier-free, could be plated on a 3.5 mm long plastic rod.", "However, U.S. Pat.", "No.", "3,351,049 does not disclose any method of plating carrier-free 103Pd onto the plastic rod.", "As discussed hereafter, the uniform distribution of carrier-free 103Pd on a substrate has been an ongoing and unsolved problem.", "U.S. Pat.", "No.", "4,323,055 discloses brachytherapy seeds comprising a coating of radioactive silver iodide on a silver wire encapsulated inside a titanium container.", "WO 97/19706 discloses the immobilization of a radioactive powder within a polymeric matrix.", "The seeds disclosed in prior patents comprise a tiny sealed capsule having an elongate cavity containing the radioisotope, e.g., iodine-125 (125I) or palladium-103 (103Pd), adsorbed onto a substrate.", "Such seeds are suitable for use with radioisotopes that emit radiation capable of penetrating the capsule walls.", "Therefore, the seeds generally contain radioisotopes that emit γ-radiation or low-energy X-rays, as opposed to β-emitting radioisotopes.", "Because of the low energy X-rays emitted by 125I and 103Pd, and the short half-life of 125I and 103Pd, the seeds can remain implanted in the tissue of a patient indefinitely without excessive damage to surrounding healthy tissue or excessive exposure to other individuals near the patient.", "In order to function effectively, radiation emitting from the radioisotope within the seed should not be blocked or otherwise unduly attenuated.", "Seeds based on metal wire substrates have the disadvantage that a portion of the radioactivity is absorbed by the wire substrate itself, i.e.", "the radioactive emissions from the seed are attenuated by the wire.", "The amount of radioactivity absorbed by the wire increases as the atomic number (i.e.", "Z) of the metal wire substrate increases.", "The precise amount of attenuation is related to the identity and the dimensions of the wire substrate.", "For example, silver iodide-125 coated on an 0.5 mm diameter silver wire has up to about 40-50% of the radioactivity absorbed by the silver wire.", "Therefore, in the manufacture of a radioactive seed of a preselected activity, additional 125I is loaded onto the wire to account for the absorption of radioactivity by the wire and also by the seed capsule.", "As the preselected radioactivity of the seed increases, the cost of the extra amount of radioisotope which is loaded onto the wire substrate also increases.", "Radiation emitted from the radioisotope also should be distributed uniformly from the seed in all directions, i.e.", "an isotropic radial distribution.", "Providing a uniform distribution of radiation from a seed has been difficult to accomplish.", "For example, present-day seeds have a radioisotope adsorbed onto a carrier substrate, which is placed into a metal casing that is welded at the ends.", "The most advantageous materials of construction for the casing which encapsulates the radioisotope-laden carrier are stainless steel, titanium, and other low atomic number metals, with titanium and titanium alloys being preferred.", "However, problems exist with respect to sealing casings made from these materials.", "In particular, metallic casings typically are sealed by welding, but welding of such small casings is difficult because welding can locally increase the casing wall thickness, or can introduce higher atomic number materials at the ends of the casing where the welds are located.", "The presence of such localized anomalies can significantly alter the geometrical configuration at the welded ends, resulting in undesirable shadow effects in the radiation pattern emanating from the seed.", "Such seeds also have the disadvantage of providing a non-homogeneous radiation dose to the target due to their construction, i.e.", "the relatively thick ends attenuate the emanating radiation more than the relatively thin body of the seed.", "Problems also have been encountered in homogeneously applying the radioisotope to the substrate.", "Brachytherapy seeds are small in size, and the amount of radioisotope present in each seed is extremely small, e.g.", "less than 1×10−6 g of radioactive isotope per seed.", "The amount of radioisotope present in each seed necessarily decreases as the specific activity of the isotope increases.", "This presents severe handling and manufacturing problems with respect to homogeneously applying a small chemical amount of radioisotope onto the substrate, especially when the radioisotope is carrier-free.", "These problems, together with safety problems, increase in scope as the radioactivity of the isotope increases.", "Several patents are directed to implantable radioactive seeds for use in brachytherapy.", "Examples of such patents include Kubiatowicz U.S. Pat.", "No.", "4,323,055; Suthanthiran U.S. Pat.", "No.", "4,891,165; Russell, Jr. et al.", "U.S. Pat.", "Nos.", "4,784,116 and 4,702,228; Lawrence U.S. Pat.", "No.", "3,351,049; Good U.S. Pat.", "No.", "5,342,283; Carden, Jr. U.S. Pat.", "No.", "5,405,309; and Langton et al.", "U.S. Pat.", "No.", "5,460,592.U.S.", "Pat.", "No.", "5,405,309 addresses the previously mentioned problem of uniformly distributing carrier-free 103Pd on a substrate.", "The specific problem addressed by U.S. Pat.", "No.", "5,405,309 is that carrier-free radioisotopes are present in a seed at vanishingly small amounts, and that use of an extremely dilute carrier-free radioisotope solution presents significant handling problems, in addition to safety problems associated with an intensely radioactive composition.", "U.S. Pat.", "No.", "5,405,309 teaches that 103Pd can be applied more easily, evenly, and safely to a substrate by admixing non-radioactive palladium metal (i.e.", "carrier Pd) with carrier-free 103Pd to increase the physical mass of the palladium and facilitate application of the palladium onto the substrate.", "Because 103Pd is expensive to produce, it is important that application of the 103Pd radioisotope onto the substrate is as efficient and reproducible as possible.", "The method of U.S. Pat.", "No.", "5,405,309 utilizes electroplating, i.e.", "a process involving passage of an electric current, to achieve a homogeneous distribution of 103Pd on the substrate.", "However, the addition of non-radioactive palladium metal, i.e.", "carrier Pd, to facilitate electroplating of 103Pd attenuates the low energy X-ray emissions of the 103Pd adsorbed onto the substrate by providing an additional high Z material that attenuates radiation emanating from radioactive 103Pd.", "The result is that additional 103Pd must be applied to the substrate to attain at least a threshold radioactive dose.", "This adds to the cost of such 103Pd seeds.", "It is a major disadvantage to prepare costly carrier-free 103Pd (eg.", "using a high energy cyclotron), then to use a process that provides a product wherein a portion of the radioactive emissions are effectively lost by dilution of carrier-free 103Pd with carrier Pd.", "Although the above patents illustrate improvements in seeds for use in brachytherapy, the art still suffers from the problem of providing a 103Pd seed that, simultaneously, is easy to manufacture and has a uniform distribution of radioisotope on the substrate, while minimizing attenuation of radioactivity emanating from the seed.", "The present invention is directed to providing 103Pd brachytherapy seeds having these attributes.", "SUMMARY OF THE INVENTION The present invention is directed to 103Pd-containing brachytherapy seeds.", "More particularly, the present invention is directed to carrier-free 103Pd brachytherapy seeds, wherein the carrier-free 103Pd is homogeneously deposited on a substrate to provide a uniform radiation dose in the treatment of a disease, like cancer.", "Accordingly, one aspect of the present invention is to provide brachytherapy seeds comprising carrier-free 103Pd adsorbed on the surface of a suitable substrate.", "Preferably, the seed has a total activity of about 10 to 75 MBq (ca.", "0.3 to 2 mCi), preferably 25 to 50 MBq (ca.", "0.7 to 1.4 mCi), and more preferably 30 to 45 MBq (ca.", "0.8 to 1.2 mCi).", "Another aspect of the present invention is to provide a method of manufacturing a brachytherapy seed comprising carrier-free 103Pd adsorbed onto a substrate.", "The method comprises an electroless deposition of carrier-free 103Pd onto a substrate.", "Yet another aspect of the present invention is to provide a substrate for a carrier-free 103Pd brachytherapy seed, wherein, in one embodiment, the substrate comprises a core of a radiopaque metal (e.g.", "silver), a sheath of a radiotransparent metal (e.g.", "aluminium) surrounding the core, and a coating of a material suitable for an electroless deposition of 103Pd (e.g.", "copper or nickel) applied over the radiotransparent metal sheath.", "In a preferred embodiment, the outer metal coating comprises nickel.", "In another embodiment, the core of the substrate comprises a radiotransparent core, such as a polymer, graphite, or a low atomic number (Z) metal, such as aluminium.", "The radiotransparent core is coated with a material suitable for an electroless deposition of 103Pd.", "These and other aspects of the invention will become apparent from the following detailed description of the preferred embodiments, taken in conjunction with the Figures.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a partially cut-away side view of a present brachytherapy seed; FIG.", "2 is a cut-away side view of another embodiment of a present brachytherapy seed; and FIG.", "3 is a cross-sectional view of one embodiment of a substrate for a present brachytherapy seed.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Brachytherapy is a form of radiation therapy wherein a radioactive source is positioned near, or within, a radiation target, e.g.", "a tumor.", "The radioactive source is delivered in the form of a seed containing a radioisotope that has been applied to a substrate.", "The radioisotope-laden carrier is encapsulated by, and sealed within, a suitable metal casing.", "Various radioisotopes have been used in brachytherapy, and several factors are considered when deciding which radioisotope is used for a particular therapy.", "These factors include the type and intensity of radiation emanating from the radioisotope, the half-life of the radioisotope, and the particular disease or condition being treated.", "The decision also encompasses considerations of efficacy of the therapy, safety to surrounding healthy tissue, and safety to medical personnel that handle and implant the brachytherapy seeds.", "The threshold radiation dose required to treat a particular disease, such as a cancer, is an important parameter in designing a brachytherapy seed.", "A threshold dosage must reach the target site for effective treatment, but a significant percentage of the radiation emitted from a brachytherapy seed is attenuated and unavailable for therapeutic purposes.", "For example, the substrate absorbs a significant portion of the emitted radiation, and the casing further attenuates radiation emanating from the seed.", "In addition, prior seed designs can suffer in having the radioisotope unevenly distributed on the substrate.", "This results in an uneven radiation dose emanating from the seed as a whole.", "Accordingly, the amount of radioisotope applied to the carrier is increased such that the seed as a whole emits at least the threshold radiation dose necessary to treat the disease.", "It would be desirable, therefore, to provide a brachytherapy seed wherein attenuation of radiation emanating from the radioisotope is reduced.", "Reducing the amount of attenuation, while maintaining the threshold radiation dose to treat the disease, has several advantages, including a reduced gross amount of radioisotope applied to the substrate, an increased safety to personnel that manufacture, handle, and implant the seeds, and a significant cost savings.", "In a first aspect, the present invention provides: a brachytherapy seed which comprises: (a) a substrate having a surface suitable for the electroless deposition of palladium, (b) carrier-free 103Pd disposed on said surface of said substrate to provide a carrier-free 103Pd-laden substrate; and (c) a biocompatible casing for encapsulating the carrier-free 103Pd-laden substrate.", "The present invention, therefore, is directed to carrier-free 103Pd seeds, i.e.", "seeds free of non-radioactive palladium and other metals, and methods of manufacturing such seeds.", "As used herein, “carrier-free 103Pd” is defined as palladium-103 which does not include non-radioactive palladium metal or other palladium isotopes added, or other non-radioactive metals, as a carrier.", "The present process utilizes electroless plating of carrier-free 103Pd onto a substrate.", "By the term “electroless” is meant a process that does not utilize the passage of an electric current.", "As a result, seed manufacture is simplified, and no additional carrier is present to attenuate 103Pd emissions.", "The present invention provides a simple and efficient method of depositing carrier-free 103Pd onto a suitable substrate, which in turn is loaded into a seed casing.", "A preferred method utilizes electroless deposition of carrier-free 103Pd onto a surface of a “wire” or “pin” (i.e.", "a short metallic rod) substrate.", "By the term “surface suitable for the electroless deposition of palladium” is meant a surface material which, on contact with palladium ions in solution, in the presence of a reducing agent, reacts to deposit palladium metal on the surface in an autocatalytic chemical reduction.", "In particular, carrier-free 103Pd is deposited on a catalytic substrate surface from a solution containing 103Pd and a reducing agent.", "Preferred such surface materials suitable for the electroless deposition of palladium are nickel, copper or mixtures thereof.", "Nickel is especially preferred.", "Suitably, the substrate is a cylindrical rod or wire having a treated surface to which the carrier-free 103Pd is applied.", "The substrate serves primarily as a solid support on which the carrier-free 103Pd is uniformly distributed.", "A substrate utilized in the present invention can be constructed of any material that serves as a solid support for the carrier-free 103Pd.", "It is preferred that the substrate is constructed from a material that is detectable by X-rays, i.e.", "is radiopaque, to serve as an X-ray marker.", "This enables medical personnel to properly position the seeds near or in the target site, and to scan the patient at a later date to determine whether the seeds have moved from the target site.", "The substrate, therefore, is constructed from any material onto which the requisite therapeutic amount of carrier-free 103Pd can be attached, and preferably, that is detectable by X-rays or other detection means.", "Silver and copper are preferred materials for the substrate, because these metals provide good X-ray visualization.", "Also, carrier-free 103Pd also can be easily attached to a copper surface.", "Other X-ray opaque metals, such as gold and iron, for example, can be used as a substrate.", "The substrate also can be a radiotransparent metal, or non-metallic material such as a polymer or graphite rod, that is capable of having carrier-free 103Pd deposited thereon by an electroless method.", "When the substrate comprises a radiopaque metal, such substrates have the advantage that they function both as a carrier for the radioisotope and as a marker for detection of the seeds.", "Furthermore, because the substrate generally conforms to the shape of the casing, the exact location and orientation of the seed in the tissue can be determined from X-ray photographs, for example.", "Preferred substrates include (a) a simple radiopaque metal rod or (b) a radiotransparent metal, polymer, or graphite rod.", "Suitable and preferred substrates are described in detail below.", "The substrates of the present invention are generally acicular in shape, and have a circular cross section.", "They are of a suitable length and diameter for easy disposition into a seed casing, and preferably occupy a substantial portion of the casing cavity.", "The substrate preferably is about 3 mm long and 0.5 mm in diameter (maximum) when used in a standard casing having a length of 4.5 mm and an exterior diameter of 0.8 mm.", "A 3 mm long substrate results in minimum shifting within the casing while allowing adequate room to weld the ends of the casing without adversely affecting the substrate.", "The diameter of the substrate is about 0.1 mm to about 0.7 mm (the maximum inside diameter of a conventional casing).", "The preferred diameter is about 0.5 mm, which, if needed, provides good X-ray visibility, is relatively easy to handle during seed manufacture, and slides easily into the seed casing without abrading against the interior walls of the casing.", "The substrate also can be spherical in shape.", "The brachytherapy seed of the present invention uniformly emits radiation over its entire geometry because of a homogeneous distribution of carrier-free 103Pd on the substrate.", "A uniform radiation emission from the seed reduces the amount of radioisotope needed to provide a therapeutic dose because the seed does not have any relatively “cold” spots attributed to an uneven distribution of 103Pd on the substrate.", "Hence, the seed does not require as large an excess of carrier-free 103Pd to provide a threshold radioactive dose, and the seed as a whole provides a therapeutic radioactive dose to the target.", "Prior 103Pd seeds utilized non-radioactive, carrier palladium to dilute the 103Pd, which permitted a uniform distribution of 103Pd on the substrate.", "For example, see U.S. Pat.", "No.", "5,405,309 which adds carrier Pd to carrier-free 103Pd, and U.S. Pat.", "No.", "4,702,228 which utilizes 103Pd-enriched palladium that also contains non-radioactive Pd carrier.", "In contrast, the present invention utilizes carrier-free 103Pd.", "The carrier-free 103Pd-laden substrates of the present invention may be prepared by an electroless deposition process.", "Electroless deposition of metals, including palladium, is disclosed, for example, in Heugh et al.", "U.S. Pat.", "No.", "4,255,194; Abys U.S. Pat.", "No.", "4,424,421; Josso et al.", "U.S. Pat.", "No.", "5,085,693; Das et al.", "US 5,264,288; and Feldstein et al.", "U.S. Pat.", "No.", "5,420,477, each incorporated herein by reference.", "The present invention shows that carrier-free 103Pd can be deposited onto a substrate to provide a seed product having an apparent activity of about 37 MBq (1 mCi) 103Pd.", "The carrier-free 103Pd can be deposited by an electroless method using either (a) an acid electroless plating solution containing carrier-free 103Pd in 0.1 to 0.001 N HCl, (b) an alkaline electroless plating solution containing carrier-free 103Pd, ammonium sulfate (13.2 g/L), ammonium citrate (24.3 g/L), and ammonium hydroxide (50 mL/L).", "After deposition, the carrier-free 103Pd-laden substrate is sealed within a biocompatible casing.", "By the term “biocompatible” is meant a material that does not corrode when in contact with body fluids, and is non-toxic when implanted in the patient's body.", "A brachytherapy seed casing typically is manufactured from a cylindrical tube of a metal that provides adequate thin wall strength, and that readily allows radiation to pass uniformly through the material.", "The thin walls permit a larger substrate to be disposed in the seed, and reduces attenuation of emitted radiation.", "Suitable casing materials are metals, and typically low atomic numbered metals, such as stainless steel alloy, titanium, or titanium alloy.", "Higher atomic number metals, such as gold or platinum, attenuate too much radiation emanating from the carrier-free 103Pd-laden substrate to be useful per se.", "However, higher atomic numbered metals are useful as a plating over various low atomic number materials, such as beryllium, which otherwise is too toxic if used without an outer biocompatible coating.", "Other suitable casing materials include, but are not limited to, tantalum, nickel alloys, copper alloys, and aluminium alloys.", "If the seed is designed for detection by ultrasound, as opposed to X-rays, the casing can be manufactured from an echogenic material, e.g.", "the casing can be predominantly aluminium.", "Suitable casing materials also include inert synthetic materials, for example, Teflon™.", "The casing is completely sealed so there is no danger of leakage.", "Titanium, which has a low atomic number and a high strength-to-weight ratio, is the preferred casing material.", "Titanium is exceptionally corrosion-resistant, and is satisfactory from the standpoint of biocompatibility.", "Preferably, the titanium is a pure alloy to assure good working properties.", "The wall thickness of a titanium casing can be about 0.025 to about 0.127 mm, with radiation attenuation being about 7% per 0.025 mm.", "An optimum wall thickness for a titanium casing is about 0.051 mm.", "Examples of casing designs are illustrated in FIGS.", "1 and 2.A single casing can contain one radiolabeled substrate that occupies substantially all of the cavity inside the casing.", "Alternatively, each casing can contain two or more such substrates, for example, optionally separated by a suitable spacer.", "The substrate arrangement is such that there is a uniform radiation field emanating from the seed.", "An example of a present brachytherapy seed is illustrated in FIG.", "1, wherein a seed [1] contains a therapeutic amount of carrier-free 103Pd [2] disposed on a substrate [3].", "The carrier-free 103Pd-laden substrate [3] is disposed in a cavity [5] of a cylindrical casing [4].", "Casing [4] is sealed at ends [6] and [7], typically by welding.", "FIG.", "2 illustrates another embodiment of a present brachytherapy seed [10] having a carrier-free 103Pd-laden substrate [12] encapsulated by a casing [14].", "Casing [14] is a tube having a centre portion [16] and two end portions [18].", "Centre portion [16] has a diameter [d1] that is substantially larger than the diameter [d2] of end portions [18].", "Ends [20] and [22] are sealed, for example by plasma discharge welding.", "In FIG.", "2, a cylindrical tube, such as a titanium tube, of uniform diameter is swaged at each end to provide a tube having a centre portion of a first diameter and end portions of a second diameter, wherein the second diameter is substantially less than the first diameter.", "In particular, the diameter of each end portion of the swaged tube, independently, is about 25% to about 80% less than the diameter of the center portion of the tube.", "The casing ends are sealed by standard techniques such as plasma discharge, laser, electron beam, or tungsten inert gas (TIG) welding.", "The overall size of a brachytherapy seed casing is designed for implantation by perforate penetration or injection, e.g.", "by hypodermic needle or similar device especially designed for positioning brachytherapy seeds.", "Therefore, the casing has a relatively narrow maximum outer diameter of about 0.25 to about 1 mm, and about 0.25 to about 25 mm in length.", "For permanent implantation, as by hypodermic injection, the outside diameter of the seed is preferably about 0.80 mm and is small enough to pass through a 17-gauge hypodermic needle.", "The seed typically is about 4 to 5 mm long.", "Such seeds exhibit minimal movement in the tissue and do not migrate from the area where they are implanted.", "Carrier-free 103Pd is produced by a high current cyclotron bombardment of a rhodium target at an energy of about 15 to about 20, and typically about 18 MeV (mega electron volts).", "103Pd produced in a cyclotron is carrier-free, unlike nuclear reactor-produced 103Pd.", "103Pd produced in a nuclear reactor results from the bombardment of 102Pd.", "The abundance of 102Pd is 1.0% of naturally-occurring palladium.", "The most enriched 102Pd which can be used in the 103Pd reactor process contains about 75-80% 102Pd with the remaining 20-25% being other Pd and non-Pd isotopes.", "The maximum specific activity of 103Pd produced in a nuclear reactor is about 12,950 GBq/g (ca.", "350 Ci/g).", "In contrast, carrier-free 103Pd produced in a cyclotron has a specific activity of about 2,775,000 GBq/g (ca.", "75,000 Ci/g).", "The term “specific activity” as used herein means the total activity of 103Pd per gram of material.", "The term “therapeutic or apparent activity” as used herein means the 103Pd activity as determined from measuring the X-ray intensity outside the seed.", "This is the therapeutic activity that treats the disease or condition, and, therefore the activity used when developing a treatment plan for a patient.", "It can be seen that utilizing carrier-free 103Pd as the radioisotope can greatly reduce the gross amount of Pd adsorbed onto the substrate because of the extremely high specific activity of carrier-free 103Pd.", "By adsorbing only carrier-free 103Pd onto a substrate, attenuation of 103Pd emissions is reduced because non-radioactive carrier Pd is not present on the substrate.", "In addition, the step of adding carrier Pd to carrier-free 103Pd is eliminated, thereby eliminating one manipulation step involving a very highly radioactive material.", "Carrier-free 103Pd can be prepared, for example, by depositing rhodium metal onto a suitable substrate, such as a copper or a silver substrate.", "The resulting rhodium target is then placed in a charged particle accelerator, such as a cyclotron, and bombarded with protons or deuterons.", "The energy of the impacting particle, i.e.", "about 18 MeV, is selected such that essentially the only Pd atoms created on the rhodium target are 103Pd, that is, the 103Pd is carrier-free.", "The rhodium metal target containing the carrier-free 103Pd is then treated to remove the rhodium metal from the substrate, for example, by etching with an acid, such as nitric acid (HNO3).", "This removal step often is accomplished by mechanically disrupting the continuity of the rhodium layer on the substrate, for example, by perforating the rhodium surface with a sharply pointed tool.", "The exposed (i.e.", "non deposit-containing) substrate surface is protected by an inert covering layer and the perforated target is immersed in an HNO3 bath.", "A mixture containing rhodium flakes results, which is filtered to recover the solid rhodium flakes containing 103Pd.", "The recovered rhodium flakes are rinsed on the filter, and the flakes together with the filter are placed in a crucible and heated to decompose the filter and leave the rhodium metal flakes containing the 103Pd.", "The rhodium metal flakes are then partially dissolved in molten NaHSO4 (sodium bisulfate), and the resulting NaHSO4/rhodium flake mixture is dissolved in dilute hydrochloric acid (HCl).", "This procedure is repeated several times in order to dissolve any remaining rhodium metal containing carrier-free 103Pd.", "This method of producing carrier-free 103Pd is fully disclosed in Carden, Jr., U.S. Pat.", "No.", "5,405,309, incorporated herein by reference.", "Alternatively, carrier-free 103Pd can be produced by the high current cyclotron bombardment of a natural rhodium target with protons at an energy of approximately 18 MeV.", "The rhodium cyclotron target is produced by electroplating natural rhodium onto a copper support.", "Following bombardment, the rhodium-electroplated region of the copper support is removed from the bulk of the copper support, i.e.", "the rhodium-electroplated region is punched out from the support.", "The rhodium region then is placed in a 50% nitric acid solution to remove the remaining copper backing.", "The rhodium metal next is dissolved in concentrated hydrochloric acid.", "Hydrogen tetrachloroaurate trihydrate can be added (e.g.", "at 2 to 3 times the weight of rhodium, preferably the stoichiometric amount of rhodium) to speed dissolution.", "In addition, dissolution can be facilitated by heating the hydrochloric acid solution, and applying an alternating current with graphite electrodes.", "The resulting solution contains rhodium metal, rhodium isotopes, and 103Pd in approximately 6N hydrochloric acid.", "The solution is transferred to an extractor, and an organic compound, e.g.", "0.25% α-furyldioxime in 20% aqueous ethanol, then is added to produce an organic-soluble 103Pd metal complex.", "This complex is extracted with chloroform (3×50 ml).", "The combined chloroform extracts are washed with dilute hydrochloric acid, then the chloroform is evaporated to dryness.", "The residue is treated with a mixture of aqua regia (4:1 HCl:HNO3) and 30% hydrogen peroxide to decompose any remaining organic materials.", "The remaining carrier-free 103Pd then is solubilized in dilute hydrochloric acid.", "In a second aspect, the present invention provides a substrate in a form suitable for use in a brachytherapy seed, which has a surface coating suitable for the electroless deposition of palladium and further comprises a radiopaque metal core chosen from silver, platinum, iridium, palladium, thallium, copper, iron or lead or a mixture thereof, surrounded by a sheath of a radiotransparent metal.", "In one preferred embodiment, the substrate has concentric zones comprising the radiopaque metal core/a radiotransparent metal sheath/a coating suitable for electroless deposition of palladium, (eg.", "carrier-free 103Pd), as illustrated in FIG.", "3 and described in detail hereafter.", "For such a concentric substrate, a preferred radiotransparent metal sheath comprises aluminium, and a preferred radiopaque core material is silver.", "One preferred substrate is illustrated in FIG.", "3.In FIG.", "3, substrate [30] comprises a radiopaque metal core [32] surrounded by a radiotransparent metal sheath [34].", "The radiotransparent metal sheath [34] is covered by a coating [36] of a material suitable for an electroless deposition of carrier-free 103Pd, for example, a metal like copper or nickel.", "The core [32] is preferably radiopaque to X-rays and therefore useful for imaging implanted seeds, because the casing and metal sheath [34] are relatively transparent to X-rays.", "Sheath [34] comprises a low atomic number material, like aluminium, to further minimize attenuation of the carrier-free 103Pd emissions.", "The metal coating [36] has electrochemical properties that facilitate electroless deposition of carrier-free 103Pd onto the substrate.", "The electroless 103Pd plating is performed using carrier-free 103Pd in aqueous acid or base solution, preferably in the presence of a buffer and/or a reducing agent, e.g.", "hydrazine.", "Substrate [30] typically has a length of about 2.5 to about 5 mm.", "The outer diameter of substrate [30] is about 0.25 to about 0.8 mm, typically about 0.5 mm.", "Core [32] has a diameter of about 0.05 mm to about 0.3 mm, and metal sheath [36] has a thickness of about 0.1 to about 0.3 mm.", "A radiopaque core [32] comprises a metal having a high Z, for example silver, gold, platinum, iridium, palladium, thallium, copper, iron, or lead.", "Radiotransparent sheath [34] comprises a low Z metal, like aluminium.", "The presence of a low Z metal in sheath [34] reduces attenuation of radiation emitted from the carrier-free 103Pd.", "A radiotransparent core, either free of or having a radiotransparent sheath, also reduces attenuation of radiation emitted by the carrier-free 103Pd.", "In most preferred embodiments, substrate [30] is coated with a thin coating [36] of a material suitable for an electroless deposition of 103Pd, such as a metal like nickel or copper, about 2×10−3 to about 5×10−3 mm thick.", "Coating [36] facilitates electroless coating of carrier-free 103Pd onto substrate [30].", "In most preferred embodiments, coating [36] comprises nickel.", "In a third aspect, the present invention provides a substrate in a form suitable for use in a brachytherapy seed, which comprises a radiotransparent core with a surface coating, wherein the surface coating is suitable for the electroless deposition of palladium.", "The radiotransparent core may suitably comprise: a polymer, graphite, or a low Z metal, having a coating of a material suitable for electroless deposition of 103Pd.", "Substrates of this type are particularly useful when means other than X-ray visualisation are utilized to detect and position the seeds near or in a target site, because a radiopaque core is no longer essential.", "For example, if the seeds are detected using ultrasound, and the material of construction of the casing is echogenic, then the substrate can be radiotransparent.", "Alternatively, substrates of this type may be chosen such that the thickness of the metallic coating on the radiotransparent core exceeds about 0.05 mm, so that the coating itself ensures X-ray visualisation.", "Such substrates can be prepared by depositing a suitable metal (chemically or by using “sputtering” and “ion plating” techniques) onto a substrate other than a metal, e.g.", "a polypropylene filament.", "In a fourth aspect, the present invention provides a method of preparing a carrier-free 103Pd-laden substrate which comprises: immersing a substrate having a surface suitable for the electroless deposition of palladium into an electroless bath comprising carrier-free 103Pd for a sufficient time to deposit a pre-selected amount of carrier-free 103Pd onto the surface suitable for the electroless deposition of palladium of said substrate.", "The surface suitable for the electroless deposition of palladium preferably comprises nickel or copper or a mixture thereof.", "The electroless bath comprises either (a) an acid electroless plating solution containing carrier-free 103Pd in 0.1 to 0.001 N HCl, (b) an alkaline electroless plating solution containing carrier-free 103Pd, ammonium sulfate (13.2 g/L), ammonium citrate (24.3 g/L), and ammonium hydroxide (50 mL/L).", "In a further aspect, the carrier-free 103Pd seeds of the present invention can be used to treat conditions responsive to brachytherapy.", "Such conditions include: head and neck cancers, melanoma, brain cancers, non-small cell lung cancer, breast cancer, and ovarian, uterine, and cervical cancer, and other diseases including proliferative diseases, arthritis, urethral stricture, and fibroid uterine tumors.", "The present carrier-free 103Pd seeds can thus be used in a method of treating a disease or condition that is responsive to radiation therapy, for example, a cancer, which comprises the permanent or temporary placement of a seed comprising an amount of carrier-free 103Pd adsorbed on the surface of a suitable substrate, at the site to be treated within a patient for a sufficient period of time to deliver a therapeutically effective dose.", "The therapeutically effective dose can be easily determined by persons skilled in the art based on the condition or disease that is being treated, the severity of the disease or condition, the individual patient, and the strength of the radiation emanating from the carrier-free 103Pd seeds.", "The amount of carrier-free 103Pd required to provide a therapeutically effective dose depends in part on the amount of radiation absorbed by the substrate and by the casing.", "The amount of attenuation in any given case can be readily determined by a skilled person, for example, by trial-and-error experimentation or by calculation, and the amount of carrier-free 103Pd adsorbed onto the carrier can be adjusted accordingly.", "For example, the ratio of the specific activity of the substrate to the apparent activity of the seeds may be about 1.5 to about 2 to 1, due to absorption by a silver substrate and a titanium casing.", "The invention is illustrated by the following Examples.", "Example 1 shows how a coating suitable for the electroless deposition of palladium (in this case nickel), can be deposited onto a substrate.", "Example 2 shows the ability of an electroless method to deposit non-radioactive palladium onto a metal substrate.", "The electroless plating of non-radioactive palladium from dilute acid onto a copper substrate was accomplished in less than one hour.", "In addition, the Pd remained adhered to the copper substrate after a 24-hour soak in distilled water.", "Similar tests using an aluminium substrate indicated that the addition of a reducing agent to the bath facilitated Pd deposition.", "Example 3 shows the electroless deposition of carrier-free 103Pd onto a substrate.", "EXAMPLE 1 Coating of a Substrate with Nickel A substrate can be coated with nickel by immersing the substrate in an electroless bath containing 4.7 g/L nickel sulfate hexahydrate, 3.0 g/L sodium hypophosphite hydrate, 8.1 g/L sodium citrate dihydrate, and 4.0 g/L ammonium chloride, at pH 9.25.About 5 mL of this nickel electroless bath was added to a small glass vial containing 200 zincated aluminium pins having a silver core.", "The vial was rotated at 75° C. for 15 minutes.", "The resulting substrates were nickel-plated, silver-cored aluminium pins.", "EXAMPLE 2 Electroless Deposition of Non-Radioactive Palladium The electroless deposition of 7 μg (micrograms) of non-radioactive palladium from different electroless baths onto 700 cleaned copper wires was evaluated.", "The results are summarized below: Bath Composition % Bath Depletion1) 0.1 N HCl 91.3% Ammonium sulfate/citrate2) 0% 0.001 N HCl 97.3% 1)percent of Pd plated from the solution; and 2)13.2 g/L ammonium sulfate, 24.3 g/L ammonium citrate, and 50 mL/L ammonium hydroxide.", "EXAMPLE 3 Electroless Deposition of 103Pd The electroless deposition of carrier-free 103Pd onto a nickel-coated substrate of Example 1 was carried out using an alkaline bath.", "The alkaline bath contained carrier-free 103Pd, ammonium hydroxide (50 mL/L), optional citrate (24.3 g/L) (as a buffer), and a hydrazine (12 mL/L) or a hypophosphite (4.1 g/L) (as a reducing agent).", "The tests showed a reproducible bath depletion of carrier-free 103Pd of over 90%." ] ]

[ [ "Method for saving computer data", "The invention relates to a method for saving computer data which consist in transferring the data to be saved from a client computer to a buffer storage formed by the hard disc of a backup server, organised in a plurality of volumes of predetermined size, and in then transferring the data from said buffer storage onto a final medium (for example magnetic cartridge).", "The invention is characterised in that it consists in recording on said permanent medium, after transferring the volumes from the buffer storage onto the final medium, a single sequence of data specific to each of said volumes (basic data), the method further comprising a step which consists in constituting a database wherein are recorded the basic data." ], [ "1.A method for saving computer data which consist of transferring the data to be backed up from a client computer to a buffer memory formed by the hard disk of a backup server, organised into a plurality of volumes with predetermined sizes, and then transferring the data from the said buffer memory onto the permanent medium (for example a magnetic cartridge), characterised in that a unique sequence of data for each of the said volume (basic data) is recorded on the said permanent medium after each of the volumes and after the transfer onto the permanent medium, the method also comprising a step in which a database is created in which the basic data are saved.", "2.A method for saving computer data according to claim 1, characterized in that the basic data include the logical address on the volume start cartridge and the logical address on the volume end cartridge.", "3.A method for saving computer data according to claim 1, characterized in that the basic data include the approximate size of the volume.", "4.A method for saving computer data according to claim 2, characterized in that the basic data include the approximate size of the volume.", "5.A method for saving computer data according to claim 1, characterized in that the basic data include the number of the reader used to transfer the volume from the disk cache to the cartridge and the number of the disk on which the volume is located at the time that it is transferred to the cartridge.", "6.A method for saving computer data according to claim 1 characterized in that the basic data include the number of the disk partition in which the volume is located before it is transferred to the cartridge.", "7.A method for saving computer data according to claim 1, characterized in that the basic data include the processor number used to transfer the volume of the disk cache to the cartridge.", "8.A method for saving computer data according to claim 1, characterized in that the basic data include the bar code of the cartridge containing the volume and/or the name of the cartridge.", "9.A method for saving computer data according to claim 1, characterized in that the basic data include the hexadecimal code indicating the cartridge type, the total size of data stored on the cartridge, the maximum capacity of the cartridge, the state of the cartridge load counter, the hexadecimal code indicating the state of the volume, the number of the host machine in the configuration to which the volume belongs, the character code used in the volume header, the date of the most recent write or modification made by the host machine, the time of the most recent write or modification made by the host machine, the date of the most recent read access of the volume by the host machine, the time of the most recent read access of the volume by the host machine, the date on which the volume in the disk cache was transferred to the cartridge, and the time at which the volume in the disk cache was transferred to the cartridge.", "10.A method for saving computer data according to claim 1, characterized in that the data transferred from the permanent support to the buffer memory when data are restored to the client computer do not include the basic data.", "11.Equipment for saving data comprising a backup server controlling a buffer memory organised into volumes of a predetermined size, and a station for reading-writing permanent media, the server comprising a communication interface with at least one client computer for data exchanges, characterised in that the server also comprises a database for saving basic data and means of associating the basic data with data recorded temporarily in the volumes of the buffer memory, and for deleting the basic data when the data are transferred from the permanent media to the buffer memory." ], [ "<SOH> FIELD OF THE INVENTION <EOH>This invention relates to the domain of saving computer data." ], [ "<SOH> BRIEF DESCRIPTION OF THE PRIOR ART <EOH>The state of the art includes equipment for making computer backups, comprising a server with a buffer memory, for example a hard disk, and control means for a reader-writer of permanent media.", "U.S. Pat.", "No.", "5,809,511 discloses a system for transferring data between a host station and complementary equipment comprising a cache memory and robot controlled equipment for management of backup media.", "U.S. Pat.", "No.", "5,963,971 discloses a storage system on a medium forming a virtual disk.", "The purpose of this invention is to provide a solution that optimises the size of permanent media and facilitates reconstruction of data." ], [ "CROSS-REFERENCE TO RELATED APPLICATIONS This application is a national phase filing of and claims the benefit of priority of International Application Number PCT/FR01/02381, filed Jul.", "20, 2001, entitled or “Procede de Sauvegarde de Donnees Informatiques,” which translates to “Method for Saving Computer Data”.", "The entire disclosure contained in the above-mentioned patent application is incorporated by reference as if set forth at length herein.", "STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable REFERENCE OF A “MICROFICHE APPENDIX” Not applicable FIELD OF THE INVENTION This invention relates to the domain of saving computer data.", "BRIEF DESCRIPTION OF THE PRIOR ART The state of the art includes equipment for making computer backups, comprising a server with a buffer memory, for example a hard disk, and control means for a reader-writer of permanent media.", "U.S. Pat.", "No.", "5,809,511 discloses a system for transferring data between a host station and complementary equipment comprising a cache memory and robot controlled equipment for management of backup media.", "U.S. Pat.", "No.", "5,963,971 discloses a storage system on a medium forming a virtual disk.", "The purpose of this invention is to provide a solution that optimises the size of permanent media and facilitates reconstruction of data.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood after reading the description of a non-restrictive example embodiment in which: FIG.", "1 shows an aspect of the present invention constructed according to the teachings herein.", "FIG.", "2 shows a further aspect of the present invention constructed according to the teachings herein.", "DETAILED DESCRIPTION OF THE INVENTION In its most general form, the invention relates to a process for backing up computer data which consists of transferring the data to be backed up from a client computer to a buffer memory formed by the hard disk of a backup server, organised into a plurality of volumes with predetermined sizes, and then transferring the data from the said buffer memory onto the permanent medium (for example a magnetic cartridge), characterised in that a unique sequence of data for the said volume (basic data) is recorded on the permanent medium after each of the volumes and after the transfer onto the permanent medium, the method also comprising a step in which a database is created in which the basic data are saved.", "The basic data include the logical address on the volume start cartridge and the logical address on the volume end cartridge.", "The basic data include the approximate size of the volume.", "Advantageously, the basic data include the number of the drive used to transfer the volume from the disk cache to the cartridge and the number of the disk on which the volume is located at the time that it is transferred to the cartridge.", "The basic data include the number of the disk partition in which the volume is located before it is transferred onto the cartridge.", "The basic data include the number of the processor used to transfer the volume of the disk cache to the cartridge.", "The basic data include the bar code of the cartridge containing the volume and/or the name of the cartridge.", "The basic data contain at least part of the data including the hexadecimal code indicating the cartridge type, the total size of data stored on the cartridge, the maximum capacity of the cartridge, the state of the cartridge loading counter, the hexadecimal code indicating the state of the volume, the number of the host machine in the configuration to which the volume belongs, the character code used in the volume header, the date of the most recent write or modification made to the volume by the host machine, the time of the most recent write or modification made by the host machine, the date of the most recent read access of the volume by the host machine, the time of the most recent read access of the volume by the host machine, the date on which the volume in the disk cache was transferred to the cartridge, and the time at which the volume in the disk cache was transferred to the cartridge.", "Advantageously, data transferred from the permanent medium to the buffer memory when data are restored to the client computer do not include the basic data.", "The invention also relates to equipment for backing up data comprising a backup server controlling a buffer memory organised into volumes of a predetermined size, and a station for reading-writing permanent media, the server comprising a communication interface with at least one client computer for data exchanges, characterised in that the server also comprises a database for saving basic data and means of associating the basic data with data recorded temporarily in the volumes of the buffer memory, and for deleting the basic data when the data are transferred from the permanent media to the buffer memory.", "In one operating mode, intended particularly for management of mainframes, data volumes written by host machines are firstly created by the backup equipment according to the invention in a buffer disk space.", "These volumes have a maximum size fixed at the time of the configuration of the backup equipment system according to the invention, that is usually fairly low and is of the order of 250 Mbytes.", "Secondly, the volumes are transferred by the backup equipment according to the invention to cartridges, making one or several copies.", "Only really significant data are transferred to the tape.", "Thus, a maximum volume of 250 Mbytes may for example only contain 10 Mbytes of data.", "In this case, only these 10 Mbytes will be transferred to tape, to optimise tape space.", "The backup equipment uses a database to internally manage the list of known volumes, storing a certain amount of data such as: the name of the volume the medium on which it is stored (disk, cartridge), the position on the medium (number of the disk partition, or logical start and end addresses on the cartridge) etc.", "This data is used to find a volume.", "At the time that data are transferred from the disk cache to cartridges, the backup equipment according to the invention adds private data called “Basic data”, at the end of the transfer of each volume.", "These data are only written onto the cartridges, and are ignored during transfers in the reverse direction, in the case in which a volume is transferred from a cartridge to the disk cache, for example to be restored by the host machine.", "Therefore, they are entirely managed internally by the backup equipment according to the invention and transparently for host machines.", "The basic data for a given volume are written in the form of an ASCII character string with the following structure: Title CR LF VolumeStartposition VolumeEndposition VolumeSize ReaderChannel \\ DiskChannel DiskPartition ProcessorNumber BarCode CartridgeName CartridgeType SizeUsed CartridgeSize LoadCounter VolumeName VolumeStatus HostCode CodingType Writedate Writetime Readdate Readtime EmptyDate EmptyTime CR LF In this string, fields are separated from each other by a space, the \\ character indicates that the rest of the string is described in detail on the next line, for page layout reasons only.", "\\ characters do not appear in the genuine basic data.", "Description of Basic Data Fields Title: title indicating the meaning of the following main fields in abbreviated form.", "CR: ASCII character code 0x13 (hexadecimal) LF: ASCII character code 0x10 code (hexadecimal) VolumeStartPosition: logical address of the start of the volume on the cartridge.", "VolumeEndPosition: logical address of the end of the volume on the cartridge VolumeSize: approximate size of the volume in kbytes.", "ReaderChannel: number of the reader (defined in the backup equipment configuration according to the invention) used to make the transfer from the disk cache volume to the cartridge DiskChannel: number of the disk (defined in the backup equipment configuration according to the invention) in which the volume is located at the time that it is transferred to the cartridge.", "DiskPartition: number of the disk partition in which the volume is located before it is transferred to the cartridge.", "ProcessorNumber: number of the processor used to transfer the volume of the disk cache to the cartridge.", "BarCode: bar code of the cartridge containing the volume.", "CartridgeName: cartridge name, as declared on the backup equipment according to the invention.", "This name is independent of the bar code.", "CartridgeType: hexadecimal code indicating the cartridge type.", "The possible values are as follows: 0x0000001L operating cartridge 0x00000010L cartridge with read access 0x00000020L cartridge with write access 0x00000080L cartridge being reorganised 0x00000100L cartridge to be reorganised 0x00000200L cartridge not to be reused 0x00000400L blocked empty cartridge 0x00000800L reorganised cartridge 0x00001000L archive type cartridge 0x00002000L mirror type cartridge 0x00010000L cartridge for DLT reader 0x00020000L cartridge for Exabyte reader 0x00040000L cartridge for 3480 reader 0x00080000L cartridge for 3590 reader 0x01F00000L mask for number of the archive pool or mirror to which the cartridge belongs.", "The code used for the CartridgeType field may possibly be a combination of the previous values.", "SizeUsed: total size of data stored on the cartridge, in Megabytes.", "CartridgeSize: maximum capacity of the cartridge, in MegaBytes.", "LoadCounter: cartridge load counter.", "Indicates the number of times that the cartridge was loaded in a reader.", "These data are used to determine cartridge wear.", "VolumeName: volume name, as it is known by the host machine.", "VolumeStatus: hexadecimal code indicating the volume status.", "This code is a combination of indicators for which the access masks and possible values are as follows: 0x0000001L 1 if the volume is valid, and 0 if it is invalid (old version or logically erased volume) 0x0000008L 1 if the volume is of the mirror type 0x00000010L 1 if the volume has a mirror copy on another cartridge 0x00000020L 1 if a copy of this volume is to be made on a mirror cartridge 0x00001000L 1 if the volume is of the archive type 0x00002000L 1 if the volume is shared between several host systems 0x00010000L 1 if the volume must always be copied on DLT cartridges 0x00020000L 1 if the volume must always be copied on Exabyte cartridges 0x00040000L 1 if the volume must always be copied on 3480 cartridges 0x00080000L 1 if the volume must always be copied on 3590 cartridges.", "0x01F00000L number of the archive pool or mirror (from 0 to 31) HostCode: number of the host machine to which the volume belongs, in the backup equipment configuration according to the invention.", "CodingType: character code used in the volume header (0=ASCII, 1=EBCDIC) WriteDate: date of the most recent write or modification of the volume by the host machine, in the form yyyy-mm-dd WriteTime: time of the most recent write or modification of the volume by the host machine, in the form hh:mm:ss ReadDate: date of the most recent read access of the volume by the host machine, in the form yyyy-mm-dd ReadTime: time of the most recent read access of the volume by the host machine, in the form hh:mm:ss EmptyDate: date on which the disk cache volume was transferred to the cartridge, in the form dd-mm-yyyy EmptyTime: time at which the disk cache volume was transferred to the cartridge, in the form hh:mm:ss Accumulation of Basic Data Basic data is cumulative, in order to accelerate the analysis of cartridges in order to reconstruct the database.", "As shown in FIG.", "1, assume that a tape contains volumes V1, V2, V3, V4 and V5.The basic data associated with each of these volumes are called B1, B2, B3, B4 and B5.The basic data B1 only contain data related to volume B1.The basic data B2 contain the accumulated data for B1 and data about volume V2 in a single data record.", "Therefore B2 contains data for V1 and V2.Basic data B3 contain the accumulated data for B2 and data about volume B3 in a single data record.", "Therefore B3 contains data for V1, V2 and V3.etc.", "Therefore the final basic data on the cartridge, that is B5, contain an accumulated total of all data about volumes present on the cartridge.", "If a cartridge contains a very large number of volumes, the accumulated basic data may be large.", "In order to limit this increase in size, a maximum size has been arbitrarily fixed at 132 kbytes.", "When the standard construction of basic data for a volume exceeds 132 kbytes, the backup equipment according to the invention assigns reduced basic data to this volume, to contain only basic data for this new volume without accumulating data for previous volumes.", "For subsequent volumes, the standard mechanism for accumulating data for the current volume with data for the previous volume will be repeated.", "In the reduced basic data, the VolumeAddress field for the single volume described (volume start logical address on the cartridge) contains an address other than 0.Referring to FIG.", "2, assume that a cartridge contains n+1 volumes.", "The nth volume (Vn) contains complete basic data denoted Bn, which therefore contain a list of data for the n first volumes.", "The first volume described in Bn, V1, is indicated with a VolumeAddress field equal to 0.After volume Vn and its basic data Bn, the cartridge also contains volume Vn+1 and basic data Bn+1 that we assume are reduced.", "This means that Bn+1 only contains data for volume Vn+1 with a VolumeAddress field indicating the position of volume Vn+1 on the cartridge at an address greater than 0, since the volume Vn+1 is not located at the beginning of the cartridge.", "Therefore, by analysing the final basic data on the cartridge (Bn+1), it is very easy to detect that these data are reduced (since an+1>0) and it can be deduced that the previous basic data (Bn) are located on the cartridge immediately before address an+1.Retrieving Basic Data If the database on the backup equipment system according to the invention is entirely lost, these basic data can be used to completely reconstruct the database.", "A function integrated into the code of the backup equipment processors according to the invention is used to analyse a cartridge to extract the most recent basic data from it.", "This analysis can also be made by external software; all that is necessary is to move to the end of the tape, to move back one record and to read the last data record.", "The basic data thus retrieved at the end of the cartridge contain a description of the volumes on the cartridge.", "As described in the previous section, if the VolumeAddress field in the first volume contains a value not equal to zero, this means that the first volume is not at the beginning of the tape.", "The conclusion is that the basic data are reduced.", "In this case all that is necessary is to move to the address VolumeAddress on the cartridge, and then move back by one record to read the basic data for the previous volume.", "These data are then accumulated with the basic data for previous volumes.", "The cartridge can be read backwards repeatedly until basic data are found in which the VolumeAddress field is equal to 0 for the first volume.", "All volumes in the cartridge can then be known by accumulating all retrieved basic data.", "Reconstruction of the Database from Basic Data Reconstruction of the database requires that all basic data stored on all cartridges in the library are retrieved and then analysed using an appropriate software.", "These data include the data necessary to reconstruct the database.", "The first step in this operation is to draw up a list of all volumes contained on all cartridges, and also to determine whether or not each volume on a cartridge is valid for the host machine.", "A single volume (same name, same host system) may be stored on several different cartridges, or at several different locations on the same cartridge.", "This can occur for the following reasons: either there are several different versions of the same volume that have been updated by the host machine several times or the data are the same and they have been moved internally by the backup equipment according to the invention.", "In any case, the analysis of the WriteDate and WriteTime basic data for all occurrences of this volume shows which is the most recent copy, and therefore the only valid copy.", "If the most recent version is present at several locations (same WriteDate and WriteTime data), then any one of these occurrences can be used to become the valid version of the volume in the new database.", "All that is necessary then is to recreate an empty database for the backup equipment according to the invention and to fill in all the tables with the collected data.", "CONCLUSION Having now described a preferred embodiment of the invention, it should be apparent to those skilled in the art that the foregoing is illustrative only and not limiting, having been presented by way of example only.", "All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same purpose, and equivalents or similar purpose, unless expressly stated otherwise.", "Therefore, numerous other embodiments of the modifications thereof are contemplated as falling within the scope of the present invention as defined by the appended claims and equivalents thereto.", "Moreover, the techniques may be implemented in hardware or software, or a combination of the two.", "Preferably, the techniques are implemented in computer programs executing on programmable computers that each include a processor, a storage medium readable by the processor (including volatile and non-volatile memory and/or storage elements), at least one input device and one or more output devices.", "Program code is applied to data entered using the input device to perform the functions described and to generate output information.", "The output information is applied to one or more output devices." ] ]

[ [ "Sport and Transport Device", "The invention relates to a sport and transport device comprising at least one top panel (2), at least two wheels, and at least one drive device (2.4, 3).", "The top panel (2) is connected to a frame (1) in such a way that it can pivot about at least one pivot axis and the frame is connected to the wheels.", "The device (2.4, 3) is actuated by the movement of the top panel (2) about the pivot axis in order to drive at least one of the wheels (6).", "The invention is characterized in that the actuating means (5.1, 11) are disposed on the top side of the top panel (2), enabling devices provided on the frame (1) or the wheels (6) to be actuated in each position of the top panel (2).", "The device also comprises a specially designed suspension system between the front axle (10) and the frame (1)." ], [ "1.Sport and transport device which has at least one top panel (2), at least two wheels (6), and at least one actuating means (5.1) located above the top panel (2) for one foot of a person operating the device; characterized in that the actuating means (5.1) is designed as an actuating plate supported on the top panel (2), which actuating plate has a device for a foot arrangement and which is displaceable in a horizontal direction relative to the surface of the top panel (2).", "2.Sport and transport device according to claim 1, characterized in that the actuating means (5.1, 11) and the respective apparatus are connected by Bowden cables.", "3.Sport and transport device according to claim 1, characterized in that the apparatus and associated actuating means are a gearshift system.", "4.Sport and transport device according to claim 3, characterized in that the gearshift system comprises a derailleur.", "5.Sport and transport device according to claim 3, characterized in that the gearshift system comprises a toothed-belt gearshift.", "6.Sport and transport device according to claim 3, characterized in that the gearshift system comprises a slide-type gearshift.", "7.Sport and transport device according to claim 5, characterized in that the gearshift system has a shift cage 14.8.Sport and transport device according to claim 1, characterized in that the apparatus and associated actuating means are a brake system 5.9.Sport and transport device according to claim 1, characterized in that the device comprises an electric motor which drives at least one wheel (6).", "10.Sport and transport device according to claim 1, characterized in that the frame (1) comprises a steerable front axle (10).", "11.Sport and transport device according to claim 1, characterized in that the wheels are detachably connected to the frame (1).", "12.Sport and transport device according to the preamble of claim 1, characterized in that a wheel axle (10) is coupled by a suspension system (120) to a frame (1) of the device, the suspension system (120) having elastomer elements (138) as spring elements which are gripped between square tubes (130, 136).", "13.Sport and transport device which comprises at least one top panel (2), at least two wheels, and at least one drive apparatus (2.4, 3) wherein the top panel (2) is pivotably attached to a frame (1) which is connected to the wheels, and the drive apparatus (2.4, 3) is actuated by movement of the top panel (2) about the pivot axis in order to drive at least one of the wheels (6), characterized in that the drive apparatus (2.4, 3) has a rack (2.4) which is provided on opposite sides with teeth, the first teeth engaging a first gear (3.8) and the second teeth engaging another gear (3.8)." ], [ "The invention relates to a sport and transport device.", "A plurality of devices are known in the area of sport and transport devices.", "Skateboards and snowboards, for example, are both used depending on the type of terrain.", "These devices have the disadvantage that they can be propelled, accelerated, or braked only by the slope of the surface on which they are used, or by the physical exertion of the user—for example, by removing a foot from the device and pressing down on the surface.", "German Patent 196 25 948 A1 describes a sport and transport device with a stand-on board, at least three road wheels, and a steering apparatus in which shifting the body weight from one leg to the other causes a motion that sets at least one wheel in motion through an appropriate kinematic drive unit, thus effecting motion.", "Similar sport and transport devices with a stand-on board are known from German Patent 88 08 3 66 U1, German Patent 692 00 850 T2, and from WO 92/06753 A1.These devices also have an actuating apparatus above the top panel for a foot of the person riding on the device.", "A common feature of these known actuating devices is the fact that the person riding the device must move his foot in a vertical direction relative to the top panel or the stand-on panel.", "The goal of the invention is to create a sport and transport device that may be ridden safely, is easy to steer, yet is of simple design.", "This goal is achieved by a sport and transport device with the features of claim 1.Modifications of the invention are the subject of the subclaims.", "By providing actuating means on the top side of the top panel according to the invention, the invention allows the rider or user of the device to operate this top panel when underway without removing his feet from the top panel.", "Due to the drive function of the top panel, i.e., the pivoting of the top panel about the pivot axis, the top panel occupies ever-changing positions.", "In order to permit operation of the apparatus, which may be provided on the frame or on the wheels, during the entire course of travel, i.e., independently of the current position of the top panel, the actuating means and corresponding apparatus are preferably connected by Bowden cables.", "In an embodiment of the sport and transport device according to the invention, the apparatus and associated actuating means form a gearshift system.", "Using the gearshift system, different gear speeds may be generated in one gear system by which at least one of the wheels is driven.", "The gear system is preferably attached to the frame.", "Use of the gearshift enables the rider to adjust the handling characteristics of the device to the driving surface conditions, specifically to the grade.", "The gearshift system may comprise a derailleur, a toothed-belt gearshift, or a slide-type gearshift.", "In this last case, the gear system may comprise a shift cage.", "Depending on the type of transmission between the gear system and the wheel driven by it, i.e., the chain or toothed belt, at least two speeds may be actuated by an appropriate gearshift system.", "In another embodiment, the apparatus and associated actuating means may be a braking system.", "In this embodiment, the advantages of the inventive design of the sport and transport device are particularly apparent, since in order to ensure the safety of the rider and other riders, the rider must be able to operate the brake at any time and in any position of the top panel, and this brake must engage reliably.", "The brake system may be designed in various ways.", "Preferably, it has the following principal components: one brake plate one rotary bushing two brake nipples two Bowden cables two thrust bearings one sleeve The design concept also encompasses equipping the sport and transport device with an electric motor that drives at least one wheel.", "This motor may be integrated into the gear system.", "The motor may be employed, as desired, to transmit power directly to the drive wheel or provide a power boost to the motion generated by the pivot motion of the top panel.", "Electric power may be supplied by a battery that may be accommodated in the frame of the device.", "Controlling the direction of the sport and transport device according to the invention is effected by the rider's shifting his weight in a lateral direction.", "To this end, the frame preferably includes a steerable front axle.", "This axle may be flanged onto a main support tube of the frame and is preferably designed as a spring element so as to enhance riding comfort.", "The wheels of the sport and transport device are preferably connected detachably to the frame.", "This detachable connection feature provides the sport and transport device according to the invention with a high degree of flexibility in terms of the area of utilization.", "The device may thus easily be converted to a winter sports device.", "To make this change, the wheels must simply be removed and replaced by runners or by wheels with spikes.", "A modification of the sport and transport device has an elastomer suspension apparatus that elastically couples the wheel axle to the frame of the device.", "The suspension here is selected so that the turn-in of the axle, i.e., the steering angle, is directly determined by a connection angle located between the front axle and the tubular joint axis.", "The following discussion describes the invention based on the attached figures which show embodiments of the subject of the invention.", "FIG.", "1 is a side view of an embodiment of the sport and transport device according to the invention; FIG.", "2 is a perspective top view of an embodiment of the sport and transport device according to the invention; FIG.", "3 is a perspective bottom view of an embodiment of the sport and transport device according to the invention; FIG.", "4 is a front view of an embodiment of the sport and transport device according to the invention; FIG.", "5 is a rear view of an embodiment of the sport and transport device according to the invention; FIG.", "6 is a schematic top view of an embodiment of the sport and transport device according to the invention with a gearshift; FIG.", "7 is a bottom view of an embodiment of the sport and transport device according to the invention with a braking system; FIG.", "8 is a perspective view of an embodiment of the sport and transport device according to the invention with winter equipment; FIG.", "9 is a schematic representation of the engagement of a rack with the drive gear system; FIG.", "10 is a perspective representation of the engagement of a rack with the drive gear system; FIG.", "11 is a perspective view of a cross section of a gear system of a sport and transport device according to the invention without gearshift; FIG.", "12 is a perspective view of a cross section of a gear system of a sport and transport device according to the invention with a derailleur; FIGS.", "13 and 13a are a perspective cross-sectional view and a cross-sectional view, respectively, of a gear system of a sport and transport device according to the invention with a slide gearshift in a first position; FIGS.", "14 and 14a are a perspective cross-sectional view and a cross-sectional view, respectively, of a gear system of a sport and transport device according to the invention with a slide gearshift in a second position; FIGS.", "15 through 17 are perspective views of one embodiment of a gear system of the sport and transport device with a motor according to the invention; FIG.", "18 is a perspective view, obliquely from below, of another embodiment of a sport and transport device showing an articulation system joining the frame to the front axle of the device; FIG.", "19 is an enlarged top view from the side of the articulated axle system; FIG.", "20 is partial cross-sectional view of the articulated axle system; FIG.", "21 is a perspective top view from the front of the front axle of the device including road wheels and suspension system; FIG.", "22 is a view similar to that of FIG.", "21, however of the frame seen from above; FIG.", "23 is a view of the frame plus the suspension system from below; FIG.", "24 is a view similar to that of FIG.", "19, however with an exploded view of the suspension system.", "The design of the sport and transport device according to the invention is described based on FIGS.", "1 through 5.A tubular frame 1 serves as the base that includes a main support tube 1.3 and forms a fork 1.1, 1.2 in the rear section.", "Tubular frame 1 is bent, at least in the area of main support tube 1.3.A steerable front axle 10 is flanged on at the front end of main support frame 1.3, i.e., at the end opposite fork 1.1, 1.2.The principle of the front axle 10 corresponds to that of the front axles of known skateboards.", "Due to the fact that only one steerable axle is present, however, the steering angle has been doubled.", "The geometry of axle 10 was designed to achieve a corresponding suspension effect even over rough terrain.", "In addition, the wheels of front axle 10 have been mounted with a camber to enhance the handling stability.", "The camber may, for example, be 5°.", "The embodiment of the sport and transport device shown has three wheels, with only one wheel acting as the drive wheel 6.This wheel is mounted at the other end of main support tube 1.3 in fork 1.1, 1.2.That section of the drive apparatus which is a drive gear system 3 is also mounted in fork 1.1, 1.2.Drive wheel 6 is mounted and braced in fork 1.1, 1.2.As described above, fork 1.1, 1.2 functions as the receiving apparatus for the positive-engagement support of gear system 3, and also as the support for drive wheel 6.This embodiment has the advantage that gear system 3 is supported by positive engagement, and thus does not require any additional attaching elements.", "In addition, the simultaneous use of fork 1.1, 1.2 as the support for drive wheel 6 represents a simplification in design.", "Wheel 6 and gear system 3 by which wheel 6 is driven are attached by the same component 1.1, 1.2 in this design—with the result that no relative displacement may occur between wheel 6 and gear system 3 that might cause failures such as breakage of the chain.", "Drive wheel 6 is preferably driven by a chain or toothed belt 7 through drive gear system 3.Top panel 2, which may have the shape of a skateboard for example, is pivotably connected to tubular frame 1.This connection may, for example, be of the design shown in FIGS.", "1 through 5.Here a joint plate 2.1 is attached to the bottom side of top panel 2.Top panel 2 is pivotably attached at joint bushing 1.4 to main support tube 1.3 by joint plate 2.1.Tubular frame 1 preferably has a bent shape so as not to inhibit top panel 2 from pivoting about this axis.", "In addition, top panel 2 may have a shape in which a kink 2.2 is provided in the area of the pivot axis and, as a result, top panel 2 extends from this kink 2.2 upward to the respective ends of panel 2.In addition to this V-shaped design, the top panel may, for example, also have two kinks (not shown) between which an initially horizontal surface extends, with the panel extending upward at a slight angle to the horizontal surface from the kinks to the ends.", "The shape of top panel 2 is selected based on ergonomic requirements such as stability under load, steerability, braking and shifting of the device, as well as the input of power.", "Kinking top panel 2 enhances its stiffness and provides the necessary means for the input of power.", "The panel is preferably designed with concave sides, which design enhances the stiffness of the board and saves weight.", "The bent shape of frame 1 and the kinked shape of top panel 2 thus ensure that top panel 2 can be moved frontward and rearward, up and down, in response to swiveling or seesawing motions.", "Top panel 2 receives this seesawing motion from the shifting weight of the operator.", "A means 2.4 is attached on the underside of top panel 2 in the area, in the installed condition, above gear system 3; this means forming a drive apparatus along with gear system 3.In the embodiment shown, this means is a rack 2.4.As a result of the seesawing motions of top panel 2, rack 2.4 alternately descends into and emerges from drive gear system 3, thereby driving gear system 3.Gear system 3 will be explained more precisely below referring to FIGS.", "9 through 17.However, this system is preferably designed such that output wheel 6 is caused to rotate permanently, i.e., in a forward direction, as the rack moves up and down.", "FIG.", "8 shows an embodiment of the sport and transport device according to the invention in which the front wheels are replaced by runners and drive wheel 6 is provided with spikes.", "The remainder of the device design corresponds to that shown in FIGS.", "1 through 5 and described above.", "Gear system 3 is mounted by positive engagement between forks 1.1 and 1.2.Retention is provided by a gear system housing which preferably has two gear system shells 3.1, 3.2.Projections may be provided on gear system housing 3.1, 3.2 which match the shape of the fork 1.1, 1.2.In addition, gear system 3 has a drive shaft and counter-rotating shaft.", "The drive shaft and counter-rotating shaft are mounted in gear system shells 3.1 and 3.2 on pivots 3.5 and 3.6.The drive shaft is mounted in housing sections 3.1 and 3.2 by bearings 3.18.The function of gear system 3 engaged by rack 2.4 engages will be described in more detail with reference to FIGS.", "9 through 11.As FIG.", "9 shows, rack 2.4 runs by positive engagement between two pinions 3.7, 3.8 which are interconnected by a toothed belt 3.11, thereby driving these forward and backward.", "As is evident from FIG.", "10, pinions 3.7 and 3.8 are each supported on pivots 3.5 and 3.6.Two synchronizer rotors 3.9 and 3.10 are also located opposite on the two pivots 3.5 and 3.6, and are interconnected by toothed belt 3.11.Synchronizer rotor 3.10 and pinion 3.8 are rigidly interconnected on the counter-rotating shaft and supported on pivots 3.6 by needle bearings.", "The output rotor 3.14 is connected by friction with the bearing sleeve so as to transmit the driving power.", "Synchronizer rotor 3.9 and pinion 3.7 are supported on bearing sleeve 3.15 by freewheels 3.16 and 3.17.A toothed belt 7 or chain 7 runs via output rotor 3.14 to drive wheel 6 on a synchronizer rotor 6.2 or a sprocket 6.2 (see FIGS.", "2, 3, and 5), thereby transmitting and converting the up and down motion imparted by rack 2.4 to gear system 3, into a rotational motion of drive wheel 6.Wheel 6 is supported by roller bearings on rear axle 6.3.The drive axle is force-fitted into the wheel body, with a synchronizer rotor or sprocket 6.2 with a freewheel being supported on said axle, thereby receiving the transmission of power.", "Propulsion is effected by toothed belt 7 or chain 7 via the drive shaft of the gear system.", "In the embodiment shown, the drive wheel is the rear wheel of the device.", "By modifying the freewheel arrangement, however, the drive direction may also be reversed so that the drive wheel is not in front and the steered axle in back.", "The drive function through the top panel remains unchanged here.", "In a preferred embodiment, the sport and transport device may have a gearshift.", "A gear system with gearshift is shown in FIGS.", "12 through 14.FIG.", "12 illustrates one option for a shift function, namely a derailleur.", "Synchronizer rotor 3.14 is replaced by chain wheels 4 and 5.Toothed belt 3.11 is replaced by a chain 7.Chain 7 drives drive wheel 6 through chain wheels 4 and 5 via a sprocket 6.2.Different transmission ratios are possible based on the different diameters of the chain wheels 4 and 5 on which chain 7 is shifted by a shift unit.", "The aforementioned shift unit comprises a tumbler 8 (see FIG.", "6) which is displaceably mounted on fork 1.1 and 1.2.Tumbler 8 is actuated by a two-arm shift lever 9 which is rotatably mounted in top panel 2 about rotational axis 13.Tumbler 8 is actuated by two shift nipples 11 through a Bowden cable.", "Shift nipples 11 are preferably of a pin-type design and are attached to the top side of shift lever 9.Tumbler 8, the Bowden cable, and shift nipples 11 are interconnected by friction.", "Shift nipples 11 of shift lever 9 project upward through curved grooves 12 from top panel 2.Actuating shift nipples 11 with the front foot (heel or toes) on which the rider is standing, i.e., by moving nipples 11 within grooves 12, rotates the shift lever, thereby shifting the corresponding drive transmission ratio.", "Shift lever 9 itself is designed as a spring element acting against the shift direction.", "This means that the user may step on shift nipples 11 without wanting to shift.", "These are pressed down without causing any shift action.", "Shift lever 9 is attached at its end position by a bead.", "As a result, the gearshift may be actuated by the foot at any time, or in any position occupied by top panel 2.No riding operation, propulsion operation, or steering operation need be interrupted or changed.", "FIGS.", "13, 13a and 14, 14a show another possible type of two-stage gearshift.", "The rack here consists of two different tooth segments arranged in tandem and spaced.", "The segments have different widths and may be interconnected by a cross brace to enhance stability.", "The rack is attached to top panel 2 as in the embodiment described previously.", "FIG.", "13 shows a first shift position.", "A shift cage 14, in which four pinions are integrated—of which two each have a smaller diameter 16 and two each have a larger diameter 17, is displaceable on the drive and gear system axle.", "Shifting, or displacement of the shift cage, occurs in precisely the same way as with the derailleur.", "In other words, when shift cage 14 is in the position shifted to the rear shown in FIGS.", "13 and 13a, the smaller rack segment, the width of which matches the distance between the two larger pinions 15, engages the two larger pinions 15 mounted on opposite pivots.", "If shift cage 14 is moved by the Bowden cable in the direction of the arrow, the resulting position is that shown in FIGS.", "14, 14a in which the larger rack segment, the width of which matches the distance between the two smaller pinions 16, engages the two smaller pinions 16, while the smaller rack segment no longer engages larger pinions 15.FIGS.", "15 through 17 show an embodiment in which an electric motor is integrated into the gear system.", "Through a speed-reduction gear system, the electric motor along with the motor pinion engages the counter-rotating shaft via an intermediate gear, thus driving this shaft.", "Just as with the manual drive, power is transmitted to the drive shaft by toothed belts through the toothed rotor of the counter-rotating shaft, and thereby transmitted to drive wheel 6.Since it must be possible when riding to apply the brake at any time, and since top panel 2 occupies ever-changing positions due to its drive function, no rigid type of brake unit can be selected.", "For this reason, a brake system was developed which duplicates all the motions of top panel 2.Specific features of the brake system are found in FIGS.", "7 and 2.The brake system is integrated into top panel 2.It has a symmetrical design so that the device may be operated at different riding positions (left foot back or right foot back) without any additional components.", "The brake system consists of the following principal components: brake plate (5.1) rotary bushing (5.2) brake nipple (5.3) Bowden cable (5.5) thrust bearing (5.6) sleeve (5.7) Two holes 2.4 to accept rotary bushings 5.2 are incorporated symmetrically into top panel 2.Sleeve 5.7 is installed in one of these two holes as a pivot bearing for brake plate 5.1.The other side is free.", "Brake plate 5.1 is installed along with both rotary bushings 5.2 in top panel 2 from above and secured from below.", "Brake plate 5.1 rests flat on top panel 2.Due to the one free hole, brake plate 5.1 may be either rotated or displaced as a function of the freedom of movement.", "Brake nipple 5.3 is frictionally installed in brake plate 5.1, into which brake plate Bowden cable 5.5 is inserted.", "This cable is supported by thrust bearing 5.6 attached to top panel 2 and routed to the brake 4.1 on the brake wheel.", "Drive wheel 6 is preferably equipped with a drum brake which is controlled by the brake system.", "The wheel brake function may also be achieved by various rim-based and tire-based brake systems.", "The user stands on the brake plate, which is equipped with support ribs 5.4 for the foot arrangement, and is able to rotate brake plate 5.1 with brake nipple 5.3 by rotating his foot.", "This initiates the braking action and achieves the braking effect in accordance with the amount of torsional force.", "Although the above discussion speaks in terms of rotating brake plate 5.1, brake plate 5.1 may also be simply laterally displaceable.", "In this case, brake plate 5.1 is moved by the rider's foot in the appropriate direction to achieve the braking effect.", "FIGS.", "18 through 24 show a second embodiment of a sport and transport device according to the invention with a top panel 2.In contrast to the embodiment shown in FIGS.", "1 through 17, the mechanical linkage between frame 1 and front axle 10 is achieved in this device in a special way by means of a suspension system using elastomers.", "FIG.", "18 is a perspective view seen obliquely from below, with the device facing the viewer from its front axle 10.The reference numerals explained above continue to apply to the same components.", "The center segment of frame 1 is connected through a suspension system 100 to front axle 10, on the left and right ends of which are attached road wheels 6.The center segment of Y-shaped frame 1 is rigidly connected to a tubular joint 104.At its front end facing front axle 10, this tubular joint 104 has a square hole in which a suspension apparatus using elastomers rests, this apparatus being explained more fully below.", "The front segment of tubular joint 104 is designed with a downward bend of angle {dot over (α)} relative to the road surface.", "A connection component 102 is attached to front axle 10.This connection component 102 rests flat on the top free surface of the front section of tubular joint 104.Front axle 10 is movably connected to frame 1 by this connection component 102 through suspension system 120 and tubular joint 104.The mechanical coupling of frame 1 and front axle 10 by suspension system 120 is especially evident in the cross-sectional diagrams of FIG.", "20 and FIG.", "24.A bolt 122 sits stationary in a hole of connection component 102.The stationary connection may, for example, be achieved simply by a screwed connection indicated by reference numeral 142.This bolt 122 is attached in connection component 102 such that this component extends rearward at an oblique angle as seen from the direction of travel.", "Bolt 122 sits rotatably movably in a slide bushing 140 of tubular joint 104.Bolt 122 has a square cross section.", "A square tube 136 sits on this square cross section of bolt 122.An outer square tube 130 is located with a certain clearance around inner square tube 136.The square tube 136 simultaneously contacts the outer wall of suspension system 120.Elastomer strips 138 sit offset at 90° angles between outer square tube 130 and inner square tube 136.Suspension system 120 is closed by a cover plate 142 and retained by nuts 144 which are screwed onto the end of bolt 122 pointing away from connection component 102.The functional principle of the suspension system is as follows.", "Tilting the Y-tube of frame 1 via top panel 2 causes tubular joint 104 and front axle 10 to rotate toward each other.", "At the same time, bolt 122 also rotates with its square component, which is rigidly connected to front axle 10, thereby also rotatably carrying along inner square tube 136.Since outer square tube 130 is mounted in the square hole of tubular joint 104 and thus cannot rotate, front axle 10 is oriented in the resting state at right angles to the center tube of frame 1, namely, β equals 90°.", "When the sport and transport device is tilted to the left or right via top panel 2 by shifting weight, suspension system 102 experiences a load by which front axle 10 is adjustable for a defined steering.", "The possible steering angle β is a direct function of connection angle α between tubular joint 104 and front axle 10 of the device.", "The larger angle α, the smaller is steering angle β, and visa versa.", "The angle α may be adjusted here for the specific design of the device, depending on the area of utilization for the device, whether on outdoor terrain, on smooth ground, for competition, or other areas of utilization.", "It must be mentioned in this regard that the described articulated axle system with suspension system 100 using elastomers, while extremely advantageous, does not necessarily have to be employed.", "Other suspension systems are also possible for coupling frame 1 to front axle 10.Suspension system 120 may be advantageously designed to be interchangeable.", "In this way, suspension systems 120 having different elasticities may be employed, namely, an operator may at any time exchange the suspension system 120 in order to adjust the handling and steering characteristics of the device to his specific wishes or his physical requirements, such as body weight and riding skill.", "The aforementioned elastomers 138 are inserted prestressed between square tube 136 and outer square tube 130." ] ]

[ [ "Route guidance system", "Provided is a route guidance system that provides a route in premises in order to perform service of route guidance in the premises, which includes: a route guidance apparatus, which is comprised of visible region calculation means that calculates a range where a user can see, signpost extraction means that extracts a signpost to be guided from a visible region, route search means that searches the route from a starting point to a destination, route information generation means that comprises guidance map generation means that generates a map for guidance and guidance sentence generation means that generates the guidance sentence, route leading means that obtains current positional information to send appropriate guidance information to a portable terminal, and position determination means that performs coordinate conversion to information from a position detection apparatus of the user; a portable terminal apparatus including a user interface, which displays an image or a route guidance sentence; a position detection apparatus that obtains current positional information of the user; a map DB that stores data/signpost information regarding the route; a guidance sentence DB that stores basic data for generating the guidance sentence; and route information data that stores guidance data output from the route information generation means." ], [ "1.A route guidance apparatus, which is comprised of: position determination means that detects a current position of a user; visible region calculation means that calculates a range where the user can see from the current position detected; route search means that searches a route from a starting point to a destination; traveling direction calculation means that calculates a traveling direction from a visible region and the route; signpost extraction means that extracts a signpost to be guided from the visible region; and route guidance sentence generation means that generates a route guidance sentence of the route found.", "2.A route guidance apparatus, which is comprised of: position determination means that detects a current position of a user; visible region calculation means that calculates a range where the user can see from the current position detected; route search means that searches a route from a starting point to a destination; traveling direction calculation means that calculates a traveling direction from a visible region and the route; mark extraction means that extracts a mark to be guided from the visible region; image data retrieval means that retrieves binary image data or illustration image data, which are specified to guide a point to pass on said route; guidance sentence synthesis means for synthesizing a guidance sentence to guide the point to pass.", "3.A route guidance apparatus, which is comprised of: route search means that searches a route from a starting point to a destination; first frame map generation means that cuts out points to pass from map data in a size, which can be displayed on a display screen of a portable terminal, in order to guide the point to pass, and cuts out such that connection points of the route in each of frames match in cutting out; guidance sentence synthesis means that synthesizes a guidance sentence to guide the point to pass.", "4.A route guidance apparatus, which is comprised of: route search means that searches a route from a starting point to a destination; second frame map generation means that cuts out point to pass from map data in a size, which can be displayed on a display screen of a portable terminal, in order to guide the point to pass, and cuts out such that the route in each of frames is displayed in the center of the screen in cutting out; guidance sentence synthesis means that synthesizes a guidance sentence to guide the point to pass.", "5.A route guidance apparatus of claim 3, wherein the guidance sentence synthesis means synthesizes a guidance sentence using a moving direction of a user as a reference in order to guide point to pass.", "6.A route guidance apparatus of claim 3, wherein the guidance sentence synthesis means synthesizes a guidance sentence using a railway direction as a reference in order to guide point to pass on a platform of a train station.", "7.A route guidance apparatus of claim 3, wherein the guidance sentence synthesis means does not perform guidance to turn right/left at a first direction turning point taking into consideration an error of position determination means at a starting point when the starting point is near the point where a direction turn is made for the first time, but guides a mark showing a direction turning direction from the direction turning point and performs guidance to turn right/left from a second direction turning point.", "8.A route guidance apparatus of claim 3, wherein the guidance sentence synthesis means provides information of a route such that it guides positional information of a vicinity of a tag when it detects the tag for a vision-impaired person, in guidance regarding the route, guidance sentences regarding mark information along the route, which are severally punctuated by punctuations, are arranged in a route order and a user performs a button operation for each guidance sentence to read out the sentences sequentially.", "9.A route guidance apparatus according to claim 1, comprising: route leading means that displays a frame view, a binary image or an illustration image, which corresponds to a tag, and a route guidance sentence on a terminal or reads out a guidance sentence, synchronously with detecting a tag placed in premises.", "10.A route guidance apparatus according to claim 1, comprising: route leading means that displays a frame view, a binary image or an illustration image, which corresponds to a tag, and a route guidance sentence on a terminal or reads out a guidance sentence, in accordance with input of the user.", "11.A route guidance apparatus, which is comprised of: position determination means that detects a current position of a user; visible region calculation means that calculates a visible region which is a range where the user can see from the current position detected; route search means that searches a route from a starting point to a destination; traveling direction calculation means that calculates a traveling direction from the visible region and the route; mark extraction means that extracts a mark to be guided from the visible region; and guidance sentence generation means that generates a route guidance sentence of the route found.", "12.A route guidance apparatus according to claim 11 wherein when an intersecting point exists between the route searched by said route search means and a boundary of the visible region calculated by said visible region calculation means, the traveling direction calculation means calculates a direction of the intersecting point as a traveling direction, and the mark extraction means extracts a mark seen from said user in a direction that exists within a visible region calculated by said visible region calculation means and that is near the direction calculated by said traveling direction calculation.", "13.A route guidance apparatus, which is comprised of: position determination means that detects a current position of a user; visible region calculation means that calculates a range where the user can see from the current position detected; route search means that searches a route from a starting point to a destination; traveling direction calculation means that calculates a traveling direction from a visible region and the route; mark extraction means that extracts a mark to be guided from the visible region; image data search means that searches viewpoint image data specified in order to guide point to pass on said route; and guidance sentence generation means for generating a guidance sentence in order to guide the point to pass.", "14.A route guidance apparatus, which is comprised of: route search means that searches a route from a starting point to a destination; and first frame map generation means that cuts out a map around a point to pass as a frame from a map database in order to guide the point to pass, and cuts out each frame such that a part of the route in each frame overlaps in cutting out; and guidance sentence generation means that generates a guidance sentence to guide the point to pass.", "15.A route guidance apparatus, which is comprised of: route search means that searches a route from a starting point to a destination; second frame map generation means that cuts out a map around a point to pass as a frame from a map database in order to guide the point to pass, and cuts out such that the route in each frame is displayed in the center of a screen in cutting out; and guidance sentence generation means that generates a guidance sentence to guide the point to pass.", "16.A route guidance apparatus according to claim 11, wherein the guidance sentence generation means generates the guidance sentence using a moving direction of the user as a reference in order to guide the point to pass.", "17.A route guidance apparatus according to claim 11, wherein in the case where the route guidance apparatus guides a route passing the points in a platform of train station premises, said guidance sentence generation means generates the guidance sentence using a direction of a railway for the user in the train station platform as a reference in order to guide the point to pass.", "18.A route guidance apparatus according to claim 11, wherein the guidance sentence generation means does not perform guidance to turn right/left at a first direction turning point when the starting point is near the point where a direction turn is made for the first time but generates the guidance sentence of a mark showing a direction of turning direction at the direction turning point.", "19.A route guidance apparatus according to claim 11, comprising: tag detection means that detects a tag, and characterized in that the guidance sentence generation means generates the guidance sentence that guides positional information near the tag detected by the tag detection means.", "20.A route guidance apparatus according to claim 11, wherein the guidance sentence generation means has a guidance sentence request acceptance function that accepts a guidance sentence request being a request for reading out the guidance sentence, and the guidance sentence generation means arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially when it accepts a guidance sentence request based on the guidance sentence request acceptance function.", "21.A route guidance apparatus according to claim 19, wherein the apparatus arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially, synchronously with detecting the tag by the tag detection means.", "22.A route guidance apparatus according to claim 11, wherein the guidance sentence generation means has a user characteristic information acceptance function that accepts user characteristic information being information regarding a user's characteristic, and the guidance sentence generation means provides route information in accordance with user characteristic information accepted based on the user characteristic information acceptance function.", "23.A route guidance apparatus according to claim 11, wherein the guidance sentence generation means, in the case where the user characteristic information accepted based on the user characteristic information acceptance function shows that the user is a vision-impaired person, said guidance sentence generation means arranges the guidance sentences for each mark information along the route in the route order and reads out each guidance sentence sequentially.", "24.A route guidance apparatus according to claim 11, comprising: tag detection means that detects tags placed in premises; and route leading means that displays a frame view or a viewpoint image, which corresponds to a tag position, and the guidance sentence or reads out the guidance sentence, synchronously with detecting the tag by the tag detection means.", "25.A route guidance apparatus according to claim 11, wherein the position detection means detects the current position of the user by a tag, which comprises: user request obtaining means that obtains a user's request; and route leading means that displays a frame view or a viewpoint image, which corresponds to the tag, and the guidance sentence on a terminal or reads out the guidance sentence in a route order in accordance with the user's request obtained by said user request obtaining means.", "26.A visible region calculator, wherein, in the case where a viewpoint and one or a plurality of polygons are in one polygon, a visible region from the viewpoint regarding an outermost polygon is computed first, an area seen from the viewpoint only for each polygon is computed next as in a row of continuous line segments regarding the polygons other than the outermost polygon, the row of the line segments computed is arranged in an argument order, an area of the line segment seen from the viewpoint is computed in an arrangement order taking in consideration a positional relation of the row of the line segments with a visible region generated at the point where the processing of the row of the line segments and a previous row of line segments ends, and the range seen from the viewpoint is calculated by the processing to find a new visible region by connecting the area computed and the visible region.", "27.A visible region calculator that calculates a range seen from a viewpoint in the case where the viewpoint and one or a plurality of polygons are in an outermost polygon having the following configuration: a) an outermost visible region calculation section that finds a visible polygon being a range seen from the viewpoint inside the outermost polygon and calculates a first temporary visible polygon; b) a visible polygonal line calculation section that computes a visible polygonal line being a part seen from said viewpoint of the polygon regarding each polygon inside the outermost polygon; c) a visible polygonal line rearrangement section that arranges the visible polygonal lines in the order of the argument of a visible limit point, which has a smaller argument between a reference line that is a half line drawn from the viewpoint and the end points of the visible polygonal line; d) a temporary visible polygon update section that performs the following processing 1 and 2 regarding the visible polygonal line in an arrangement order: 1) the processing where, when both ends of the visible polygonal line are set as A and B, wherein A is set as the visible limit point, in the case where A and B are inside the temporary visible polygon, half lines severally passing A and B are drawn from the viewpoint to the temporary visible polygon, A′ and B′ that are intersecting points between the visible lines and the temporary visible polygon are computed, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B and B to B′ are sequentially connected to form a new temporary visible polygon; and 2) the processing where, in the case where A is outside the temporary visible polygon and B is inside, an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line being the half line passing B is drawn from the viewpoint, B′ being the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "28.A visible region calculation program for calculating a range seen from a viewpoint in the case where the viewpoint and one or a plurality of polygons are in an outermost polygon, wherein it allows a computer to execute the following steps: a) an outermost visible region calculation step where a visible polygon being a range seen from the viewpoint inside the outermost polygon is computed and a first temporary visible polygon is calculated; b) a visible polygonal line calculation step in which a visible polygonal line being an area seen from said viewpoint of the polygon is computed regarding each polygon inside the outermost polygon; c) a visible polygonal line rearrangement step where the visible polygonal lines are arranged in the order of the arguments of a visible limit point, which has a smaller argument between a reference line that is a half line drawn from the viewpoint and the end points of the visible polygonal line; d) a temporary visible polygon update step where the following processing 1 and 2 is performed regarding the visible polygonal line in an arrangement order: 1) the processing where, when both ends of the visible polygonal line are set as A and B, wherein A is set as the visible limit point, in the case where A and B are inside the temporary visible polygon visible lines that are half lines severally passing A and B are drawn from the viewpoint to the temporary polygon, A′ and B′ that are the intersecting points between the visible lines and the temporary polygon are computed, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B, and B to B′ are sequentially connected to form a new temporary visible polygon; and 2) the processing where, in the case where A is outside the temporary visible polygon and B is inside, an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line being the half line passing B is drawn from the viewpoint, B′ being the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B and B to B′ are sequentially connected to form a new temporary visible polygon.", "29.A route information generator, comprising: visible region calculation means that calculates a range seen from a user; route search means that searches a route from a point where the user is now or a starting point that the user enters to a destination that the user enters; mark extraction means that extracts a mark to be guided out of a visible region from a current position or a point where guidance is given; guidance map generation means that generates a map for the route guidance, on which said route, the mark extracted, the current position and the point where the guidance is given are displayed; and guidance sentence generation means that generates a guidance sentence of the route.", "30.A route information generator according to claim 29, wherein said visible region calculation means is a visible region calculator, wherein, in the case where a viewpoint and one or a plurality of polygons are in one polygon, a visible region from the viewpoint regarding an outermost polygon is computed first an area seen from the viewpoint only for each polygon is computed next as in a row of continuous line segments regarding the polygons other than the outermost polygon, the row of the line segments computed is arranged in an argument order, an area of the line segment seen from the viewpoint is computed in an arrangement order taking in consideration a positional relation of the row of the line segments with a visible region generated at the point where the processing of the row of the line segments and a previous row of line segments ends, and the range seen from the viewpoint is calculated by the processing to find a new visible region by connecting the area computed and the visible region.", "31.A route guidance system, comprising: a route guidance apparatus having a route information generator comprising: visible region calculation means that calculates a range seen from a user; route search means that searches a route from a point where the user is now or a starting point that the user enters to a destination that the user enters; mark extraction means that extracts a mark to be guided out of a visible region from a current position or a point where guidance is given; guidance map generation means that generates a map for the route guidance, on which said route, the mark extracted, the current position and the point where the guidance is given are displayed; and guidance sentence generation means that generates a guidance sentence of the route, and position determination means that detects a current position of a terminal; a route information database that accumulates information regarding a route obtained; a terminal that displays route guidance information; a position detection apparatus that obtains current positional information; a map database that accumulates map data; and a guidance sentence database to accumulate data for generating a guidance sentence.", "32.A route leading unit to allow a terminal to display a mark on a map to lead a user, wherein it obtains a current position of the terminal at a predetermined time interval, and switches the map displayed on the terminal to a map on which the next mark in route data is displayed when it detects approach to a mark that exists in the route data.", "33.A route guidance apparatus according to claim 11, wherein the position determination means obtains the current position of the terminal at a predetermined time interval, and the guidance sentence generation means generates the guidance sentence of the route regarding the next mark to the terminal when it detects approaching to a mark that exists in the route searched by the route search means.", "34.A route guidance system, comprising: a route guidance apparatus having a route leading unit to allow a terminal to display a mark on a map to lead a user, wherein it obtains a current position of the terminal at a predetermined time interval, and switches the map displayed on the terminal to a map on which the next mark in route data is displayed when it detects approach to a mark that exists in the route data, and position determination means that detects a current position of a terminal; a route information database that accumulates information regarding a route obtained; a terminal that displays route guidance information; a position detection apparatus that obtains current positional information; a map database that accumulates map data; and a guidance sentence database that accumulate data for generating a guidance sentence.", "35.A route guidance system, comprising: a route guidance apparatus having a route information generator comprising: visible region calculation means that calculates a range seen from a user; route search means that searches a route from a point where the user is now or a starting point that the user enters to a destination that the user enters; mark extraction means that extracts a mark to be guided out of a visible region from a current position or a point where guidance is given; guidance map generation means that generates a map for the route guidance, on which said route, the mark extracted, the current position and the point where the guidance is given are displayed; and guidance sentence generation means that generates a guidance sentence of the route, a route leading unit to allow a terminal to display a mark on a map to lead a user, wherein it obtains a current position of the terminal at a predetermined time interval, and switches the map displayed on the terminal to a map on which the next mark in route data is displayed when it detects approach to a mark that exists in the route data, and position determination means that detects a current position of a terminal; a route information database that accumulates information regarding a route obtained; a terminal that displays route guidance information; a position detection apparatus that obtains current positional information; a map database that accumulates map data; and a guidance sentence database that accumulate data for generating a guidance sentence.", "36.A route guidance apparatus of claim 4, wherein the guidance sentence synthesis means synthesizes a guidance sentence using a moving direction of a user as a reference in order to guide point to pass.", "37.A route guidance apparatus of claim 4, wherein the guidance sentence synthesis means synthesizes a guidance sentence using a railway direction as a reference in order to guide point to pass on a platform of a train station.", "38.A route guidance apparatus of claim 4, wherein the guidance sentence synthesis means does not perform guidance to turn right/left at a first direction turning point taking into consideration an error of position determination means at a starting point when the starting point is near the point where a direction turn is made for the first time, but guides a mark showing a direction turning direction from the direction turning point and performs guidance to turn right/left from a second direction turning point.", "39.A route guidance apparatus of claim 4, wherein the guidance sentence synthesis means provides information of a route such that it guides positional information of a vicinity of a tag when it detects the tag for a vision-impaired person, in guidance regarding the route, guidance sentences regarding mark information along the route, which are severally punctuated by punctuations, are arranged in a route order and a user performs a button operation for each guidance sentence to read out the sentences sequentially.", "40.A route guidance apparatus according to claim 13, wherein the guidance sentence generation means generates the guidance sentence using a moving direction of the user as a reference in order to guide the point to pass.", "41.A route guidance apparatus according to claim 14, wherein the guidance sentence generation means generates the guidance sentence using a moving direction of the user as a reference in order to guide the point to pass.", "42.A route guidance apparatus according to claim 15, wherein the guidance sentence generation means generates the guidance sentence using a moving direction of the user as a reference in order to guide the point to pass.", "43.A route guidance apparatus according to claim 13, wherein in the case where the route guidance apparatus guides a route passing the points in a platform of train station premises, said guidance sentence generation means generates the guidance sentence using a direction of a railway for the user in the train station platform as a reference in order to guide the point to pass.", "44.A route guidance apparatus according to claim 14, wherein in the case where the route guidance apparatus guides a route passing the points in a platform of train station premises, said guidance sentence generation means generates the guidance sentence using a direction of a railway for the user in the train station platform as a reference in order to guide the point to pass.", "45.A route guidance apparatus according to claim 15, wherein in the case where the route guidance apparatus guides a route passing the points in a platform of train station premises, said guidance sentence generation means generates the guidance sentence using a direction of a railway for the user in the train station platform as a reference in order to guide the point to pass.", "46.A route guidance apparatus according to claim 13, wherein the guidance sentence generation means does not perform guidance to turn right/left at a first direction turning point when the starting point is near the point where a direction turn is made for the first time but generates the guidance sentence of a mark showing a direction of turning direction at the direction turning point.", "47.A route guidance apparatus according to claim 14, wherein the guidance sentence generation means does not perform guidance to turn right/left at a first direction turning point when the starting point is near the point where a direction turn is made for the first time but generates the guidance sentence of a mark showing a direction of turning direction at the direction turning point.", "48.A route guidance apparatus according to claim 15, wherein the guidance sentence generation means does not perform guidance to turn right/left at a first direction turning point when the starting point is near the point where a direction turn is made for the first time but generates the guidance sentence of a mark showing a direction of turning direction at the direction turning point.", "49.A route guidance apparatus according to claim 13, comprising: tag detection means that detects a tag, and characterized in that the guidance sentence generation means generates the guidance sentence that guides positional information near the tag detected by the tag detection means.", "50.A route guidance apparatus according to claim 14, comprising: tag detection means that detects a tag, and characterized in that the guidance sentence generation means generates the guidance sentence that guides positional information near the tag detected by the tag detection means.", "51.A route guidance apparatus according to claim 15, comprising: tag detection means that detects a tag, and characterized in that the guidance sentence generation means generates the guidance sentence that guides positional information near the tag detected by the tag detection means.", "52.A route guidance apparatus according to claim 13, wherein the guidance sentence generation means has a guidance sentence request acceptance function that accepts a guidance sentence request being a request for reading out the guidance sentence, and the guidance sentence generation means arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially when it accepts a guidance sentence request based on the guidance sentence request acceptance function.", "53.A route guidance apparatus according to claim 14, wherein the guidance sentence generation means has a guidance sentence request acceptance function that accepts a guidance sentence request being a request for reading out the guidance sentence, and the guidance sentence generation means arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially when it accepts a guidance sentence request based on the guidance sentence request acceptance function.", "54.A route guidance apparatus according to claim 15, wherein the guidance sentence generation means has a guidance sentence request acceptance function that accepts a guidance sentence request being a request for reading out the guidance sentence, and the guidance sentence generation means arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially when it accepts a guidance sentence request based on the guidance sentence request acceptance function.", "55.A route guidance apparatus according to claim 49, wherein the apparatus arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially, synchronously with detecting the tag by the tag detection means.", "56.A route guidance apparatus according to claim 50, wherein the apparatus arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially, synchronously with detecting the tag by the tag detection means.", "57.A route guidance apparatus according to claim 51, wherein the apparatus arranges the guidance sentences for each of mark information along the route in the route order and reads out each guidance sentence sequentially, synchronously with detecting the tag by the tag detection means.", "58.A route guidance apparatus according to claim 13, wherein the guidance sentence generation means has a user characteristic information acceptance function that accepts user characteristic information being information regarding a user's characteristic, and the guidance sentence generation means provides route information in accordance with user characteristic information accepted based on the user characteristic information acceptance function.", "59.A route guidance apparatus according to claim 14, wherein the guidance sentence generation means has a user characteristic information acceptance function that accepts user characteristic information being information regarding a user's characteristic, and the guidance sentence generation means provides route information in accordance with user characteristic information accepted based on the user characteristic information acceptance function.", "60.A route guidance apparatus according to claim 15, wherein the guidance sentence generation means has a user characteristic information acceptance function that accepts user characteristic information being information regarding a user's characteristic, and the guidance sentence generation means provides route information in accordance with user characteristic information accepted based on the user characteristic information acceptance function.", "61.A route guidance apparatus according to claim 13, wherein the guidance sentence generation means, in the case where the user characteristic information accepted based on the user characteristic information acceptance function shows that the user is a vision-impaired person, said guidance sentence generation means arranges the guidance sentences for each mark information along the route in the route order and reads out each guidance sentence sequentially.", "62.A route guidance apparatus according to claim 14, wherein the guidance sentence generation means, in the case where the user characteristic information accepted based on the user characteristic information acceptance function shows that the user is a vision-impaired person, said guidance sentence generation means arranges the guidance sentences for each mark information along the route in the route order and reads out each guidance sentence sequentially.", "63.A route guidance apparatus according to claim 15, wherein the guidance sentence generation means, in the case where the user characteristic information accepted based on the user characteristic information acceptance function shows that the user is a vision-impaired person, said guidance sentence generation means arranges the guidance sentences for each mark information along the route in the route order and reads out each guidance sentence sequentially.", "64.A route guidance apparatus according to claim 13, comprising: tag detection means that detects tags placed in premises; and route leading means that displays a frame view or a viewpoint image, which corresponds to a tag position, and the guidance sentence or reads out the guidance sentence, synchronously with detecting the tag by the tag detection means.", "65.A route guidance apparatus according to claim 13, wherein the position detection means detects the current position of the user by a tag, which comprises: user request obtaining means that obtains a user's request; and route leading means that displays a frame view or a viewpoint image, which corresponds to the tag, and the guidance sentence on a terminal or reads out the guidance sentence in a route order in accordance with the user's request obtained by said user request obtaining means.", "66.A route information generator according to claim 29, wherein said visible region calculation means is a visible region calculator that calculates a range seen from a viewpoint in the case where the viewpoint and one or a plurality of polygons are in an outermost polygon having the following configuration: a) an outermost visible region calculation section that finds a visible polygon being a range seen from the viewpoint inside the outermost polygon and calculates a first temporary visible polygon; b) a visible polygonal line calculation section that computes a visible polygonal line being a part seen from said viewpoint of the polygon regarding each polygon inside the outermost polygon; c) a visible polygonal line rearrangement section that arranges the visible polygonal lines in the order of the argument of a visible limit point, which has a smaller argument between a reference line that is a half line drawn from the viewpoint and the end points of the visible polygonal line; d) a temporary visible polygon update section that performs the following processing 1 and 2 regarding the visible polygonal line in an arrangement order: 1) the processing where, when both ends of the visible polygonal line are set as A and B, wherein A is set as the visible limit point, in the case where A and B are inside the temporary visible polygon, half lines severally passing A and B are drawn from the viewpoint to the temporary visible polygon, A′ and B′ that are intersecting points between the visible lines and the temporary visible polygon are computed, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B and B to B′ are sequentially connected to form a new temporary visible polygon; and 2) the processing where, in the case where A is outside the temporary visible polygon and B is inside, an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line being the half line passing B is drawn from the viewpoint, B′ being the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "67.A route guidance system as claimed in claim 31 wherein said visible region calculation means is a visible region calculator, wherein, in the case where a viewpoint and one or a plurality of polygons are in one polygon, a visible region from the viewpoint regarding an outermost polygon is computed first, an area seen from the viewpoint only for each polygon is computed next as in a row of continuous line segments regarding the polygons other than the outermost polygon, the row of the line segments computed is arranged in an argument order, an area of the line segment seen from the viewpoint is computed in an arrangement order taking in consideration a positional relation of the row of the line segments with a visible region generated at the point where the processing of the row of the line segments and a previous row of line segments ends, and the range seen from the viewpoint is calculated by the processing to find a new visible region by connecting the area computed and the visible region.", "68.A route guidance system as claimed in claim 31 wherein said visible region calculation means is a visible region calculator that calculates a range seen from a viewpoint in the case where the viewpoint and one or a plurality of polygons are in an outermost polygon having the following configuration: a) an outermost visible region calculation section that finds a visible polygon being a range seen from the viewpoint inside the outermost polygon and calculates a first temporary visible polygon; b) a visible polygonal line calculation section that computes a visible polygonal line being a part seen from said viewpoint of the polygon regarding each polygon inside the outermost polygon; c) a visible polygonal line rearrangement section that arranges the visible polygonal lines in the order of the argument of a visible limit point, which has a smaller argument between a reference line that is a half line drawn from the viewpoint and the end points of the visible polygonal line; d) a temporary visible polygon update section that performs the following processing 1 and 2 regarding the visible polygonal line in an arrangement order: 1) the processing where, when both ends of the visible polygonal line are set as A and B, wherein A is set as the visible limit point, in the case where A and B are inside the temporary visible polygon, half lines severally passing A and B are drawn from the viewpoint to the temporary visible polygon, A′ and B′ that are intersecting points between the visible lines and the temporary visible polygon are computed, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B and B to B′ are sequentially connected to form a new temporary visible polygon; and 2) the processing where, in the case where A is outside the temporary visible polygon and B is inside, an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line being the half line passing B is drawn from the viewpoint, B′ being the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "69.A route guidance system as claimed in claim 35 wherein said visible region calculation means is a visible region calculator, wherein, in the case where a viewpoint and one or a plurality of polygons are in one polygon, a visible region from the viewpoint regarding an outermost polygon is computed first, an area seen from the viewpoint only for each polygon is computed next as in a row of continuous line segments regarding the polygons other than the outermost polygon, the row of the line segments computed is arranged in an argument order, an area of the line segment seen from the viewpoint is computed in an arrangement order taking in consideration a positional relation of the row of the line segments with a visible region generated at the point where the processing of the row of the line segments and a previous row of line segments ends, and the range seen from the viewpoint is calculated by the processing to find a new visible region by connecting the area computed and the visible region.", "70.A route guidance system as claimed in claim 35 wherein said visible region calculation means is a visible region calculator that calculates a range seen from a viewpoint in the case where the viewpoint and one or a plurality of polygons are in an outermost polygon having the following configuration: a) an outermost visible region calculation section that finds a visible polygon being a range seen from the viewpoint inside the outermost polygon and calculates a first temporary visible polygon; b) a visible polygonal line calculation section that computes a visible polygonal line being a part seen from said viewpoint of the polygon regarding each polygon inside the outermost polygon; c) a visible polygonal line rearrangement section that arranges the visible polygonal lines in the order of the argument of a visible limit point, which has a smaller argument between a reference line that is a half line drawn from the viewpoint and the end points of the visible polygonal line; d) a temporary visible polygon update section that performs the following processing 1 and 2 regarding the visible polygonal line in an arrangement order: 1) the processing where, when both ends of the visible polygonal line are set as A and B, wherein A is set as the visible limit point, in the case where A and B are inside the temporary visible polygon, half lines severally passing A and B are drawn from the viewpoint to the temporary visible polygon, A′ and B′ that are intersecting points between the visible lines and the temporary visible polygon are computed, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B and B to B′ are sequentially connected to form a new temporary visible polygon; and 2) the processing where, in the case where A is outside the temporary visible polygon and B is inside, an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line being the half line passing B is drawn from the viewpoint, B′ being the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B, and B to B′ are sequentially connected to form a new temporary visible polygon." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to an apparatus and a system for providing a person, who is not good at reading a map, with guidance including landmark information and route information regarding a direction where the moving person needs to proceed when moving in an architectural structure such as a building with a method using an image or the like such that the user does not became lost in an open space.", "Further, the present invention also relates to a calculation apparatus of a visible region and a program thereof, which are necessary in extracting a land mark to support the movement of the moving person.", "2.Description of the Prior Art In the prior art, there has existed the following known references regarding a system that performs route guidance in premises or in a building.", "The invention disclosed in Japanese Patent Laid-open No.", "8-190688 is one that provides a pedestrian in the premises, who is a pedestrian having difficulty to obtain whereabouts of himself/herself or guidance information to a destination, of appropriate guidance information.", "A user reads information of radio wave reaction apparatuses embedded in the premises using an information reader, and the information to be output changes according to user's information set in a user information setting section although its contents are fixed.", "Further, a pedestrian guidance apparatus can be provided inexpensively.", "Furthermore, in the invention disclosed in Japanese Patent Laid-Open No.", "11-276516, it is one that provides a vision-impaired person in a train station with information such as a route to a destination and a landmark through a portable terminal by audio input/output, detects the position of the handicapped person by a tag, and lends/guides the vision-impaired person safely and accurately.", "However, the following problems have not been solved in the prior art.", "There have been the problems: (1) In the prior art, a movement instruction at an intersection such as ‘Turn right at the intersection of .", ".", ".", "’ is shown based on route data.", "But, in the premises in the case of diagonally crossing an open space such as an atrium, a vault and a concourse although giving route guidance based on landmarks is more simple, the prior art gives a guidance so as to go around the sides of the open space, for example, along the route data on the map; (2) In the route guidance using the portable terminal of a low resolution and a small screen, a color image is hard to watch when it is directly sent to the terminal.", "In addition, the color image requires a long transfer time due to a large data quantity.", "Further, in an image of low compress ion effect even by a binary image, many route guidance points cannot be displayed when the route becomes long; (3) In the case of guiding a route with a map divided in frames, there are individual variations as to where to display the route to be more understandable, some users feel that the route is hard to read if it is always displayed in the center because of lack of continuity between frames and other users feel it is better to display the route always in the center, therefore, display must be changed for each user; (4) In the case of performing route leading, since current positional information of the user from position detection means includes an error, the current position goes off from a position where the user actually is when the current position is guided based on the positional information from the position detection means.", "Further, when a traveling direction of the route is guided based on the positional information from the position detection means, it is different from the actual traveling direction of the user; and (5) In the case of performing position detection using the tag, there is a case where a reader cannot detect the tag, and thus appropriate route leading cannot be made.", "Furthermore, as shown in Japanese Patent Laid-open No.", "9-167297 ‘Intersection guidance apparatus’, in a system that performs route guidance taking in consideration things that come into view, there is a case where an intersection guided cannot be seen from the current position in guiding information regarding the intersection such as ‘Turn right at the next intersection’ in a car navigation system.", "In this case, whether or not a range of vision from the current position to the intersection guided is blocked is calculated, and the guidance is announced at the point when a vehicle has traveled to a point where vision is not blocked.", "However, in Japanese Patent Laid-Open No.", "9-1672 97, whether or not a straight line from the current posit ion to the intersection crosses a polygon, which constitutes landmarks near the intersection is only deter mined, and since this is not a processing to determine what is in the range of vision, it is impossible to extract the landmark in the range of vision and guide the route utilizing the landmark.", "Further, as a visible region calculation algorithm in the case of a plurality of polygons as shown in ‘Computational geometry and geographical information processing’ (the second edition) (4.6.2.problem of visibility), when a set of n pieces of line segments, which do not cross with each other except for an end point, and a viewpoint are given, there exists a method that calculates by a task of O(n log n) assuming that the sum of sides of an polygons by a plane scanning method is n, in which a half line from the viewpoint is made rotate once.", "A processing outline is shown as follows.", "In the processing, the visible region is calculated while finding the sets of line segments (S 0 , S 1 , S 2 , .", ".", ".", ").", "(Pre-processing) The end points that constitute each line segment are arranged in an argument order.", "Herein, the argument is an angle formed by a half line l from the viewpoint, which shows a reference direction, and a half line drawn from the viewpoint to the end point of the line segment.", "A result where the end points of the line segments are arranged in the argument order is shown as L, and the number of L elements is shown as N. An order is put to the end points of each line segment in an anti-clockwise order around the viewpoint, which is defined as an origin and an ending point.", "Further, the L elements can be brought out in the order where they have been arranged from the natural number of 0.", "(1) The set S 0 of the first line segments that cross the half line l from the viewpoint, which shows a reference direction, is obtained.", "Among line segments that cross the half line l, an inters acting point with a line segment nearest the viewpoint is stored as an origin Ps of a visible polygon.", "(2) It is assumed that i=0.", "(3) The i-th element of L is brought out.", "If the line segment having the i-th element as the end point is included in S i , the line segment is pulled out from S i , and the line segment is added if it is not included in S i .", "The result is set as S i+1 .", "There exists the foregoing method.", "Assuming from the foregoing, the processing is considered to proceed as follows.", "Specifically, the line segments of S i+1 are sorted from the one nearest the viewpoint, that is, from the one having the intersecting point with the half line drawn from the viewpoint to an end point of the i-th element, which is nearest the viewpoint.", "i) The followings are performed when the number of the L elements is two or more.", "When the top element is pulled out regarding S i , the line segment from the origin Ps of the visible polygon to a point Pc of the element is drawn because the element is the ending point.", "Further, a point P x where the half line from the viewpoint, which passes the element Pc, crosses the line segment being the second element from the top of S i is obtained, the line segment from Pc to P x is drawn, and P x is set as Ps.", "When the top element is added regarding S i , a point P x where the half line from the viewpoint, which passes the element Pc, crosses the line segment being the top element of S i is obtained because the element is the origin, the line segment from Ps to P x is drawn and the line segment from P x to Pc is drawn.", "Pc is set as Ps.", "ii) The following is performed, because when the number of the L elements is less than two, it is the outer most line segment.", "When the top element is pulled out regarding S i , the line segment from Ps to the point Pc of the element is drawn because the element is the ending point of the line segment.", "Pc is set as Ps.", "When the top element is added regarding S i , the line segment from Ps to Pc is drawn because the element is the origin of the segment.", "Pc is set as Ps.", "(4) i=i+1.That is, i is incremented by one.", "The processing stops when i=N.", "(If not, it proceeds to iii).", "A specific example of the foregoing processing will be described: assuming the viewpoint shown by ⋆, the half line 1 showing the reference direction, and the line segment are given as in FIG.", "98 (a line segment having the end points z 0 and z 1 shall be expressed in z), the processing proceeds as follows.", "The case of i=0: Since a is included in S 0 ={b,a,g}, it is pulled out and sorted, and S 1 ={b,g} is set.", "Drawing is not performed because a is not the top of a list.", "The case of i=1: Since b is included in S 1 ={b,g}, it is pulled out and sorted, and S 2 ={g} is set.", "The line segment b x b 1 g x is drawn from be that is Ps, and g x is stored as Ps.", "The case of i=2: Since g is included in S 2 ={g}, it is pulled out and sorted, and S 3 ={ } is set.", "The line segment g x g 1 is drawn from g x that is Ps, and g 1 is stored as Ps.", "The case of i=3: Since h is not included in S 3 ={ }, it is added and sorted, and S 4 ={h} is set.", "The line segment g 1 h 0 is drawn from g 1 that is Ps, and h 0 is stored as Ps.", "The case of i=4: Since c is not included in S 4 ={h}, it is added and sorted, and S 5 ={c,h} is set.", "The line segment h 0 h x c 0 is drawn from h 0 that is Ps, and c 0 is stored as Ps.", "The case of i=5: Since c is included in S 5 ={c,h}, it is pulled out and sorted, and S 6 ={h} is set.", "The line segment c 0 c 1 h x is drawn from c 0 that is Ps, and h x is stored as Ps.", "However, in the method by the plane scanning method in which the half line from the viewpoint is made rotate once, although n log n is certainly enough for the processing of (3), S 0 needs to be obtained after an optimal line segment 1 is decided in the processing of (1), and there has existed a problem that a calculation amount in deciding the half line l was large and it was not necessarily performed by a small task considering the processing of (1).", "The present invention solves the problem, forms the visible region faster than before, and provides the visible landmark when the user moves on the route based on the visible region formed even in the case where the open space has an obstacle that blocks the range of vision, and its object is to provide a route guidance system that includes the following a to k: a. visible region calculation means that calculates a region where the moving person can look over in the open space even in the case where the obstacle exists; b. route search means that searches the route from a starting point to the destination; c. guidance sentence generation means that generates a route guidance sentence; d. guidance map generation means that generates a map used in the route guidance; e. signpost extraction means that extracts a signpost to be guided out of the foregoing visible region; f. route information generation means; g. position determination means that converts the positional information obtained from position detection means into a coordinate on the map; h. route leading means; i. position detection means that identifies a plane where the moving person is; j. a portable terminal by which the moving person receives the route guidance information; and k. a route map database where route data is stored." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is characterized in that it comprises: the position determination means that detects the current position of the user; the visible region calculation means that calculates the range where the user can see from the current position detected; the route search means that searches the route from the starting point to the destination; traveling direction calculation means that calculates a traveling direction from the visible region and the route; the signpost extraction means that extracts the signpost to be guided out of the visible region; and route guidance sentence generation means that generates the route guidance sentence of the route found, and the invention is further characterized in that it comprises: image data retrieval means that retrieves binary image data or illustration image data, which are specified to guide a point passing the route; and guidance sentence synthesis means to synthesize guidance sentences to guide the point to pass, and the invention is further characterized in that it comprises: a position detection apparatus that identifies the place where the moving person is; route leading means 1 that performs route guidance every time when a position is detected by a tag; route leading means 2 that performs route guidance by a key operation of the user; and position estimation means that estimates the position of the user when the tag cannot be read.", "With the foregoing, the region where the user can look over is calculated by the positional information of the user, the route guidance that guides the signpost regardless of how the route data is formed on the database is enabled, and it is enabled that the route guidance is performed without interruption either by estimating the position or the key operation of the user even in the case where the position detection means fails to detect the tag.", "Furthermore, the present invention comprises: the visible range calculation means that calculates the range where the user can see; target extraction means that extracts a target to be guided out of the visible region; the route search means that searches the route from the starting point to the destination; route information generation means including the guidance map generation means that generates the map for guidance and the guidance sentence generation means that generates the guidance sentence; the route guidance apparatus comprises the route leading means that obtains current positional information to send appropriate guidance information to the portable terminal and the position determination means that converts the information from a user's position detection apparatus into a coordinate; a terminal having a user interface, which displays an image or the route guidance sentence; the position detection apparatus that obtains user's current positional information; a map database that stores data regarding the route and landmark information; a guidance sentence database that stores basic data to generate the guidance sentence; and a route information database that stores the guidance data output from the route information generation means.", "With the foregoing, the route information easy for the user to understand can be provided.", "Further, in the present invention, a visible region calculator is pro vided.", "In which it calculates a visible range regarding the outermost visible region first and then calculates the visible region in the order where the line segments constituting the obstacles are rearranged based on the argument.", "Accordingly, the visible region can be calculated more efficiently than a conventional plane operation method, and the route guidance processing thus can be efficiently performed." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to an apparatus and a system for providing a person, who is not good at reading a map, with guidance including landmark information and route information regarding a direction where the moving person needs to proceed when moving in an architectural structure such as a building with a method using an image or the like such that the user does not became lost in an open space.", "Further, the present invention also relates to a calculation apparatus of a visible region and a program thereof, which are necessary in extracting a land mark to support the movement of the moving person.", "2.Description of the Prior Art In the prior art, there has existed the following known references regarding a system that performs route guidance in premises or in a building.", "The invention disclosed in Japanese Patent Laid-open No.", "8-190688 is one that provides a pedestrian in the premises, who is a pedestrian having difficulty to obtain whereabouts of himself/herself or guidance information to a destination, of appropriate guidance information.", "A user reads information of radio wave reaction apparatuses embedded in the premises using an information reader, and the information to be output changes according to user's information set in a user information setting section although its contents are fixed.", "Further, a pedestrian guidance apparatus can be provided inexpensively.", "Furthermore, in the invention disclosed in Japanese Patent Laid-Open No.", "11-276516, it is one that provides a vision-impaired person in a train station with information such as a route to a destination and a landmark through a portable terminal by audio input/output, detects the position of the handicapped person by a tag, and lends/guides the vision-impaired person safely and accurately.", "However, the following problems have not been solved in the prior art.", "There have been the problems: (1) In the prior art, a movement instruction at an intersection such as ‘Turn right at the intersection of .", ".", ".", "’ is shown based on route data.", "But, in the premises in the case of diagonally crossing an open space such as an atrium, a vault and a concourse although giving route guidance based on landmarks is more simple, the prior art gives a guidance so as to go around the sides of the open space, for example, along the route data on the map; (2) In the route guidance using the portable terminal of a low resolution and a small screen, a color image is hard to watch when it is directly sent to the terminal.", "In addition, the color image requires a long transfer time due to a large data quantity.", "Further, in an image of low compress ion effect even by a binary image, many route guidance points cannot be displayed when the route becomes long; (3) In the case of guiding a route with a map divided in frames, there are individual variations as to where to display the route to be more understandable, some users feel that the route is hard to read if it is always displayed in the center because of lack of continuity between frames and other users feel it is better to display the route always in the center, therefore, display must be changed for each user; (4) In the case of performing route leading, since current positional information of the user from position detection means includes an error, the current position goes off from a position where the user actually is when the current position is guided based on the positional information from the position detection means.", "Further, when a traveling direction of the route is guided based on the positional information from the position detection means, it is different from the actual traveling direction of the user; and (5) In the case of performing position detection using the tag, there is a case where a reader cannot detect the tag, and thus appropriate route leading cannot be made.", "Furthermore, as shown in Japanese Patent Laid-open No.", "9-167297 ‘Intersection guidance apparatus’, in a system that performs route guidance taking in consideration things that come into view, there is a case where an intersection guided cannot be seen from the current position in guiding information regarding the intersection such as ‘Turn right at the next intersection’ in a car navigation system.", "In this case, whether or not a range of vision from the current position to the intersection guided is blocked is calculated, and the guidance is announced at the point when a vehicle has traveled to a point where vision is not blocked.", "However, in Japanese Patent Laid-Open No.", "9-1672 97, whether or not a straight line from the current posit ion to the intersection crosses a polygon, which constitutes landmarks near the intersection is only deter mined, and since this is not a processing to determine what is in the range of vision, it is impossible to extract the landmark in the range of vision and guide the route utilizing the landmark.", "Further, as a visible region calculation algorithm in the case of a plurality of polygons as shown in ‘Computational geometry and geographical information processing’ (the second edition) (4.6.2.problem of visibility), when a set of n pieces of line segments, which do not cross with each other except for an end point, and a viewpoint are given, there exists a method that calculates by a task of O(n log n) assuming that the sum of sides of an polygons by a plane scanning method is n, in which a half line from the viewpoint is made rotate once.", "A processing outline is shown as follows.", "In the processing, the visible region is calculated while finding the sets of line segments (S0, S1, S2, .", ".", ".", ").", "(Pre-processing) The end points that constitute each line segment are arranged in an argument order.", "Herein, the argument is an angle formed by a half line l from the viewpoint, which shows a reference direction, and a half line drawn from the viewpoint to the end point of the line segment.", "A result where the end points of the line segments are arranged in the argument order is shown as L, and the number of L elements is shown as N. An order is put to the end points of each line segment in an anti-clockwise order around the viewpoint, which is defined as an origin and an ending point.", "Further, the L elements can be brought out in the order where they have been arranged from the natural number of 0.", "(1) The set S0 of the first line segments that cross the half line l from the viewpoint, which shows a reference direction, is obtained.", "Among line segments that cross the half line l, an inters acting point with a line segment nearest the viewpoint is stored as an origin Ps of a visible polygon.", "(2) It is assumed that i=0.", "(3) The i-th element of L is brought out.", "If the line segment having the i-th element as the end point is included in Si, the line segment is pulled out from Si, and the line segment is added if it is not included in Si.", "The result is set as Si+1.There exists the foregoing method.", "Assuming from the foregoing, the processing is considered to proceed as follows.", "Specifically, the line segments of Si+1 are sorted from the one nearest the viewpoint, that is, from the one having the intersecting point with the half line drawn from the viewpoint to an end point of the i-th element, which is nearest the viewpoint.", "i) The followings are performed when the number of the L elements is two or more.", "When the top element is pulled out regarding Si, the line segment from the origin Ps of the visible polygon to a point Pc of the element is drawn because the element is the ending point.", "Further, a point Px where the half line from the viewpoint, which passes the element Pc, crosses the line segment being the second element from the top of Si is obtained, the line segment from Pc to Px is drawn, and Px is set as Ps.", "When the top element is added regarding Si, a point Px where the half line from the viewpoint, which passes the element Pc, crosses the line segment being the top element of Si is obtained because the element is the origin, the line segment from Ps to Px is drawn and the line segment from Px to Pc is drawn.", "Pc is set as Ps.", "ii) The following is performed, because when the number of the L elements is less than two, it is the outer most line segment.", "When the top element is pulled out regarding Si, the line segment from Ps to the point Pc of the element is drawn because the element is the ending point of the line segment.", "Pc is set as Ps.", "When the top element is added regarding Si, the line segment from Ps to Pc is drawn because the element is the origin of the segment.", "Pc is set as Ps.", "(4) i=i+1.That is, i is incremented by one.", "The processing stops when i=N.", "(If not, it proceeds to iii).", "A specific example of the foregoing processing will be described: assuming the viewpoint shown by ⋆, the half line 1 showing the reference direction, and the line segment are given as in FIG.", "98 (a line segment having the end points z0 and z1 shall be expressed in z), the processing proceeds as follows.", "The case of i=0: Since a is included in S0={b,a,g}, it is pulled out and sorted, and S1={b,g} is set.", "Drawing is not performed because a is not the top of a list.", "The case of i=1: Since b is included in S1={b,g}, it is pulled out and sorted, and S2={g} is set.", "The line segment bxb1gx is drawn from be that is Ps, and gx is stored as Ps.", "The case of i=2: Since g is included in S2={g}, it is pulled out and sorted, and S3={ } is set.", "The line segment gxg1 is drawn from gx that is Ps, and g1 is stored as Ps.", "The case of i=3: Since h is not included in S3={ }, it is added and sorted, and S4={h} is set.", "The line segment g1h0 is drawn from g1 that is Ps, and h0 is stored as Ps.", "The case of i=4: Since c is not included in S4={h}, it is added and sorted, and S5={c,h} is set.", "The line segment h0hxc0 is drawn from h0 that is Ps, and c0 is stored as Ps.", "The case of i=5: Since c is included in S5={c,h}, it is pulled out and sorted, and S6={h} is set.", "The line segment c0c1hx is drawn from c0 that is Ps, and hx is stored as Ps.", "However, in the method by the plane scanning method in which the half line from the viewpoint is made rotate once, although n log n is certainly enough for the processing of (3), S0 needs to be obtained after an optimal line segment 1 is decided in the processing of (1), and there has existed a problem that a calculation amount in deciding the half line l was large and it was not necessarily performed by a small task considering the processing of (1).", "The present invention solves the problem, forms the visible region faster than before, and provides the visible landmark when the user moves on the route based on the visible region formed even in the case where the open space has an obstacle that blocks the range of vision, and its object is to provide a route guidance system that includes the following a to k: a. visible region calculation means that calculates a region where the moving person can look over in the open space even in the case where the obstacle exists; b. route search means that searches the route from a starting point to the destination; c. guidance sentence generation means that generates a route guidance sentence; d. guidance map generation means that generates a map used in the route guidance; e. signpost extraction means that extracts a signpost to be guided out of the foregoing visible region; f. route information generation means; g. position determination means that converts the positional information obtained from position detection means into a coordinate on the map; h. route leading means; i. position detection means that identifies a plane where the moving person is; j. a portable terminal by which the moving person receives the route guidance information; and k. a route map database where route data is stored.", "SUMMARY OF THE INVENTION The present invention is characterized in that it comprises: the position determination means that detects the current position of the user; the visible region calculation means that calculates the range where the user can see from the current position detected; the route search means that searches the route from the starting point to the destination; traveling direction calculation means that calculates a traveling direction from the visible region and the route; the signpost extraction means that extracts the signpost to be guided out of the visible region; and route guidance sentence generation means that generates the route guidance sentence of the route found, and the invention is further characterized in that it comprises: image data retrieval means that retrieves binary image data or illustration image data, which are specified to guide a point passing the route; and guidance sentence synthesis means to synthesize guidance sentences to guide the point to pass, and the invention is further characterized in that it comprises: a position detection apparatus that identifies the place where the moving person is; route leading means 1 that performs route guidance every time when a position is detected by a tag; route leading means 2 that performs route guidance by a key operation of the user; and position estimation means that estimates the position of the user when the tag cannot be read.", "With the foregoing, the region where the user can look over is calculated by the positional information of the user, the route guidance that guides the signpost regardless of how the route data is formed on the database is enabled, and it is enabled that the route guidance is performed without interruption either by estimating the position or the key operation of the user even in the case where the position detection means fails to detect the tag.", "Furthermore, the present invention comprises: the visible range calculation means that calculates the range where the user can see; target extraction means that extracts a target to be guided out of the visible region; the route search means that searches the route from the starting point to the destination; route information generation means including the guidance map generation means that generates the map for guidance and the guidance sentence generation means that generates the guidance sentence; the route guidance apparatus comprises the route leading means that obtains current positional information to send appropriate guidance information to the portable terminal and the position determination means that converts the information from a user's position detection apparatus into a coordinate; a terminal having a user interface, which displays an image or the route guidance sentence; the position detection apparatus that obtains user's current positional information; a map database that stores data regarding the route and landmark information; a guidance sentence database that stores basic data to generate the guidance sentence; and a route information database that stores the guidance data output from the route information generation means.", "With the foregoing, the route information easy for the user to understand can be provided.", "Further, in the present invention, a visible region calculator is pro vided.", "In which it calculates a visible range regarding the outermost visible region first and then calculates the visible region in the order where the line segments constituting the obstacles are rearranged based on the argument.", "Accordingly, the visible region can be calculated more efficiently than a conventional plane operation method, and the route guidance processing thus can be efficiently performed.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a configuration view of a route guidance system of the present invention.", "FIG.", "2 is a system configuration view of the route guidance means 1.FIG.", "3 is a configuration view of a portable terminal apparatus.", "FIG.", "4 is an entire view of premises used in embodiment 2.FIG.", "5 is a flowchart of the route guidance means 1.FIG.", "6 is a view of a visible polygon used in embodiment 1.FIG.", "7 is a view where the visible polygon and a route are superposed near a destination, which is used in embodiment 1.FIG.", "8 is a system configuration view of the route guidance means 2.FIG.", "9 a flowchart of the route guidance means 2.FIG.", ", is a view used in embodiment 2, where the visible polygon is calculated from E in a premises view.", "FIG.", "11 is a view of a binary image at point U in FIG.", "4.FIG.", "12 is a view of a binary image at point Q in FIG.", "4.FIG.", "13 is a view of a binary image at point R in FIG.", "4.FIG.", "44 is a view of a binary image at point S in FIG.", "4.FIG.", "15 is a view of a binary image at point T in FIG.", "4.FIG.", "16 is a system configuration view of a route guidance means 3.FIG.", "17 is a flowchart of the route guidance means 3.FIG.", "18 is a flow chart of frame map generation means 1.FIG.", "19 is a view showing an example of a display screen size of the portable terminal used in embodiment 3.FIG.", "20 is an entire premises view used in embodiment 3.FIG.", "21 is a flow chart of frame map generation means 2.FIG.", "22 is a view in which the entire premises view used in embodiment 3 is cut into the display screen size of the portable terminal.", "FIG.", "23 is a view of an illustration image at point U in FIG.", "4.FIG.", "24 is a view of an illustration image at point Q in FIG.", "4.FIG.", "25 is a view of an illustration image at point R in FIG.", "4.FIG.", "26 is a view of an illustration image at point S in FIG.", "4.FIG.", "27 is a view of an illustration image at point T in FIG.", "4.FIG.", "28 is a flowchart of the route leading means 1.FIG.", "29 is a view showing an example of a route and frame views divided along the route, which are used in embodiment 6.FIG.", "30 is a flowchart of the route leading means 2.FIG.", "31 is an exemplary view of a table in which tags, coordinate data, and tag ID's are correlated and accumulated.", "FIG.", "32 is an exemplary view of a table in which destinations and coordinate data are correlated and accumulated.", "FIG.", "33 is a view showing the guidance sentence.", "FIG.", "34 is a view showing examples of the route guidance sentence.", "FIG.", "35 is an exemplary view of guidance mark data.", "FIG.", "36 is an exemplary view of the guidance mark data.", "FIG.", "37 is an exemplary view of a table in which passage orders of nodes, the binary image data, and the illustration image data are correlated and accumulated.", "FIG.", "38 is an exemplary view of a table in which the passage orders of nodes and guidance sentence data are correlated and accumulated.", "FIG.", "39 is an exemplary view of a frame-route elements row correspondence table.", "FIG.", "40 is an exemplary view of a tag-node/facilities correspondence table.", "FIG.", "41 is an exemplary view of the frame-route elements row correspondence table.", "FIG.", "42 is an view showing the guidance sentences.", "FIG.", "43 is an exemplary view of a table where a terminal ID, a terminal status, an ID of the frame view displayed, an ID of the guidance sentence, an ID of the tag picked up immediately previously, an element row of the routes, a row of the frame view ID's, and a row of the guidance sentence ID's are stored.", "FIG.", "44 is an exemplary view of a table where the terminal ID, the terminal status, a tag row, the ID of the guidance sentence, and the row of the guidance sentence ID's are stored.", "FIG.", "45 is a view showing an example of the guidance sentence data.", "FIG.", "46 is a function block diagram of the route guidance apparatus in embodiment 7.FIG.", "47 is an exemplary view of a table where starting points and destinations are correlated and accumulated.", "FIG.", "48 is an exemplary view of a table where posit ions and coordinates thereof are correlated and accumulated.", "FIG.", "49 is an exemplary view of a table where marks and coordinates thereof are correlated and accumulated.", "FIG.", "50 is a function block diagram of the route guidance apparatus in embodiment 8.FIG.", "51 is an exemplary view of a table where points to pass and image data are correlated and accumulated.", "FIG.", "52 is an exemplary view of a table where the points to pass and guidance sentence data are correlated and accumulated.", "FIG.", "53 is a function block view of the route guidance apparatus in embodiments 9 and 10.FIG.", "54 is a flow chart explaining the processing of the route guidance apparatus in embodiment 9.FIG.", "55 is a flowchart explaining the generation processing of guidance sentence by the guidance sentence generation means in embodiment 13.FIG.", "56 is a flow chart explaining the processing of the route guidance apparatus in embodiment 14.FIG.", "57 is a view showing a part of a function block diagram of the route guidance apparatus in embodiment 15.FIG.", "58 is a flow chart explaining the processing of the route guidance apparatus in embodiment 15.FIG.", "59 is a flow chart explaining the operations of the position determination means, user request obtaining means, and the route leading means.", "FIG.", "60 is a function block diagram of the route guidance apparatus in embodiment 17.FIG.", "61 is a flowchart explaining the operation of a visible region calculation apparatus in embodiment 18.FIG.", "62 is a function block diagram of the visible region calculator in embodiment 19.FIG.", "63 is a flowchart explaining the operation of a visible region calculator in embodiment 18.FIG.", "64 is a flow chart explaining an operation when the visible region calculator in embodiment 19 calculates a temporary visible region.", "FIG.", "65 is an exemplary view showing the relation between the temporary visible region and a half line drawn from the viewpoint.", "FIG.", "66 is an exemplary view showing the relation between the visible region and the half line drawn from the viewpoint.", "FIG.", "67 is a flowchart explaining the operation of the visible region calculator in embodiment 19.FIG.", "68 is an exemplary view showing the relation between a visible polygonal line and a temporary visible polygon.", "FIG.", "69 is an exemplary view showing the relation between the visible polygonal line and the temporary visible polygon.", "FIG.", "70 is a function block diagram of a route information generator in embodiment 20.FIG.", "71 is a flow chart explaining the processing of the route information generator in embodiment 20.FIG.", "72 is a function block diagram of the route guidance system in embodiment 21.FIG.", "73 is a flowchart explaining the operation of the route guidance system in embodiment 21.FIG.", "74 is a flowchart explaining the processing of a route leader in embodiment 22.FIG.", "75 is a function block diagram of the route guidance system in embodiment 23.FIG.", "76 is an exemplary view explaining a state where the starting points and the destinations are correlated and accumulated in the route information database.", "FIG.", "77 is an exemplary view of a table where the routes, the maps, and the guidance sentences are correlated and accumulated.", "FIG.", "78 is a function block diagram of the route guidance system in embodiment 24.FIG.", "79 is a view explaining the argument at the end point of the line segment.", "FIG.", "80 is a block diagram showing a configuration example of the route guidance system in embodiment 25.FIG.", "81 is a block configuration view of the route information generation means in embodiment 25.FIG.", "82 is a block configuration view of the route leading means in embodiment 25.FIG.", "83 is a flow chart explaining the processing of the route information generation means in embodiment 25.FIG.", "84 is a flow chart explaining the processing of the route leading means in embodiment 26.FIG.", "85 is an exemplary view of the map that shows the current position of the user and the route to go.", "FIG.", "86 is an exemplary view of the map that shows the visible region of the user at the starting point.", "FIG.", "87 is an exemplary view of the map that shows the visible region when the user has moved to reach a corner.", "FIG.", "88 is an exemplary view of the map that shows the visible region after the user has moved.", "FIG.", "89 is an example of the premises view used in embodiment 25.FIG.", "90 is a view of the visible region calculated at point a, which is used in embodiment 25.FIG.", "91 is a view of the visible region calculated at point b, which is used in embodiment 25.FIG.", "92 is a view of the visible region calculated at point c, which is used in embodiment 25.FIG.", "93 is a map for guidance, which is used in embodiment 25 and embodiment 26 and generated in embodiment 25.FIG.", "94 is a map for guidance, which is used in embodiment 25 and embodiment 26 and generated in embodiment 25.FIG.", "95 is a map for leading, which is displayed on the terminal used in embodiment 26.FIG.", "96 is a map for leading, which is displayed on the terminal used in embodiment 26.FIG.", "97 is a map for leading, which is displayed on the terminal used in embodiment 26.FIG.", "98 is an exemplary view of the viewpoint and the line segment to explain an example of the conventional plane scanning method.", "PREFERRED EMBODIMENT OF THE INVENTION FIG.", "1 is the block diagram showing the configuration example of the premises route guidance system according to the present invention, which is comprised of: a position detection apparatus 20 that informs the user's position; a portable terminal apparatus 30 that transmits position detection information from the position detection apparatus 20 and user information to a premises route guidance apparatus 10 to display a photographed image of the route or the mark; the map database (hereinafter, referred to as DB40) in which data of the map is accumulated; a guidance sentence DB60 where the guidance sentences, in which route guidance is performed using the direction of railway in a train station as a reference, of the route is accumulated; an must ration image DB80 where the illustration photographed images regarding guidance points are accumulated; a binary image DB70 where binary images regarding the guidance points are accumulated; route data 24 where the route search results are stored; a correspondence table 44 between tags and each guidance sentence; a frame view image 95; a guidance image DB90; a correspondence table 44 between the tags and each guidance sentence; and the premises route guidance apparatus 10 that processes the position detection (tag) information and photographed image information to guide route.", "Among them, the position detection apparatus 20 transmits positional coordinate to the user.", "The portable terminal shown in FIG.", "3 is equipped with user infer nation 33, position detection (tag) reading means 31, route data reading-out means 32, image data display means 34, and illustration image data extraction means 35, and it displays or reads out the image or text data processed by the route guidance apparatus on the portable terminal.", "The premises route guidance apparatus 10 comprises: position determination means 11 that determines where the user is in the premises by the positional information; user determination means 12 that receives the characteristics of the user such as a vision-impaired person, a hearing-impaired person, and an able-bodied person to determine a para meter for route search, or decides output means such that it outputs the binary image or the illustration image when a terminal type used is a monochrome terminal and outputs a color image when it is a color terminal; speech synthesis means 13 that pronounces the route guidance sentence output; route guidance means 1(14); route guidance means 2(16); route guidance means 3(17); and route leading means 1(18) and route leading means 2(19), which leads the route.", "FIG.", "2(a) is the block configuration view regarding the route guidance means 1 in the route guidance apparatus, which is comprised of: visible polygon calculation means 142 as an example of visible region calculation means that calculates a range that can be seen from a current position; route search means 141 that searches the route to the destination; traveling direction calculation means 143 that calculates which direction the traveling direction of the route is from the current position; signpost extract ion means 144 that extracts a mark showing the traveling direction; and route guidance sentence generation means 145 that generates the route guidance sentence.", "FIG.", "2(b) is the block configuration diagram regarding the route leading means 1(18) in the route guidance apparatus, which is comprised of: the position detection apparatus (20) that detects tag information; the portable terminal apparatus (30) that receives the positional information to provide the route information to the user; the position determination means 11 that determines the current position from the tag; route search means (170) that performs route search; the route data 24 that stores the route search results; image data synthesis means 181 that synthesizes the image data of frame view used in the route guidance; guidance sentence synthesis means 183 that synthesizes the guidance sentence that corresponds to each frame view; guidance image retrieval means 184 that retrieves a guidance image provided to the user every time the tag is detected; guidance sentence retrieval means 191 that retrieves the guidance sentence provided to the user every time the tag is detected; the map DB40; a frame view image DB95 that stores images of frame views, which are a source for synthesizing the guidance images; the guidance image DB90 that stores the guidance images synthesized; the guidance sentence DB60 that stores the guidance sentences and guidance sentence generation rules that generate the guidance sentence; the tag-frame view correspondence table 45; and the tag-guidance sentence correspondence table 44.FIG.", "2(c) is the block configuration diagram regarding the route leading means 2(19), which is comprised of: the position detection apparatus (20) that detects the tag information; the portable terminal apparatus (30) that receives the positional information to provide the route information to the user; the position determination means 11 that determines the position of the tag; the route search means (170) that performs route search; the guidance sentence retrieval means 191 that retrieves the guidance sentence that corresponds to the tag; guidance sentence synthesis means 193 that synthesizes the route guidance sentence near the tag; the guidance sentence DB60 that stores the guidance sentences and the guidance sentence generation rules that generate the guidance sentence; the route data 24 that stores the route search results; and the tag-guidance sentence correspondence table 44.FIG.", "7 is the block configuration view regarding the route leading means 1(18), which is comprised of: the portable terminal apparatus (30) that detects the tag information; the position determination means 11 that determines the position from the tag; image data retrieval means 181 that retrieves data of frame view that corresponds to the current position; and the guidance sentence synthesis means 183 that synthesizes the guidance sentence that corresponds to each frame view.", "FIG.", "8 is the block configuration view regarding the route leading means 2(19), which is comprised of: the portable terminal apparatus (30) that detects the tag information; the position determination means 11 that determines the position of the tag; the guidance sentence retrieval means 191 that retrieves the guidance sentence that corresponds to the tag; and the guidance sentence synthesis means 193 that synthesizes the route guidance sentence near the tag.", "Embodiment 1 The route guidance means 1 will be described based on an example.", "Herein, consideration will be given to the case of passing in the premises from A to B as shown by the solid line arrow.", "The route data is drawn in the premises in dotted lines on the map DB40.The user loses his/her way at point A, and rang a system using the portable terminal 30 first.", "The user enters a destination B through the user interface of the portable terminal 30.A portable terminal Pa reads an ID in Ta from the posit ion detection Ta, and transmits data: a tag ID; the terminal being a PDA type terminal; and the destination being B to the route guidance apparatus 10.The processing will be described along the flowchart of the route guidance means 1 shown in FIG.", "5.In the route guidance means 1, the position determination means 11 outputs a user's position A(15,23) in the premises (step 51), the route search means 141 set the current position A as a variable (Entrance) (step 52), and performs route search from the current position A and the destination B to output the route where the user should proceed (step 53).", "The visible polygon calculation means 142 calculates the region in the premises that can be seen from A, which is shown in a dot-meshed region in FIG.", "6, using the information of the current point A (step 54).", "In the traveling direction calculation means 143, the visible polygon and the route are superposed to calculate the intersecting point between them and obtains an intersecting point D of the destination (step 55), and D is bound to a variable (Exit) when there is an intersecting point (step 56).", "A vector AD shown by a broken line arrow in FIG.", "6 is set as a direction where the user should proceed.", "In the signpost calculation means 144, a vector having the smallest angle is extracted from the traveling direction vector of the user and a vector connecting the tag and a target (double-line arrow in FIG.", "6) in the visible polygon as the signpost and sets an Ld in FIG.", "6 as a guidance mark, and an angle θ made, by an immediately preceding traveling direction and the double-line arrow vector is compartmentalized in a 45-degree step to register a corresponding sentence ‘Left ahead’ (step 57).", "An entity of the variable (Exit) is set as an entity of a new Entrance (step 58), and the processing returns to step 54.When the user arrives D in FIG.", "7, the visible polygon is as shown in dot-mesh in FIG.", "7, and the destination B is set as Exit since the mark B is at the place of destination (step 59).", "The destination B and the direction of the destination that is ‘Ahead’ are registered as the guidance marks (step 60).", "In the route guidance sentence generation means 145, arriving all guidance marks registered, the direction and the guidance mark in the guidance mark data shown in FIG.", "35 and FIG.", "36 are respectively applied to X and Y of a template, which is ‘Y of X is the destination.’ in the case of destination and ‘Walk toward Y of X.’ otherwise, and the guidance sentence shown in FIG.", "33 is generated (step 61).", "In the case of this example, the route guidance sentences that depend on the route network are shown in FIG.", "34.The foregoing embodiment 1 calculates the region where the user can look over by the user's positional information in the open space in the premises, and the route guidance that guides the signpost regardless of how the route data is formed on a database is enabled.", "Embodiment 2 FIG.", "8 is the block diagram showing the configuration example of the route guidance means 2 in the present invention, and it comprises: the map DB40 where the route data for searching the route in the premises; the route search means 170 that searches the route in the premises; the guidance point illustration image DB70 where the illustration images of the guidance points are accumulated; the guidance point binary image DB80 where the binary images of the guidance points are accumulated; image data retrieval means 171 that retrieves the image of the guidance point based on a route search result; the guidance sentence DB60 where the guidance sentences are accumulated; guidance sentence generation means 173 that generates the guidance sentence from the guidance sentence DB based on the route search result; visible polygon calculation means 174 as an example of the visible region calculation means that calculates a range that can be seen from the current position; traveling direction calculation means 175 that calculates which direction the traveling direction of the route is from the current position; and signpost extraction means 176 that extracts a nark showing the traveling direction.", "The processing of the embodiment of the route guidance means 2 will be described along the flowchart of the route guidance means 2 shown in FIG.", "9.Herein, consideration will be given to the case of passing in the premises from E to F as shown by the broken line arrow.", "In the route guidance means 2, when the route search means 170 is activated (step 90), the starting point E, the destination F and the parameter of an output image are entered (step 91).", "The route search is executed to output a node row of E, U, Q, R, S, T and F is output as the route search result (step 92).", "Similarly to the processing (step 53 to step 60) of the flowchart of the route guidance means 1 in embodiment 1, the visible polygon is calculated in the route order to find guidance points of u, q, r, s and t. FIG.", "10 shows the view in which the visible polygon from E was calculated to find the guidance point u (step 93 to step 98).", "Herein, when a threshold value n of the route guidance point is set to 10, the route guidance point is smaller than n since the route guidance point of the node row EU QRSTF is 7 (step 99).", "Referring to the correspondence table between the passage orders of nodes and the binary image data show n in FIG.", "37, the binary images to which parameters are sped fled are retrieved from the binary image data DB, and they are arranged in the order of the route search result (step 101).", "When the route guidance point is larger than n, the correspondence table between the passage orders of nodes and the illustration image data shown in FIG.", "37 is referred to and the illustration images to which parameters are specified are retrieved from the illustration image data DB, and they are arranged in the order of the route search result (step 102).", "Referring to the correspondence table between the passage orders of nodes and the guidance sentence data shown in FIG.", "38, the guidance sentence is added to the image (step 103).", "The images shown in FIG.", "11 to FIG.", "15 and the guidance sentence are sequentially output to the port able terminal 30 (step 104), the illustration image data extraction means (35) shown in FIG.", "4 extracts the illustration image data FIG.", "23 to FIG.", "27 in the case of the illustration image (step 105), and the processing ends (step 106).", "The invention described in embodiment 2 can perform the route guidance easier to understand for the user who has difficulty in reading the route on the map by performing the route guidance that uses the image data, in which an arrow is added to the binary image or the illustration image for the guidance point in the traveling direction of the user, in the route guidance in the premises.", "Embodiment 3 FIG.", "16 is the block diagram showing a configuration example of the route guidance means 2 in the present invention, and it comprises: the map DB40 where the route data for searching the route in the premises is accumulated; route search means 161 that searches the route in the premises; frame map generation means 1 that matches connection points of the route in each frame map to generate the frame map; the guidance sentence DB60 where the guidance sentences are accumulated; and guidance sentence generation means 163 that gene rates the guidance sentence from the guidance sentence DB based on the route search result.", "The processing of the embodiment of the route guidance means 2 will be described along the flowchart of the route guidance means 2 shown in FIG.", "17 and the flowchart of the frame map generation means 162 shown in FIG.", "18.Herein, consideration will be given to the case of passing in the premises from E to F as shown by the broken line arrow.", "In the route guidance means 3, when the route search means 3 is activated (3000), the starting point E, the destination F and the parameter of the output image are entered (step 171).", "The route search is executed to output the node row of E, U, Q, R, S, T and F is output as the route search result (step 172).", "The frame map generation means 162 is activated according to the parameter of the output image entered (step 173).", "In the frame map generation means 162, a circumscribed rectangle of the route is calculated first (step 174).", "The circumscribed rectangle is expanded to an integral multiplication of the aspect ratio of the portable terminal screen (step 175).", "The circumscribed rectangle is cut into frames and the frame that does not include the route is abandoned (step 183).", "The frames are arranged in the route order and are output (refer to FIG.", "20).", "At this point, the premises entire view shown in FIG.", "20 is made such that the frame maps do not overlap with each other along the route to fit the screen of the display screen size of the portable terminal shown in FIG.", "19, the routes at the boundaries between each of the frame map match, and thus, the frame views of 1 to 4 are formed, that is, as shown in the one-dot chain line.", "Referring to the correspondence table between the passage orders of nodes and the guidance sentence data shown in FIG.", "38, the guidance sentence is added to the image (step 175).", "The image and the guidance sentence are sequentially output to the port able terminal 30 in the order of route search to end the processing (steps 176, 177).", "The invention described in embodiment 3 can provide the route guidance, in which a pedestrian does not lose his/her direction by matching the display direction of the frame view in the route guidance and the direction of a railway and by displaying the frame maps while matching the connection points between each of the maps, in the route guidance in the premises, particularly in the station.", "Embodiment 4 FIG.", "16 is the block diagram showing a configuration example of the route guidance means 2, and it comprises: the map DB40 where the route data for searching the route in the premises is accumulated; the route search means 161 that searches the route in the premises; the frame map generation means 162 that matches connection points of the route in each frame map to generate the frame map; frame map generation means 164 that displays the route in each frame map in the center of each frame; the guidance sentence DB60 where the guidance sentences are accumulated; and the guidance sentence generation means 163 that generates the guidance sentence from the guidance sentence DB based on the route search result.", "The processing of the embodiment of the route guidance means 3 will be described along the flowchart of the route guidance means 3 shown in FIG.", "17 and the flowchart of the frame map generation means 2 shown in FIG.", "21.Herein, consideration will be given to the case of passing in the premises from E to F as shown by the broken line arrow.", "In the route guidance means 3, when the route search means 3 is activated (step 170), the starting point E, the destination F and the parameter of the output image are entered (step 171).", "The route search is executed to output the node row of E, U, Q, R, S, T and F is output as the route search result (step 172).", "The frame map generation means 164 is activated according to the parameter of the output image entered (step 174).", "The nodes having a distance L (10 meters, for example) or more are selected among each of the nodes E, U, Q, R, S, T and F on the route (step 211).", "The premises entire view shown in FIG.", "22 is cut out such that each of the nodes E, U, Q, R, S, T and F is positioned in the center of the frame map as shown in the one-dot chain line along the route, that is.", "Into the frame views of 1 to 5 (step 212).", "The frame views are arranged in the order of each node E, U, Q, R, S, T and F (step 213).", "Refer ring to the correspondence table between the passage orders of nodes and the guidance sentence data shown in FIG.", "38, the guidance sentence is added to the image (step 175).", "The image and the guidance sentence are sequentially output to the portable terminal 30 in the order of route search to end the processing (steps 177).", "The invention described in embodiment 4 can perform the route guidance with the frame map easier to watch by matching the display direction of the frame view in the route guidance and the direction of the railway and by displaying the route guidance points such as the mark and the route node in each frame maps in the center of the frame view.", "Embodiment 5 The embodiment of the route leading means 1 will be described based on the flowchart of the route leading means 1 shown in FIG.", "28 and the block diagram of the route leading means 1 shown in FIG.", "7.Herein, description will be made for the case of passing the route as shown in FIG.", "29.In the route leading means 1, when the user activates the route leading means 1(18) (1800), the position determination means 11 converts the starting point automatically detected by the position detection apparatus 20 into the coordinate on the map data, and the map determination means 11 further converts the destination entered by the user into the coordinate on the map data.", "The converted coordinates of the starting point and the destination are input to the route search means 170 to perform route search, and the row of facilities and nodes on the route is output in the route order (1810).", "The row of the nodes/facilities output in the example of FIG.", "2(b) is shown as follows.", "m: mark; t: tag; and n: node.", "[m0, n1, m1, m2, n2, n3, m3, n4, n5, m5] .", ".", ".", "(an example of output of route search result) The image data synthesis means 181 checks with the element-frame view correspondence table shown on table 16 in the map DB40 to find which frame view the node and the facilities belong to, a corresponding row of the frame views is retrieved from the frame view image 95 to draw the coordinate row of the route search results as the route on the corresponding frame view, it is stored in the frame view image DB90, and a frame-route element row correspondence table is generated as shown in FIG.", "39 (1815).", "Referring to the tag-node/facilities correspondence table of FIG.", "40 in the map DB40, the tags on route are inserted in the row of the nodes and the facilities (1820).", "The result where they have been inserted is as follows.", "[m0, n1, m1, t1, m2, n2, n3, t2, m3, n4, n5, m5] .", ".", ".", "(a: an example of an element row when the tag information is inserted) The foregoing is the example where t2 and t3 compete and t2 has been selected.", "The nodes t2 and t3 competed are previously stored in the map DB40 as a competition list such as in (t2, t3).", "This enables it to deal with the case where t3 is received instead of t2 while the route leading is performed.", "Regarding the frame with the tag, the row of the nodes/facilities that corresponds to the frame is divided by the tag (1825).", "Information of the mark and the node and corresponding guidance sentence are generated in each frame view, and they are respectively stored in the image DB90 and the guidance sentence DB60.ID's of the guidance sentence and the guidance image are stored in the route data 24 in the form of FIG.", "43 (1830).", "In FIG.", "43, an ID of each terminal, a status of the terminal specified by a terminal code, an ID of the frame view currently displayed, and ID of the guidance sentence currently displayed, an ID of the tag picked up immediately previously, an element row of the routes, and ID of the frame view displayed, and an ID of the guidance sentence corresponding to each frame are respectively stored in a terminal code, a current status, a field of a frame displayed, a sentence for display, an immediately previous tag, a route, a row of frames, and a row of guidance sentences, the current status is updated when the status changes, the frame for display and the sentence for display are updated when the frame and the guidance sentence displayed on the terminal change, and the immediately previous tag is updated every time the tag is detected.", "Next, the route leading begins (1835).", "At the starting point, the frame of 1.1 in FIG.", "29(b), which corresponds to {circle around (1)} of FIG.", "41, is transmitted and displayed (1840).", "The positional information sent from the position detection apparatus 20 is checked by the position determination means 11 (1845), it checks whether or not the tag detected is the same as the immediately previous tag of FIG.", "43 (1850) if a tag has been detected, and ignores the tag detected if they are the same and return to (1845).", "If they are different, the frame of 1.2 in FIG.", "29(b), which corresponds to {circle around (2)} and {circle around (3)} of FIG.", "41, is transmitted, {circle around (2)} is displayed first, and the tag t1 detected is stored as a new tag (1855).", "Whether or not the new tag t1 is the last tag t2 or the competing t3 in the row of the route fields of FIG.", "43 (1860) is checked, and the frame of 2 in FIG.", "29(b), which corresponds to {circle around (3)}, is displayed by a button operation if it is not the last tag.", "If it is the last tag, the route leading ends (1865).", "If a tag is not detected in (1845), the position determination means estimates the user's posit ion, it generates the guidance sentence such as ‘You are between track no.", "7 and no.", "2 now.’ and sends it to the terminal (1847), and returns to (1845).", "The terminal displays the guidance sentence.", "Now, in step (1855), when t3 is received instead of the tag t2, a correspondence relation between t2 and t3 is found out by referring to the competition list, and the frame 3 is displayed.", "In generating the guidance sentence that shows a direction to proceed next at the node n1 and the node, which are the first direction turning points, when the node n1 is within a range of error of the position detection by radio wave of the tag from the starting point S, a relative directional indication such as ‘Right turn’ that cannot be shown unless the starting point is assured is inappropriate and should be avoided, and the positional information of the node n1 and the guidance sentence of the mark showing the next traveling direction are generated as in the top line of FIG.", "42.FIG.", "42 is the example of the guidance sentences of embodiment 5.Regarding the frame view, the map on which the illustration image, the binary image, and a town block shape are described may be used corresponding to each frame of FIG.", "29.The invention described in embodiment 5 synchronizes a guidance frame view of the route with tag detection in performing the route guidance, and assures the user of receiving information regarding the traveling direction near the tag before detecting the tag without fail even when there is an error in the range where the radio wave reach in the tag, and furthermore, the first traveling direction turning point and the mark that can be seen from the first traveling direction turning point are guided even when the positional information of the first traveling direction turning point includes an error, and thus information provision where the route information is not complex can be performed.", "Embodiment 6 The embodiment of the route leading means 2 will be described based on the flowchart of the route leading means 2 shown in FIG.", "30 and the block diagram of the route leading means 2 shown in FIG.", "8.Herein, description will be made for the case of passing the route as shown in FIG.", "29.In the route leading means 2, when the user activates the route leading means 2(16) (1900) from the terminal 30, the starting point automatically detected by the position detection means 20 and the destination that the user enters by the portable terminal 30 are input to the route search means 170 to perform the route search, and the row of nodes and facilities on the route is output in the route order (1905).", "The row of the nodes/facilities output in the example of FIG.", "29(a) is shown as follows.", "m: mark; t: tag; and n: node.", "[m0, n1, m1, m2, n2, n3, m3, n4, n5, m5] .", ".", ".", "(example of output of route search result) Referring to the row of the node s/facilities and the tag-node/facilities correspondence table of FIG.", "40, the row of the tags on the route is generated in the route order is generated (1915).", "The guidance sentence synthesis means 193 generates the guidance sentences shown in FIG.", "45, which correspond to the row of the nodes and the facilities, from the guidance sentence DB60 and the gene ration rule in the guidance sentence DB, and the guidance sentences and the row of the nodes and the facilities, which is the route search result, are stored in the route data 24 in the form shown in FIG.", "44 in the route order (1915).", "At the same time, the route leading begins (1920).", "Firstly, guidance of a route out line is sent to the speech synthesis means 13, and the guidance by voice is performed from the terminal (1925).", "When the position detection apparatus 20 detects the tag (1945), the guidance sentence from the tag t1 detected to before the tag t2 out of the guidance sentence data of FIG.", "45 is sent to the terminal (1950).", "Whether or not t1 is the last tag on the route is checked by referring to the route field of FIG.", "44 (1955).", "Command waiting from the portable terminal is performed if it is not the last tag, and the route leading ends if it is the last tag (1960).", "When the user performs the button operation on the portable terminal (1930), the fields of the rows of the sentence for display and the guidance sentence of FIG.", "44 are referred to, the guidance sentence before or after the sentence for display is sent to the terminal in accordance with the button operation, and the field of the sentence for display is updated (1935).", "Whether or not the sentence for display updated is the last guidance sentence is checked by referring to the row of the guidance sentences, the command waiting of the portable terminal is performed if it is not the last one, the route leading ends if it is the last one (1960), and the processing ends (1965).", "An Example of the Guidance Sentence: FIG.", "45 is the example in the case where the tag t1 is detected after the mark m2.The invention described in embodiment 6 provides the vision-impaired person of a provision method of the route information and an information provision method of the current position separately, the current position is provided when the tag is detected and the information of the traveling direction is provided by the button operation of the user, and thus information provision where the route information is not intricate can be performed.", "Embodiment 7 FIG.", "46 exemplifies the function block diagram of the route guidance apparatus in embodiment 7 of the present invention.", "In FIG.", "46, a route guidance apparatus 4600 comprises: position determination means 4601; visible region calculation means 4602; route search means 4603; traveling direction calculation means 4604; mark extraction means 4605; and guidance sentence generation means 4606.The position determination means 4601 detects the current position of the user.", "For example, a tag that emits the radio wave including its own number is embedded in a wall or a floor in the premises, the portable terminal that the user has is designed to receive the radio wave from the tag, and the position determination means 4601 receives the number of the tag, which the portable terminal transmits, and detects the current position of the user from the number of the tag.", "Alternatively, the portable terminal that the user has emits the radio wave, a receiving apparatus installed on the wall or the floor receives the radio wave, and the current position may be detected depending on which receiving apparatus has received the radio wave of the portable terminal.", "The visible region calculation means 4602 calculates the visible region that is the range where the user can see from the current position detected.", "Specifically, it calculates the range where the user can see from the current posit ion of the user detected by the position determination means 4601.When obstacles that block the range of vision of the user are approximated by polygons and their positions are stored, the range where the user can see can be calculated by a visible region calculation method by the conventional plane scanning method.", "Or other methods may be used.", "The route search means 4603 searches the route from the starting point to the destination.", "FIG.", "47 exemplifies an example of the table used in the route search, and FIG.", "47 expresses that from which point to which point one can directly reach.", "For example, the first line of the table of FIG.", "47 means that one can directly reach from point A to point C. By using such a table, to obtain the route from any starting point given to any destination, a line where the first starting point given appears in the row of the starting points is retrieved, a value of the row of a final destinations on the line is obtained, a line that has the value obtained as the starting point is retrieved, and a value of the row of the final destinations on the line is obtained, which are repeated and continued until the destination is obtained as the value of the row of the final destinations.", "For example, when A is set as the starting point and G is set as the destination, the line that has A as the value of the row of the starting points is retrieved, the first line of FIG.", "47 is obtained, and C is obtained as the final destination.", "Next, the line where C appears as the value of the row of the starting points, the third line, the fourth line, and the fifth line are obtained and D, E and F are obtained as the final destination, the lines where each of the three appears in the row of the starting points are retrieved, the sixth line is obtained as the line where D appears in the row of the starting points when the row of the final destinations is obtained, the processing ends because its final destination is G, and finally, a route A→C→D→G is obtained.", "The traveling direction calculation means calculates the traveling direction from the visible region and the route.", "Specifically, it calculates the traveling direction where the user should proceed from the range, where the user can see, calculated by the visible region calculation means 4602 and the route searched by the route search means 4603.As a calculation method, in which direction the direction of a posit ion to be proceeded next from the starting point is within the visible region is calculated if the current position of the user detected by the position determination means 4601 is the starting point of the route searched by the route search means 4603.For example, the starting point is A and when the route search means 4603 has searched that the position to be proceeded next is C, in which direction the direction from A to C is within the visible region is calculated.", "For this calculation, the coordinate position of each point is stored in the route guidance apparatus 4600 as shown in FIG.", "48, and the direction may be calculated from the coordinate positions.", "Alternatively, the route searched by the route search means 4603 is expressed in the row of the line segments that has the position on the route as the end point, the intersecting point between the row of the line segments and the visible region calculated by the visible region calculation means 4602 is obtained, and the direction that directs from the current position to the intersecting point may be the traveling direction.", "The mark extraction means 4605 extracts the mark to be guided from the visible region.", "Specifically, it extracts the mark to be guided out of the marks within the visible region calculated by the visible region calculation means 4602.In this processing of extraction, the mark near the traveling direction calculated by the traveling direction calculation means 4604 is extracted.", "The mark that exists within the visible region calculated by the visible region calculation means 4602 can be extracted by correlating the marks and their coordinate positions as shown in FIG.", "49, and further the mark near the traveling direction calculated by the traveling direction calculation means 4604 can be extracted.", "If it does not exist in the mark near the traveling direction calculated by the traveling direction calculation means 4604, the mark in the direction opposite to the traveling direction may be calculated or the mark in the direction 90 degrees in the left or 90 degrees in the right may be extracted.", "The guidance sentence generation means 4606 gene rates the route guidance sentence of the route found.", "Specifically, it generates the guidance sentence that guides the route in the case of proceeding the route searched by the route search means 4603 while relying on the traveling direction calculated by the traveling direction calculation means 4604 or the mark extracted by the mark extraction means 4605.To generate this guidance sentence, a template of the guidance sentence that includes variables as ‘Please proceed while looking at A on B.’ is prepared, the name of the mar k is inserted in ‘A’, the direction where the mark can be seen is inserted in ‘B’, and thus the guidance sentence may be generated.", "For example, by the mark extraction means 4605, when a mark called Ld has been extracted by the mark extraction means and calculation has been made that the mark was on the left side of the traveling direction, Ld is substituted for ‘A’ and ‘Left ahead’ is substituted for ‘B’ to generate the sentence as ‘Please proceed while looking at Ld on left ahead’.", "Alternatively, complete sentences as shown in FIG.", "33 are prepared from the beginning, searching is performed depending on the mark and the direction where it can be seen, and the guidance sentence on the first line of the table may be obtained.", "Further, when it is found out that the destination is within the visible reed, on and it is ahead of the user, the second line of the lines of FIG.", "33 is obtained.", "In this embodiment, the visible region calculation means 4602 may calculate not only the range that can be see n from the current position of the user, which has been detected by the position determination means 4601, but also the range that can be seen from the point on the route, which has been searched by the route search means 4603.FIG.", "5 is the flow chart that explains the operation of the route guidance apparatus 4600 in such a case.", "Firstly the positional information, that is, the cur rent position of the user is detected by the position determination means (51), and the current position is set as Entrance.", "Specifically, the coordinate of the current position is substituted for a variable called Entrance (52).", "Next, the route from the cur rent position to the destination is searched by the route search means 4603 (53).", "The visible polygon from Entrance to the direction of the destination is calculated (54).", "Specifically, the visible polygon, which is the visible range seen from the point substituted for the variable called Entrance, is calculated using the visible region calculation means 4602.Next, determination is made whether or not there is the intersecting point between the route search result and the visible polygon (55).", "Specifically, whether or not there is the intersecting point between the route searched by the route search means 4603 and the visible region calculation means 4602 is checked, and the intersecting point between the route search result and the visible polygon is set as Exit if there is the intersecting point (56).", "In other words, the intersecting point is substituted for the variable called Exit.", "The direction from the point substituted for Entrance to the point substituted for Exit is found by the traveling direction calculation means 4604, the mark having the minimum direction difference is extracted by the mark extraction means 4605, and the direction and the mark found are registered (57).", "Next, the point substituted for Exit is substituted for Entrance to return to step 54.If there is no intersecting point at step 55, the destination is substituted for Exit when the destination is in the visible polygon that is the visible region (59).", "Then, the destination and the direction of the destination are registered (60), and all the marks and the directions registered are output by inserting them in the guidance sentence (61).", "With this embodiment, the route that can reach the destination faster can be guided even if the route search means has searched a circuitous route.", "Further, the position determination means may obtain the current position of the terminal at a regular time interval, and the guidance sentence generation means may gene rate the guidance sentence of the route regarding the next mark to the terminal when determination is made that it has approached the mark that exists in the route searched by the route search means.", "Specifically, one in which the guidance sentence of the route regarding the next mark is generated is transmitted to the terminal.", "As described, the direction or the like when the user has approached the next mark can be previously informed to the user by generating the guidance sentence regarding the next mark to the terminal for the user, and smooth route guidance is thus made possible.", "Embodiment 8 FIG.", "50 exemplifies the function block diagram of the route guidance apparatus in embodiment 8 of the present invention.", "In FIG.", "50, the route guidance apparatus is one in which an image data retrieval means 5001 is added to the route guidance apparatus 4600 of embodiment 7.The image data retrieval means 5001 searches viewpoint image data specified to guide points to pass on the route.", "Specifically, it retrieves the image data to guide the route in the case of proceeding the route searched by the route search means 4603 while relying on the traveling direction calculated by the traveling direction calculation means 4604 or the mark extracted by the mark extraction means 4605.The image data in this case is the viewpoint image data.", "The viewpoint image data is data of an image when a landscape is see n from a particular viewpoint, and is image data where the landscape is expressed three-dimensionally.", "For example, the image expressed by perspective or the image in which the landscape is viewed in bird's-eye is cited.", "The image data retrieval means 5001 performs retrieval of the image data from the user's position and the direction calculated by the traveling direction calculation means 4604 or in accordance with the user's position and the mark extracted by the mark extraction means 4605, for example.", "FIG.", "51 is the table where ‘points to pass’ and ‘image data’ are correlated, in which the ‘points to pass’ comprises three elements, in which the first element, the second element, and the third element respectively express the starting point, a point to pass through in halfway, and a point to be reached after passing through, or the first element is the position, the second element and the third element respectively express the direction and the mark, and it expresses that the image data stored in the row of ‘image data’ is retrieved for the three elements.", "FIG.", "52 exemplifies the table in which the data used for generating the guidance sentence by the guidance sentence generation means 4606 in this embodiment is stored.", "In FIG.", "52, although the same row as the row of ‘points to pass’ in the table of FIG.", "51 exists in FIG.", "52 in order to gene rate the guidance sentence while synchronizing with the image data retrieved by the image data retrieval means 5001, it is not limited to the point where the guidance sentence is generated while synchronizing with the image data retrieved by the image data retrieval means 5001, and it may generate the guidance sentence of the contents to complement the image data or the contents more detail than the image data.", "According to the route guidance apparatus by this embodiment, provision of the guidance sentence with the viewpoint image data expressed three-dimensionally to the user guided is made possible, and guidance service easier to understand can be provided to the user.", "Embodiment 9 FIG.", "53 exemplifies the function block diagram of the route guidance apparatus according to embodiment 9 of the present invention, and a route guidance apparatus 5300 includes: route search means 5301; frame map generation means 5302; a map database 5303; and guidance sentence generation means 5304.The route search means 5301 searches the route from the starting point to the destination as described in embodiment 7 and embodiment 8.Frame map generation means 5302 cuts out maps around the points to pass as the frame map from the map database 5303 in order to guide the points to pass, and it cuts out each frame such that a part of the route in each frame overlaps in cutting out them.", "The ‘point to pass’ is the point to be passed in moving an the route searched by the route search means 5301.The map database 5301 holds the maps around the point to be passed when moving on the route, and it is one in which the map around a coordinate is retrieved when the coordinate of the point is given, for example.", "‘A part of the route in each frame overlaps in cutting out the m.’ means that when a map is cut out as a frame and the frame of the map cut out next is compared with it, the routes displayed on the both frames have caiman areas.", "The guidance sentence generation means 5304 gene rates the guidance sentence to guide the point to pass.", "As in embodiment 8, the guidance sentence may be generated while synchronizing with the frame generated in the frame map generation means 5302, or the guidance sentence of the contents to complement the contents of the frame or the contents more detail than the frame may be generated.", "FIG.", "54 is the flowchart that explains the processing of the route guidance apparatus 5300, mainly the processing of the frame map generation means.", "Firstly, the route is searched by the route search means 5301 to obtain the route (S5401).", "Next, the starting point is substituted for a variable L (S5402).", "Substituting the starting point means to substitute the coordinate of the starting point.", "Then, determination is made whether or not L is the destination (S5403).", "Specifically, determination is made whether or not the coordinate of L is the coordinate of the destination, the processing ends when it is the destination, and the map around L is obtained when it is not (S5404).", "In other words, the map around the coordinate substituted for L is obtained as the frame from the map database 5303.When the frame is obtained, it is displayed for the user or it is correlated with the coordinate position substituted for L and accumulated, and the frame is displayed when detection is made that the user has reached the position of the coordinate.", "When the map of the vicinity is obtained, L is moved along the route (S5405).", "Specifically, the coordinate position substituted for L is updated to the coordinate after it has moved along the route.", "At this point, by setting the coordinate position substituted for L to the position on the route, which is displayed on the map of the frame, ‘A part of the route in each frame overlaps in cutting out them.’ is done.", "After L is moved, the processing returns to step S5403.As described, by overlapping a par t of the route in each frame in cutting out the frames, the current position of the user, which is displayed on the frame, does not change largely on the screen when the next frame is displayed for the user, and displaying easy to see for the user is made possible.", "Embodiment 10 In embodiment 9 of the present invention, when the frame nap generation means 5302 cuts out the frames in embodiment 8, they are cut out so that the route in each frame is displayed in the center of the screen instead of overlapping the route in each frame.", "For example, in the case where the route search means 5301 has searched the one that passes the points E, U, Q, R, S, T and F as shown in FIG.", "22, the points that are remote by a predetermined distance L (10 m for example) or more are selected among them, each of the points E, U, Q, R, S, T and F is cut into the frame views of 1 to 5 as shown in the one-dot chain line of FIG.", "22, and arranging the frame views in the order of E, U, Q, R, S, T and F is performed.", "As described, by cutting out such that route is displayed in the center of the screen, the points to be passed on the route are displayed on the center of the frame views, and performing the route guidance by the frame view easier to see is made possible.", "Embodiment 11 In embodiment 7 to embodiment 10, the guidance sentence generation means may generate the guidance sentence based on the user's moving direction to guide the point to pass.", "The user's moving direction is the direction where the user moves when the user travels on the route, where the route has bee n searched by the route search means.", "By generating the guidance sentence in this manner, when the route is bent at a certain point, the guidance sentence regarding which way to turn for the moving direction until the user reaches the point is generated.", "For example, assuming that the route from S to G shown in FIG.", "29(a) has been searched by the route search means, although the route is bent at the point of n1, the guidance sentence that says ‘Please turn right at n1.’ is generated because the user who moves from the point of m0 to n1 turns right when the guidance sentence to turn at n1 is generated.", "As described, by generating the guidance sentence based on the user's moving direction, guiding of the user by the guidance sentence easier to understand than the guidance sentence such as ‘At n1, please turn to the direction where m1 exists.’ for example, is made possible.", "Embodiment 12 In embodiment 7 to embodiment 11, in the case where the route guidance apparatus guides the route that passes the points in a platform of the train station premises, the guidance sentence generation means may generate the guidance sentence using the direction of the railway for the user in the station platform as a reference in order to guide the points to pass.", "Since the railway usually extends in a straight line and it is a noticeable structure, the guidance easy for the user to understand is made possible when the guidance sentence such as ‘Please turn right for the rail way.’ or ‘Please to straight directly along the railway.’ are generated using the direction of the railway as a reference.", "Embodiment 13 In embodiment 7 to embodiment 12, the guidance sentence generation means does not give guidance of turning right or left at the first direction turning point when the starting point is near the point where a direction turn is made for the first time, but may generate the guidance sentence of the mark that shows a direction turning direction from the direction turning point.", "For example, assume that the route search means has searched the route shown in FIG.", "29(a) as the route having S as the starting point and G as the destination and that the starting point S and the first direction turning point n1 are near.", "If the starting point S is the posit ion detected by using the radio wave of the tag or the like, it is possible that the user is not actually at S but at the opposite side of S when seen from n1 in FIG.", "29(a) because the position is detected with an error.", "If the user is actually at the opposite side of S when seen from n1, the user moves to the opposite direction to m1 at n1 when the guidance sentence generation means assumes that the user is at S to generate the guidance sentence as ‘Please turn right at n1.’.", "Therefore, when the starting point and the first direction turning point are near, the guidance of turning right or left is not performed but the guidance sentence of the mark that shows the direction turning direction from the direction turning point is generated.", "FIG.", "55 is the flowchart that explains the processing of the guidance sentence generation by the guidance sentence generation means in this embodiment.", "Firstly, whether or not the guidance to the destination has been per formed is determined at step S5501.Specifically, whether or not the guidance sentence to the destination has been generated is determined, the processing ends if the guidance sentence to the destination has been generated, the processing moves to the step S5502 if not and whether or not the point to be guided is the direction turning point is determined.", "If it is not the direction turning point, the processing moves to step S5503 to generate the guidance sentence, and returns to step S5501.If it is the direction turning point, the processing returns to step S5504, whether or not the direction turning point is near the starting point, that is, whether or not they are within an error range since this direction turning point is the first direction turning point, and the processing moves to step S5505 if they are within the error range and the guidance sentence of the mark that shows the direction turning direction is generated.", "For example.", "In the case of FIG.", "29(a), the guidance sentence as ‘Please turn to the direction where m1 can be seen.’ is generated at n1.If the first direction turning point and the starting point are not within the error range at step S5504, the processing moves to step S5506 and the guidance sentence that guides the direction of the direction turn is generated.", "Hereinafter, in step S5507 and step S5508, generation of the guidance sentence in a regular manner is repeated until the guidance sentence to the destination is generated.", "As described, when the first direction turning point is near the starting point, the movement of the user to a totally different direction can be prevented by generating the guidance sentence of the mark that shows the direction turning direction.", "Embodiment 14 In embodiment 7 to embodiment 13, the route guidance apparatus may include the tag detection means, and the guidance sentence generation means may generate the guidance sentence that guides the positional information near the tag detected by the tag detection means.", "As the positional information near the tag, information such as where the tag is or what is around the tag is cited as an example.", "FIG.", "56 is the flowchart that explains the processing of the route guidance apparatus in this embodiment.", "In step S5601, whether or not the tag has been detected by the tag detection means is determined.", "If the tag is not detected, the processing moves to step S5602 and whether or not the guidance has ended is determined.", "As the case where the guidance ends, a case where the user has pushed a button for guidance end or the like is cited.", "The processing ends if the guidance has ended, and returns to step S5601 if not.", "When the tag has been detected in step S5601, the processing moves to step S5603 to obtain the position of the tag.", "To obtain the position of the tag, it may be obtained from the contents where the ID's of tags and coordinate positions are correlated and stored, as shown in FIG.", "31.In step S5604, the guidance sentence that guides the positional information near the position obtained is gene rated, and the processing returns to step S5601.As described, when the tag is detected, the guidance sentence that guides the positional information near the position thereof is generated, and thus the user guided can know whether or not he/she is moving to a right direction.", "Further, in a museum or the like, the user can be informed of the guidance sentence regarding an art object when he/she moves in front of the art object.", "Furthermore, in this embodiment, the guidance sentences for each of mark information along the route are arranged in the route order and each guidance sentence may be read out synchronously with the detection of a tag by the tag detection means.", "Specifically, to give guidance such that the user moves on the route, the information regarding the marks is arranged in the order of the marks that app ear as the user travels on the route, and each of information may be read out every time the tag is detected.", "Embodiment 15 In embodiment 7 to embodiment 14, the guidance sentence generation means may have a guidance sentence request acceptance function, and the guidance sentence generation means may arrange the guidance sentences for each of the mark information along the route in the route order and may read out each guidance sentence when it accepts a guidance sentence request based on the guidance sentence request acceptance function.", "FIG.", "57 shows a part of the function block diagram of the route guidance apparatus in this embodiment, in which the guidance sentence generation means 5304 has a guidance sentence request acceptance function 5701, and the guidance sentence request acceptance function 5701 accepts the guidance sentence request that is a request for reading out the guidance sentence.", "The guidance sentence request acceptance function 5701 includes input means such as a button, voice recognition, and a touch panel.", "When the guidance sentence request acceptance function 5701 accepts the guidance sentence request, the guidance sentence generation means arranges the guidance sentences for each of the mark information, which is the information regarding the marks to guide the user along the route in the route order, and reads out each guidance sentence sequentially.", "In other words, the guidance sentence generation means arranges and accumulates the guidance sentences for each of the mark information in the route order, and performs reading out of each guidance sentence when the guidance sentence request is accepted.", "FIG.", "58 is the flowchart that explains the processing of the guidance sentence generation means 5304 in this embodiment.", "In step S5801, whether or not the guidance sentence request has been accepted by the guidance sentence request acceptance function 5701 is determined, the processing moves to step S5802 if it has been accepted, and the guidance sentence of the mark information is read out.", "For example, reading out of the guidance sentence regarding the mark seen from the current position is performed such that in which direction it can be seen and such as which way to proceed for the direct ion.", "When reading out is finished, the processing returns to step S5801.In the case where the guidance sentence request has not been accepted in step S5801, the processing moves to step S5803 to determine whether or not the guidance has ended, the processing ends if it has ended, and returns to step S5801 if not.", "Whether or not the guidance has ended is determined, for example, by detecting the operation of the button or the like by the user or detecting that the user has reached the destination with the position determination means.", "As described, the route guidance apparatus accepts the guidance sentence request with the guidance sentence request acceptance function 5701 and reads out the guidance sentence, and thus provision of the guidance sentence when the user wants the guidance sentence is made possible.", "Further, the guidance sentence generation means 5304 may have a user characteristic information acceptance function, and the guidance sentence generation means 5304 may provide the route information in accordance with user characteristic information that is information regarding a user's characteristic accepted based on the user characteristic information acceptance function.", "The user characteristic information acceptance function is a function to accept the user characteristic information that is the information regarding the user's characteristic, and it accepts a characteristic to be considered in performing the route guidance such as the user's age, whether or not the user is a foreigner, which language his/her mother tongue is if he/she is not a Japanese, whether or not the user is a vision-impaired person, a hearing-impaired person, a walking-impaired person or an able-bodied person, for example.", "The guidance sentence generation means provides the route information in accordance with the user characteristic information accepted by the user characteristic information acceptance function.", "For example, reading out of the guidance sentence is performed slowly or characters included in a map are made large in the case of displaying the map when the user is a child or an aged person, and Chinese characters are used as little as possible in the case of a child.", "When the user is a foreigner, reading out of the guidance sentence and displaying of the map in his/her mother tongue is performed.", "Particularly, reading out of the route information is not performed but the route information is provided by displaying characters when the user is a hearing-impaired person, and the route information is not displayed but reading out is performed in the case of a vision-impaired person.", "Further, if there is an obstacle such as stairs is on the route, the guidance sentence that informs of it in advance may be generated and read out.", "More over, the guidance sentence regarding the marks meaningful particularly to the vision-impaired person, such as where textured paving blocks are, which direction to proceed for a direct ion from which a specific sound is heard, what kind of a feeling and a sound are made when poking a road surface with a stick, or how they change, and where a large object, a noticeable light source, and a guidance display in braille are, may be generated and read out.", "Further, when the user characteristic information that the user feels it difficult to move on the same passage as the able-bodied person or the user characteristic information that he/she walks slower than the able-bodied person, the information such as in which direction a slope and an elevator are instead of stairs and how far they are may be pro vided, or an existence of a route where people's movement is slow may be provided.", "Embodiment 16 In embodiment 16 of the present invention, the route guidance apparatus in embodiment 7 or 8 further includes the tag detection means and route leading means.", "The tag detection means detects tags planed in the premises, the posit ion of the tag is obtained and the position where the apparatus is now is detected by detecting the tag by the tag detection means.", "The route leading means performs displaying of the frame view or the viewpoint image, which correspond to the tag position, and the route guidance sentence or reading out of the guidance sentence synchronously with detecting the tag by the tag detection means.", "As described, since an appropriate frame view or viewpoint image and the route guidance sentence are displayed and the guidance sentence is read out as the user moves on the route by performing detection of the position with the tag detection and per forming displaying of the frame view or the viewpoint image and the route guidance sentence or reading out of the guidance sentence, service of the route guidance easy to understand for the user can be provided.", "Embodiment 17 In this embodiment of the present invention, the position determination means 4601 in embodiment 7 or 8 detects the current position of the user, and user request obtaining means and route leading means are further included.", "FIG.", "60 shows the function block diagram of the route guidance apparatus in this embodiment, and user request obtaining means 6001 and route leading means 6002 are added to FIG.", "46.The user request obtaining means 6001 obtains the user's request.", "Specific ally, it obtains a request that the user wants to receive provision of the route information, and it includes input means such as the button, the voice recognition, and the touch panel, for example.", "The route leading means 6002 performs displaying of the frame view or the viewpoint image, which correspond to the tag, and the route guidance sentence on the terminal or reading out of the guidance sentence in the route order in accordance with the user's request that the user request obtaining means 6001 obtains.", "Specifically, it stores which tag has detected with the movement of the user to recognize the current position, displays the frame view or the viewpoint image, which correspond to the current position recognized, and the route guidance sentence an the terminal or reads out the guidance sentence, and performs leading such that the user moves an the route when the user's request is made.", "FIG.", "59 is the flowchart that explains the operation of the position determination means 4601, the user request obtaining means 6001, and the route leading means 6002 in this embodiment.", "Firstly, on step S5901, whether or not the user request has been obtained is deter mined on the user request obtaining means 6001.If it has been obtained, the processing moves to step S5902 to obtain the position detected by the tag.", "The position detected by the tag is detecting an identifier of the tag by receiving the radio wave from the tag and detecting the position by information where the identifier of the tag and the position are corresponded with each other.", "In step S5903, obtaining and displaying of the frame view or the viewpoint image, which correspond to the position, is performed (step S5903 is not executed if the image data search means does not exist.).", "In step S5904, reading out of the guidance sentence that corresponds to the position is performed, and the processing returns to step S5901.In the case where it is determined that the user request has not been obtained in step S5901, the processing moves to step S5905 to determine whether or not the tag has been detected by the position determination means 4601, and the processing moves to step S5906 to store the tag if the tag has bee n detected.", "Specifically, the identifier of the tag is stored to make it possible to detect the position in step S5902.Then, the processing returns to step S5901.In step S5905, whether or not the guidance has ended is determined if the tag is not detected, the whole process ends if it has ended, and the processing returns to step S5901 if not.", "Whether or not the guidance has ended is performed by detecting whether or not the tag detected in the position determination means 4601 is the one of the destination or by detecting that the user performs d a button operation of a guidance end.", "Embodiment 18 This embodiment relates to the visible region calculator.", "In this embodiment, the visible region calculator, when the viewpoint and one or a plurality of polygons are in one polygon, the visible region from the viewpoint regarding an outermost polygon is computed first.", "Next, a part seen from the viewpoint only for each polygon is computed as in a row of continuous line segments regarding the polygons other than the outermost polygon, the row of the line segments computed is arranged in the argument order, the row of the line segments, a part of the line segment seen from the viewpoint is computed in the order of arrangement taking in consideration a positional relation between the row of the line segments and a visible region generated at the point where the processing of a previous row of line segments ends, and the range seen from the viewpoint is calculated with the processing to find a new visible region by connecting the area computed and the visible region.", "FIG.", "61 is the flowchart that explains the operation of the visible region calculator in this embodiment.", "In step 2110, the visible region regarding the outer most polygon is computed.", "The visible region calculator in this embodiment is one to calculate the visible region when the viewpoint and one or a plurality of polygons are in one polygon, and the ‘one polygon’ that includes the ‘viewpoint and one or a plurality of polygons’ inside thereof is the outermost polygon.", "In this step, the visible region that is a visible range seen from the viewpoint regarding the outermost polygon is calculated.", "When the outermost polygon is a convex figure, the outermost polygon becomes the visible region as it is, but when the outermost polygon is a concave figure, the range narrower than the outermost polygon sometimes becomes the visible region.", "In step 2120, the visible polygonal line of only each facilities is calculated regarding the facilities.", "The ‘each facilities’ means the polygon inside the outermost polygon, and the ‘visible polygonal line’ is the continuous line segments of the part seen from the viewpoint of the polygon.", "In step 2130, regarding the row of line segments called the visible polygonal lines calculated in step 2120, the visible polygonal line is sorted in the order that the argument for either end point of the visible polygonal line is small.", "The ‘argument’ is an angle in which a predetermined direction is specified and the direction is set as a reference.", "In step 2140, 0 is substituted for a variable I in order to bring out sequentially the visible polygonal line sorted.", "Steps 2145, 2150 and 2160 are steps that correspond to the foregoing ‘a part of the line segment seen from the viewpoint is calculated taking in consideration a positional relation between the row of the line segments and a visible region generated at the point where the processing of a previous row of line segments ends, and the range seen from the viewpoint is calculated with the processing to find a new visible region by connecting the area calculated and the visible region’.", "Firstly, step 2145 determines whet her or not both ends are inside or outside the visible region regarding an I-th visible polygonal line.", "The processing of step 2160, 2170 or 2150 is executed respectively if the both ends are inside, the both ends are outside, or either one is inside.", "Step 2150 is the processing when either one of the end points of the visible polygonal line is inside the visible region, in which the intersecting point between the visible polygonal line and the visible region and the intersecting point between the half line from the viewpoint, which passes another end of the visible polygonal line, and the visible region are calculated, a new visible region is generated such that the coordinates that constitute the two intersecting points, the visible polygonal line within the visible region, and the visible region become counter-clockwise, and a pointer to the visible polygonal line is stored.", "Specifically, in FIG.", "69, a line segment 6901 is the I-th visible polygonal line, point B is inside the visible region, and the end point of the line segment 6901, which is not point B, is outside the visible region.", "A polygonal line 6902 is a side of the polygonal line that constitutes the visible region.", "In this case, intersecting point C between the line segment 6901 that is the visible polygonal line and the visible region is calculated.", "Further, intersecting point B′ between the half line from the viewpoint, which passes point B that is another end of the line segment 6901 as the visible polygonal line, and the visible region is compute d. The new visible region is gene rated such that the coordinates between the two intersecting points C and B′ and the visible polygonal line BC within the visible region become counter-clockwise.", "In other words, a new visible region having a polygonal line rat lad CBB′ as a boundary is generated.", "The pointer of CBB′ that is the boundary of the new visible region is stored.", "The ‘pointer’ is one that indicates a thing by its position, and the pointer of CBB′ is the one that indicates the position of the polygonal line called CBB′.", "For example, when data of sides called CB and BB′ is accumulated in a memory of a computer, a memory address where the data of sides is accumulated is stored.", "When this processing ends, it moves to step 2170.Step 2160 is the processing when both of the end points of the visible polygonal line are inside the visible region, in which two line segments that connect the intersecting point, where two half lines passing the both ends of the visible region from the viewpoint cross the visible region, and the both ends of the visible region, which correspond to each of the intersecting points, and the visible polygonal line are set as new three line segments, a new visible region is generated such that the coordinates that constitute the new line segments and the visible region become counter-clockwise, and the pointer to the visible polygonal line is stored.", "FIG.", "68 exemplifies the case where the both of the end points of the visible polygonal line are inside the visible region, where a side 6801 having end points as A and B is the visible polygonal line and the boundary of the visible region is a line segment 6802.In this case, a half line passing A is drawn from the viewpoint, and a point that crosses the line segment 6802 is computed, which is set as A′.", "Further, a half line passing B is draw n from the viewpoint and a point that crosses the line segment 6802 is computed, which is set as B′.", "Accordingly, AA′ and BB′ are obtained as the two line segments that connect to the both ends of the visible region, and A′ABB′ are connected to generate a new visible region such that the coordinates that constitute the line segments and the visible polygonal line 6801 become counter-clockwise.", "The pointer of A′ABB′ that is the boundary of the new visible region is store d. Sped finally, when the data of the sides called A′A, AB, and BB′ is accumulated in the memory, the memory address accumulated is stored.", "When this processing ends, it moves to step 2170.Step 2170 is the processing executed after the processing of step 2160 ends after the processing of step 2150 ends in the case where the bot h ends of the I-th visible polygonal line are outside the visible region, the value of I is increased only by 1, the processing moves to step 2180 since the processing of all the visible polygonal lines has ended if I becomes equal to the number of the polygonal line s, it moves to step 2145 if not, and the processing of the next visible polygon al line is performed.", "In step 2180, since the processing of all the visible polygonal lines has ended and the visible region has been computed, outputting of the visible region computed polygon data and the pointers of all the visible polygonal lines that has become a part of the visible region is performed.", "Specifically, to output the visible region as the polygon data, the pointer of all the visible polygonal lines that constitute the visible region are output.", "In such an embodiment, since the direction that becomes a reference to decide the argument is arbitrarily specified and the visible region can be calculated, the range see n from the viewpoint can be calculated faster than using a conventionally known method when the viewpoint and the polygons exist inside the outermost polygon.", "Embodiment 19 The visible region calculator in this embodiment is one that calculates the range seen from the viewpoint in the case where the viewpoint and one or a plurality of polygons are inside one polygon (hereinafter, referred to as the ‘outermost polygon’).", "FIG.", "62 shows the function block diagram of the visible region calculator in this embodiment, and a visible region calculator 6200 includes: an outermost visible region calculation section 6201; a visible polygonal line calculation section 6202; a visible polygonal line rearrangement section 6203; and a temporary visible polygon update section 6204.The outermost polygon visible region calculation section 6201 finds the visible polygon that is the range seen from the viewpoint of the outermost polygon, and calculates the first temporary visible polygon.", "The outermost polygon and the temporary visible polygon match when the outermost polygon is the convex figure, and the flowchart that explains the calculation processing of the temporary polygon in a general shape is exemplified in FIG.", "64.Firstly, in step S6401, the half lines are drawn from the view point to each vertex of the outermost polygon.", "Steps from step S6402 are the processing performed, where each one of the half lines drawn are brought out, and whether or not a half line is left is determined in step S6402.The processing ends if it is not left, and the polygon at this point is the temporary visible polygon.", "The processing moves to step S6403 if a half line is left to brought out.", "In step S6404, whether or not the half line crosses at an intersecting point other than the vertex of the outermost polygon is determined, the processing returns to step S6402 if not, the vertex of the outermost polygon is set as P and another intersecting point is set as Q if the half line crosses at the intersecting point other than the vertex of the outermost polygon, and the processing moves to step S6405.In step S6405, whether or not P is nearer the viewpoint than Q.", "If P is not nearer the viewpoint than Q, it is the state as shown in FIG.", "65 and at least the vicinity of Q can be seen from the viewpoint, and thus the processing returns to step S6402 without performing anything.", "If P is nearer the viewpoint than Q, it is in the situation as shown in FIG.", "66, the processing thus moves to step S6406, and the area from P to Q of the outermost polygon is removed to add a line segment PQ.", "In other words, in FIG.", "66, since the region surrounded by a line segment 6601 and a line segment 6602 cannot be seen from the viewpoint, the area from P to Q of the outermost polygon, that is, the line segment 6601 and the line segment 6602 are removed from a side 6600 of the outermost polygon to add the line segment PQ, and thus the temporary visible polygon is formed.", "Then, the processing returns to step S6402 to perform the processing of the next half line.", "The visible polygonal line calculation section 6202 computes the visible polygonal line that is a part seen from the viewpoint for each polygon inside the outermost polygon ignoring the existence of other polygons.", "Specifically, regarding each polygon in the outer most polygon, it is assumed that only the polygon exists and selecting of visible sides out of the ones of the polygon is performed.", "In this processing, the half line is drawn from the viewpoint to a point on each side of the polygon and if the half line crosses an other side of the polygon before it crosses the side, the side cannot be seen from the viewpoint, and thus it does not become the visible polygonal line, and on the contrary.", "If the half line does not cross another side before it crosses the side, the side can be seen from the viewpoint, and it becomes the visible polygonal line.", "Note that the visible polygonal line calculation section 6202 may compute individual line segment that constitutes the visible polygonal line that is a polygonal line, instead of the polygonal line where the line segments called the visible polygonal lines are connected at the end points.", "Hereinafter, the individual line segment that constitutes the visible polygonal line is simply called the ‘visible polygonal line’ to simplify description.", "The visible polygonal line rearrangement section 6203 arranges the visible polygonal line computed in the visible polygonal line calculation section 6202 in the argument order of a visible limit point, which has a smaller argument to a reference line that is the half line drawn from the viewpoint and the end points of the visible polygonal line.", "For example, in the case where there are the viewpoint and a visible polygonal line AB as exemplified in FIG.", "79, when the reference line that is the half line drawn from the viewpoint is a half line 7901, the argument of end point A is 6 that is an angle formed by the half line drawn from the viewpoint to A and the reference line, the argument of end point B is τ similarly, and thus the end point A is the visible limit point of the visible polygonal line AB because θ is smaller than x.", "The visible polygonal rearrangement section 6203 arranges the visible polygonal line in the argument order of the visible limit point.", "In the following description, it is assumed that the visible polygonal line is arranged in the order of the smaller argument of the visible limit point.", "The temporary visible polygon update section 6204 performs the foil owing first processing and second processing regarding the visible polygonal line in the order where the visible polygonal line has been arranged in the visible polygonal line rearrangement section.", "Firstly, the first processing is the processing in the case where A and B are inside the temporary visible polygon when the both ends of the visible polygonal line are set as A and B (A is set as the visible limit point), in which visible lines that are the half lines severally passing A and B are drawn from the viewpoint to the temporary polygon.", "A′ and B′ that are the intersecting points between the visible lines and the temporary polygon are corrupted, an area of the side from A′ to B′ of the temporary visible polygon is removed, and A′ to A, A to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "Specifically, the case where the first processing is performed is the case where the visible polygonal line 6801 having A and B as the end points is inside the temporary visible polygon 6802, which is a side where the viewpoint exists, as shown in FIG.", "68.In this case, the visible line that is the half line passing A is drawn from the viewpoint to find the intersecting point with the temporary visible polygon 6802, and it is set as A′.", "Similarly regarding B, the visible line that is the half line passing B is draw n from the viewpoint to find the intersecting point with the temporary polygon 6802, and it is set as B′.", "In finding a new temporary visible polygon, 6803 and 6804 that are an area of the side from A′ to B′ of the temporary visible polygon are removed, and adding of a side A′A, a side AB and a side BB′ is performed.", "The second is the processing in the case where A is outside the temporary visible polygon and B is inside where the both ends of the visible polygonal line are set as A and B (A is set as the visible limit point), and it is the processing in which an intersecting point C between the visible polygonal line and the temporary visible polygon is computed, the visible line that is the half line passing B is drawn, B′ that is the intersecting point with the temporary visible polygon is computed, an area of the side from C to B′ of the temporary visible polygon is removed, and C to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "Specifically, the case where the sec and processing is performed is the case where the end point B of the visible polygonal line 6901 is inside the temporary visible polygon and another end point is outside the temporary visible polygon as shown in FIG.", "69.In this case, the visible polygonal line 6901, the temporary visible polygon, and the intersecting point C are computed.", "Further, the visible line that passes from the viewpoint to B is drawn to find B′ that is the intersecting point with the temporary visible polygon.", "Then, 6901 and 6902 that are an area of the side from C to B′ of the temporary visible polygon are removed, and adding of a side CB and a side BB′ is performed.", "Note that a case can be considered where the both ends of the visible polygonal line exist outside the temporary visible polygon, but the temporary visible polygon is formed after the visible polygonal line is rearranged according to the argument of the visible limit point and an area see n from the viewpoint does not exist in the visible polygonal line in this case, and thus there is no need to perform processing to such a visible polygonal line.", "FIG.", "63 and FIG.", "67 are the flowcharts that explain the operation of the visible region calculator 6200, and they can be regarded as the flowcharts that explain steps executed by the computer in order to realize the visible region calculator 6200 by the computer.", "In the case of realizing the visible region calculator 6200 by the computer, the function block diagram of the visible region calculator exemplified in FIG.", "62 can be regarded as the diagram that shows a module configuration of a program executed by the computer.", "Step S6301 is an outermost visible region calculation step, and computation of the first temporary visible region is obtained in the outermost visible region calculation section 6201.Step S6302 is a visible polygonal line calculation step, and the visible polygonal line is calculated by the visible polygonal line calculation section 6202.Step S6303 is a visible polygonal line rearrangement step, and arranging of the visible polygonal line according to the argument of the visible limit point is performed by the visible polygonal line rearrangement section 6203.Step S6304 to step S6306 are steps in which the visible polygonal line is brought out by one to perform processing, whether or not there is no visible polygonal line is determined in step S6304, the processing ends when there is no visible polygonal line, and the temporary visible region at this point is the visible polygon.", "If the visible polygonal line is left, the processing moves to step S6305 to bring out the visible polygonal line.", "Step s6306 is a temporary visible polygon update step, and the temporary visible polygon is updated by the temporary visible polygon update section 6204.The processing of updating the temporary visible polygon is shown by the flowchart in FIG.", "67, and whether or not the both ends of the visible polygonal line are inside the temporary visible polygon is determined first in step S6701.If so, the processing moves to step S6702, and moves to step S6704 if not.", "Step S6702 is executed when the both ends of the visible polygonal line are inside the temporary visible polygon, which is the case of FIG.", "68.In this case, the half lines are severally drawn from the viewpoint to the end points A and B of the visible polygonal line, an intersecting point A′ and the half line passing A and the temporary visible polygon is computed, and B′ is computed similarly regarding B.", "In step S6703, an area from A′ to B′ of the temporary visible polygon, that is, 6803 and 6804 are removed, and A′ to A, A to B, and B to B′ are sequentially connected to form a new temporary visible polygon.", "Step S6704 is executed is when neither both ends of the visible polygonal line are inside the temporary visible polygon, whether or not the end point of the visible polygon al line, which is not the visible limit point, is inside the temporary visible polygon is determined, the processing moves to step S6705 if so, and the processing of the flowchart in FIG.", "67 ends if not.", "The case where the processing has moved to step 6705 is the case exemplified in FIG.", "69, and C that is the intersecting point between the temporary visible polygon and the visible polygonal line is computed.", "In step S6706, the half line passing B is draw n from the viewpoint to find B′ that is the intersecting point with the temporary visible polygon.", "In step S6707, an area of the side from C to B′ of the temporary visible polygon is remove d, and C to B and B to B′ are sequentially connected to form a new visible polygon.", "According to the visible region calculator or the visible region calculation program by this embodiment.", "It is possible that the reference line is specified in any direction to start calculation of the visible region, and the visible region can be computed more efficiently than to find by the conventionally known method.", "Embodiment 20 FIG.", "70 shows the function block diagram of the route information generator of embodiment 20 in the present invention.", "In this embodiment, a route information generator 7000 has: visible region calculation means 7001; route search means 7002; mark extraction means 7003; guidance map generation means 7004; and guidance sentence generation means 7005.The visible region calculation means 7001 calculates the range seen from the user.", "As the visible region calculation means 7001, calculation means using a conventionally known algorithm or the visible region calculator in embodiment 18 or embodiment 19 may be used.", "The route search means 7002 searches the route from the point where the user is now or the starting point that the user enters to the destination that the user enters.", "To obtain the point where the user is now, a GPS (global positioning system) is used or the identifier of the tag is obtained by receiving the radio wave emitted from the tag embedded in the wall surface or the floor, and the position is obtained from the identifier.", "Further, the point where the user is now may be found by detecting an acceleration and integrating it.", "Further more, the map may be displayed on the touch panel to allow the user to instruct the starting point and the destination, the starting point and the destination may be accepted by using an input device such as a keyboard, or the starting point and the destination may be accepted by the voice recognition.", "A table in which the starting points and the destinations are correlated is pre pared as FIG.", "47 to search the route, and to obtain the route from the starting point give n to any destination, a line where the starting point given appears in the row of the starting points is retrieved, a value of the row of the final destinations on the line is obtained, a line that has the value obtained as the starting point is retrieved, and a value of the row of the final destinations on the line is obtained, which are repeated and continued until the destination is obtained as the value of the row of the final destinations.", "By performing such processing, a row of two pairs that consist of a point (origin) and another point (ending point) is obtained.", "The mark extraction means 7003 extracts the mark to be guided out of the visible region from the current position or the point where the guidance is given.", "Specifically, the visible region, which is the range seen from the current position or the point where the guidance is given, is calculated by the visible region calculation means 7001 such that the user proceeds on the route searched by the route search means 7002, the landmark to be guided to, that is, the landmark that shows the direction where the user should proceed next is extracted out of the visible region.", "For this extraction, using the table where the points on the route and their coordinate positions are correlated as shown in FIG.", "48 and the table where the marks and their coordinate positions are correlated shown in FIG.", "49, when the user has reached a point on the route, the direction from the point to a point where he/she should proceed is calculated, and finding of the landmark seen in the direction near the direction is performed.", "The guidance map generation means 7004 generates the map for route guidance, on which the route, the mark extracted, and the point to be guided are displayed.", "Specifically, it generates the map where the route searched by the route search means 7002, the landmark extracted by the mark extracting means 7003, and the point to be guided, that is, the point where the user should go next are displayed.", "For this purpose, the table where the points and their coordinates are correlated shown in FIG.", "48 and the table where the land marks and their coordinates are correlated shown in FIG.", "49 are used, and out of the coordinate of the landmark, the coordinate of the point to be guided, and the coordinate of the point to go to next, the map including these coordinates may be searched from the map database.", "The guidance sentence generation means 7005 gene rates the guidance sentence of the route.", "For example, the table as shown in FIG.", "38 where two or three points, which continuously constitute the route searched by the route search means 7002, and the guidance sentence data are correlated is prepared, and the guidance sentence is generated referring to the table, which is cited as one example.", "FIG.", "71 is the flowchart that explains the processing of the route information generator 7000 in this embodiment.", "In step S7101, the route from the point where the user is now or the starting point where the user enters to the destination where the user enters is searched by using the route search means 7002.A loop is formed after step 7102, and from step S7103 to step S7107 are repeated until it is determined that the route is not left in step S7102.In step S7103, the route is brought out.", "Specifically, a pairs of the origin and the ending point is brought out.", "In step S7104, the visible region from the origin is calculated by the visible region calculation means 7001.In step S7105, the mark is extracted by the mark extraction means 7003 from the visible region calculated at step S7104.In step S7106, the map is generated by the guidance map generation means 7004.In step S7107, the guidance sentence is generated by the guidance sentence generation means.", "The map and the guidance sentence generated in this manner are accumulated once by accumulation means inside or outside the route guidance information generator, and are provided to the user when necessary, or moving of the user on the route is detected and, for example, in the case where passing of the point to be guided is detected, the map and the guidance sentence regarding the point to be guided next may be generated and provided to the user.", "Embodiment 21 FIG.", "72 exemplifies the function block diagram of the route guidance system according to embodiment 21 of the present invention.", "In this embodiment, the route guidance system has: a route guidance apparatus 7201; a route information database 7205; a terminal 7204; a position detection apparatus 7206; a map database 7207; and a guidance sentence database 7208.The route guidance apparatus 7201 has the route information generator 7000 and the position determination means 7202.The route information generator 7000 is the one described in embodiment 20.The position determination means 7202 detects the current position of the terminal 7204, that is, a current terminal position.", "As a detection method, a method in which the user operates the terminal and the current position entered is received, or the tag is used as the position detection apparatus 7206, in which the identifier of the tag is obtained by receiving the radio wave emitted from it, and the terminal 7204 may transmit it to the position determination means 7202.Conversely, the terminal emits the radio wave and the position detection apparatus 7206 receives it, and then the position determination means 7202 may obtain which position detection apparatus has received to detect the position of the terminal 7204.The route information database 7205 accumulates the information regarding the route obtained.", "The ‘information regarding the route obtained’ is the information regarding the route obtained by the route information generator 7000, and the map for route guidance, which has been generated by the guidance map generation means 7004 of the route information generator 7000, and the guidance sentence of the route, which has been generated by the guidance sentence generation means 7005, are accumulated.", "At this point, the point searched by the route search means 7002, the map, and the guidance sentence are correlated and accumulated.", "The position detection means 7206 is means for obtaining the current positional information.", "The ‘current positional information’ is the position where the terminal 7204 is now, and the tag may be used as the position detection means 7206 or the position detection means 7206 may be a satellite of the GPS.", "The map database 7207 accumulates the map data.", "Specifically, it is the database to obtain, from the current position of the terminal, which has been detected by the position determination means 7202, what is around it, in which direction it is, and in what kind of situation it is (situation of the floor or the like, situation of congestion of people, level of danger, or the like).", "The guidance sentence database 7208 accumulates the data for generating the guidance sentence.", "The data for generating the guidance sentence is the template for generating the guidance sentence, which is a sentence including variables X, Y and Z such as ‘Y of X is Z.’, for example, and it is data from which a specific guidance sentence is generated if specific values of X, Y and Z are determined.", "FIG.", "73 is the flowchart that explains the operation of the route guidance system in this embodiment.", "In step S7301, the route information generator 7000 obtains the starting point and the destination.", "For example, it obtains the starting point and the destination entered from the terminal 7204.Alternatively, it may obtain them from an input apparatus connected to the route guidance apparatus 7201.In step S7302, the route information generator 7000 searches the route, and it generates the route information to accumulate in the route information database at step S7303.From step S7304 to step S7307 form the loop executed until the guidance ends, and whether or not the guidance has ended is determined at step S7304.For example, it is determined whether or not the position determination means 7202 has detected that the terminal 7204 is at the destination, or whether or not the user has informed that he/she did not desire further guidance by pressing a button on the terminal 7204.The processing ends if the end of guidance has been detected, and it moves to step S7305 if not.", "In step S7305, the position of the terminal 7204 is detected and obtained by the position determination means 7202.In step S7306, the route information of the position, which has been obtained in step S7305, is obtained by retrieving the route information database.", "At this point, the map database 7207 and the guidance sentence database are retrieved simultaneously, what is around the position obtained from the map database 7207 or the like is obtained, the template for generating the guidance sentence is obtained from the guidance sentence database 7208, and the retrieval result of the map database 7207 may be embedded in the variable areas of the template.", "For example, ‘ahead’ as X, ‘stairs’ as Y, and ‘dangerous’ as Z are obtained from the map database 7207, the template saying ‘Y of X is Z.’ is obtained, and the guidance sentence saying ‘The stairs ahead are dangerous.’ may be generated.", "In step S7307, the route information obtained in step S7306 is transmitted to the terminal 7204.And at this point, the guidance sentence, which is obtained by embedding the retrieval result of the map database 7207 in the variable areas of the template, may be transmitted.", "Embodiment 22 FIG.", "74 is the flowchart that explains the processing of a route leading unit in the present invention.", "The route leading unit is an apparatus to allow the terminal to display the mark on the map to lead the user, and the route leading unit of the present invention obtains the current position of the terminal at a regular time interval, and switches the map displayed on the terminal to a map on which the next mark in the route data is displayed when it detects approach to a mark that exists in the route data.", "Firstly, the route leading unit waits until a predetermined time passes in step S7401 and after a predetermined time has passed, the processing moves to step S7402 to obtain the position of the terminal.", "For example, the terminal receives the radio wave of the tag and the position of the terminal is obtained based on the identifier of the tag that has received the radio wave, or the terminal receives the radio wave from GPS satellites and the position is obtained form its result.", "In step S7403, whether or not it has approached the mark is deter mined, the processing moves to step S7404 if it is determined that it has approached it, and returns to step S7401 if not.", "In step S7404, the map on which the next mark is displayed is transmitted to the terminal in order to switch to the map on which the next mark is displayed.", "The foregoing description is based on the premise that the terminal and the route leading unit are in remote positions, but the terminal may include the route leading unit.", "As described, when it is detected that it has approached the landmark, the user having the terminal can be notified beforehand of the direction to go at the next point by switching to the map on which the next mark is displayed, and route leading easy for the user to understand ca n be performed.", "Further, since an error is involved in obtaining the current position, display of the map on which the next landmark is displayed after the user has passed the landmark can be prevented, and the user can be prevented from being confused.", "Embodiment 23 FIG.", "75 is the function block diagram that explains an embodiment of the route guidance system of the present invention.", "In this embodiment, the route guidance system includes: a route guidance apparatus 7500; the route information database 7205; the terminal 7204; the position detection apparatus 7206; the map database 7207; and the guidance sentence database 7208.The route guidance apparatus 7500 includes a route leading unit 7501 and the position determination means 7202.The route leading unit 7501 is an apparatus to allow the terminal to display the mark on the map to lead the user, and the route leading unit in embodiment 22 is used in this embodiment.", "The position determination means 7202 detects the current position of the terminal 7204.The result detected is sent to the route leading unit 7501 to enable the route leading unit 7501 to obtain the current position periodically.", "The route information database 7205 accumulates the information regarding the route obtained.", "Although not shown in FIG.", "75, the route guidance apparatus has a route information generator that searches the route from the starting point and the destination and generates the information regarding the route, and the route information database 7205 accumulates the information regarding the route generated and obtained by this route information generator.", "The ‘information regarding the route’ includes the points to pass, the image data and the guidance sentence to guide the points to pass, and the like.", "FIG.", "76 and FIG.", "77 show one example of the contents of the route information database 7205, and the table of FIG.", "76 correlates the routes formed by the points to pass with the origins and the ending points and stores them.", "For example, the first line of this table means that the points to be passed are u, w and y when the starting point is A and the destination is D. FIG.", "77 is the table in which the map data and the guidance sentences to be displayed on the terminal are correlated with the point to be passed and stored, which means that map 1024 is displayed on the terminal as the map and s7055 is used as the guidance sentence as the guidance sentence when the point to be guided is g, for example.", "The route leading unit 7501 obtains the position of the terminal from the position determination means 7202 at a predetermined time interval, reads out the table of FIG.", "76 to obtain the point to be guided next when the terminal approaches the mark that exists in the route, that is, the point to be guided, and obtains the map and the guidance sentence from FIG.", "77 to transmit them to the terminal.", "The terminal 7204 displays the route guidance information.", "The route guidance information is information such as the map and the guidance sentence transmitted from the route leading unit 7501, which guides the user so as to move on the route.", "The map database 7207 accumulates the map data.", "Specifically, it is the database to obtain what is around it, in which direction it is, and in what kind of situation it is (situation of the floor or the like, situation of congestion of people, level of danger, or the like), from the current position of the terminal, which has been detected by the position determination means 7202.The guidance sentence database 7208 accumulates the data for generating the guidance sentence.", "The data for generating the guidance sentence is the template for generating the guidance sentence, which is a sentence including variables X, Y and Z such as ‘Y of X is Z.’, for example, and it is data from which a specific guidance sentence is generated if specific values of X, Y and Z are determined.", "The route leading unit 7501, after having obtained the current position of the terminal, determines whether or not the terminal has approached the mark and retrieves the map database 7207 to obtain the situation around the terminal, and generates the guidance sentence by using the data for generating the guidance sentence obtained from the guidance sentence database 7208 and transmits it to the terminal 7204.With this kind of route guidance system, when it is detected that the user has approached the mark, the user having the terminal can be notified beforehand of the direction to go at the next point by switching to the map on which the next mark is displayed, and route leading easy for the user to understand can be performed.", "Further, sine e an error is Involved in obtaining the current position, displaying of the map on which the next mark is displayed after the user has passed the mark can be prevented, and the user can be prevented from being confused.", "Furthermore, by generating an appropriate guidance sentence while the map database 7207 is retrieved to obtain a surrounding situation, the guidance service easy for the user to understand can be provided.", "Embodiment 24 FIG.", "78 shows the function block diagram of the route guidance system of embodiment 24 of the present invention.", "The difference from the route guidance system of embodiment 23 is that the route in formation generator 7000 is clarified in a route guidance apparatus 7800 and the route information generator 7000 uses the route information generator of embodiment 20.Since the visible region of the user who moves on the route is calculated to generate the route information and the guidance is given based on it by using the route information generator of embodiment 20, the guidance service easy for the user to understand can be provided.", "Embodiment 25 FIG.", "80 is the block diagram that shows a configuration example of a route guidance system in embodiment 25 according to the present invention, and it comprises: a position detection apparatus 820 that obtains the user's current positional information; a portable terminal 830 that transfers the information from the position detection apparatus to the route guidance apparatus and displays the route guidance information from the route guidance apparatus; a map database 840 where the database of map is accumulated; a guidance sentence database 860 where the data to constitute the guidance sentence of route based on the route search result; a route information database 850 where the route guidance information generated by the route information generation means is stored; and a route guidance apparatus 810 that generates the route information and leads the route based on the route in formation generated.", "The route guidance apparatus 810 comprises: position determination means 811 that converts the information of the position detection apparatus into coordinates; route information generation means 814 that generates the route information; and route leading means 818 that leads the route based on the route information generated.", "FIG.", "81 is the block diagram of the route information generation means, and it comprises: visible region calculation means 971 that calculates the range seen from the current position; route search means 970 that searches the route to the destination; signpost extraction means 981 that extracts the mark to be guided from the visible region; guidance map generation means that generates the map for route guidance based on the route search result; and guidance sentence synthesis means 983 that generates the route guidance sentence.", "FIG.", "82 is the block diagram of the route leading means, the route leading means 818 receives the user's current position from the position determination means 811 and determines the guidance sentence and the map to be displayed, obtains the route information to be displayed from the route information data 850 if the route guidance data currently displayed needs to be converted, and send it to the portable terminal 830.The processing in the route information generation means 814 will be described according to the processing shown in FIG.", "83.A case is considered where the outermost polygon, the facilities A, B and C are arranged, and the route network winds in front of the facilities A as in FIG.", "89.When the positional information detected by the position detection apparatus 820 is passed from the portable terminal 830 of FIG.", "80 to the position determination means 811 of the route guidance apparatus 810, the route information generation means 814 is activated to send the positional information if it is sent from the portable terminal to which the route leading is not performed.", "When it is sent from the portable terminal to which the route leading is performed, the positional information is sent to the route leading means 818.The route information generation means 814 is activated and the positional information of the starting point and the destination is passed from the position determination means 811, and reads the data related to the map from the map database 840 and the data to generate an amount for guidance from the guidance sentence database 860 (1800).", "The positional information of a starting point a and a destination d is passed to the route search means 970, and the route search is performed to receive a node row of a, b, c and d on the route (1810).", "The starting point a is set as the guidance point (1815).", "The visible region from the guidance point a is computed by the visible region calculation means 971 as in FIG.", "90, the mark extraction means 981 finds the facilities A, B and C that are within the visible region by tracing the pointers of the polygonal line, and the node b which has not been reached on the route within the visible region is obtained (1820).", "Whether or not the destination d is within the visible region is determined (1825).", "Since it is not within the visible region, appropriate facilities that is the facilities C, which is the facilities adjacent to the visible region and farthest from a on the route, is selected to store as the mark in this case, and the node b on the route nearest from the mark C is set as the next guidance point (1830), for example.", "The visible region calculation means 971 finds the visible region from the guidance point b as in FIG.", "91, the mark extraction means 981 finds the facilities A, B and C that are within the visible region by tracing the pointers of the polygonal line, and the node c which has not been reached on the route within the visible region is obtained (1820).", "Whether or not the destination d is within the visible region (1825).", "Since it is not within the visible region, appropriate facilities that is the facilities B, which is the facilities adjacent to the visible region and farthest from b on the route, is selected to store as the mark in this case, and the node c on the route nearest from the mark B is set as the next guidance point (1830), for example.", "The visible region calculation means 971 finds the visible region from the guidance point c as in FIG.", "92, the mark extract ion means 981 finds the facilities A, B and C that are within the visible region by tracing the pointers of the polygonal line, and the node d which has not been reached on the route within the visible region is obtained (1820).", "Whether or not the destination d is within the visible region (1825) is determined.", "Since d is within the visible region, facilities' name X of the marks C and B extracted, ‘kiosk’ and ‘coffee shop’ for example, and typical points of the fascinating are found from the map database 840 after storing D as the mark, a traveling direction Z at the nodes, that is, corners b and c are found as left turn and right turn respectively from the coordinate data of the nodes a, b, c and d. Further, a positional relation Y with the corresponding marks C and B is found as ahead and right from the coordinate data of the typical points and the coordinate data of the nodes (1835).", "The guidance map generation means 184 generates the maps for guidance, which correspond to the facilities, as in FIG.", "93 and FIG.", "94, and they are arranged in the route order, that is, in this order (1840).", "The guidance sentence synthesis means 183 selects the guidance sentence template in the guidance sentence database in accordance with types of the variables, which are the facilities name, the traveling direction, and the node position in this case, inserts the data of the marks and the direction found in (1835) into ‘Z at Y of X’ to generate the guidance sentences such as ‘Left turn at the front of the kiosk’ and ‘Right turn at the right of the coffee shop’ (1835), and arranges them in the route order, that is, in this order (1845).", "The route guidance sentence generated and the map for route guidance are output to the route information database 850.Embodiment 26 The processing in the route leading means 818 will be described according to the processing shown in FIG.", "84.A case is considered where leading is performed on the route generated in embodiment 25.At the point when the leading begins.", "FIG.", "93 and a corresponding guidance sentence are displayed, a mark currently displayed is applied to the mark C. When the positional information detected by the position detection apparatus 820 is passed from the portable terminal 830 of FIG.", "80 to the position determination means 811 of the route guidance apparatus 810, the positional information is sent to the route leading means 818 if it is sent from the portable terminal to which the route guidance is performed.", "When the route leading means 818 is activated (1900), the positional coordinate shown by an asterisk of FIG.", "95 is obtained as the position of the portable terminal 830 from the posit ion determination means 811 (1910).", "Referring to the map database 840, since there is no mark in which the positional coordinate obtained has the distance from the coordinates of the nodes showing the marks in the route information data, which becomes a thresh old value (10 meters for example) or less (1920), the map FIG.", "93 currently displayed is superposed by the current position with the asterisk, and it is displayed on the portable terminal 830 as in FIG.", "95 (1922).", "The current positional information is obtained as the position of the portable terminal 830, the positional coordinate shown by the asterisk of FIG.", "96 from the position determination means 811 (1910).", "Referring to the map database 840, since there is no mark in which the positional coordinate obtained has the distance from the coordinates of the nodes showing the marks in the route information data, which becomes the threshold value or less (1920), the map FIG.", "93 currently displayed is superposed by the current position with the asterisk, and it is displayed as in FIG.", "96 (1922).", "The current positional information is obtained as the position of the portable terminal 830, the positional coordinate shown by the asterisk of FIG.", "97 from the position determination means 811 (1910).", "Refer ring to the map database 840, there is a mark c in which the positional coordinate obtained has the di stance from the coordinates of the nodes showing the marks in the route information data, which becomes the threshold value or less, it is different from the mark C currently displayed (1920), and the mark B is not the destination (1925), therefore, a mark currently displayed is applied to the mark B and the map for guidance FIG.", "94, in which the mark B in the route information database 850 is displayed, is brought out and the current position is super posed, and it is sent to the portable terminal 830 together with the guidance sentence that corresponds to the map (1930).", "Hereinafter, as it is similarly performed and the portable terminal approaches the destination, the destination D becomes the mark (1920), therefore, the mark is determined as the destination (1925), a message that tells of reaching the destination is sent (1940), and the processing ends (1950).", "As described, the present invention has effects that performing an appropriate route guidance even in the open space is made possible by guiding the signpost or the destination to the user in the open space and that it is made possible even in the portable terminal of low resolution by the frame view, the illustration image or the binary image, where the direction is made constant in the route guidance in the premises, which has been hard to understand in a conventional frame view, regardless of how the route diagram is drawn in the open space, or without re-editing the route to the point to be guided even when a large number of destinations are in the open space.", "Further, in performing the route leading by setting the tag detection as a trigger, provision of the appropriate route information is made possible eve n in the case of causing a position detection error involved with the range where the radio wave reaches from the tag and when tag reading means fail to read the tag, or the like.", "The present invention is one to provide the route information easy for a person with normal vision to understand regardless of the state of the route network by forming the visible region in a high-speed and providing the mark seen by eyes when the user moves on the route based on the visible region formed.", "Note that the present invention is applicable not only in the premises but also outside if the region is compartmentalized to constitute the outermost polygon." ] ]

[ [ "Mono-or Poly-Quarternary Polysiloxanes", "Monoquaternary or polyquaternary polysiloxanes are useful as surface finishing components, for example, in cosmetic formulas for skin and hair care, in polishes for the treatment of hard surfaces, in formulas for the drying of automobiles and other hard surfaces after machine washing, for the treatment of textiles and textile fibers, as separate softeners for textiles following the washing whereof with nonionic or anionic/non-ionic detergent formulas, or as softeners in formulas for textile washing based on non-ionic or anionic/non-ionic surfactants, whereby amino groups are used in the form of amines or amine salts as a function of pH value." ], [ "1.Monoquaternary or polyquaternary polysiloxane of the general formula (I), S—K-Q1-K—S (I) wherein S is or R1 is C1-C22 alkyl, C1-C22 fluoroalkyl or aryl, n is 0 to 1000, Q1 is a secondary amino structure or a tertiary amino structure or a quaternary amino structure R2 is a univalent or divalent, straight chain, cyclical or branched C1-C30 hydrocarbon radical, which is interrupted by —O—, —NH—, —C(O)—, —C(S)— and can be substituted with —OH or represents a single bond to the radical K, R3 is a univalent, straight chain, cyclical or branched C1-C30 hydrocarbon radical, which is interrupted by —O—, —NH—, —C(O)—, —C(S)— and can be substituted with —OH or a structure -A-E-, with A is —CH2C(O)O—, —CH2CH2C(O)O— or —CH2CH2CH2C(O)O— and E is a polyalkylene oxide unit of the structure —[CH2CH2O]q—[CH2CH(CH3)O]r—R4 q is 1 to 200, r is 1 to 200, R4 is H, straight chain, cyclical or branched C1-C20 hydrocarbon radical, which is interrupted by —O— or —C(O)— and can be substituted with —OH and can be acetylene, olefin or aromatic, whereby, when a plurality of R3 radicals are present in the molecule, these may be identical or different, and where K is a divalent or trivalent, straight chain, cyclical or branched C2-C40 hydrocarbon radical, which is interrupted by —O—, —NH—, NR1—, —C(O)—C(S) and can be substituted with —OH or contains a Q2 unit, with Q2 is a secondary amino structure or a tertiary amino structure or a quaternary amino structure R5 is a univalent or divalent, straight chain, cyclical or branched C1-C20 hydrocarbon radical, which is interrupted by —O—, —NH—, —C(O)—, —C(S)— and can be substituted with —OH, whereby the free valence of the divalent radical R5 can bond to Q1, and when a large number of K radicals are present in the polysiloxane, these may be identical to or different from each other.", "2.Monoquaternary or polyquaternary polysiloxane according to claim 1, wherein n is between 0 and 100.3.Monoquaternary or polyquaternary polysiloxane according to claim 1, wherein q is between 1 and 50.4.Monoquaternary or polyquaternary polysiloxane according to claim 1, wherein r is between 0 and 100.5.Monoquaternary or polyquaternary polysiloxane according to claim 1, wherein R2 and R5 are —CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)5CH3, —CH2CH2OH, wherein R6 is a straight chain, cyclical or branched C1-C18 hydrocarbon radical, which is interrupted by —O—, —NH—, —C(O)—, —C(S)— and can be substituted with —OH.", "6.Monoquaternary or polyquaternary polysiloxane according to claim 1, wherein R3 is —CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)5CH3, —CH2CH2OH, wherein R6 is a straight chain, cyclical or branched C1-C18 hydrocarbon radical, which is interrupted by —O—, —NH—, —C(O)—, —C(S)— and can be substituted with —OH.", "7.Monoquaternary or polyquaternary polysiloxane according to any claim 1, wherein K is a divalent or trivalent, straight chain, cyclical or branched C3-C30 hydrocarbon radical, which is interrupted by —O—, —NH—, NR1—, —C(O)—C(S) and can be substituted with —OH or contains a Q2 unit.", "8.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 1, wherein, for the manufacture of compounds containing quaternary ammonium groups of the general structure S—K-Q1-K—S wherein Q1 is monofunctional, tertiary-amino-function-containing siloxane derivatives are alkylated with reactive, monofunctional siloxane derivatives, which are synthesized by hydrosilylation of, for example, halogenated alkenes, unsaturated halogen carbon acid esters, and epoxy-functional alkenes, with monofunctional SiH compounds of the general structures wherein the molar ratio of the tertiary amino function to the reactive alkylating group is advantageously 100:1 to 1:1.9.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 8, wherein allyl chloride and allyl bromide are used as halogenated alkenes.", "10.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 8, wherein unsaturated halogen carbon acid esters of the group consisting of chloroacetic acid allyl ester, chloroacetic acid propargyl ester, 3-chlorpropionic acid allyl ester, and 3-chlorpropionic acid propargyl ester are used as unsaturated halogen carbon acid esters.", "11.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 8, wherein vinyl cyclohexene oxide and allyl glycidyl ether are used as epoxy-functional alkenes.", "12.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 8, wherein, for the manufacture of tertiary-amino-function-bearing compounds of the general structure S—K-Q1-K—S wherein Q1 is secondary-amino-function-bearing unsaturated structures are directly bonded to the monofunctional Si—H siloxane through hydrosilylation and subsequently, along with monofunctional, reactive, alkylating siloxane intermediates, are converted into tertiary-amino-structure-bearing compounds, wherein the stoichiometry of the secondary amine to the reactive, alkylating siloxane is advantageously 1:1.13.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 12, wherein n-methyl allylamine or CH2═CHCH2OCH2CH(OH)CH2NHCH3 are used as secondary-amino-function-bearing unsaturated structures.", "14.Process for the manufacture of monoquaternary or multiquaternary polysiloxanes according to claim 1, wherein, for the manufacture of tertiary-amino-function-bearing compounds of the general structure S—K-Q1-K—S wherein Q1 is and K and S have the meanings according to claim 1, di-secondary amines along with monofunctional, reactive, alkylating siloxane intermediates, are converted into tertiary-aminostructure-bearing compounds, wherein the stoichiometry of the di-secondary amine to the reactive, alkylating siloxane is advantageously 1:2.15.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 1, wherein, for the manufacture of equimolar quantities of tertiary-amino-function- and quaternary-ammonium-group-containing compounds of the general structure S—K-Q′-K—S secondary-tertiary diamines along with monofunctional, reactive, alkylating siloxane intermediates, are converted into di-tertiary-aminosiloxane-structure-bearing compounds, wherein the stoichiometry of the secondary-tertiary diamine to the reactive, alkylating siloxane is advantageously 1:1, and subsequently, the di-tertiary aminosiloxane structures, along with one mole of a monofunctional, reactive, alkylating siloxane compound, are converted to the tertiary-ammonium-group- and quaternary-ammonium-group containing siloxane derivatives.", "16.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 1, wherein, for the manufacture of tertiary-amino-function- and quaternary-ammonium-group-containing compounds of the general structure S—K-Q1-K—S di-secondary amines along with monofunctional, reactive, alkylating siloxane intermediates, are converted into tertiaryamino-structure-bearing compounds, whereby the stoichiometry of the di-secondary amine to the reactive, alkylating siloxane is advantageously 1:2, and subsequently alkylation with epoxides in the presence of acids, alkyl halogenides or halogen carbon acid esters, takes place, wherein the molar ratio of the tertiary amino groups to the alkylating agents is advantageously 100:1 to 11.17.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 1, wherein, for the manufacture of quaternary-ammonium-group- and tertiary-amino-function-containing compounds of the general structure S—K-Q1-K—S secondary amines or secondary-tertiary diamines along with monofunctional, reactive, alkylating siloxane intermediates are converted into tertiary- or di-tertiary-aminosiloxane-structure bearing compounds, whereby the stoichiometry of the secondary amine or the secondary-tertiary diamine to the reactive, alkylating siloxane is advantageously 1:1, and subsequently, the thus formed tertiary- or di-tertiary-aminosiloxane structures, along with a difunctional alkylating agent are converted into quaternary-ammonium-group-containing, or quaternary-ammonium-group- and simultaneously tertiary-amino-structure-containing siloxane derivatives.", "18.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes wherein the halogen carbon acid esters are used on low-molecular, oligomeric and polymeric alkylene oxides of the general structure HO[CH2CH2O]q[CH2CH(CH3)O]r—R4 wherein q, r and R4 have the meanings according to claim 1.19.Process for the manufacture of monoquaternary or polyquaternary polysiloxanes according to claim 1, wherein halogen carbon acid esters from the group consisting of oligoethylene glycols with molar weights of 400, 600 and 800 g/mol are used as halogen carbon acid esters on low-molecular, oligomeric and polymeric alkylene oxides.", "20.A cosmetic composition comprising monoquaternary or polyquaternary polysiloxanes, in which two siloxane units are bonded to each other by means of amino or ammonium units, according to claim 1." ], [ "The invention concerns monoquaternary or polyquaternary polysiloxanes, their manufacture and use as surface finishing components.", "EP-A-0 441 530 describes a textile softener made of polysiloxane, which contains tertiary amine groups in silk chains.", "Also described is the reaction of α,ω-epoxy-modified siloxanes with piperazine, which depends upon the piperazine mixture used, to produce oligomeric or polymeric structures with tertiary amine functions in the main chains, such as described in U.S. Pat.", "No.", "4,847,154.The further introduction of ethylene oxide/propylene oxides as hydrophilic components leads to an improvement of the effect.", "To this end it is proposed on the one hand, to position alkylene oxides and tertiary amine groups in silk chains, which are bonded to ester structures by the main siloxane chain, as described in U.S. Pat.", "No.", "5,591,880 and U.S. Pat.", "No.", "5,650,529.The drawback here is the complicated esterification in the presence of amino groups.", "The alternative to this is known, to bring about a reaction between α,ω-epoxy-modified siloxanes and polyalkylene oxides having secondary amine functions, as described in U.S. Pat.", "No.", "5,981,681.Branched alkaline oxide-modified quaternary polysiloxanes are synthesized from α,ω-OH terminated polysiloxanes and trialkoxysilanes by means of condensation.", "U.S. Pat.", "No.", "5,602,224 describes quaternary ammonium structures, to which silanes are introduced, where the quaternary nitrogen atom is replaced by alkylene oxide units.", "Strictly comb-like alkylene oxide-modified polysiloxane quaternary compounds are similarly described in U.S. Pat.", "No.", "5,098,979.The hydroxyl groups of the comb-structured substituted polyethersiloxanes were transformed with epichlorohydrin into the corresponding chlorohydrin derivative.", "This is followed by a quaternation with tertiary amines.", "A drawback of this strategy is that it requires dealing with epichlorohydrin, and the relatively slight reactivity of the chlorohydrin group during quaternation.", "For this reason, the hydroxyl groups of comb-structured substituted polyethersiloxanes are instead esterized with chloroacetic acid.", "Through the carbonyl activation the final quaternation can be more easily achieved, as described in U.S. Pat.", "No.", "5,153,294 and U.S. Pat.", "No.", "5,166,297.WO 01/41719 and WO 01/41720, published after the priority day of this announcement, describe quaternary polysiloxane compounds for use in cosmetic preparations.", "α,ω-biquaternary polysiloxanes are described in U.S. Pat.", "No.", "4,891,166.Synthesis occurs by a reaction of α,ω-diepoxides with tertiary amine groups in the presence of acids.", "U.S. Pat.", "No.", "4,833,225 describes linear polyquaternary polysiloxanes, which are produced by a reaction of α,ω-diepoxides with ditertiary amines in the presence of acids.", "Alternatively, it is possible to transform α,ω-halogen alkyl modified siloxanes with ditertiary amines into polymer polyquaternary compounds, such as described in U.S. Pat.", "No.", "4,587,321.The substances according to U.S. Pat.", "No.", "4,891,166, U.S. Pat.", "No.", "4,833,225 and U.S. Pat.", "No.", "4,587,321 have a marked tendency to shrink on solid body surfaces.", "With the compounds described here, it is a question of the nature of either α,ω-bisfunctional polysiloxanes, corresponding chain-like (AB)η copolymers, comb-like functionalized siloxane or rather products with a portion in branching positions of siloxane chains.", "In DE-OS 43 18 536, DE-OS 44 37 886 and the publications of R. Wagner, L. Richter, B. Weiland, J. Reiners, J. Weissmüller, Appl.", "Organomet.", "Chem.", "(1996), 437 as well as R. Wagner, L. Richter, Y. Wu, J. Weissmüller, A. Kleewein.", "E. Hengge, Appl.", "Organomet.", "Chem.", "12 (1998), 265, saccharide-modified siloxane derivatives having available two silicon groups moving independently of each other are described.", "No statements were made with regard to suitability as textile softeners or for finishing other surfaces.", "Furthermore, it was felt to be disadvantageous to have to include the step of saccharin addition into the synthetic process.", "It is therefore the objective of the present invention to make available structures which do not have the disadvantages of the state of the art.", "The objective was accomplished by compounds composed of two independently mobile siloxane groups and a connecting amine or ammonium element.", "The objective is accomplished in accordance with the invention through monoquaternary or polyquaternary polysiloxane derivatives of the general formula (I): S—K-Q1-K—S (I) where S or R1 C1-C22-Alkyl, C1-C22-Fluoroalkyl or Aryl, n 0 to 1000, Q1 a secondary amine structure or tertiary amine structure or quaternary ammonium structure R2 represents a branched or bivalent straight chain, cyclical or branched C1-C30-hydrocarbon radical, which is interrupted by —O—, —NH—C(O)—, —C(S)— and can be substituted with —OH or represents a single bond to the K radical, R3 a simple straight chain, cyclical or branched C1-C30-hydrocarbon radical, which is interrupted by —O—, —NH—C(O)—, —C(S)— and can be substituted with —OH or an -A-E structure with A —CH2C(O)O—, CH2CH2C(O)O— or —CH2CH2CH2C(O)O and E a polyalkylene oxide group of the following structure —[CH2CH2O]q—[CH2CH(CH3)O]r—R4 q 1 to 200 r 0 to 200, R4 corresponds to H, straight chain, cyclical or branched C1-C20-hydrocarbon radical, which is interrupted by —O—, or —C(O)— and can be substituted with —OH and can be acetyleneic, olefinic or aromatic, whereby, when a number of R3 radicals in the molecule are present, these can be the same or different, as well as K is a bivalent or trivalent straight chain, cyclical or branched C2-C40-hydrocarbon radical, which is interrupted by —O—, —NH—, —N R1— —C(O)—, —C(S)— and can be substituted by —OH, or contain a group Q2, with Q2 secondary amine structure or tertiary amine structure or quaternary ammonium structure R5 a monovalent or bivalent straight chain, cyclical or branched C1-C20-hydrocarbon radical, which can be interrupted by —O—, —NH—C(O)—, —C(S)— and substituted by —OH, where the free valence of the bivalent radical R5 can bind to Q1, and when a majority of radicals K occur in the polysiloxanes, these can be identical or different from one another.", "In one embodiment of the invention, polysiloxane compounds were prepared according to the Formula (I′): S—K-Q1-K—S (I′) wherein S S R1 C1-C22-Alkyl, C1-C22-Fluoroalkyl or Aryl, n 0 to 1000 Q1 secondary amine structure or tertiary amine structure or quaternary ammonium structure R2 a monovalent or bivalent straight chain, cyclical or branched C1-C30-hydrocarbon radical, which can be interrupted by —O—, —NH—C(O)—, —C(S)— and substituted with —OH, or has a single bond with K, R3 a monovalent straight chain, cyclical or branched C1-C30-hydrocarbon radical, which can be interrupted by —O—, —NH—C(O)—, —C(S)— and substituted by —OH, or by an -A-E-, structure.", "A —CH2C(O), —CH2CH2C(O)— or —CH2CH2CH2C(O)O— and E a polyalkylenoxide entity of the following structure —[CH2CH2O]q—[CH2CH(CH3)O]r—R4 q 1 to 200, r 0 to 200, R4 H, straight chain, cyclical or branched C1-C20-hydrocarbon radical, which is interrupted by —O—, or —C(O)— and can be substituted by —OH and can be acetyleneic, olefinic or aromatic, as well as K a bivalent or trivalent straight chain, cyclical or branched C2-C40-hydrocarbon radical, which is interrupted by —O—, —NH—, —N R1—, —N—, —C(O)—, —C(S)— and can be substituted by —OH, or contain a group Q2, with Q2 secondary amine structure or tertiary amine structure or quaternary ammonium structure R5 a monovalent or bivalent straight chain, cyclical or branched C1-C20-hydrocarbon radical, which can be interrupted by —O—, —NH—C(O)—, —C(S)— and substituted with —OH, or a has single bond to Q1, or R2 and R5—CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)5CH3, —CH2CH2OH, R6 a monovalent straight chain, cyclical or branched C1-C18-hydrocarbon radical, which can be interrupted by —O—, —NH—, —C(O)—, —C(S)— and substituted by —OH.", "The possibility a trivalent substructure for K means that K can be branched, and hence can participate with two compounds in the quaternation of Q1 over the bivalent radical R2.The possibility of a bivalent substructure for R2 means that it in these cases, it is a question of a structure forming a cyclical system, in which process R2 is then a single bond to K, especially to one exhibiting tertiary amine structure, or to a quaternary structure Q2 over R5.In a further embodiment, the present application signifies R1 C1-C18-alkyl, C1-C18-fluoroalkyl and aryl, and the radicals n, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further embodiment, the present application signifies R1 C1-C18-alkyl, C1-C6-fluoroalkyl and aryl, and the radicals n, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In further embodiment, the present application signifies R1 C1-C6-Alkyl, C1-C4-fluoroalkyl and phenyl, and the radicals n, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In further embodiment, the present application signifies R1 methyl, ethyl, trifluoropropyl and phenyl, and the radicals n, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further embodiment of the present application, K signifies a bivalent or trivalent straight chain, cyclical or branched C2-C30-hydrocarbon radical, which is interrupted by —O—, NH—, —NR1—, —C(O)—, —C(S)— and can be substituted by —OH, or contain a group Q2, and the radicals n, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further embodiment of the present application, n means 0 to 100, preferably 0 to 80 and especially preferably 10 to 80, and the radicals R1, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further embodiment of the present application, q means 1 to 50, preferably 2 to 50, and the radicals R1, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a preferred embodiment of the present application, q would be 2 to 20 and especially favored 2 to 10 and the radicals R1, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, n and r, have the aforementioned meaning.", "In a further embodiment of the present application, r means 0 to 100, preferably 0 to 50 and the radicals R1, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and n, have the aforementioned meaning.", "In a further preferred embodiment of the present application, r means 0 to 20 and especially preferably 0 to 10, and the radicals R1, R2, R3, R4, R5, R6, K, A, 3E, Q1, Q2, q and n, have the aforementioned meaning.", "In a further embodiment of the present application, R2 and R5 signify —CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)5CH3, —CH2CH2OH, with R6 a monovalent straight chain, cyclical or branched, C1-C18-hydrocarbon radical, which can be interrupted by —O—, —NH—, —C(O)—, —C(S)— and substituted by —OH.", "In a further embodiment of the present application, R3 signifies —CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)5CH3, —CH2CH2OH, wherein R6 is a monovalent straight chain, cyclical or branched, C1-C18-hydrocarbon radical, which can be interrupted by —O—, —NH—, —C(O)—, —C(S)— and substituted by —OH.", "In a further preferred embodiment of the present application, R4 means a bivalent or trivalent straight chain, cyclical or branched C1-C18-hydrocarbon radical, which can be interrupted by —O—, —NH—C(O)—, —C(S)— and can be substituted with —OH, or make a single bond with Q1, and the radicals n, R1, R2, R3, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further preferred embodiment, R4 means C1-C6-alkyl, —CH2CH═CH2, —CH2CH(OH)CH2OCH2CH═CH2, —CH2C≡CH, —C(O)CH3, —C(O)CH2CH3 and the radicals n, R1, R2, R3, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further preferred embodiment, K means and the radicals n, R1, R2, R3, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In a further preferred embodiment of the present invention, R6 means unsubstituted C5-C17-hydrocarbon radicals, which are derived from the corresponding saturated or unsaturated fatty acids, and the radicals n, R1, R2, R3, R5, R6, K, A, 3E, Q1, Q2, q and r, have the aforementioned meaning.", "In the context of the present invention, the concept of “C1-C22-Alkyl or C1-C30-hydrocarbon radical” means aliphatic hydrocarbon compounds with 1 to 22 carbon atoms or 1 to 30 carbon atoms which might be in a straight chain or branched.", "Cited by way of example are methyl, ethyl, propyl, n-butyl, pentyl, hexyl, heptyl, nonyl, decyl, undecyl, isopropyl, neopentyl, and 1,2,3 trimethylhexyl.", "In the framework of the present invention, the concept of “C1-C22-Fluoralkyl” means aliphatic hydrocarbon compounds with 1 to 22 carbon atoms which might be straight or branched, in which at least one fluorine atom is substituted.", "Examples cited are monofluoromethyl, monofluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, 1,1,1-trifluoropropyl, 1,2,2-trifluorobutyl.", "Within the framework of the invention, the concept “aryl” means unsubstituted phenyl, or phenyl substituted one or more times by OH, F, CL, CF3, C1-C6-alkyl, C1-C6-alkoxy, C3-C7-cycloalkyl C2-C6-alkenyl or phenyl.", "The expression can also mean naphthyl if necessary.", "A further object of the present invention is to make available a process for the production of the compounds of the general formula (I) or (I′).", "The point of departure for the synthesis in accordance with the invention compounds is monofunctional H-siloxane of the general structure where R1 and n have the meanings given above.", "Since these compounds are not commercially available, these siloxanes, especially the longer-chain derivatives, can be manufactured according to known procedures (Silicone, Chemie und Technologie, Vulkan-Verlag, Essen 1989, pp.", "82-84).", "The acid-catalyzed equilibriation of trimethylsilyl-terminated siloxanes, for example, hexamethyldisiloxane (MM), with dimethylsiloxy-rich compounds, for example octamethylcyclotetrasiloxane (D4), [takes place] in the presence of a corresponding mixture containing SiH, but not a siloxane deriving from SiH delivered product, in which the SiH function is located within the chain.", "In the equilibriation balance all the relevant products are formed, which per molecule have available either none, or more than one SiH function.", "The acid catalyzed equilibriation of the α-SiH compounds, for example pentamethyldisiloxane (MMH) with dimethylsiloxane-rich compounds, or for example octamethylcyclotetrasiloxane (D4) delivers monofunctional products with terminal SiH function.", "Pentamethyldisiloxane can for example be substituted by equimolar mixtures of hexamethyldisiloxane (MM) and tetramethyldisiloxane (MHMH).", "In equilibriation balance there are additional products formed, which per molecule have none or two terminal SiH functions.", "The equilibriation of cyclic siloxanes, such as hexamethylcyclotrisiloxane (D3) or octamethylcyclotetrasiloxane (D4) with alkaline trimethyl silanolates, e.g., potassium trimethyl silanolate, produces oligo siloxanolates, which react with dimethylchlorosilane with the corresponding monofunctional compounds with terminal SiH function.", "In the equilibriation balance, additional products are formed, which per molecule have available either none, or only two terminal silanolate functions.", "In consequence, there are also products present which have available none, or two terminal SiH functions.", "In the framework of the invention, there were described, besides strictly defined monofunctional compounds, also mixtures, treated as monofunctional SiH compounds.", "Reactive, alkylating, monofunctional siloxane compounds are synthesized through hydrosilylation by, for example, halogenated alkyls, especially allylic chloride and allylic bromide, unsaturated carboxylic haloacid esters, preferably chloroacetic acid allylic esters, chloroacetic acid propargyl esters and 3-chloropropionic acid allylic esters and epoxy-functional alkenes, for example vinylcyclohexenoxide and allylic glyco ether, with the here described monofunctional SiH compounds.", "Hydrosilylation in general, with the substances from the cited groups, is likewise known (B. Marciniec, Comprehensive Handbook on Hydrosilylation, Pergamon Press, Oxford 1992, p. 116-121, 127-130, 134-137, 151-155).", "The subsequent synthesis of compounds having secondary amine functions of the types ABA (ABA [cut off] means that two polysiloxane groups are bonded by a bridging amino- or ammonium structure) whose general structure is S—K-Q1-K—S in which K and S have the aforementioned meanings, occurs preferably through alkylization of two primary amine exhibiting amino groups, for example α,ω-alkylenediamines, preferably ethylenediamine, 1,3-propylenediamine, 1,6-hexylenediamine, short-chain ethylenoxide/propylenoxide groups containing diprimary amines, especially Jeffamine® (Huntsman Corp.) of the type Jeffamine EDR 148, Jeffamine ED 600, Jeffamine D 230, Jeffamine D 400, with reactive, alkylating, in the sense of the invention, monofunctional siloxane intermediate products.", "The stochiometry of the reaction between the diprimary amine and the monofunctional siloxane has a ratio of 1:2.The synthesis of tertiary amine functions containing ABA type compounds of the general structure S—K-Q1-K—S in which K and S have the aforementioned meanings, occurs preferably in two ways.", "On the one hand, it is possible to first of all directly bind the secondary amine function containing unsaturated structures, for example, N=methylallyl amine or CH2═CHCH2OCH2CH(OH)CH2NHCH3, through hydrosilylation, to the monofunctional Si—H siloxane.", "This process is generally known, and is, for example, described by B. Marciniec, Comprehensive Handbook on Hydrosilylation, Pergamon Press, Oxford 1992, pp.", "122-124).", "These secondary amine structures that are produced, can be transformed in a following step, using reactive alkylation siloxane intermediates, into polymers containing tertiary amine structures.", "The stochiometry of this reaction has a ratio of aminosiloxane to monofunctional siloxane of about 1:1.As an alternative to the step-wise synthesis detailed above, it is possible to produce tertiary amine functionalized polymers in one reaction step.", "The point of departure for this is in the handling of the reactive, alkylation siloxane intermediate steps, preferably the epoxy derivative, especially the allylic glycide ether species.", "This might be transformed, by reacting with primary amines, for example methylamine, in a molar ratio of preferably 2:1 into tertiary amines.", "It is also possible to use difunctional secondary amines, for example piperazine, for this reaction.", "In this case, molar ratio of the secondary amine group to the alkylation group, preferably to one epoxy group, would be preferably 1:1.Among the results of carrying out such reactions, products were obtained in which two tertiary amine groups are to be found between the two siloxane blocks.", "The synthesis of monoquaternary or polyquaternary polysiloxanes of the types ABA of the general structure S—K-Q1-K—S in which Q1 means Occurs in various ways beginning with tertiary amino function-bearing siloxane derivatives.", "On the one hand, transforming the above-described reactants, monofunctional siloxane derivatives, preferably the epoxy functional derivatives, into tertiary amines is preferred, using secondary amines, for example, dimethyl amine or morpholine which then in a follow-up step would react with a second mole of reactive, monofunctional siloxane compound to the quaternary products.", "For both reaction steps, the preferred molar ratio is 1:1.The application of secondary-tertiary diamines opens the possibility of creating regioselective combinations of tertiary amines and quaternary structures.", "The alkylation of amines of types N-methylpiperazine with preferably one mole epoxy-functional siloxane produces ditertiary aminosiloxane, which for example, are quaternated from a second mole of reactive, monofunctional siloxane compounds, for example a halogen carboxylic acid ester derivative, into methylated nitrogen atoms.", "A surplus of the reactive, monofunctional siloxane compounds, or an addition of a further alkylation agent, leads to an incipient alkylation of the second nitrogen atom.", "The secondary amines, produced by alkylation, for example dimethylamine, or secondary-tertiary diamines, for example N-methylpiperazine, with preferably one mole epoxy-functional siloxane accessible tertiary or ditertiary aminosiloxanes, might in a preferred embodiment with difunctional alkylation agents in a molar ratio 2:1.As a result of such a reaction, two quaternary ammonium groups, or two quaternary ammonium groups in the neighborhood, in any given case of a tertiary amine group, are bonded with each other over a single-chained spacer.", "Dihalogen-alkanes, diepoxy-compounds in the presence of acids, α,ω-dihalogen oligoalkylene oxides or dihalogen carboxylic acid esters of alkylene oxides are suitable alkylation substances for this purpose.", "Preferred starting materials for α,ω-dihalogen alkylene oxides and dihalogen carboxylic acid esters are lower molecular oligomers and polymers, alkylene oxide of the general compound HO[CH2CH2O]q—[CH2CH(CH3)O]rH in which q and r have the aforementioned meanings.", "Preferred reactants are diethyleneglycol, triethyleneglycol, tetraethyleneglycol, the oligoethyleneglycols with a molecular weight of 300 to 1000 g/mole, preferably, 400, 600, and 800, as well as dipropyleneglycol.", "α,ω-dihalogenalkylene oxides can be produced in the usual way, e.g.", "through halogenation with thionyl chloride.", "Esterization takes place in the familiar way (Organikum, Organisch-chemisches Grundpraktikum [Organikum: Organic Chemistry Basic Practical Course], 17.Auflage, VEB Deutscher Verlag der Wissenschaften, Berlin 1988, pp.", "402-408), through reaction with C2-C4 carboxylic haloacids, their anhydrides, or acid chlorides.", "The process described in the present document, primarily based in piperazine-based derivatives with two tertiary amino groups between two siloxane blocks, can also be transferred to quaternary ammonium salts.", "The degree is quaternation is steered by the molar ratio of the two tertiary amino groups, which are bonded between the two siloxane blocks, to the alkylation agents.", "It is preferable, when working on an equimolar basis, to synthesize products, in which all the tertiary amines are transformed into quaternary ammonium functions.", "On the other hand, it can be advantageous to preserve a part of the tertiary amine functions through the selective deficiency in alkylation agents to preserve a part of the tertiary amine functions.", "Examples of advantageous alkylation agents are epoxy derivatives in the presence of acids, alkyl halogenides or carboxylic haloacid esters, preferably carboxylic haloacid esters with alkylene oxide.", "Preferred starting materials for these alkylations means are lower molecular, oligomer and polymer alkylene oxides of the general compound HO[CH2CH2O]q—[CH2CH(CH3)O]rR4 where q, r and R4 is as cited above.", "Preferred reactants are the corresponding monosubstituted derivatives of diethylene glycol, triethylene glycol, tetraethylene glycol, the oligoethylene glycols with molar weight of 300 to 1000 g/mole, preferably 400, 600, and 800, as well as dipropylene glycol.", "The production of these ethers and esters takes place in a known manner by acid- or alkali catalyzed addition of ethylene oxide and/or propylene oxide with the corresponding alcohol (Organikum, Organisch-chemisches Grundpraktikum, 17.Auflage, VEB DeutscherVerlag der Wissenschaften, Berlin 1988, p. 259; U.S. Pat.", "No.", "5,625,024) or carboxylic acids (E. Sung, W. Umbach, H. Baumann, Fette Seifen Anstrichmittel [Fats, Soaps, Paints] 73, 88 [1971]).", "The following syntheses of carboxylic haloacid esters follow the known manner (Organikum, Organisch-chemisches Grundpraktikum, 17.Auflage, VEB Deutscher Verlag der Wissenschaften, Berlin 1988, pp.", "402-408) through reaction with the C2-C4-halogen-carboxylic acids, whose anhydrides or acid chlorides.", "The selective synthesis of hydroxyfunctional carboxylic haloacid esters, in which R4 stands for hydrogen, is attained by the addition of ethylene oxide and/or propylene oxide to the corresponding carboxylic haloacids under acid conditions.", "When more than one tertiary amino function is introduced between the siloxane blocks, e.g., through piperazine structures, it becomes possible to bring to bear the hydrophilic and the surfactant properties within broader limits, through the relationship of the tertiary amines to the quaternary structure.", "It lies within the framework of the invention, to bring about a reaction of a number of siloxane components and/or alkylation agents while maintaining the desired general overall stochiometry.", "This opens up the possibility, for example, of creating a desired length of siloxane chain, employing a single siloxane component, or otherwise through the selective mixing of several siloxane components.", "Anions coming into consideration are primarily those which were formed during the quaternation of halogenated iodides, especially chloroiodide.", "Other anions can also be employed through ion exchange reactions.", "Examples cited are organic anions, such as carboxylates, sulfonates, sulfates, polyethercarboxylates and polyethersulfates.", "Alkylation reactions are preferably carried out in polar organic solvents.", "Suitable for this are for example alcohols from the group consisting of methanol, ethanol, i-propanol and n-butanol; glycols form the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, methyl-, ethyl- and butylether of the cited glycols, 1,2-propylene glycol, and 1,3-propylene glycol, ketones such as acetone, and methylethylketone, esters, such as ethylacetate, butylacetate and 2-ethylhexylacetate, ethers such as tetrahydrofuran and nitro-compounds, such as nitromethane.", "The choice of solvents is focussed essentially on the solubility of the reaction partner, and the target reaction temperature.", "The reactions take place in the range of 20° C. to 130° C., preferably 40° C. to 100° C. Products of the invention combining the softening of the characteristics of the siloxane structures and the tendency of amino structures or quaternary, ammonium groups to adsorption on negatively charged solid-body-surfaces, might be successfully used in cosmetic formulations for skin- and hair-care, in cleaning agents for treating and handling hard surfaces, in formulas for drying automobiles and other hard surfaces after machine-washing, for use with textiles and textile phases, as a separate softener after the washing of textiles with non-ionic or anionic/non-ionic detergent formulas, as a softener in non-ionic or anionic/non-ionic washing of textiles based on tenside formulas.", "Along with this, amino derivatives might be used, depending on the pH value, in the form of amine or amine salts.", "The invention concerns the broadening of the application of the polysiloxane compounds described herein, in cosmetic formulas for skin- and hair care, in cleaning agents for treating and handling hard surfaces, in formulas for drying automobiles and other hard surfaces, for example, after machine-washing, for use with textiles and textile phases, as a separate softener after the washing of textiles with non-ionic or anionic/non-ionic detergent formulas, as softeners for non-ionic or anionic/non-ionic washing of textiles based on tenside formulas, as well as a means for preventing or reversing textile wrinkling.", "The invention regards the broader application of the herein-described polysiloxane compounds as wash-resistant hydrophilic softeners for initial textile finishing.", "Further, the invention concerns compounds containing at least one polysiloxane compound together with at least one additional ingredient typical for the composition.", "Below there are given some typical examples of compositions of this type in which the polysiloxane compounds of the invention can be employed with advantage.", "Typical catalysts in such kinds of compounds are for example, the substances, which are described in A. Domsch: Die kosmetischen Präparate [Cosmetic Preparations], Vol.", "I and II, 4th edition.", "Verl.", "für chem.", "Industrie, H. Ziolkowsky K G, Augsburg, as well as the International Cosmetic Ingredient Dictionary and Handbook 7th Edition 1997, by J.", "A. Wenninger, G. N. McEwen Vol.", "1-4, by The Cosmetic, Toiletry and Fragrance Association of Washington D.C. or under http://www.cosmetic-world.com/inci/Incialf.htm.", "Anionic Shampoo.", "The formulation given here is conceived of as a basic formulation.", "Anionic shampoos usually contain the following ingredients, without being limited to them: Alkylsulfate, alkylethersulfate, sodium lauryl sulfate, sodium lauryl ether sulfate, ammonium lauryl sulfate, ammonium lauryl ether sulfate, TEA-laurylsulfate, TEA-lauryl-ethersulfate, alkyl benzol sulfonate, α-olefinsulfonate, paraffinsulfonate, sulfosuccinate, N-acyl tauride, sulfate-glyceride, sulfated alkalonamide, carboxylate salts, N-acyl-amino-acid-salts, silicones, etc.", "Components % Ammonium lauryl sulfate 10.00-30.00 Ammonium lauryl ether sulfate 5.00-20.00 Cocamidopropyl betaine 0.00-15.00 Lauramide DEA 0.00-5.00 Cocamide Mea 0.00-5.00 Dimethicone copolyol 0.00-5.00 (dimethylsiloxane glycol polymer) Cyclopentasiloxane 0.00-5.00 Polysiloxane compound 0.50-5.00 of the invention Polyquaternium-10 0.00-2.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Sodium chloride q.s.", "Non-Ionized Shampoo The composition example is intended as a basic formulation.", "Non-ionized shampoos, generally speaking, contain (without being limited to) the following components: Monoalkanolamides, monoethanolamides, monoisopropanolamides, polyhydroxy derivatives, sucrose monolaurate, polyglycerin ester, amino oxides, polyethoxylated derivatives, sorbitan derivatives, silicone, etc.", "Components % Lauramide DEA 10.00-30.00 Lauramide oxide 5.00-20.00 Cocamide Mea 0.00-5.00 Dimethicone copolyol 0.00-5.00 Polysiloxane compound 0.50-5.00 of the invention Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Sodium chloride q.s.", "Amphoteric Shampoo The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: N-alkyl-iminodipropionate, n-alkyl-iminopropionate, amino acids, amino acid derivatives, amino betaines, imidazolinium derivatives, sulfobetaine, sultaine, betaine, silicone, etc.", "Components % PEG-80 sorbitan laurate 10.00-30.00 Lauroamphoglycinate 0.00-10.00 Cocamidopropyl hydroxysultaine 0.00-15.00 PEG-150 distearate 0.00-5.00 Lauryl ether-13 carboxylate 0.00-5.00 Polysiloxane compound 0.50-5.00 of the invention Scents 0.00-5.00 Deionized water q.s.", "100% Sodium chloride q.s.", "Cationic Shampoo The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Bis-quaternary ammonium compounds, bis-(trialkyl ammonium acetyl) diamine, amidoamine, ammonium alkyl ester, silicone, etc.", "Components % Lauryl ether-13 carboxylate 10.00-30.00 Isopropyl myristate 5.00-20.00 Cocamidopropyl betaine 0.00-15.00 Lauramide DEA 0.00-5.00 Cocamide Mea 0.00-5.00 Polysiloxane compound 0.50-5.00 of the invention Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Sodium chloride q.s.", "Solidifying Agents The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, etc.", "Components % Ceteareth-20 0.10-10.00 Steareth-20 0.10-10.00 Stearyl alcohol 0.10-10.00 Stearamidopropyl dimethylamine 0.00-10.00 Dicetyl dimonium chloride 0.00-10.00 Polysiloxane compound 0.50-5.00 of the invention Cyclopentasiloxane 0.00-5.00 Dimethicone 0.00-5.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% “Clear Rinse Off” Solidifying Agents The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, etc.", "Components % Glycerin 0.10-10.00 Cetrimonium chloride 0.00-10.00 Polysiloxane compound 0.50-5.00 of the invention Hydroxy ethyl cellulose 0.00-5.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Solidifying Agents for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, butane, propane, isobutane, CFCs, fluorinated aerosol propellants, dimethyl ether, compressed gases, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Nonoxynol-15 0.00-2.00 Nonoxynol-20 0.00-2.00 Scents 0.00-5.00 Aerosol propellants 0.00-20.00 Preservatives 0.00-0.50 Deionized water q.s.", "100% Pump Spray (Solidifying Agent) for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Cyclomethicone 0.00-80.00 Ethanol 0.00-80.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Solidifying Agent Spray for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, butane, propane, isobutane, CFCs, fluorinated aerosol propellants, dimethyl ether, compressed gases, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Cyclomethicone 0.00-80.00 Ethanol 0.00-50.00 Aerosol propellants 0.00-50.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Gel Solidifying Agents for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Thickening agents, cellulose derivatives, acryl acid derivatives, fixative polymers, conditioning chemicals, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Hydroxyethyl cellulose 0.00-2.00 Scents 0.00-5.00 Preservatives 0.00-0.50 Citric acid 0.00-2.00 Deionized water q.s.", "100% Styling Gel for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fixative polymers, lacquer, acryl acid derivatives, cellulose derivatives, vinyl derivatives, conditioning chemicals, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Fixatives 0.10-10.00 Hydroxy ethyl cellulose 0.00-2.00 Scents 0.00-5.00 Citric acid 0.00-2.00 Deionized water q.s.", "100% Styling Spray for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Fixative polymers, lacquer, vinyl derivatives, fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, butane, propane, isobutane, CFCs, fluorinated aerosol propellants, dimethyl ether, compressed gases, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Cyclomethicone 0.00-80.00 Fixatives 0.10-10.00 Ethanol 0.00-50.00 Aerosol propellants 0.00-50.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% Pump Spray (Styling) for Hair The composition example is intended as a basic formulation.", "Formulas of this category, generally speaking, contain (without being limited to) the following components: Vinyl derivatives, fixative polymers, lacquer, fatty acids, fatty acid esters, ethyloxylated fatty acids, ethyloxylated fatty acid esters, fatty alcohols, ethyloxylated fatty alcohols, glycols, glycol esters, glycerin, glycerin esters, lanolin, lanolin derivatives, mineral oil, petrolatum, lecithin, lecithin derivatives, waxes, wax derivatives, cationic polymers, proteins, protein derivatives, amino acids, amino acid derivatives, humectants, thickening agents, silicone, solvents, ethanol, isopropanol, isoparaffin solvents, butane, propane, isobutane, CFCs, fluorinated aerosol propellants, dimethyl ether, compressed gases, etc.", "Components % Polysiloxane compound 0.50-5.00 of the invention Fixatives 0.10-10.00 Cyclomethicone 0.00-80.00 Ethanol 0.00-50.00 Preservatives 0.00-0.50 Scents 0.00-5.00 Deionized water q.s.", "100% The use of polysiloxane derivatives of the invention, when applied in the area of hair cosmetics, leads to favorable effects with regard to setting, sheen, hold, body, volume, moisture regulation, color retention, protection against the effects of the environment (UV, salt water, etc.", "), capacity for reshaping, anti-static properties, capacity for dyeing, etc.", "EXAMPLES The following examples serve to explain the present invention in greater detail, but without limiting it in any way.", "Example 1 1a) 3.37 g (0.1 mol) of an epoxysiloxane with the formula and 10.1 g (0.1 mol) n-methyl piperazine were dissolved in 40 ml i-propanol and heated at reflux temperature for 7 hours.", "The solvent was distilled off, following the conclusion of the reaction, in a water jet vacuum and then in an oil pump vacuum.", "39 g of a clear, light brown fluid of the following structure: were obtained.", "According to a gas chromatography analysis, the epoxide was quantitatively transferred into the piperazine derivative.", "1b) 497 g (8.87 mol) CH CCH2OH were placed under nitrogen at room temperature.", "Under intensive agitation, 955 g (8.45 mol) chloroacetic acid chloride was dripped in over 1 hour.", "During the dripping process, the temperature increased to 60° C. and intensive HCl development took place.", "The preparation took on a black color.", "After the conclusion of the dripping process, the preparation was heated for 1 hour at 130° C. Fractionated distillation resulted in a principal yield of 891 g of a light yellowish oil with the structure CH CCH2OC(O)CH2Cl with a boiling point of 179-181° C. The purity of the ester, determined by gas chromatography, was 99%.", "13C-NMR: Shift Substructure (ppm) ClCH2C(O)OCH2C CH 40.4 ClCH2C(O)OCH2C CH 166.5 ClCH2C(O)OCH2C CH 53.1 ClCH2C(O)OCH2C CH 76.4 ClCH2C(O)OCH2C CH 75.6 1c) 26.5 g (0.2 mol) of the chloroacetic acid ester according to Example 1 b and 44 mg of a 3.43% Lamoreaux catalyst solution according to U.S. Pat.", "No.", "3,220,972 were placed under nitrogen at room temperature.", "Over a period of 30 minutes, 48.8 g (0.22 mol) 1,1,1,3,5,5,5 heptamethyl trisiloxane (M2DH) were dripped in and the temperature was increased to 60° C. Subsequently, the preparation was heated for 4 hours at 100° C. After distilling of all components which boiled at up to 120° C. and at 2 hPa, 64.5 g of a yellowish fluid were obtained.", "According to gas chromatography analysis, the product contained 85% target product and 15% heptamethyl trisiloxane ester of chloroacetic acid.", "13C-NMR of the Si—C linked target product Substructure Shift (ppm) ClCH2C(O)OCH2CH═CH—Si 40.3 ClCH2C(O)OCH2CH═CH—Si 166.7 ClCH2C(O)OCH2CH═CH—Si 67.8 ClCH2C(O)OCH2CH═CH—Si 144.4 ClCH2C(O)OCH2CH═CH—Si 126.6 1d) 21.8 g (0.05 mol) of the siloxanyl modified piperazine derivative according to Example 1a) and 17.7 g (0.05 mol) of the chloroacetic acid ester derivative according to Example 1c) were absorbed in 50 ml methyl propyl ketone under nitrogen and heated for 6 hours at reflux temperature.", "Following the conclusion of the reaction, all components which boiled at up to 100° C. and at 4 hPa were removed under vacuum.", "35.7 g of a ductile, brown mass of the following structure: were obtained.", "13C-NMR of the Si—C linked target product Shift Substructure (ppm) —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 65.7 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 51.2 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 46.4 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 60.3 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 52.8 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 61.0 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 169.0 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 66.5 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 144.1 —CH(OH)CH2NCH2CH2N+(CH3)CH2C(O)OCH2CH═CH—Si 126.0 Example 2 2a) 238 g (2.24 mol) diethylene glycol were placed under nitrogen at room temperature.", "Under intensive agitation, 558 g (4.93 mol) chloroacetic acid chloride was dripped in over 1 hour.", "During the dripping process, the temperature increased to 82° C. and intensive HCl development took place.", "After the conclusion of the dripping process, the preparation was heated for 30 minutes at 130° C. Subsequently, all components which boiled at up to 130° C. and at 20 hPa were removed.", "The result was 566 g of a light yellowish oil with the structure ClCH2C(O)OCH2CH2OCH2CH2OC(O)CH2Cl The purity of the ester, determined by gas chromatography, was 99.2%.", "13C-NMR: Substructure Shift (ppm) ClCH2— 40.7 ClCH2C(O)— 167.1 ClCH2C(O)OCH2— 65.2 ClCH2C(O)OCH2CH2— 68.6 2b) 21.8 g (0.05 mol) of the siloxanyl modified piperazine derivative according to Example 1a) and 6.46 g (0.025 mol) of the chloroacetic acid ester derivative according to Example 2a) were dissolved in 100 ml i-propanol and heated at reflux temperature for 10 hours.", "Subsequently, all components which boiled at up to 70° C. and at 20 hPa were removed.", "The result was 26.1 g of a hard, amorphous mass with the following formula: (The compound corresponds to the following definition of the claim: R1=methyl n=0 K (left side)= Q1= with R3=methyl and R2=bond to K K (right side)= Q2<K′ with Q2= with R3=methyl and R5=—CH2—CO—O—CH2CH2OCH2CH2O—CO—CH2— K′= 13C-NMR: Substructure Shift (ppm) —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 66.0 —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 52.5 —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 45.6 —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 60.4 —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 61.3 —CH(OH)—CH2—N—CH2—CH2—N+—CH2—C(O)— 169.2/169.8 CH3—N+ 52.9 Example 3 110 g (0.03 mol) of an epoxy modified siloxane of the following statistical composition and 1.3 g (0.015 mol) piperazine were dissolved in 120 ml i-propanol and heated at reflux temperature for 5 hours.", "Following the conclusion of the reaction, all components which boiled at up to 100° C. and at 4 hPa were removed under vacuum.", "109.7 g of a light yellow oil of the following structure: were obtained.", "13C-NMR: Substructure Shift (ppm) —CH(OH)CH2NCH2 66.0 —CH(OH)CH2NCH2 60.5 —CH(OH)CH2NCH2 53.2 Example 4 As proof of the softening properties as an internal softener during the washing process, strips of bleached cotton which had not undergone any further surface treatment were subject to a washing process in the presence of Ariel Futur®, Dash 2 in 1® containing bentonite, and the aminosiloxane described in Example 2.The following boundary conditions were maintained: Strip 1 Strip 2 Strip 3 Strip weight 13.40 13.55 13.29 (g) Water quantity 669 679 665 (ml) Detergent 0.66 g Ariel 0.68 g Ariel 0.64 g Dash Futur ® Futur ® 2 in 1 ® Siloxan 0.2 g — — Example 2 Average grade 1.5 2.8 1.7 The water was heated to 60° C.; the detergents—and, in the case of cotton strip 1, also the aminosiloxane according to Example 2—were dissolved.", "Subsequently, the cotton strips were washed in these solutions for 30 minutes.", "After that, the strips were rinsed five times with 600 ml water each time, after which they were dried for 30 minutes at 120° C. 14 test persons evaluated the three cotton strips for softness to the touch.", "The grade of 1 was given to the softest strip and the grade of 3 was given to the strip which was perceived as hardest.", "As a result of the evaluation, cotton strip 1 received an average grade of 1.5.Cotton strip 2 received an average grade of 2.8; cotton strip 3, which had been treated with bentonite, received an average grade of 1.7." ] ]

[ [ "Reducing the level of bacteria and viruses in aquaculture", "A method of reducing the level of bacteria and viruses in a volume of water during aquaculture comprises: (a) providing the volume of water to be stocked with farmed aquatic organisms or eggs thereof; (b) prior to the stocking, introducing into the water an aqueous solution of glutaraldehyde in an amount such as to provide in the water from 0.1 to 2 ppm of glutaraldehyde; (c) stocking the water with the farmed aquatic organisms or eggs thereof at a time when the concentration of glutaraldehyde is 0.1 to 2 ppm.", "(d) allowing the farmed aquatic organisms or eggs thereof to grow; and (e) optionally during a period of the growth, introducing into the water at least one further portion of an aqueous solution of glutaraldehyde in an amount such as to maintain the concentration of the glutaraldehyde at 0.1 to 2 ppm." ], [ "1.A method of reducing the level of bacteria and viruses in a volume of water during aquaculture, which method comprises: (a) providing the volume of water to be stocked with farmed aquatic organisms or eggs thereof; (b) prior to the stocking, introducing into the water an aqueous solution of glutaraldehyde in an amount such as to provide in the water from 0.1 to 2 ppm of glutaraldehyde; (c) stocking the water with the farmed aquatic organisms or eggs thereof at a time when the concentration of glutaraldehyde is 0.1 to 2 ppm.", "(d) allowing the farmed aquatic organisms or eggs thereof to grow; and (e) optionally during a period of the growth, introducing into the water at least one further portion of an aqueous solution of glutaraldehyde in an amount such as to maintain the concentration of the glutaraldehyde at 0.1 to 2 ppm.", "2.A method according to claim 1, wherein the concentration of glutaraldehyde in each of steps (b), (c) and (e) is within the range 0.5 to 1.5 ppm.", "3.A method according to claim 1 or claim 2, applied to bacteria of the species Vibrio.", "4.A method according to claim 3, wherein the bacteria of the species Vibrio are selected from Vibrio parahaemolyticus and Vibrio harveyi.", "5.A method according to claim 1 or claim 2, applied to viruses selected from White Spot Syndrome Baculovirus (WSBV) complex, Yellow Head Virus and Taura Syndrome Virus.", "6.A method according to claim 5, wherein the viruses are WSBV complexes selected from HABV; RV-JP; and SEMBV.", "7.A method according to any preceding claim, wherein the farmed aquatic organisms or eggs thereof are crustacea or eggs thereof.", "8.A method according to claim 7, wherein the crustacea or eggs thereof are shrimps, prawns or eggs thereof.", "9.A method according to claim 7 or claim 8, wherein the crustacea are selected from Litopenaeus vannamei, Litopenaeus setiferus, Litopenaeus stylirostris, Litopenaeus aztecus, Litopenaeus chinensis, Litopenaeus duorarum, Penaeus monodon and Penaeus vannamei.", "10.A method according to any preceding claim, which includes the step (e), given and defined in claim 1.11.A method according to claim 10, wherein at least two portions of the glutaraldehyde are introduced into the water, with an interval of from 5 to 10 days between respective additions thereof.", "12.A method according to any preceding claim, which includes, in addition to step (e), a further final period of growth during which no further glutaraldehyde is added.", "13.A method according to claim 12, wherein the said further final period is at least thirty days.", "14.A method according to any preceding claim, which is carried out using a sealed aerated pond.", "15.A method according to any preceding claim, which is carried out in a marine environment." ], [ "This invention relates to a method of reducing the level of bacteria and viruses in a volume of water during aquaculture, especially shrimp farming.", "More specifically the threat comes from virulent, difficult to treat viruses, for example Taura Syndrome Virus (TSV), Yellow Head Virus (YHV) and White Spot Syndrome Baculovirus (WSBV).", "Shrimp are also prone to diseases caused by bacteria such as Vibrio parahaemolyticus.", "The above viruses all effect shrimp in the growing out stage of farming.", "TSV typically affects the shrimp Litopenaeus vannamei in its juvenile stage (0.1-5.0 g weight) within 2-4 weeks of stocking into grow out ponds.", "YHV especially affects shrimps in the late juvenile stage (5-15 g weight).", "WSBV is particularly virulent, often affecting early post larval stages, but equally capable of affecting shrimp in the later stages of culture.", "These viruses are examples of the better characterised pathogens.", "A large number of viral pathogens have been described which have damaging effects on shrimp yields.", "For controlling the growth of ectoparasites (protozoa) during shrimp farming in tanks it is known to add to the tank 2.5 ppm of glutaraldehyde; see Conference paper, 8th Scientific Compress, Faculty of Veterinary Medicine, Assuit University, Egypt, 15-17 Nov. 1998.However, in this paper, the use of formalin was preferred.", "Fitzpatrick et al, Progressive Fish-Culturist (1995), 57, 153-155, evaluated three candidate fungicides for treatment of adult spring chinook salmon, including glutaraldehyde, applied in respective amounts of 31.2 and 6.2 mg/l in a flow-through system.", "Although treatment with glutaraldehyde was regarded as showing promise, another alternative, hydrogen peroxide was recommended.", "Articles by each of Salvesen et al, Aquaculture International (1997), 5, 249-258, and Olsen et al, Aquaculture (1999), 176, 3-13, disclose the disinfection of the eggs of Atlantic halibut and turbot with 400-1200 mg/l of glutaraldehyde.", "However, at such high levels, glutaraldehyde may be toxic to certain forms of aquatic life; again see Fitzpatrick et al where levels as low as 62 mg/l were toxic to spring chinook salmon.", "We have now found surprisingly that a highly effective reduction in both viruses and bacteria harmful to aquatic life, especially those harmful to shrimps, can be obtained by application, to a pond in which farmed aquatic organisms and eggs thereof are to grow, of very low concentrations of glutaraldehyde.", "Thus, the invention provides a method of reducing the level of bacteria and viruses in a volume of water during aquaculture, which method comprises: (a) providing the volume of water to be stocked with farmed aquatic organisms or eggs thereof; (b) prior to the stocking, introducing into the water an aqueous solution of glutaraldehyde in an amount such as to provide in the water from 0.1 to 2 ppm of glutaraldehyde; (c) stocking the water with the farmed aquatic organisms or eggs thereof at a time when the concentration of glutaraldehyde is 0.1 to 2 ppm.", "(d) allowing the farmed aquatic organisms or eggs thereof to grow; and (e) optionally during a period of the growth, introducing into the water at least one further portion of an aqueous solution of glutaraldehyde in an amount such as to maintain the concentration of the glutaraldehyde at 0.1 to 2 ppm.", "Preferably, the concentration of glutaraldehyde used in each of steps (b), (c) and (e) is within the range 0.5 to 1.5 ppm.", "The method is especially suitable for reducing the level of bacteria, especially the Vibrio spp, in particular, Vibrio parahaemolyticus and Vibrio harveyi.", "The method is also especially suitable for reducing the level of White Spot Syndrome Baculovirus (WSBV) complex, (also known as China Virus) e.g.", "HHNBV; RV-PJ and SEMBV Yellow Head Virus (also known as Hua Leung) and Taura Syndrome Virus.", "The method is especially suitable for the farming of crustacean especially shrimps or prawns, eggs thereof, for example Litopenaeus vannamei, Litopenaeus setiferus, Litopenaeus stylirostris, Litopenaeus aztecus, Litopenaeus chinensis, Litopenaeus duorarum, Penaeus monodon and, especially Penaeus vannamei (Black Tiger Shrimps).", "It is preferred, during a period of growth to introduce into the water at least one further portion of an aqueous solution of glutaraldehyde in an amount such as to maintain the concentration of the glutaraldehyde at 0.1 to 2 ppm.", "More preferably, at least two portions of the glutaraldehyde are introduced into the water, with an interval of from 5 to 10 days between respective additions thereof.", "In addition to step (e), it is preferred to provide a further final period of growth, more preferably at least thirty days, during which no further glutaraldehyde is added.", "The aquaculture is most preferably carried out using a sealed aerated pond and is also most preferably carried out in a marine environment.", "Thus, by treating the water of a grow-out pond with from 0.1 to 2 ppm of glutaraldehyde, any opportunist pathogen present may be controlled and the yield of a shrimp or prawn farming operation dramatically improved, as later demonstrated.", "Likewise, it is also possible to utilize the method in the farming of other crustaceans, shellfish and fish.", "The glutaraldehyde is preferably administered as a 50% solution thereof in water, a convenient form which is commercially available.", "The pond is treated with glutaraldehyde prior to stocking.", "The amount of glutaraldehyde to be used will differ from pond to pond depending on size and depth.", "It is recommended that 20 kg of a 50% aqueous solution of glutaraldehyde are added per hectare per metre of depth (i.e.", "at a glutaraldehyde concentration of 1 ppm).", "The correct amount of product should be evenly distributed throughout the pond using a suitable means of dispersion.", "It is acceptable to make a pre-dilution even of the 50% aqueous solution, in water prior to dispersion.", "Once the pond is stocked, the glutaraldehyde may be added, at 20 kg/ha/metre depth, once each week, for a maximum of twelve weeks.", "Treatment is preferably stopped 30 days prior to harvesting the shrimp.", "The above recommendations can be used as a general guide.", "Local conditions such as sea temperature, climate and pond water composition may affect the efficacy.", "It is recommended that the health of the shrimp is checked regularly to ascertain that there are no adverse effects.", "Preferred embodiments of the invention will now be described in more detail with reference to the following Example.", "EXAMPLE Two ponds were chosen in the Far East.", "Pond number 1 had an area of 6000 m2 and a depth of 1 metre.", "It was stocked at a level of 30 post-larvae/m2 and therefore regarded as simulating intensive farming.", "Pond number 2 had an area of 7000 m2 and also a depth of 1 metre.", "It was stocked with 10 post-larvae/m2 and therefore considered as representative of semi-intensive farming.", "A 50% aqueous solution of glutaraldehyde as active ingredient was added to each pond prior to stocking and treated on 30 days out of the 107 days prior to harvest.", "The target dose for each treatment was 1.0-1.4 ppm of the aqueous solution (i.e.", "0.5-0.7 ppm glutaraldehyde), which amounts to 10-14 kg aqueous solution/ha/metre depth (1 ha=10,000 m2).", "In this manner, apart from the last 30 days before harvest, the concentration of glutaraldehyde was maintained within the range 0.5-0.7 ppm.", "The results of this study are shown in Table 1 below.", "Results TABLE 1 Yield (tonnes/ha.", "equivalent) from test ponds following treatment with glutaraldehyde solution (GDA) Treatment Area Depth Stock Length Target Dose Yield Pond (m2) (m) (Post-larvae/m2) Water treatment (days) (ppm) [kg/ha] (Tonnes/ha) Pond 1 6000 1 30 GDA 30 (1.0-1.4) [10-14] 7 Pond 2 7000 1 30 GDA 30 (1.0-1.4) [10-14) 1.36 The yield from Pond 1 (intensive) was 4.2 tonnes in 107 days from 6000 m2, a yield equivalent to 7 tonnes/ha.", "The yield from Pond 2 (semi-intensive) was 0.95 tonnes in 107 days from 7000 m2 equivalent to 1.36 tonnes/ha.", "Note: The normal yield of semi intensive scale farming in Vietnam is about 0.7 tonnes/ha.", "From the above study it can be seen that, even at concentrations as low as 0.5-0.7 ppm, glutaraldehyde (as active ingredient) showed a highly beneficial effect in treating and preserving the water against harmful diseases on a shrimp farm.", "The method enables efficient pond management, which may start with a clean unstocked pond which has been treated with glutaraldehyde to reduce the risk of disease.", "Hygienic water treatment with glutaraldehyde will help prepare the water for introduction of post-larval stage shrimp.", "It can be used to control pathogens at levels which are not harmful to these shrimp.", "It is possible, using the method to ensure that the microbial population of the pond is controlled throughout the growing season.", "This is desirable because micro-organisms may be latent, or introduced by the mechanics of the farming process.", "The method of the invention can be safely used throughout the season helping to reduce this risk.", "When used correctly, a method in accordance with the invention is unlikely to be harmful to the shrimp while providing an excellent margin of safety for the consumer." ] ]

[ [ "Adhesive material based on block copolymers having a p(a)-p(b)-p(a) structure", "A pressure sensitive adhesive comprised of P(A)-P(B)-P(A) block copolymers, wherein P(A) has a glass transition temperature of 0° C. or below, P(B) has a glass transition temperature of 20° C. or above, and P(A) and P(B) are immiscible." ], [ "1.A pressure sensitive adhesive comprised of P(A)-P(B)-P(A) block copolymers, each block copolymer having one middle (co)polymer block P(B) and two end (co)polymer blocks P(A), wherein P(A) represents a (co)polymer block of a component A which is comprised of at least one monomer A1, the (co)polymer block P(A) having a glass transition temperature of 0° C. or below, P(B) represents a (co)polymer block of a component B which is comprised on at least one monomer B1, the (co)polymer block P(B) having a glass transition temperature of 20° C. or above, the (co)polymer block P(B) is insoluble in the (co)polymer block P(A) and the (co)polymer blocks P(A) and P(B) are immiscible.", "2.The pressure sensitive adhesive of claim 1, wherein component A is composed of at least two monomers A1 and A2.3.The pressure sensitive adhesive of claim 1, wherein the monomer A2 has at least one functional group which behaves inertly in a free-radical polymerization reaction and which serves to raise the cohesion of the block copolymer by bonds between the individual block copolymers, the functional group of at least one copolymerized monomer A2 of one block copolymer macromolecule entering into interaction with at least one further block copolymer macromolecule.", "4.The pressure sensitive adhesive as claimed in claim 1, wherein the (co)polymer P(A) has a glass transition temperature of between −80° C. and 0° C., or the (co)polymer block P(B) has a glass transition temperature of between 25° C. and 180° C., or both.", "5.The pressure sensitive adhesive of claim 1, wherein component A comprises at least one monomer A1 of the formula where R1=H or CH3 and R2 is selected from the group consisting of branched or unbranched, saturated alkyl groups having 4 to 14 carbon atoms.", "6.The pressure sensitive adhesive of claim 1, wherein component A comprises at least one monomer A2 of the following formula where R3=H or CH3 and —OR4 represents or contains the functional group for increasing the cohesion.", "7.The pressure sensitive adhesive of claim 1, wherein the cohesion-increasing functional group is selected from the group consisting of hydroxyl, carboxyl, epoxy, acid amide, isocyanato amino, a group containing a photoinitiator for UV crosslinking, and an unsaturated group.", "8.The pressure sensitive adhesive of claim 1, wherein component B comprises at least one monomer B1 which results in (co)polymer blocks P(B) which are capable of forming a two-phase domain structure with the (co)polymer blocks P(A).", "9.The pressure sensitive adhesive of claim 1, having an average molecular weight of between 25,000 and 750,000 g/mol.", "10.The pressure sensitive adhesive of claim 1, wherein (co)polymer blocks P(B) represent between 10 and 60% by weight of the entire block copolymer.", "11.The pressure sensitive adhesive of claim 1, wherein the weight ration of A2 to A1 is between 0.1 and 20.12.The pressure sensitive adhesive of claim 1, further comprising up to 50% by weight of additives selected from the group consisting of resins, crosslinkers, aging inhibitors, light stabilizers, ozone protectants, fatty acids, plasticizers, nucleators, blowing agents, accelerators, fillers, and combinations thereof.", "13.An adhesive tape provided with the pressure sensitive adhesive of claim 1 on one or both sides.", "14.The pressure sensitive adhesive of claim 3, wherein said interaction is a cross-linking reaction.", "15.The pressure sensitive adhesive of claim 9, wherein said average molecular weight is between 100,000 and 500,000 g/mol.", "16.The pressure sensitive adhesive of claim 10, wherein said (co)polymer blocks P(B) represents between 15 and 40% by weight of the entire block.", "17.The pressure sensitive adhesive of claim 11, wherein said weight ratio is between 0.5 and 10.18.The pressure sensitive adhesive of claim 12, wherein said amount is from 20 to 40% by weight.", "19.The adhesive tape of claim 13, wherein said adhesive tape is formed by applying said pressure sensitive adhesive as a melt to one or both sides of a backing." ], [ "The invention relates to pressure sensitive adhesives based on block copolymers of the general type P(A)-P(B)-P(A).", "In the field of pressure sensitive adhesives (PSAs) continuing technological developments in the coating technique mean that there is an ongoing need for new developments.", "Within the industry, hotmelt processes with solventless coating technology are of increasing importance in the preparation of PSAs, since the environmental regulations are becoming ever more stringent and the prices of solvents continue to rise.", "Consequently, solvents are to be eliminated as far as possible from the manufacturing operation for PSA tapes.", "The associated introduction of the hotmelt technology is imposing ever-greater requirements on the adhesives.", "Acrylic PSAs in particular are the subject of very intensive investigations aimed at improvements.", "For high-level industrial applications, polyacrylates are preferred on account of their transparency and weathering stability.", "In addition to these advantages, however, these acrylic PSAs must also meet stringent requirements in respect of shear strength and bond strength.", "This profile of requirements is met by polyacrylates of high molecular weight and high polarity, and, subsequent to the preparation, of highly efficient crosslinking.", "These high shear strength, polar PSAs, however, possess the disadvantage that they are not well suited to the hotmelt extrusion operation, because high application temperatures are necessary and because, furthermore, shearing within the extruder lowers the molecular weight of the polymer.", "This damage significantly reduces the level of the adhesive properties.", "The bond strength and the tack are generally low, since owing to the polar fractions in the adhesives the glass transition temperature is relatively high.", "The shear strengths of the hotmelt-coated acrylic PSAs, in particular, fall distinctly in comparison to the original, solvent-coated PSA.", "At the present time, therefore, different concepts aimed at reducing the flow viscosity and thereby facilitating extrusion coating of these PSAs are being investigated.", "One possibility for this is the very efficient crosslinking of a low viscosity, apolar acrylic PSA not until it is on the backing, where appropriate by copolymerization of UV photoinitiators into the polyacrylate chain.", "For example, benzoin acrylate has been used as a comonomer and the crosslinking has been conducted on the backing using UV light [DE 27 43 979 A1].", "In U.S. Pat.", "No.", "5,073,611, on the other hand, benzophenone and acetophenone are used as copolymerizable monomers.", "Moreover, very efficient chemical crosslinking takes place by radiation in the case of polyacrylates containing double bonds [U.S. Pat.", "No.", "5,741,543].", "Styrene-isoprene-styrene (SIS) block copolymers, in contrast, are widespread elastomers for hotmelt-processable PSAs [preparation processes: U.S. Pat.", "No.", "3,468,972; U.S. Pat.", "No.", "3,595,941; application in PSAs: U.S. Pat.", "No.", "3,239,478; U.S. Pat.", "No.", "3,935,338].", "Good processing properties are achieved by virtue of a relatively low molecular weight and by virtue of a specific morphology [EP 0 451 920 B1], which raises the shear strength.", "These PSAs can be crosslinked very effectively with UV light in the presence of photoinitiators (incorporated by copolymerization, where appropriate) or with electron beams, since the middle blocks contain a large number of double bonds.", "Nevertheless, these elastomers possess disadvantages, such as, for example, severe aging under UV light and in an atmosphere containing oxygen/ozone, and also a relatively low thermal shear strength, so that these PSAs are not suitable for relatively long-term outdoor bonds or for applications in relatively high temperature ranges.", "An improvement in the problem of aging, hotmelt processability, high cohesion, and efficient chemical crosslinking by radiation is provided by the combination of SIS polymers with polyacrylates.", "U.S. Pat.", "No.", "5,314,962 describes A-B-A block copolymers as elastomers for adhesives, but these lead to an increase in cohesion of the PSA merely owing to A-domain formation and therefore lack great shear strength, especially at high temperatures.", "EP 0 921 170 A1 describes A-B-A block copolymers which have been modified with additions of resin.", "Here, no crosslinking has been carried out, so that in this case as well the shear strength of the PSAs described is very low.", "It is an object of the invention, therefore, to provide improved pressure sensitive adhesives based on polyacrylate which exhibit the disadvantages of the prior art only to a reduced extent, if at all, and with which it is possible to achieve an increase in the cohesion, and which, in particular, are suitable for processing by the hotmelt process and for use as hotmelt adhesives, without losing the properties which are advantageous for use as a PSA.", "This object is achieved, surprisingly and unforeseeably, by the pressure sensitive adhesive as described in the main claim.", "The subclaims relate to improved developments of the pressure sensitive adhesive, and to their use.", "Claim 1 relates accordingly to a pressure sensitive adhesive based on block copolymers of the general type P(A)-P(B)-P(A), each block copolymer being composed of one middle (co)polymer block P(B) and two end (co)polymer blocks P(A), characterized in that P(A) represents a (co)polymer block of a component A which is composed of at least one monomer A1, the (co)polymer block P(A) possessing a glass transition temperature of 0° C. or below, P(B) represents a (co)polymer block of at least one component B which is composed of at least one monomer B1, the (co)polymer block P(B) possessing a glass transition temperature of 20° C. or above, the (co)polymer block P(B) is insoluble in the (co)polymer block P(A), the blocks P(B) and P(A) are immiscible.", "In one first advantageous embodiment of the inventive pressure sensitive adhesive, component A is composed of at least two monomers A1 and A2.In another outstanding embodiment, component A2 includes at least one functional group which behaves inertly in a free-radical polymerization reaction and which serves to raise the cohesion of the block copolymer; in particular, by bonds between the individual block copolymers, the functional group of component A2 of one block copolymer macromolecule entering into interaction with at least one further block copolymer macromolecule; in particular, by a crosslinking reaction.", "This crosslinking is initiated advantageously by means of high-energy irradiation, examples being electron beams and UV light.", "It is also appropriate to supply thermal energy which induce crosslinking reaction, corresponding to the choice of the respective functional groups.", "The selection of the appropriate energy supply for a respective crosslinking reaction in the case of a given functional group is prior art and is known to the skilled worker.", "Bonds between the individual block copolymers in the above sense are all bonds ranging from purely physical forces of attraction through to bonds originating from a chemical reaction (for example, covalent bonds, ionic bonds, van der Waals bonds).", "Included here, in addition to the above-described crosslinking reactions, for example, are dipole-dipole interactions and/or hydrogen bonds.", "It may be mentioned here that the function of forming bonds may also be served by interlinks, interloops, interhooks or the like between the macromolecules or side chains located thereon.", "In one very favorable development of the inventive PSAs the block P(A) possesses a glass transition temperature of between −80° C. and 0° C. and/or the block P(B) a glass transition temperature of between 25° C. and 180° C. As monomers A1 it is possible to use acrylic monomers or vinyl monomers which, alone or in combination with monomer A2, lower the glass transition temperature to below 0° C. In the inventive context it has proven very advantageous to use, as monomer A1, at least one compound of the following general formula where R1=H or CH3 and R2 is chosen from the group of the branched or unbranched, saturated alkyl groups having from 4 to 14 carbon atoms, very preferably having from 4 to 9 carbon atoms.", "Specific examples of such (modified) acrylic and methacrylic esters are n-butyl acrylate, n-pentyl acrylate, n-hexyl acrylate, n-heptyl acrylate, n-octyl acrylate, n-nonyl acrylate, and branched isomers thereof, an example being 2-ethylhexyl acrylate.", "Additionally it is favorable in the context of the invention to use, optionally, as monomers A1, vinyl monomers from the following groups: vinyl esters, vinyl ethers, vinyl halides, vinylidene halides, vinyl compounds with aromatic rings and aliphatic rings in hetero-position.", "Nonexclusive examples of these compounds are vinyl acetate, vinylformamide.", "As monomer A2 it is advantageous to use acrylic monomers or vinyl monomers which, alone or in combination with monomer A1, lower the glass transition temperature of the block copolymer to below 0° C. and carry at least one functional group for crosslinking.", "In one advantageous variant of the process of the invention use is made, as monomer A2, of one or more compounds of the following general formula where R3=H or CH3 and the radical —OR4 represents or contains the functional group for raising the cohesion of the pressure sensitive adhesive.", "For the functional group it is advantageous to choose a hydroxyl, a carboxyl, an epoxy, an acid amide, an isocyanato or an amino group, a group containing a UV photoinitiator for UV crosslinking, or an unsaturated group.", "Particularly preferred examples of component A2 are hydroxyethyl acrylate, hydroxypropyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, acrylic acid, methacrylic acid, allyl alcohol, maleic anhydride, itaconic anhydride, itaconic acid, benzoin acrylate, acrylated benzophenone, acrylamide, dimethylacrylamide, and glyceridyl methacrylate, this list not being conclusive.", "In addition to acrylic monomers it is also possible to use vinyl compounds having double bonds which do not react during the polymerization.", "Particularly preferred examples thereof are isoprene and butadiene.", "For acrylates modified with double bonds, allyl acrylate and acrylated cinnamic esters are especially advantageous.", "Further very advantageous methods of introducing unsaturated compounds are described in U.S. Pat.", "No.", "5,741,543.As monomer B1 use ought to be made of at least one monomer such that the resulting (co)polymer blocks P(B) are capable of forming a 2-phase domain structure with the (co)polymer blocks P(A).", "Examples hereof are vinylaromatics, methyl methacrylates, cyclohexyl methacrylates, isobornyl methacrylates; especially methyl methacrylate and styrene.", "In one preferred embodiment of the inventive block copolymers the pressure sensitive adhesive possesses an average molecular weight of between 25,000 and 750,000 g/mol, in particular between 100,000 and 500,000 g/mol.", "It is further of advantage if the fraction of the polymer blocks P(B) lies between 10 and 60 percent by weight of the entire block copolymer, more preferably between 15 and 40 percent by weight.", "The weight fraction of component A2 in relation to component A1 lies preferably between 0.1 and 20, in particular between 0.5 and 5.For preparing the block copolymers of the invention it is possible to make use of any controlled-growth polymerizations which proceed in accordance with free-radical mechanisms.", "Also suitable are the polymerization reactions which proceed in accordance with anionic mechanisms, particularly for those block copolymers of the invention where there is no crosslinking-capable group or where this group is inert toward reactions in which ions are involved or in which ions are formed during the course of the reaction.", "Examples of advantageous preparation processes are ATRP (atom-transfer radical polymerization), polymerization controlled by nitroxide or TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy pyrrolidinyloxyl) and/or its derivatives, or polymerization by the RAFT process (rapid addition-fragmentation chain transfer).", "For the preparation it is possible, for example, to use a difunctional initiator which in one step initiates the polymerization of the component B and then in a second step polymerizes components A (or A1 and A2), as end blocks (II), it being possible as an option to isolate the intermediate.", "I-R-I in the reaction equation which follows represents the difunctional initiator containing the functional groups I.", "In addition, the triblock copolymer may be prepared by free-radical recombination of the macromonomers P(A)-P(B)* (III).", "2P(A)-P(B)*→P(A)-P(B)-P(B)-P(A) (III) For polymerizing the block copolymers it is possible with preference to use nitroxide regulators for free-radical control.", "The polymerization may be conducted in the presence of one or more organic solvents and/or in the presence of water or without solvent.", "It is preferred to use as little solvent as possible.", "The polymerization time, depending on conversion rate and temperature, is between 6 and 48 h. In the case of solution polymerization, preferred solvents used are esters of saturated carboxylic acids (such as ethyl acetate), aliphatic hydrocarbons (such as n-hexane or n-heptane), ketones (such as acetone or methyl ethyl ketone), special boiling point spirit or mixtures of these solvents.", "For polymerization in aqueous media or in mixtures of organic and aqueous solvents, it is preferred to add emulsifiers and stabilizers known to those skilled in the art for the polymerization.", "Polymerization initiators used include customary radical-forming compounds such as, for example, peroxides, azo compounds, and peroxosulfates.", "Mixtures of initiators can also be used.", "For free-radical stabilization use is made of nitroxides of type (IVa) or (IVb) where R1, R2, R3, R4, R5, R6, R7 and R8 represent identical or different compounds or atoms: one or more halides, such as chlorine, bromine or iodine, for example the group of the linear, branched cyclic, saturated or unsaturated hydrocarbons represent from the group of the esters —COOR9 or alkoxides —OR10 or phosphonates —PO(OR11)2, where R9, R10 or R11 representatively stand for groups of the linear, branched cyclic, saturated or unsaturated hydrocarbons.", "The compounds (IVa) or (IVb) may also be attached to polymer chains of any kind and may therefore be utilized for synthesizing the block copolymers, as macroradicals or macroregulators.", "More preferred are controlled regulators for the polymerization of compounds of the following type: 2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (generally known and commercially available as PROXYL), 3-carbamoyl-PROXYL, 2,2-dimethyl 4,5-cyclohexyl-PROXYL, 3-oxo-PROXYL, 3-hydroxylimine-PROXYL, 3-aminomethyl-PROXYL, 3-methoxy-PROXYL, 3-t-butyl-PROXYL, 3,4-di-t-butyl-PROXYL 2,2,6,6-tetramethyl-1-piperidinyloxy pyrrolidinyloxyl (generally known and commercially available as TEMPO), 4-benzoyloxy-TEMPO, 4-methoxy-TEMPO, 4-chloro-TEMPO, 4-hydroxy-TEMPO, 4-oxo-TEMPO, 4-amino-TEMPO, 2,2,6,6-tetraethyl-1-piperidinyloxyl, 2,2,6-trimethyl-6-ethyl-1-piperidinyloxyl N-tert-butyl-1-phenyl-2-methyl propyl nitroxide N-tert-butyl-1-(2-naphthyl)-2-methyl propyl nitroxide, N-tert-butyl-1-diethylphosphono-2,2-dimethyl propyl nitroxide N-tert-butyl-1-dibenzylphosphono-2,2-dimethyl propyl nitroxide N-(1-phenyl-2-methylpropyl)-1-diethylphosphono-1-methyl ethyl nitroxide di-t-butyl nitroxide diphenyl nitroxide T-butyl t-amyl nitroxide.", "As a further controlled polymerization method use is made of atom transfer radical polymerization ATRP, in which case as initiator it is preferred to use monofunctional or difunctional secondary or tertiary halides and, for the obstruction of the halide(s), complexes of Cu, of Ni, of Fe, of Pd, of Pt, of Ru, of Os, of Rh, of Co, of Ir, of Cu, of Ag or of Au [EP 0 824 111; EP 0 826 698; EP 0 824 110; EP 0 841 346; EP 0 850 957].", "The various possibilities of ATRP are furthermore described in patents U.S. Pat.", "No.", "5,945,491, U.S. Pat.", "No.", "5,854,364 and U.S. Pat.", "No.", "5,789,487.As a further preferred variant, the RAFT process (reversible addition fragmentation chain transfer) is carried out.", "The process is described in detail in patents WO 98/01478 and WO 99/31144.Suitable with particular advantage for preparing block copolymers are trithiocarbonates [Macromolecules 2000, 33, 243-245], which in a first step randomly copolymerize monomers of the middle block, subsequently can be isolated or can be used directly for the subsequent copolymerization of monomers of the end blocks.", "In order to prepare a pressure sensitive adhesive the block copolymers described so far are processed furthers in solution or from the melt.", "Suitable solvents are one or more organic solvents.", "In order to produce a pressure sensitive adhesive tape the block copolymer must be modified with resins.", "Examples of resins which can be used include terpene resins, terpene phenol resins, C5 and C9 hydrocarbon resins, pinene resins, indene resins, and rosins, alone and also in combination with one another.", "In principle, however, it is possible to use all resins which are soluble in the corresponding polyacrylate; reference may be made in particular to all aliphatic, aromatic, alkylaromatic hydrocarbon resins, hydrocarbon resins based on pure monomers, hydrogenated hydrocarbon resins, functional hydrocarbon resins, and also natural resins.", "The weight fraction of the resins within the block copolymer may be between 0 and 50% by weight, more preferably between 20 and 40% by weight.", "It is additionally possible to add plasticizers, various fillers (for example, carbon black, TiO2, solid or hollow beads of glass or other materials, nucleators), blowing agents, compounding agents and/or aging inhibitors.", "In an advantageous development, crosslinker substances compatible/soluble in P(A) are added.", "Examples of suitable crosslinkers include polyfunctional acrylates, polyfunctional hydroxides, polyfunctional expoxides, polyfunctional amines or polyfunctional isocyanates.", "This list does not make any claim to completeness.", "In one advantageous development, UV photoinitiators are added to the block copolymers, especially to those with groups capable of crosslinking and, in this context, especially those having groups capable of crosslinking by initiation through UV light.", "Suitable and advantageous photoinitiators are, for example, benzoin ethers, such as benzoin methyl ether and benzoin isopropyl ether, for example, substituted acetophenones, such as 2,2-diethoxyacetophenone (available as Irgacure 651 from Ciba Geigy), 2,2-dimethoxy-2-phenyl-1-phenylethanone, dimethoxyhydroxyacetophenone, substituted alpha-ketols, such as 2-methoxy-2-hydroxypropiophenone, for example, aromatic sulfonyl chlorides, such as 2-naphthylsulfonyl chloride, for example, and photoactive oximes, such as 1-phenyl-1,2-propanedione 2-(o-ethoxycarbonyl) oxime.", "A feature of one further development, which makes the process of the invention particularly advantageous for the production of adhesive tapes, for example, is that the pressure sensitive adhesive is processed further from the melt, and that it is applied in particular to a backing.", "As backing material, for adhesive tapes for example, it is possible in this context to use the materials which are customary and familiar to the skilled worker, such as films (polyesters, PET, PE, PP, BOPP, PVC), nonwovens, foams, wovens, and woven films, and also release paper (glassine, HDPE, LDPE).", "This list should not be conclusive.", "The crosslinking of the hotmelt pressure sensitive adhesives of the invention is accomplished by brief UV irradiation in the range of 200-400 nm using standard commercial high-pressure or medium-pressure mercury lamps with an output, for example, of from 80 to 200 W/cm or ionizing radiation, such as electron beam curing, for example.", "For UV crosslinking it may be appropriate to adapt the lamp output to the web speed or to carry out partial shading of the web, while running it slowly, in order to reduce the thermal stress to which it is subjected.", "The irradiation time is governed by the construction and output of the respective lamps.", "Crosslinking may also be initiated or promoted by thermal energy, particularly at a temperature of 70-140° C. For testing, depending on the sample, PET films or siliconized release papers are coated with an application rate of 50 g/m2.The invention further relates to the pressure sensitive adhesive obtained by the process of the invention or by one of its developments.", "The content of the invention is furthermore the use of the pressure sensitive adhesive thus obtained for an adhesive tape, in which case the acrylic pressure sensitive adhesive is present as a single-side or both-sides film on a backing.", "The intention is to illustrate the invention below by a number of examples, without thereby wishing to subject it to any unnecessary restriction.", "As a function of the desired technical adhesive properties of the acrylic hotmelts, a selection of acrylic and vinylic monomers is made.", "Quantities, proportions, and percentage fractions are based on the total amount of the monomers.", "EXAMPLES Test Methods The following test methods were employed for evaluating the technical adhesive properties of the PSAs prepared.", "Shear Strength (Test A1, A2) A strip of the adhesive tape, 13 mm wide, was applied to a smooth, cleaned steel surface.", "The application area was 20 mm×13 mm (length×width).", "The following procedure was then undertaken: Test A1: At room temperature, a 1 kg weight was fastened to the adhesive tape and the time recorded until the weight fell off.", "Test A2: At 70° C., a 1 kg weight was fastened to the adhesive tape and the time recorded until the weight fell off.", "The shear stability times measured are each reported in minutes and correspond to the average of three measurements.", "180° Bond Strength Test (Test B) A strip, 20 mm wide, of an acrylate pressure adhesive applied as a film to polyester was applied to steel plates.", "The PSA strip was pressed onto the substrate twice using a 2 kg weight.", "The adhesive tape was then immediately peeled from the substrate at 300 mm/min and at an angle of 180°.", "The steel plates were washed twice with acetone and once with isopropanol.", "All measurements were conducted at room temperature under climate-controlled conditions.", "The results of the measurements are reported in N/cm and are averaged from three measurements.", "Determination of the Gel Fraction (Test C) After careful drying, the solvent-free samples of adhesive are welded into a pouch of polyethylene nonwoven (Tyvek web).", "The difference in the weight of the samples before extraction and after extraction with toluene is used to determine the gel index as a percentage of the toluene-insoluble weight fraction of the polymer.", "Preparation of the Samples The acrylates, methacrylates, and styrene used are available commercially and were purified by distillation before being used.", "Trithiocarbonate (V) as a regulator was prepared in accordance with Macromolecules 2000, 33, 243-245 and Synth.", "Commun.", "1988, 18, 1531-1536.Procedure for the Polymerizations The polymerization was generally conducted in two stages.", "In the first step the polyacrylate blocks were prepared, in the second step the polystyrene and/or polymethyl methacrylate blocks.", "Example 1 A 2,000 ml Schlenk vessel was charged with 800 g of n-butyl acrylate, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=412,000 g/mol, Mw/n=1.67).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 30 weight fractions of Foral 85™ (Hercules) and 5 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was spread from solution at 50 g/m2 onto a siliconized release paper and then dried at 120° C. for 15 minutes.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 2 A 2,000 ml Schlenk vessel was charged with 800 g of 2-ethylhexyl acrylate, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=401,000 g/mol, Mw/n=1.70).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of Foral 85™ (Hercules) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was freed from the solvent and then coated from the melt through a slot die at 50 g/m2 onto a siliconized release paper and then irradiated with an electron beam dose (EB dose) of 60 kGy with an acceleration voltage of 230 kV.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 3 A 2,000 ml Schlenk vessel was charged with 650 g of 2-ethylhexyl acrylate, 150 g of N-tert-butylacrylamide, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=384,000 g/mol, Mw/n=1.73).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of Foral 85™ (Hercules) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was freed from the solvent and then coated from the melt through a slot die at 50 g/m2 onto a siliconized release paper and then irradiated with a EB dose of 60 kGy with an acceleration voltage of 230 kV.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 4 A 2,000 ml Schlenk vessel was charged with 400 g of 2-ethylhexyl acrylate, 400 g of n-butyl acrylate, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "The reaction mixture was then cooled to room temperature and the polymer was precipitated from 6.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "After isolation through a cooled frit and drying under reduced pressure, 400 g of the polymer were again introduced into a 2,000 ml Schlenk vessel, 500 ml of toluene, 0.25 g of 1,1′-azobis(1-cyclohexanecarbonitrile) (Vazo 88™, DuPont), 150 g of methyl methacrylate were added, the vessel was degassed three times and then the polymerization was carried out under argon at 80° C. with a reaction time of 8 h, with stirring.", "For isolation, the reaction mixture was cooled to RT, the polymer was dissolved in 700 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=445,000 g/mol, Mw/n=1.61).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of Foral 85™ (Hercules) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was freed from the solvent and then coated from the melt through a slot die at 50 g/m2 onto a siliconized release paper and then irradiated with a EB dose of 60 kGy with an acceleration voltage of 230 kV.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 5 A 2,000 ml Schlenk vessel was charged with 760 g of n-butyl acrylate, 40 g of acrylic acid, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=430,000 g/mol, Mw/n=1.76).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of Foral 85™ (Hercules) and 3 weight fractions of Catenex 945™ (Shell) and 0.6 weight fractions of aluminum acetylacetonate were added.", "The compounded composition was spread from solution at 50 g/m2 onto a siliconized release paper and then dried at 120° C. for 20 minutes.", "To analyze the technical adhesive properties, test methods A1, A2 and B were carried out.", "Example 6 A 2,000 ml Schlenk vessel was charged with 780 g of 2-ethylhexyl acrylate, 20 g of hydroxyethyl acrylate, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=405,000 g/mol, Mw/n=1.71).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of Foral 85™ (Hercules) and 3 weight fractions of Catenex 945™ (Shell) and 0.6 weight fractions of Desmodur N75™ (Bayer) were added.", "The compounded composition was spread from solution at 50 g/m2 onto a siliconized release paper and then dried at 120° C. for 20 minutes.", "To analyze the technical adhesive properties, test methods A1, A2 and B were carried out.", "Example 7 A 2,000 ml Schlenk vessel was charged with 796 g of 2-ethylhexyl acrylate, 4 g of acrylated benzophenone Ebecryl 36™ (UCB), 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=422,000 g/mol, Mw/n=1.65).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of RX-207™ (Cray Valley) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was spread from solution at 50 g/m2 onto a siliconized release paper and then dried at 120° C. for 15 minutes.", "For curing, these samples were irradiated at 20 m/min using a medium-pressure mercury lamp (120 W/cm) with 4 passes through the lamp.", "As a reference, the unirradiated PSA tape was likewise tested (example 71).", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 8 A 2,000 ml Schlenk vessel was charged with 770 g of 2-ethylhexyl acrylate, 20 g of N-tert-butylacrylamide, 4 g of benzoin acrylate, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=397,000 g/mol, Mw/n=1.73).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of RX-207™ (Cray Valley) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was spread from solution at 50 g/m2 onto a siliconized release paper and then dried at 120° C. for 15 minutes.", "For curing, these samples were irradiated at 20 m/min using a medium-pressure mercury lamp (120 W/cm) with 4 passes through the lamp.", "As a reference, the unirradiated PSA tape was likewise tested (example 8′).", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 9 A 2,000 ml Schlenk vessel was charged with 750 g of 2-ethylhexyl acrylate, 40 g of methyl acrylate, 10 g of acrylated cinnamic ester, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 160 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=402,000 g/mol, Mw/n=1.78).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of RX-207™ (Cray Valley) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was concentrated and then coated from the melt through a slot die onto a siliconized release paper.", "For curing, the adhesive tape sample was irradiated with an EB dose of 20 kGy with an acceleration voltage of 230 kV.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Example 10 A 2,000 ml Schlenk vessel was charged with 750 g of 2-ethylhexyl acrylate, 40 g of methyl acrylate, 10 g of acrylated cinnamic ester, 400 ml of toluene, 0.156 g of the trithiocarbonate (V) and 0.12 g of azoisobutyronitrile (AIBN), the vessel was degassed three times and then the polymerization was conducted under argon.", "For initiation the reaction mixture was heated to 60° C. and polymerized for 8 h with stirring.", "Then, under reduced pressure, the solvent and the remaining monomers were separated off by distillation and 250 ml of toluene and also 240 g of styrene were added.", "After a further reaction time of 24 h at 90° C., for isolation, the reaction mixture was cooled to RT and the polymer was dissolved in 800 ml of dichloromethane and then precipitated from 8.0 L of methanol (cooled to −78° C.) with vigorous stirring.", "The precipitate was filtered off on a cooled frit and then analyzed via GPC (Mn=455,000 g/mol, Mw/n=1.89).", "100 g of the block copolymer were dissolved in 200 g of toluene and then 20 weight fractions of RX-207™ (Cray Valley) and 3 weight fractions of Catenex 945™ (Shell) were added.", "The compounded composition was concentrated and then coated from the melt through a slot die onto a siliconized release paper.", "For curing, the adhesive tape sample was irradiated with an EB dose of 20 kGy with an acceleration voltage of 230 kV.", "To analyze the technical adhesive properties, test methods A1, A2, B and C were carried out.", "Results The table below lists the technical adhesive properties of examples 1 to 4.TABLE 1 BS-steel Gel index SST RT SST 70° C. [N/cm] [%] Example Test A1 Test A2 Test B Test C 1 390 2 13.5 0 2 +10000 1020 5.1 34 3 +10000 4635 4.6 40 4 +10000 2270 5.0 32 Application rate: 50 g/m2.SST: Shear stability times [min] RT: Room temperature BS: Bond strength on steel Example 1 shows that with the use of the block copolymer of the invention it is possible to produce pressure sensitive adhesives of very high bond strength.", "Through electron beam crosslinking it is possible to achieve a marked increase in the shear strength—especially under hot conditions.", "Example 3 demonstrates that the thermal shear strength can be increased further by raising the glass transition temperature, by means of N-tert-butylacrylamide in the end blocks.", "With a PMMA middle block, as well, good cohesion is achieved (example 4).", "Table 2 shows the results of the technical adhesive evaluations of examples 5 to 10.TABLE 2 BS-steel Gel index SST RT SST 70° C. [N/cm] [%] Example Test A1 Test A2 Test B Test C 5 +10000 2350 5.8 — 6 +10000 1755 5.7 — 7 +10000 1090 6.4 49 7′ 765 15 12.7 0 8 +10000 4550 6.0 54 8′ 465 5 12.2 0 9 +10000 1105 5.7 52 10 +10000 3005 5.4 55 Application rate: 50 g/m2.SST: Shear stability times [min] RT: Room temperature BS: Bond strength on steel Examples 5 and 6 demonstrate that both acrylic acid and hydroxyethyl acrylate are suitable for inserting into the block copolymer functional groups which can be utilized for thermal crosslinking with metal chelates or polyfunctionalized isocyanates, respectively, and which therefore make possible PSAs of high shear strength.", "Examples 7 and 8 show that, in addition, photoinitiators can be copolymerized and after UV irradiation lead to gel formation.", "The comparison with the unirradiated specimens (examples 7′ and 8′) provides significantly higher figures for the irradiated specimens, both in terms of shear strength at room temperature and in terms of the thermal shear strength, which correlates in turn with a marked increase in cohesion.", "Examples 9 and 10 demonstrate that through the incorporation of double bonds electron beam crosslinkability is facilitated and thus it is possible to crosslink acrylic block copolymers efficiently.", "This is a 371 of PCT/EP01/08739 filed 27 Jul.", "2001 (international filing date)." ] ]

[ [ "Surface-attached polyfunctional polymer networks for sensor chips", "The invention relates to polyfunctional polymer networks comprising an assembly of cross-linked polymer subchains attached to a surface, with each polymer subchain comprising a multitude of identical or different repeating units carrying one or more functional groups which allows an interaction with a sample or probe molecule." ], [ "1.A polyfunctional polymer network comprising an assembly of cross-linked polymer subchains covalently attached to a surface, wherein each polymer subchain comprises a multitude of identical or different repeating units carrying one or more functional groups which allow an interaction of the polymer with a sample or probe molecule.", "2.A polyfunctional polymer network according to claim 1, wherein said one or more functional groups allow a covalent interaction of the polymer with said sample or probe molecule.", "3.A polyfunctional polymer network according to claim 1, wherein said one or more fuctional groups are chosen from carboxylic acids, maleinimides, N-hydroxy succinimides, epoxides, isothiocyanates, isocyanates or azides.", "4.A polyfunctional polymer network according to claim 1, wherein said sample molecule or probe molecule is chosen from proteins, peptides, polysaccharides or nucleic acids and derivatives thereof.", "5.A polyfunctional polymer network according to claim 1, wherein said polymer subchains comprises segments that make said polymer network water-swellable.", "6.A polyfunctional polymer network according to claim 5, wherein said water-swellability is provided by monomers chosen from acrylic acid, methacrylic acid, dimethyl acrylamide or vinyl pyrrolidon.", "7.A polyfunctional polymer network according to claim 1, further comprising a multitude of identical or different probe molecules immobilized at said polymer subchains via a reaction with said functional groups.", "8.A polyfunctional polymer network according to claim 7, wherein said probe molecules are selected from nucleic acids, PNAs, polysaccharides, proteins and peptides.", "9.A surface carrying a polyfunctional polymer network according to claim 1.10.A surface according to claim 9, wherein said polyfunctional polymer network forms patterned arrays.", "11.A process for the production of a polyfunctional polymer network according to claim 1, comprising the steps of: a) covering a surface with a monolayer of a polymerization initiator which comprises one or more functional groups suitable for covalent attachment to the surface; and b) initiating and carrying out a crosslinking polymerization reaction in the presence of monomers carrying functional groups which allow a coupling reaction, preferably a covalent coupling reaction, of the obtained polymer subchains with specific sample molecules or probe molecules and in the presence of cross-linkers.", "12.A process according to claim 11, wherein said initiator comprises a chlorosilane, an alkoxysilane, a disulphide or a thiol group.", "13.A process according to claim 11 wherein said initiator comprises a group chosen from azo groups, peroxo groups, or a ketone group in conjugation with an aromatic system.", "14.A process according to claim 13, wherein said initiator comprises a group chosen from aromatic ketones or aromatic ketones containing sulphur.", "15.A process according to claims 11, wherein said cross-linker is selected from the group consisting of bisacrylates, bismethacrylates and bisacrylamides.", "16.A process for the detection of sample nucleic acid molecules, using a polyfunctional polymer network according to claim 7, which comprises the steps of: (a) allowing a hybridization reaction to take place between the probe and the sample; (b) followed by removal of the non hybridized nucleic acid molecules in a washing step; and (c) detection of the hybridized nucleic acid molecules.", "17.Process for purifying a compound from a sample comprising the steps of (a) contacting said sample with a polyfunctional polymer network of any of claims 1 to 8 under conditions that allow binding of said compound to the functional groups of the polymer network or to the probe molecules, and (b) removing material from said sample that has not bound to said polyfunctional polymer network or said probe molecules.", "18.Process according to claim 17 further comprising (c) eluting the bound complex from said polyfunctional polymer network.", "19.A process according to claim 17, wherein said compound is a nucleic acid, a polysaccharide or a polypeptide or a complex thereof, preferably an antibody or a fragment or derivative thereof.", "20.An affinity matrix, comprising a surface according to claim 9.21.A sensor chip, comprising a surface according to claim 9.22.A medical or diagnostic instrument, comprising a surface according to claim 9.25.A method comprising the steps of: providing a surface according to claim 9; immobilizing a starter molecule for the formation of oligomers or polymers, on the surface; and conducting polymerization on the surface.", "26.A method according to claim 25, wherein the polymerization is nucleic acid synthesis.", "27.A method according to claim 25, wherein the polymerization is peptide synthesis.", "28.A method comprising the steps of: providing a polyfunctional polymer network according to claim 1 as a gel; adding a plurality of sample molecules to the gel; applying an electric field to the gel; and separating the sample molecules." ], [ "Due to the steadily growing importance of microtechniques in a wide variety of scientific applications, the development of systems which allow the interaction of molecules with surfaces remains a critical issue.", "Such interactions include the possibility of removing specific molecules from a sample, e.g.", "to facilitate their analysis/detection, but also of presenting molecules on a surface, thus allowing subsequent reactions to take place.", "These principles for the immobilization of molecules can be applied in sensor or chromatographic systems or for the provision of modified surfaces in general.", "In recent years there have been numerous approaches to fabricate sensor chips which are based on self-assembled monolayers (SAM's) of bifunctional molecules which directly or indirectly couple sample molecules to the sensor surface.", "Typically, these bifunctional molecules carry a silane or thiol/disulfide moiety in order to achieve a bond with the inorganic surface and an additional functional group (e.g.", "amino or epoxide groups) which interact with sample molecules, often contained in biological samples in the form of an oligonucleotide, a protein or a polysaccharide etc.", "While the formation of a direct bond between the bifunctional compound and the sample molecule is possible, the sample molecules do not necessarily interact directly with the couplers forming the monolayer.", "Alternatively, appropriate immobilized biomolecules themselves can act as probes for the detection of sample molecules.", "Such probe molecules can equally be immobilized via a reaction with the free functional groups of the monolayer.", "In particular, if biomolecules are used as probe molecules, their presence may significantly enhance the specificity of the interaction of the sample molecules with the modified surface.", "For example, in cases where the fast analysis of a sample of DNA fragments or molecules is required, the monolayers of bifunctional molecules can first be brought in contact with synthetic oligonucleotides which will thus be immobilized.", "Subsequently, the hybridization of specific molecules, such as compatible strands from a sample is detected, e.g.", "via fluorescence microscopy, if dye-labeled sample molecules are used.", "Although these techniques are well established for this purpose, the application of standard detection methods is problematic, especially in cases where the surface area available for the detection of one specific type of sample molecules is restricted, e.g.", "if a variety of molecules is to be analyzed in a parallel process, since the monolayers are limited in their graft density.", "For example, since the number of hybridized double strands per surface unit of a sensor can not easily be increased, suitable detectors have to meet very high requirements with regard to their sensitivity.", "Thus, the minimum surface area on a sensor necessary for the detection of one type of oligonucleotide can not be easily reduced.", "Moreover, the maximum density, i.e.", "one sample or probe molecule per functional group of the couplers can hardly be attained, since due to sterical hindrance on the two-dimensionally extended monolayer, only a fraction of the functional groups will be able to react with sample or probe molecules.", "Thus, the overall graft density is low and normally not well defined.", "Similar problems with regard to the limited number of reaction sites per surface unit can arise in other applications, where it is desirable to immobilize an increased amount of molecules on a surface.", "Various attempts have been made to overcome the problems outlined above.", "As regards the analysis of oligonucleotides, it has been tried to increase the graft density on the surface by using oligomers or polymers which carry an oligonucleotide strand (or a functional group for its attachment) together with a suitable group which allows the bonding of these oligomers or polymers to the surface of the sensor chip.", "Due to the increased flexibility of the oligomeric or polymeric chains, a larger fraction of the bifunctional oligomer or polymer molecules which are coupled to the surface is able to immobilize oligonucleotide probe molecules.", "However, the total oligonucleotide graft density is not significantly increased, because the graft density of the bifunctional oligomeric or polymeric molecules on the surface is limited.", "This is a consequence of the fact that the self-assembly of the oligomers or polymers is hindered for kinetic reasons, because once the sensor surface is covered with such molecules, further polymers will have to diffuse against a concentration gradient in order to reach the surface.", "A different approach to the above-mentioned self-assembled monolayers of bifunctional molecules for immobilization is using networks for DNA analysis.", "A disadvantage is that these networks are not coupled to the sensor surface and are not structured, i.e.", "do not form patterned arrays.", "Moreover, since the networks must be swellable in the used hybridisation medium there is a risc that the network is detached from the surface.", "Accordingly, it is an object of the present invention to provide a surface which is modified with a polymer layer comprising functional groups for the interaction with sample or probe molecules, wherein the number of molecules interacting per surface unit is markedly increased compared to conventional monolayers of bifunctional molecules.", "In addition, the density of available interaction sites should be higher than that obtained from the reaction of bifunctional polymers or oligomers with the surface.", "In the specific case of the detection of DNA molecules such as oligonucleotides, the object can be expressed as the provision of a surface with a graft density of synthetic oligonucleotide strands which is higher than that created by coupling the respective oligonucleotides to a functionalized monolayer of low molecular weight couplers.", "Also, the graft density should be higher then that resulting from the reaction of polymers or oligomers modified with a synthetic oligonucleotide single strand with the surface.", "This object has been achieved by a surface to which a polyfunctional polymer network comprising an assembly of crosslinked polymer subchains (forming the “meshes” of the network) (hereinafter simply also referred to as “network”) is covalently attached, which polymer subchains may each comprise a multitude of identical or different repeating units carrying one or more functional groups that allow an interaction, preferably a covalent interaction, of the polymer with sample or probe molecules.", "The number of “anchor groups” attaching or coupling the network to the surface determines the adhesion strenght of the network to the surface and the mechanical strength of the entire system.", "The number of these anchoring points is (as will be explained below) controlled via the surface density of the used immobilized initiators.", "If, for example, such a polyfunctional polymer network is used to immobilize one or more synthetic oligonucleotide probes, complementary nucleic acids can subsequently be detected from a mixture of sample molecules after a hybridization reaction has taken place.", "Surprisingly, it has been found that such a surface-bound polyfunctional polymer network does not suffer from the problems of conventional detection methods where a high functional group density at the surface could not be achieved.", "Moreover, since the flexibility of the polyfunctional polymer network allows a complete coverage of the sensor surface, surface effects, e.g.", "during laser scanning, can be avoided.", "Further, the surface-bound polyfunctional networks are stable, may easily be structured, i.e.", "may form patterned arrays, and provide an improvement in sensitivity of the sensor.", "The term “interaction”, as used in this specification includes the formation of covalent bonds, as well as attractive ionic and van-der-Waal's forces and hydrogen bonds.", "The respective functional moiety within the polyfunctional polymer network or the probe molecules, which defines the type of interaction, will be selected according to the desired application of the surface according to the invention.", "The expression “immobilize” is used hereinafter for an interaction of molecules with the polyfunctional polymer network resulting in the formation of a bond which is permanent under the chosen conditions.", "For example, probe molecules are immobilized by the polyfunctional network during their application on a sensor surface.", "However, by changing conditions (e.g.", "pH-value, addition of specific cleaving agents) an immobilization may sometimes be reversed.", "The term “sample molecule” shall be used herein for molecules which are present in a sample and which couple temporarily or permanently to the polyfunctional network according to the invention.", "The present invention includes two general principles for an interaction of the inventive polyfunctional network with the sample molecules.", "In a first embodiment, the functional groups comprised within the polyfunctional network are chosen in order to allow a direct interaction of the chains with the sample molecules.", "In a second embodiment, probe molecules are immobilized at the functional groups of the polyfunctional network, and an interaction takes place between those probe molecules and the sample molecules.", "Suitable probe molecules are molecules which are at least bifunctional, so that after their coupling to the polyfunctional polymer network new interaction sites are present in the surface-bound polyfunctional network according to the invention, which allow an interaction with sample molecules.", "Preferably, the probe molecules provide highly specific interaction sites for the sample molecules.", "They can be derived from natural or non-natural sources.", "Particularly preferred probe molecules are biomolecules such as nucleic acids, including DNA, RNA or PNA (peptide nucleic acid), most preferably oligonucleotides or aptamers, polysaccharides, proteins including glycosidically modified proteins or antibodies, enzymes, cytokines, chemokines, peptide hormones or antibiotics, and peptides.", "In order to ensure a sufficient stability, e.g.", "during a sensor application, the probe molecules are preferably covalently bound to the polyfunctional polymer network.", "Depending on use, a multitude of identical probe molecules or a mixture of two or more different probes may be immobilized.", "For example, a set of identical probe molecules is preferred for the application of the polyfunctional polymer network as an affinity matrix.", "The polyfunctional polymer network according to the present invention comprises an assembly of cross-linked polymer subchains which are attached to a surface.", "Preferably the bonds between the polyfunctional polymer network and the surface are covalent.", "The introduction of branched polymers is possible, if desired.", "In some cases addition of comonomers which allow a tayloring of the physical properties of the network depending on the desired application is appropriate.", "The minimum components of the network are the functionalized repeating units and the cross-linking units.", "For the functionalized repeating units the subchains of the polymer network contain repeating units which carry at least one of the functional groups which can interact with sample or probe molecules.", "However, in order to impart certain advantageous properties to the polyfunctional polymer network, a copolymer, formed from these monomers with specific functional groups for the interaction with sample or probe molecules (hereinafter referred to as “functionalized monomers”) together with other comonomers can be used.", "For example, the reaction of the sample or probe molecules with the polyfunctional polymer network is significantly facilitated if the polyfunctional polymer network is swellable in the solvent containing these molecules, so that comonomers should preferably be chosen which show a strong interaction with the solvent in question.", "This can be achieved by using comonomers which improve the swellability of the network.", "Since, in a most preferred embodiment of the present invention, biomolecules, which are normally present in aqueous solutions, interact with the polyfunctional polymer network, said polyfunctional polymer network is preferably water-swellable.", "The water-swellability can be adjusted over a broad range by appropriate selection of suitable monomers, as long as the swellability does not lead to detachment of the polymer layer from the support and does not negatively affect the surface properties (i.e.", "evenness or constant thickness of surface coating) needed for fluorescent analyte detection.", "Thus, for example, one or more comonomers can be used which are polar, or even soluble in water, if a homopolymer of functionalized monomers does not show sufficient interaction with water to allow a fast reaction of the molecules to be detected with the functional groups.", "Both types of monomers, functionalized as well as comonomers, preferably contain a C—C double bond which can react in a radical polymerization reaction.", "Examples for suitable comonomers which yield a water swellable polymer are acrylic acid, methacrylic acid and derivatives thereof, e.g.", "esters and amides of these acids with alcohols or amines preferably comprising 1 to 12 carbon atoms.", "Common examples of this group of monomers are hydroxyethyl methacrylate, acrylamide and dimethyl acrylamide.", "Another suitable monomer is vinyl pyrrolidon.", "It is also possible to use monomers that yield at first water insoluble polymers which can then be transferred to water soluble derivatives.", "A suitable example for this group of polymers is polyvinyl alcohol which can be obtained, for example, by saponification of polyvinyl acetate.", "If a copolymer is used, the ratio of comonomers to functionalized monomers is determined prior to the polymerization process in order to define the composition of the resulting polymer chains of the polyfunctional polymer network.", "Preferably, the ratio of the comonomers to the functionalized monomers ranges from 50/1 to 1/1, more preferably form 20/1 to 2/1.The functional groups which are necessary to allow an interaction of the polyfunctional polymer network with the sample or probe molecules are preferably present in side chains of the polymer subchains of the polyfunctional polymer network.", "A “multitude” of functional groups comprised in polymer subchains of the polyfunctional polymer network of the present invention means at least two, but preferably more than two groups per polymer subchain.", "Since the concerned functional groups are preferably comprised in repeating units forming the polymer subchains of the polyfunctional polymer network, their number may amount up to several thousand, e.g.", "up to 10000 of these groups present in a single subchain, depending on the size of the probe or sample molecule to be immobilized.", "Preferably, each chain comprises 20 to 1000 of these functional groups.", "Suitable functionalized repeating units which are present in the polymer subchains of the polyfunctional polymer network are those repeating units which comprise a polymerizable C—C double bond, as well as a further functional moiety that does not take part in the polymerization process.", "Preferably, this functional group is linked to the main polymer subchains of the polyfunctional polymer network via a C2-C10, more preferably a C3-C7 alkyl chain as a spacer.", "The spacer molecules can be part of the functionalized monomers.", "Suitable monomers for this approach include acrylic and methacrylic esters or amides of C2-C10 alcohols or C2-C10 amines.", "In order to serve as spacers, these alcohols or amines carry an additional functional group at the terminal opposite to the one forming the ester or amide bond.", "This functional group either represents the one necessary for the interaction with the sample or probe molecules, or can be transformed to such a suitable functional group in a further step.", "Alternatively, it is also possible to attach these spacer molecules to suitable reactive segments within the polymer subchains of the polyfunctional polymer network after its formation.", "In this case, reactive monomers have to be present during polymerization, such as acrylic or methacrylic acid chlorides or reactive esters thereof, as N-hydroxy succinimides or other monomers, e.g.", "maleic anhydride.", "These preferred reactive monomers can form covalent bonds to the bifunctional alcohols or amines that may be used as spacers.", "The monomers carrying the spacer unit can readily be synthesized from the respective acrylic or methacrylic acid chloride or anhydride and the ω-amino or hydroxy carboxylic acid.", "The resulting product can be transformed to the active ester derivative by using e.g.", "N-hydroxy succinimide.", "A detailed procedure for the synthesis of several examples of such monomers can be found in the literature, e.g.", "in H.-G. Batz, J. Koldehoff, Macromol.", "Chem.", "177 (1976) 683.As outlined above, it is possible to use reactive monomers which directly yield the polymer subchains of the polyfunctional polymer network according to the invention.", "Alternatively, monomers can be chosen which carry a precursor of the functional group to be used on the final surface, e.g.", "an acid chloride or an acid anhydride.", "They can subsequently be transformed to reactive groups, e.g.", "NHS ester or glycidylester groups, which allow an interaction of the polyfunctional polymer network with sample or probe molecules under the desired conditions.", "Thus, all polymerizable monomers are suitable for the purposes of the present invention, as long as they can be combined with, or comprise, functional groups necessary to allow an interaction of the polyfunctional polymer network with the sample molecules or probe molecules.", "Functional groups which can be used for the purposes of the present invention are preferably chosen according to the molecules with which an interaction is to be achieved.", "The interaction can be directed to one single type of sample molecule, or to a variety of sample molecules.", "Since one important application of the present invention is the detection of specific molecules in biological samples, the functional groups present within the polyfunctional polymer network will preferably interact with natural or synthetic biomolecules which are capable of specifically interacting with the molecules in biological samples, leading to their detection.", "Suitable functional moieties will preferably be able to react with nucleic acids and derivatives thereof, such as DNA, RNA or PNA, e.g.", "oligonucleotides or aptamers, polysaccharides, proteins including glycosidically modified proteins or antibodies, enzymes, cytokines, chemokines, peptide hormones or antibiotics or peptides or labeled derivatives thereof.", "Moreover, it will be possible to conduct the coupling reaction between the molecules to be detected or the synthetic oligonucleotides and the polyfunctional polymer network under conditions which are not detrimental to the sample or probe molecules.", "Consequently, in an nucleic acid sensor application, the reaction should be carried out in an aqueous solution, and the temperature should not be raised above 95° C. Also, the coupling reaction should proceed at a reasonable rate so that the detection can preferably be accomplished within less than 24 hours without requiring extreme pH-values in the solution.", "For the immobilization of synthetic oligonucleotide single strands, the pH should range between 7 and 11, preferably 7 to 10.During the hybridization reaction of the nucleic acid sample molecules with the probe molecules, the bond between the functional group and the synthetic oligonucleotide single strand as well as the bonds of the polyfunctional polymer network to the substrate have to be able to withstand temperatures of more than 65° C., and a pH of 6-9.In cases where DNA is used as a sample molecule, the temperatures may have to be raised up to about 95° C. in order to effect a separation of the DNA strands, which is necessary for hybridization.", "Since most of the probe molecules, especially in biological or medical applications, comprise sterically unhindered nucleophilic moieties, preferred interactions with the polyfunctional polymer network comprise nucleophilic substitution or addition reactions leading to a covalent bond between the polymer subchains and the sample or probe molecules.", "For example, synthetical oligonucleotides are usually provided with a free amine group at one end (5′ or 3′).", "Thus, exemplary functional groups provide, for example, a reactive double bond, an equivalent for a double bond (as e.g.", "an epoxy group) or a reactive leaving group.", "However, ionic or vander-Waals forces as well as hydrogen bonds can also be used to couple sample molecules to the polyfunctional polymer network if the functional groups are chosen accordingly.", "Preferred functional groups can be chosen from prior literature with respect to the classes of molecules which are to be immobilized and according to the other requirements (reaction time, temperature, pH value) as described above.", "A general list can for example be found in the text book “Bioconjugate Techniques” by G. T. Hermanson, Academic Press, 1996.In the case of the attachment of amino-terminated oligonucleotides, examples for suitable groups are so-called active or reactive esters as N-hydroxy succinimides (NHS-esters), epoxides, preferably glycidyl derivatives, isothiocyanates, isocyanates, azides, carboxylic acid groups or maleinimides.", "As preferred functional monomers which directly result in polyfunctional polymer subchains of the polyfunctional polymer network, the following compounds can be employed for the purposes of the present invention: acrylic or methacrylic acid N-hydroxysuccinimides, N-methacryloyl-6-aminopropanoic acid hydroxysuccinimide ester, N-methacryloyl-6-aminocapronic acid hydroxysuccinimide ester or acrylic or methacryl acid glycidyl esters.", "Depending on the application, there is the possibility of providing the polymer subchains of the polyfunctional polymer network with a combination of two or more different functional groups, e.g.", "by carrying out the polymerization leading to the polymer subchains in the presence of different types of functionalized monomers.", "Alternatively, the functional groups may be identical.", "An advantageous and preferred method for the preparation of the polyfunctional polymer network according to the invention is described in the following: In a first step, a surface is covalently covered by a monolayer of polymerization initiators or starter molecules.", "The groups in these initiators which allow the initiation of the polymerization are usually chosen e.g.", "from peroxo groups or azo groups if a thermally initiated radical mechanism is to be used.", "Aromatic ketones such as benzoin, benzil or benzophenone derivatives are preferably used if the polymers are formed by photochemical initiation.", "Aromatic ketones comprising sulphur may equally be used, if desired, in order to shift the suitable wavelength for photoinitation to a longer wavelength region.", "In addition to such labile groups, suitable initiators for the preferred process according to the invention carry one or more groups suitable for their attachment to the surface to be covered by the polymer subchains of the polyfunctional polymer network.", "The same or different initiators may also be present in the polymerization mixture.", "The polymer subchains of the polyfunctional polymer network according to the present invention are usually grown from the surface via a chain reaction and are cross-linked simultaneously (cross-linking polymerization using in part immobilized initiators).", "While radical mechanisms are preferred for practical reasons, the application of ionic or other polymerization techniques is also possible.", "The functional groups comprised in the initiator molecules for surface attachment have to be adapted to the sensor surface used.", "For the preparation of the initiator monolayer on metal oxides, especially silicon oxide surfaces (evaporated or sputtered SiOX layers, SiO2 surfaces of silicon wafers, glass, quartz), chlorosilane moieties or alkoxysilanes are used.", "Thiol or disulfide groups can be employed for the modification of gold surfaces.", "However, silanes are usually preferred due to their increased stability on surfaces.", "Moreover, the present invention is not restricted to inorganic surfaces.", "Organic polymer surfaces can also be used as substrates to carry the polyfunctional polymer network, and there is also the possibility to include the starters for the polymerization reaction directly into such a surface forming polymer.", "Thus, suitable surfaces according to the present invention are preferably selected from non-porous materials such as e.g.", "microscope slides, polished silicon wafers, polymer plates, vials and wells.", "Preferred examples for initiators which can be used for the purposes of the present invention are listed below, together with their structure formulae: 4,4′-Azobis-(4-cyano pentanoic acid (3′-chlorodimethylsilyl) propyl ester), compound 1 or the respective di- and trichloro or mono-, di- and trialkoxy silane analogs, 2,4′-Azo-(4-cyano pentanoic acid (3″-chlorodimethylsilyl) propyl ester), compound 2 or the respective di- and trichloro or mono-, di- and trialkoxy silane analogs; or the respective compounds with an undecyl spacer rather than an propyl spacer, or disulfide or thiol derivatives of this general type of azo compounds, 4-(3′-chlorodimethylsilyl)propyloxy) benzophenone, compound 3 or the respective di- and trichloro- or mono-, di- and trialkoxy silane analogs, silane and disulfide/thiol derivatives of arylazomalodinitriles, such as compound 4.compound number structure 1 2 3 4 Preferred examples for suitable cross-linkers are: bisacrylates, bismethacrylates, for example oligo-ethylene glycol bismethacrylates such as ethylene glycol bismethacrylate, and bisacrylamides, for example ethylene diamine bisacrylamide.", "Upon initiation of the polymerization reaction, preferably by a heating step (thermal initiation) or exposure to radiation (photoinitiation) in the presence of polymerizable functionalized monomers and cross-linkers, polymer subchains can be grown from the surface and are simultaneously cross-linked.", "The polymerization can be carried out under standard reaction conditions known in the art.", "If this technique is applied, the graft densities of the resulting polyfunctional polymer network can be controlled over a wide range, for example by variation of the polymerization time.", "Moreover, graft densities can be achieved that are inaccessible by other methods.", "Thus, the polymer subchains of the polyfunctional polymer network can be attached such that the average distance between anchoring sites on the surface is 5 nm or less, e.g.", "2 to 5 nm.", "Advantageously, such graft densities can be achieved independent of the molecular weight of the attached chains, e.g.", "even for molecular weights of 100000 g/mol or more.", "Furthermore, the preferred in-situ formation of a polyfunctional polymer network on a surface according to the present invention allows the control of the average molecular weight of the attached polymer subchains of the polyfunctional polymer network independent of the graft density.", "According to this precise control of the parameters graft density, cross-link density and molecular weight, it is possible to adapt the properties of the respective polyfunctional polymer network to a variety of applications.", "By adjusting the reaction conditions networks with thicknesses ranging from a few nanometers up to some milimeters or even more may be prepared.", "It is also possible to fine-tune the properties of the resulting polyfunctional polymer network, e.g.", "with respect to the accessibility of the functional groups for subsequently coupled probe and sample molecules which may vary considerably in their size and structure.", "The polyfunctional polymer networks obtained via the above preferred method retain a fragment of the initiator in their structure which immobilizes them on the surface, namely the portion starting with the anchoring site and leading to the predetermined point of initiation as it is known in the art for all types of initiators, in particular those mentioned in this application.", "Detailed information on the synthesis of initiator molecules, their reaction with surfaces and the preferred conditions of polymerization are described in: O. Prucker, J. Ruhe, Macromolecules, 1998, 31, 592; O. Prucker, J. Rühe, Macromolecules, 1998, 31, 602 and O. Prucker, J. Rühe, Langmuir, 1998, 24 (14), 6893.Care should be taken to remove unreacted monomers as well as non-bonded or cross-linked polymer chains with suitable solvents after polymerization.", "According to an alternative method, in a first step a polyfunctional polymer network may be pre-formed which is then covalently bound to a surface.", "A further alternative method comprises synthesizing long polymer chains with appropriate functional groups starting from immobilized (surface-bound) initiators (so-called “polymer brushes”) followed by crosslinking the latter.", "Polyfunctional polymer networks prepared according to the above methods can be applied to a wide variety of surfaces, independent of their shape.", "Even surfaces which are inaccessible for conventional surface modification methods can be provided with the polyfunctional polymer network according to the invention, since no bulky polymer molecules have to diffuse towards the surface.", "Also, it is possible to create patterned arrays of the polyfunctional polymer network by various means.", "One way are standard photolithographic processes that can either be applied after polymerization (photoablation of the polymers through masks) prior to this step (photodecomposition or photoablation of the initiator monolayer masks) or during the polymerization by means of photopolymerization through masks.", "Other possible techniques for the creation of patterned polyfunctional polymer networks are microcontact printing or related methods, which may be applied during formation of the initiator layer or during polymerization.", "Finally, ink jet techniques or other microplotting methods can be used to create patterned initiator monolayers which can subsequently be transferred to patterned polyfunctional polymer network.", "Using any of these techniques, surface structures with dimensions in the micrometer range can be created.", "The high parallel mode of signal generation and a significant improvement in the integration of analytical data is the most promising feature of such techniques, which accordingly allow the optimization of automatic analytical procedures.", "For the detection of a successful immobilization of sample or probe molecules on a polyfunctional polymer network, a variety of techniques can be applied.", "In particular, it has been found that the polyfunctional polymer networks of the present invention undergo a significant increase in their thickness which can be detected with suitable methods, e.g.", "ellipsometry.", "Mass sensitive methods may also be applied.", "If nucleic acids, for example oligonucleotides with a desired nucleotide sequence or DNA molecules in a biological sample, are to be analyzed, synthetic oligonucleotide single strands can be reacted with the polyfunctional polymer network.", "The reaction is carried out under high humidity, preferably in a buffered aqueous solution.", "The reaction temperature can be raised above room temperature, as long as it is not detrimental to the oligonucleotides.", "Preferred temperatures are in the range of 40-60° C. In this application, a multitude of identical synthetic oligonucleotide strands or a mixture of different strands can be used.", "If different strands are used, their sequences should preferably be known.", "Before the thus prepared surface is used in a hybridization reaction, unreacted functional groups are deactivated via addition of suitable nucleophiles, preferably C1-C4 amines, such as simple primary alkylamines (e.g.", "propyl or butyl amine), secondary amines (diethylamine) or amino acids (glycin).", "Upon exposure to a mixture of oligonucleotide single strands, e.g.", "as obtained from PCR, which are labled, only those surface areas which provide synthetic strands as probes complementary to the PCR product will show a detectable signal upon scanning due to hybridization.", "In order to facilitate the parallel detection of different oligonucleotide sequences, printing techniques can be used which allow the separation of the sensor surface into areas where different types of synthetic oligonucleotide probes are presented to the test solution.", "The term “hybridization” as used in accordance with the present invention may relate to stringent or non-stringent conditions.", "If not further specified, the conditions are preferably non-stringent.", "Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (1989), Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds) “Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985).", "The setting of conditions is well within the skill of the artisan and to be determined according to protocols described in the art.", "Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as for example 0.1×SSC, 0.1% SDS at 65° C. Exemplary non-stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may be set at 6×SSC, 1% SDS at 65° C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.", "The nucleic acids to be analyzed may originate from a DNA library or a genomic library, including synthetic and semisynthetic nucleic acid libraries.", "Preferably, the nucleic acid library comprises oligonucleotides.", "In order to facilitate their detection in an immobilized state, the nucleic acid molecules should preferably be labeled.", "Suitable labels include radioactive, fluorescent, phosphorescent, bioluminescent or chemoluminescent labels, an enzyme, an antibody or a functional fragment or functional derivative thereof, biotin, avidin or streptavidin.", "Antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric or single-chain antibodies or functional fragments or derivatives of such antibodies.", "The general methodology for producing antibodies is well-known and has been described in, for example, Köhler and Milstein, Nature 256 (1975), 494 and reviewed in J. G. R. Hurrel, ed., “Monoclonal Hybridoma Antibodies: Techniques and Applications”, CRC Press Inc., Boco Raron, Fla. (1982).", "Also the method taught by L. T. Mimms et al., Virology 176 (1990), 604-619, is applicable.", "As stated above, in accordance with the present invention the term “antibody” relates to monoclonal or polyclonal antibodies.", "Functional antibody fragments or derivatives provide the same specificity as the original antibody and comprise F(ab′)2, Fab, Fv or scFv fragments; see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press 1988, Cold Spring Harbor, N.Y. Preferably the antibody of the invention is a monoclonal antibody.", "Furthermore, in accordance with the present invention, the derivatives can be produced by peptidomimetics.", "Such production methods are well known in the art and can be applied by the person skilled in the art without further ado.", "Depending on the labeling method applied, the detection can be effected by methods known in the art, e.g.", "via laser scanning or use of CCD cameras.", "Also comprised by the present invention are methods where detection is indirectly effected.", "An example of such an indirect detection is the use of a secondary labeled antibody directed to a first compound such as an antibody which binds to the biological molecule (sample molecule) of interest.", "A further application of the polyfunctional polymer networks according to the invention lies in the field of affinity chromatography, e.g.", "for the purification of substances.", "For this purpose, polyfunctional polymer networks with identical functional groups or probe molecules are preferably used, which are contacted with a sample.", "After the desired substance has been immobilized by the polyfunctional polymer network, unbound material can be removed, e.g.", "in a washing step.", "With suitable eluents, the purified substance can then be separated from the affinity matrix.", "Preferred substances which may be immobilized on such a matrix are nucleic acid molecules, peptides or polypeptides (or complexes thereof), such as antibodies, functional fragments or derivatives thereof, saccharides or polysaccharides.", "A regeneration of the surfaces after the immobilization has taken place is possible, but single uses are preferred in order to ensure the quality of results.", "With the polyfunctional polymer networks of the present invention, different types of samples can be analyzed with an increased precision and/or reduced need of space in serial as well as parallel detection methods.", "The sensor surfaces to which the polyfunctional polymer networks according to the invention are attached can therefore serve in diagnostical instruments or other medical applications, e.g.", "for the detection of components in physiological fluids, such as blood, serum, sputum etc.", "Surfaces with the polyfunctional polymer networks according to the present invention can also immobilize starter molecules for synthetic applications in particular in solid phase synthesis, e.g.", "during the in situ formation of oligo- or polymers.", "Preferably, the oligo- or polymers are biomolecules and comprise peptides, proteins, oligo- or polysaccharides or oligo- or polynucleic acids.", "As immobilized initiators, a monomer of these macromolecules can be used.", "Moreover, the polyfunctional polymer networks of the present invention can be used as gels in the separation of molecules, preferably biomolecules in an electrical field.", "Generally, the present invention allows the provision of surfaces homogenically modified with polyfunctional polymer networks having superior surface adhesive properties and improved mechanical strength.", "Moreover, structured surfaces can be provided, e.g.", "by starting the polymerization from patterned arrays of initiator molecules.", "As a consequence, the polyfunctional polymer network can be adjusted optimally to the respective applications.", "The disclosure content of the documents cited throughout the specification are herewith incorporated by reference.", "The embodiments of the present invention are further illustrated in the following items: A preferred process for the detection of sample nucleic acid molecules, preferably of single stranded nucleic acid molecules, using a polyfunctional polymer network according to the invention comprises the steps of: providing a surface covalently covered with a polyfunctional polymer network according to the invention, immobilizing suitable probe molecules, preferably oligonucleotide single strands, on the polyfunctional polymer network via a reaction with the functional groups present in the polyfunctional polymer network, allowing a hybridization reaction to take place between the oligonucleotide single strands and the sample nucleic acid molecules, removing the non-hybridized nucleic acid molecules in a washing step, and detecting the hybridized nucleic acid molecules, preferably fluorometrically.", "A preferred process for purifying a compound from a sample, using a polyfunctional polymer network according to the invention comprises the steps of: providing a surface modified with a polyfunctional polymer network according to the invention, immobilizing a multitude of identical probe molecules on the polyfunctional polymer network, contacting the sample with the resulting polymer network, under conditions that allow binding of said compound to the probe molecules, and removing material from the sample that has not bound to the probe molecules.", "This process may further include the step of separating the compound from the probe molecules by use of a suitable eluent.", "The following examples illustrate the invention: Synthesis of the Initiator As an example, the preparation of compound 1 is described.", "The reaction pathway is illustrated below.", "The indices i-iii in the figure refer to the description of the various steps in the text.", "i) To a suspension of 40 g phosphorus pentachloride (PCl5) in 50 ml methylene chloride cooled with an ice-bath was added dropwise a suspension of 10 g of 4,4′-azobis-(4-cyano pentanoic acid) in 50 ml methylene chloride.", "The mixture was allowed to warm to room temperature and was stirred overnight.", "The excess PCl5 was filtered off and the remaining solution was concentrated until no more PCl5 separated.", "The mixture was filtered again and the filtrate was added to 300 ml of cold hexane, causing the separation of the acid chloride as a white solid (yield: 90%).", "ii) To a solution of 2.7 ml of allyl alcohol and 6.5 ml of pyridine in 50 ml methylene chloride at 0° C. was added dropwise a solution of 10 g of the acid chloride in 50 ml methylene chloride.", "The mixture was allowed to warm to room temperature and was stirred overnight.", "Then the solution was washed twice with 2N H2SO4, aqueous NaHCO3 and water.", "The organic layer was dried over Na2SO4 and the solvent was evaporated.", "The resulting bis allylic ester was recrystallized from methanol (yield: 90%).", "iii) To a suspension of 3 g of the bis allylic ester in 30 ml dimethyl chloro silane was added a solution of 30 mg of hexachloroplatinic acid in 0.5 ml of dimethyl ether/ethanol (1/1 v/v) and the mixture was heated to reflux for 3 h. The excess of the silane was evaporated yielding compound 1 as a pale green oil in quantitative yields.", "Residual platinum catalyst was removed by filtration of a methylene chloride solution of the product over anhydrous Na2SO4.Formation of an Initiator Monolayer The initiator synthesized under (1) was immobilized at room temperature on a glass surface under inert conditions (atmosphere of dry nitrogen) using anhydrous toluene as a solvent and dry triethylamine as a catalyst.", "The toluene solution shows a concentration of the initiator of about 50 mmol/l and triethylamine is added up to a concentration of about 10 mmol/l.", "The samples were kept in the solution overnight and then cleaned by extensive rinsing with methanol and chloroform.", "Synthesis of the Functionalized Monomer As an example, the synthesis of N-methacryloyl-6-aminocapronic acid hydroxysuccinimide ester is described.", "The reaction pathway is shown below.", "The indices i-iii in this figure refer to the description of the various steps in the text.", "i) A solution of 13.2 g 6-aminocaproic acid and 20 g NaHCO3 in 100 ml water and 50 ml 1,4-dioxane was slowly added to a solution of 10.3 ml of methacrylic acid chloride in 50 ml 1,4-dioxane.", "The solution was stirred overnight.", "Then 50 ml of water were added and the mixture was washed three times with 100 ml portions of ethyl acetate.", "The water layer was acidified (pH 2) with dilute hydrochloric acid and then extracted with three 100 ml portions of ethyl acetate.", "The combined organic layers were dried over Na2SO4, concentrated to a volume of about 50 ml and added to 350 ml of cold hexane.", "This mixture was cooled to −20° C. and the product slowly separated overnight as white crystals (yield: ca.", "14 g).", "ii) A solution of 14 g of the acid in 300 ml methylene chloride was cooled to 5° C. and 8.2 g of N-hydroxy succinimide (NHS) and 14.6 g of N,N-dicyclohexyl carbodiimide were added.", "The mixture was kept at 5° C. overnight.", "The precipitate (dicyclohexylurea) was filtered off and the solvent was evaporated.", "During this step, additional urea separated in some cases and was also filtered off.", "The crude product was recrystallized from isopropanol to yield about 15 g of the NHS ester monomer.", "Formation of a Polyfunctional Polymer Network A solution comprising the following ingredients was used: 40 mole % N,N-dimethyl acrylamide (for the water-swellable basis polymer), 10 mole % N-methacryloyl-6-aminocapronic acid hydroxysuccinimide ester (for the functionalized repeating units), 5 mole % ethylene glycol bismethacrylate (for the cross-linking units), 1 mole % azobisisobutyronitril (as an initiator), balance (to 100 mole %) ethanol.", "This solution was given into a mould and said mould pressed firmly onto a initiator-modified substrate (with immobilized initiator).", "After heating to 70° C. polymerization was performed for 10 hours.", "Thereafter, the mould was removed and the resultant surface-attached network washed with ethanol.", "Detection of Oligonucleotides Strands The obtained surface was exposed to 1 nl of a 10 μM oligonucleotide-solution and the coupling reaction was allowed to proceed at about 40-50° C. for two hours in an aqueous solution.", "The synthetic oligonucleotide was 5-amino modified and the solution was buffered with a 100 mM sodium phosphate buffer at a pH of 8.0.After the coupling reaction, the sensor surface was rinsed with the sodium phosphate buffer.", "In order to define the spatial extension of the specific types of oligonucleotide on the sensor surface for parallel detection, the reactant was printed onto the polyfunctional polymer network.", "The surface thus prepared was allowed to react with a Cy5 labeled PCR product in a buffer of 2×SSC, 10% dextrane sulphate and 50% formamide for 12 h at 28° C. The DNA content was 100 ng DNA/80 μl sample.", "After the hybridization reaction has taken place, the surface was washed in SSC-buffer and the result was detected fluorometrically via laser activation with a CCD camera.", "A fluorescence signal could only be detected for those areas which carried synthetic oligonucleotides complementary with the PCR product." ] ]

[ [ "Iso-truss structure", "An iso-truss structure (10) includes at least two helical components (30, 32) and at least one reverse helical component (34) attached thereto with opposing angular orientations.", "Each helical and reverse helical component preferably includes at least four elongate, straight segments (22) rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about a common axis (14) forming a first square cross section.", "The structure may further include at least two rotated helical components (80, 92) and at least one rotated reverse helical component (98) which are rotated with respect to the helical and reverse helical components forming a second square cross section, rotated with respect to the first.", "The structure may be straight, curved, flexible, or form angles." ], [ "1.A structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "2.A structural member in accordance with claim 1, wherein all of the helical components have continuous strands of fiber; and wherein the helical components are attached to one another at intersecting locations by over-lapping the fibers of the helical components.", "3.A structural member in accordance with claim 1, wherein the helical components define a hollow interior substantially void of material.", "4.A structural member in accordance with claim 1, wherein the helical components define openings there between.", "5.A structural member in accordance with claim 1, wherein helical components define an imaginary tubular member of square cross section.", "6.A structural member in accordance with claim 1, further comprising: a) at least two, spaced apart, rotated helical components, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, each having: 1) a common rotated longitudinal axis, 2) a common angular orientation about the rotated longitudinal axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the rotated axis; and b) at least one rotated reverse helical component, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, having: 1) a common rotated longitudinal axis with the at least two rotated helical components, 2) an opposing angular orientation with respect to the two rotated helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "7.A structural member in accordance with claim 6, wherein the longitudinal axis and the rotated longitudinal axis are concentric, and the segments of the helical components form an imaginary tubular member having a cross section of an eight-pointed star.", "8.A structural member in accordance with claim 6, wherein the longitudinal axis and the rotated longitudinal axis are concentric, and the segments form an imaginary tubular member having a cross section of two squares having a common longitudinal axis, but with one square rotated with respect to the other.", "9.A structural member in accordance with claim 1, further comprising: an end plate, attached at an end of the helical components, to attach the helical components to another object.", "10.A structural member in accordance with claim 9, wherein the helical components have continuous strands of fiber; and wherein the end plate is attached by winding the continuous strands of fiber around the end plate.", "11.A structural member in accordance with claim 9, wherein the end plate includes a perimeter, a plurality of indentations formed about the perimeter to receive strands of fiber, and a plurality of holes to attach the end plate to another object.", "12.A structural member in accordance with claim 1, wherein the helical components and segments form a repeating pattern of triangles and tetrahedrons; and further comprising: a connector, attached to the helical components and segments, to attach other objects to the helical components and segments, the connector being elongated and having a substantially triangular, cross sectional shape, the connector extending through at least two of the triangles formed by the helical components and segments.", "13.A structural member in accordance with claim 1, wherein the axes are vertically oriented, a lower end is attached to a support surface, and an upper end is located above the lower end; and further including another object attached to the upper end selected from the group consisting of: a sign placard with indicia; a horizontal utility member configured to hold utility lines; a light source.", "14.A structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; c) at least two, spaced apart, rotated helical components, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, each having: 1) a common rotated longitudinal axis, 2) a common angular orientation about the rotated longitudinal axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the rotated axis; and d) at least one rotated reverse helical component, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, having: 1) a common rotated longitudinal axis with the at least two rotated helical components, 2) an opposing angular orientation with respect to the two rotated helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "15.A structural member in accordance with claim 14, wherein the longitudinal axis and the rotated longitudinal axis are concentric, and the segments of the helical components form an imaginary tubular member having a cross section of an eight-pointed star.", "16.A structural member in accordance with claim 14, wherein the longitudinal axis and the rotated longitudinal axis are concentric, and the segments form an imaginary tubular member having a cross section of two squares having a common longitudinal axis, but with one square rotated with respect to the other.", "17.A structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and c) the at least one reverse helical component forming an angle with respect to the at least two helical components greater than 60 degrees.", "18.A structural member in accordance with claim 17, wherein the at least one reverse helical component forms an angle with respect to the at least two helical components greater than approximately 75 degrees.", "19.A structural member in accordance with claim 17, wherein each helical component includes at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "20.A flexible structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least three elongate, straight segments connected end to end in a helical configuration; and c) the helical members being laterally flexible, and are bendable between: 1) a first, straight position in which the axes are substantially straight; and 2) a second, arcuate position in which the axes are substantially arcuate.", "21.A structural member in accordance with claim 20, wherein the helical members store energy in the second, arcuate position.", "22.A structural member in accordance with claim 20, wherein the helical members are rotationally rigid about the longitudinal axes.", "23.An arcuate structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common arcuate axis, 2) a common angular orientation about the axis, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common arcuate axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration.", "24.A structural member in accordance with claim 23, wherein the arcuate axes are circular.", "25.A structural member in accordance with claim 23, wherein each helical component includes at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "26.A structural member in accordance with claim 23, wherein the arcuate axes include a first curvature, and a different second curvature.", "27.A tapering structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and c) the segments of each helical component sequentially reducing in length along the axes such that the structural member tapers.", "28.A structural member in accordance with claim 27, wherein each helical component includes at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "29.A preform member, comprising: a) at least two, spaced apart, helical components each having: at least three segments connected end to end in a helical configuration; and b) at least one reverse helical component, attached to the at least two helical components, having: at least three segments connected end to end in a helical configuration; and c) the helical components including fiber and being flexible and collapsible until impregnated with a resin matrix.", "30.A preform member in accordance with claim 29, wherein the helical components include a plurality of strands of fiber bound together.", "31.A bicycle frame, comprising: a) a handlebar location configured to attach to a handlebar and front fork; b) a seat location configured to attach to a seat; c) a peddle location configured to be attached to a peddle assembly; d) a rear wheel location configured to be attached to a rear wheel; e) a plurality of members, each extending to and between at least one of the handlebar, seat, peddle, and rear wheel locations; and f) at least one of the members including: 1) at least two, spaced apart, helical components each having: i) a common longitudinal axis, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and 2) at least one reverse helical component, attached to the at least two helical components, having: i) a common longitudinal axis with the at least two helical components, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis in an opposing angular orientation.", "32.A bicycle frame in accordance with claim 31, wherein each helical component includes at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "33.A method for forming a structural member, comprising the steps of: a) providing a mandrel; b) wrapping a fiber around the mandrel in order to create at least two helical components, each component having at least four elongated, straight segments, the at least two helical components having a common longitudinal axis, a common angular orientation about the axis, and forming a single, substantially complete rotation about the axis; c) wrapping a fiber around the mandrel in order to create at least one reverse helical component having at least four elongate, straight segments having a common longitudinal axis with the at least two helical components, but in an opposing angular orientation, and forming a single, substantially complete rotation about the axis; d) adding a matrix to the fiber; and e) curing the matrix.", "34.A method in accordance with claim 33, wherein the step of providing a mandrel further includes: providing a mandrel having an elongated core, and a plurality of heads disposed longitudinally and radially about the core, each head configured to receive and hold fiber for at least two, opposing helical components, and including four angled indentations, two angled indentations for each helical component.", "35.A method in accordance with claim 33, further comprising the step of: wrapping a fiber along a length of the mandrel in order to create at least one longitudinal component parallel with the longitudinal axes; and wherein the step of providing a mandrel further includes: providing a mandrel having an elongated core, and a plurality of heads disposed longitudinally and radially about the core, each head configured to receive and hold fiber for at least two, opposing helical components and at least one longitudinal component, and including at least six indentations, including two angled indentations for each helical component and two indentations for the longitudinal component.", "36.A method in accordance with claim 33, wherein the step of providing a mandrel further includes providing a collapsible mandrel having: a) an elongated, hollow tube including a plurality of holes, b) an elongated core, removably disposed within the tube, c) a plurality of inserts, removably disposed between the core and the tube, having a plurality of holes, d) a plurality of pins, removably disposed in the holes of the tube and inserts, and e) a plurality of heads disposed on the pins; and further including the steps of: a) removing the core from the tube after curing; b) removing the inserts from the core; c) displacing the pins through the holes into the tube; d) removing the tube; and e) removing the heads.", "37.A utility pole, comprising: a) an elongated member, vertically oriented, having a longitudinal axis and upper and lower ends, and being formed of: 1) at least two, spaced apart, helical components each having: i) a common angular orientation about the longitudinal axis, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and 2) at least one reverse helical component, attached to the at least two helical components, having: i) an opposing angular orientation with respect to the two helical components, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; b) an end plate, attached to the lower end of the elongated member, configured to attach the lower end of the elongated member to a support surface; and c) an arm, attached to the elongated member near the upper end and extending generally horizontally, configured to hold a utility line.", "38.A utility pole in accordance with claim 37, wherein the elongated member further includes: a) at least two, spaced apart, rotated helical components, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, each having: 1) a common rotated longitudinal axis, 2) a common angular orientation about the rotated longitudinal axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the rotated axis; and b) at least one rotated reverse helical component, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, having: 1) a common rotated longitudinal axis with the at least two rotated helical components, 2) an opposing angular orientation with respect to the two rotated helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "39.A utility pole in accordance with claim 37, wherein the segments of each helical component sequentially reduce in length along the axes such that the structural member tapers.", "40.A sign post, comprising: a) an elongated member, vertically oriented, having a longitudinal axis and upper and lower ends, and being formed of: 1) at least two, spaced apart, helical components each having: i) a common angular orientation about the longitudinal axis, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and 2) at least one reverse helical component, attached to the at least two helical components, having: i) an opposing angular orientation with respect to the two helical components, and ii) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; b) an end plate, attached to the lower end of the elongated member, configured to attach the lower end of the elongated member to a support surface; and c) a sign, coupled to the elongated member, including indicia.", "41.A sign post in accordance with claim 40, wherein the elongated member further includes: a) at least two, spaced apart, rotated helical components, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, each having: 1) a common rotated longitudinal axis, 2) a common angular orientation about the rotated longitudinal axis, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the rotated axis; and b) at least one rotated reverse helical component, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, having: 1) a common rotated longitudinal axis with the at least two rotated helical components, 2) an opposing angular orientation with respect to the two rotated helical components, and 3) at least four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "42.A sign post in accordance with claim 40, further comprising: a) an arcuate member having a first end attached to the upper end of the elongated member, and also including: 1) at least two, spaced apart, helical components each having: i) a common arcuate axis, ii) a common angular orientation about the axis, and iii) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and 2) at least one reverse helical component, attached to the at least two helical components, having: i) a common arcuate axis with the at least two helical components, ii) an opposing angular orientation with respect to the two helical components, and iii) at least three elongate, straight segments rigidly connected end to end in a helical configuration; and b) the sign is coupled to the arcuate member.", "43.A structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "44.A structural member in accordance with claim 43, further comprising: a) at least two, spaced apart, rotated helical components, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, each having: 1) a common rotated longitudinal axis, 2) a common angular orientation about the rotated longitudinal axis, and 3) four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the rotated axis; and b) at least one rotated reverse helical component, attached to and rotated with respect to the at least two helical components and at least one reverse helical component, having: 1) a common rotated longitudinal axis with the at least two rotated helical components, 2) an opposing angular orientation with respect to the two rotated helical components, and 3) four elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis.", "45.A structural member, comprising: a) at least two, spaced apart, helical components each having: 1) a common longitudinal axis, 2) a common angular orientation about the axis, and 3) five elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis; and b) at least one reverse helical component, attached to the at least two helical components, having: 1) a common longitudinal axis with the at least two helical components, 2) an opposing angular orientation with respect to the two helical components, and 3) five elongate, straight segments rigidly connected end to end in a helical configuration forming a single, substantially complete rotation about the axis." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.The Field of the Invention The present invention relates generally to a three-dimensional structural member which is strong and light-weight.", "More particularly, the present invention relates to a structural member having a plurality of helical components wrapped about an axis, each having straight segments connected end-to-end in a helical configuration.", "2.The Background Art The pursuit of structurally efficient structures in the civil, mechanical, aerospace and sports arenas is an ongoing quest.", "An efficient truss structure is one that has a high strength to weight ratio and/or a high stiffness to weight ratio.", "An efficient truss structure can also be described as one that is relatively inexpensive, easy to fabricate and assemble, and does not waste material.", "Trusses are typically stationary, fully constrained structures designed to support loads.", "They consist of straight members connected at joints at the end of each member.", "The members are two-force members with forces directed along the member.", "Two-force members can only produce axial forces such as tension and compression forces in the member.", "Trusses are often used in the construction of bridges and buildings.", "Trusses are designed to carry loads which act in the plane of the truss.", "Therefore, trusses are often treated, and analyzed, as two-dimensional structures.", "The simplest two-dimensional truss consists of three members joined at their ends to form a triangle.", "By consecutively adding two members to the simple structure and a new joint, larger structures may be obtained.", "The simplest three-dimensional truss consists of six members joined at their ends to form a tetrahedron.", "By consecutively adding three members to the tetrahedron and a new joint, larger structures may be obtained.", "This three dimensional structure is known as a space truss.", "Frames, as opposed to trusses, are also typically stationary, fully constrained structures, but have at least one multi-force member with a force that is not directed along the member.", "Machines are structures containing moving parts and are designed to transmit and modify forces.", "Machines, like frames, contain at least one multi-force member.", "A multi-force member can produce not only tension and compression forces, but shear and bending as well.", "Traditional structural designs have been limited to one or two-dimensional analyses resisting a single load type.", "For example, I-beams are optimized to resist bending and tubes are optimized to resist torsion.", "Limiting the design analysis to two dimensions simplifies the design process but neglects combined loading.", "Three-dimensional analysis is difficult because of the difficulty in conceptualizing and calculating three-dimensional loads and structures.", "In reality, many structures must be able to resist multiple loadings.", "Computers are now being utilized to model more complex structures." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It has been recognized that it would be advantageous to develop a structural member with enhanced performance characteristics, such as strength reduced weight, etc.", "The invention provides a three-dimensional structure or structural member, including: 1) at least two, spaced apart, helical components, and 2) at least one reverse helical component attached to the two helical components.", "The helical and reverse helical components have a common longitudinal axis, but opposing angular orientations about the axis.", "In addition, each helical and reverse helical component advantageously includes at least four elongate, straight segments rigidly connected end-to-end in a helical configuration forming a single, substantially complete rotation about the axis.", "Thus, the helical and reverse helical components form a first square-shaped cross section.", "In one aspect, the structure includes four helical components and four reverse helical components.", "In addition, the iso-truss structure can include 1) rotated helical components, and 2) rotated reverse helical components, similar to, but rotated with respect to, the helical and reverse helical components above.", "Thus, the rotated helical and rotated reverse helical components form a second square-shaped cross section, rotated with respect to the first.", "In one aspect, the structure includes four rotated helical components and four rotated reverse helical components, for a total of sixteen helical components.", "The various helical components intersect at external nodes and internal nodes.", "In one aspect, the components form eight internal and eight external nodes.", "Longitudinal or axial components may extend parallel to the axis and intersect the internal and/or external nodes.", "In one aspect, the structure includes eight external nodes.", "It has been found that such an eight node structure has unexpected structural or performance characteristics.", "In accordance with one aspect of the present invention, the structure can further include an end plate attached at an end of the helical components to attach the helical components to another object.", "In one aspect, the helical components may be formed of continuous strands of fiber, which may be wound around the end plate.", "The end plate can include a perimeter with a plurality of indentations to receive the strands of fiber.", "In accordance with another aspect of the present invention, the structure can further include a connector member attached to the helical components and segments to attach other objects to the helical components and segments.", "The connector member can include a triangular cross-sectional shape extending through triangular openings formed by the components.", "In accordance with another aspect of the present invention, the helical and reverse helical components may form an angle therebetween greater than approximately 60 degrees.", "It has been found that such angles have unexpected structural or performance characteristics.", "In accordance with another aspect of the present invention, the helical and reverse helical members can be axially and/or laterally flexible, but torsionally stiff.", "The structure may bend between a first, straight position in which the axes are substantially straight; and a second, arcuate position in which the axes are substantially arcuate.", "In addition, the structure may compress and/or expand longitudinally.", "In either case, the structure may store energy, and thus be utilized as a spring member.", "In accordance with another aspect of the present invention, the structure may be arcuate, and the components may be formed about an arcuate axis.", "Thus, the arcuate structure may form more complex shapes than a singular, linear structure, and may be better suited for certain applications.", "In accordance with another aspect of the present invention, the structure may taper.", "The segments of each helical component may sequentially reduce in length along the axes such that the structural member tapers.", "Thus, the tapering structure may form more complex shapes than a singular, linear structure, and may be better suited for certain applications.", "In accordance with another aspect of the present invention, the iso-truss structure may be utilized to hold signs, utility lines, or lights.", "The iso-truss structure further may be utilized for bicycle frames, aircraft and marine structures, etc.", "A method for forming an iso-truss structure in accordance with the present invention can include wrapping a fiber around a mandrel in order to create the two helical components and the reverse helical component.", "A matrix or resin can be added to the fiber and cured.", "The mandrel may be removed from the structure.", "The mandrel may include a plurality of heads disposed thereon to receive and hold fiber.", "The mandrel may be a collapsible or dissolvable mandrel.", "Additional features and advantages of the invention will be set forth in the detailed description which follows, taken in conjunction with the accompanying drawings, which together illustrate by way of example, the features of the invention." ], [ "BACKGROUND OF THE INVENTION 1.The Field of the Invention The present invention relates generally to a three-dimensional structural member which is strong and light-weight.", "More particularly, the present invention relates to a structural member having a plurality of helical components wrapped about an axis, each having straight segments connected end-to-end in a helical configuration.", "2.The Background Art The pursuit of structurally efficient structures in the civil, mechanical, aerospace and sports arenas is an ongoing quest.", "An efficient truss structure is one that has a high strength to weight ratio and/or a high stiffness to weight ratio.", "An efficient truss structure can also be described as one that is relatively inexpensive, easy to fabricate and assemble, and does not waste material.", "Trusses are typically stationary, fully constrained structures designed to support loads.", "They consist of straight members connected at joints at the end of each member.", "The members are two-force members with forces directed along the member.", "Two-force members can only produce axial forces such as tension and compression forces in the member.", "Trusses are often used in the construction of bridges and buildings.", "Trusses are designed to carry loads which act in the plane of the truss.", "Therefore, trusses are often treated, and analyzed, as two-dimensional structures.", "The simplest two-dimensional truss consists of three members joined at their ends to form a triangle.", "By consecutively adding two members to the simple structure and a new joint, larger structures may be obtained.", "The simplest three-dimensional truss consists of six members joined at their ends to form a tetrahedron.", "By consecutively adding three members to the tetrahedron and a new joint, larger structures may be obtained.", "This three dimensional structure is known as a space truss.", "Frames, as opposed to trusses, are also typically stationary, fully constrained structures, but have at least one multi-force member with a force that is not directed along the member.", "Machines are structures containing moving parts and are designed to transmit and modify forces.", "Machines, like frames, contain at least one multi-force member.", "A multi-force member can produce not only tension and compression forces, but shear and bending as well.", "Traditional structural designs have been limited to one or two-dimensional analyses resisting a single load type.", "For example, I-beams are optimized to resist bending and tubes are optimized to resist torsion.", "Limiting the design analysis to two dimensions simplifies the design process but neglects combined loading.", "Three-dimensional analysis is difficult because of the difficulty in conceptualizing and calculating three-dimensional loads and structures.", "In reality, many structures must be able to resist multiple loadings.", "Computers are now being utilized to model more complex structures.", "SUMMARY OF THE INVENTION It has been recognized that it would be advantageous to develop a structural member with enhanced performance characteristics, such as strength reduced weight, etc.", "The invention provides a three-dimensional structure or structural member, including: 1) at least two, spaced apart, helical components, and 2) at least one reverse helical component attached to the two helical components.", "The helical and reverse helical components have a common longitudinal axis, but opposing angular orientations about the axis.", "In addition, each helical and reverse helical component advantageously includes at least four elongate, straight segments rigidly connected end-to-end in a helical configuration forming a single, substantially complete rotation about the axis.", "Thus, the helical and reverse helical components form a first square-shaped cross section.", "In one aspect, the structure includes four helical components and four reverse helical components.", "In addition, the iso-truss structure can include 1) rotated helical components, and 2) rotated reverse helical components, similar to, but rotated with respect to, the helical and reverse helical components above.", "Thus, the rotated helical and rotated reverse helical components form a second square-shaped cross section, rotated with respect to the first.", "In one aspect, the structure includes four rotated helical components and four rotated reverse helical components, for a total of sixteen helical components.", "The various helical components intersect at external nodes and internal nodes.", "In one aspect, the components form eight internal and eight external nodes.", "Longitudinal or axial components may extend parallel to the axis and intersect the internal and/or external nodes.", "In one aspect, the structure includes eight external nodes.", "It has been found that such an eight node structure has unexpected structural or performance characteristics.", "In accordance with one aspect of the present invention, the structure can further include an end plate attached at an end of the helical components to attach the helical components to another object.", "In one aspect, the helical components may be formed of continuous strands of fiber, which may be wound around the end plate.", "The end plate can include a perimeter with a plurality of indentations to receive the strands of fiber.", "In accordance with another aspect of the present invention, the structure can further include a connector member attached to the helical components and segments to attach other objects to the helical components and segments.", "The connector member can include a triangular cross-sectional shape extending through triangular openings formed by the components.", "In accordance with another aspect of the present invention, the helical and reverse helical components may form an angle therebetween greater than approximately 60 degrees.", "It has been found that such angles have unexpected structural or performance characteristics.", "In accordance with another aspect of the present invention, the helical and reverse helical members can be axially and/or laterally flexible, but torsionally stiff.", "The structure may bend between a first, straight position in which the axes are substantially straight; and a second, arcuate position in which the axes are substantially arcuate.", "In addition, the structure may compress and/or expand longitudinally.", "In either case, the structure may store energy, and thus be utilized as a spring member.", "In accordance with another aspect of the present invention, the structure may be arcuate, and the components may be formed about an arcuate axis.", "Thus, the arcuate structure may form more complex shapes than a singular, linear structure, and may be better suited for certain applications.", "In accordance with another aspect of the present invention, the structure may taper.", "The segments of each helical component may sequentially reduce in length along the axes such that the structural member tapers.", "Thus, the tapering structure may form more complex shapes than a singular, linear structure, and may be better suited for certain applications.", "In accordance with another aspect of the present invention, the iso-truss structure may be utilized to hold signs, utility lines, or lights.", "The iso-truss structure further may be utilized for bicycle frames, aircraft and marine structures, etc.", "A method for forming an iso-truss structure in accordance with the present invention can include wrapping a fiber around a mandrel in order to create the two helical components and the reverse helical component.", "A matrix or resin can be added to the fiber and cured.", "The mandrel may be removed from the structure.", "The mandrel may include a plurality of heads disposed thereon to receive and hold fiber.", "The mandrel may be a collapsible or dissolvable mandrel.", "Additional features and advantages of the invention will be set forth in the detailed description which follows, taken in conjunction with the accompanying drawings, which together illustrate by way of example, the features of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a perspective view of an iso-truss structure in accordance with the present invention; FIG.", "2 is a side view of the iso-truss structure of FIG.", "1; FIG.", "3 is a partial perspective view of the iso-truss structure of FIG.", "1; FIG.", "4 is an end view of the iso-truss structure of FIG.", "1; FIGS.", "5a-5t are partial perspective views of the iso-truss structure of FIG.", "1 showing helical components of the present invention; FIG.", "6 is a perspective view of another iso-truss structure in accordance with the present invention; FIG.", "7 is a side view of the iso-truss structure of FIG.", "6; FIG.", "8 is an end view of the iso-truss structure of FIG.", "6; FIGS.", "9a and 9b are graphs demonstrating performance of iso-truss structures in accordance with the present invention; FIG.", "10a is an end view of the iso-truss structure of FIG.", "1; FIG.", "10b is a side view of the iso-truss structure of FIG.", "1; FIG.", "10c is an end view of another iso-truss structure of FIG.", "6; FIG.", "10d is a side view of another iso-truss structure of FIG.", "6; FIG.", "10e is an end view of another iso-truss structure; FIG.", "10f is a side view of another iso-truss structure of FIG.", "10e; FIG.", "11a is an end view of a prior art iso-truss structure; FIG.", "11b is a side view of the iso-truss structure of FIG.", "11a; FIG.", "11c is an end view of a prior art iso-truss structure; FIG.", "11d is a side view of the iso-truss structure of FIG.", "11c; FIG.", "11e is an end view of a prior art iso-truss structure; FIG.", "11f is a side view of the iso-truss structure of FIG.", "11e; FIG.", "12a is a perspective view of an end plate in accordance with the present invention; FIG.", "12b is an end view of the end plate of FIG.", "10a; FIG.", "13 is an end view of an angled plate of the present invention; FIG.", "14a is a top view of another end plate; FIG.", "14b is a cross-sectional side view of the end plate of FIG.", "14a; FIG.", "14c is a partial cross-sectional view of the end plate of FIG.", "14a; FIG.", "15 is a side view of another end plate in accordance with the present invention secured to an iso-truss structure; FIG.", "16 is a top view of the end plate of FIG.", "15; FIG.", "17a is a perspective view of an end connection in accordance with the present invention; FIG.", "17b is a top view of the end connection of FIG.", "17a with an iso-truss structure; FIG.", "18a is a perspective view of an end connection in accordance with the present invention; FIG.", "18b is a bottom view of the end connection of FIG.", "18a with an iso-truss structure; FIG.", "19a is a perspective view of an end connection in accordance with the present invention; FIG.", "19b is a bottom view of the end connection of FIG.", "19a with an iso-truss structure; FIG.", "20a is a perspective view of an end connection in accordance with the present invention with an iso-truss structure; FIG.", "20b is a side view of the end connection of FIG.", "20a; FIG.", "21a is a perspective view of an end connection in accordance with the present invention with an iso-truss structure; FIG.", "21b is a top view of the end connection of FIG.", "21a; FIG.", "22a is a perspective view of an end connection in accordance with the present invention; FIG.", "22b is a top view of the end connection of FIG.", "22a; FIG.", "23 is a perspective view of a connection in accordance with the present invention; FIG.", "24a is a perspective view of a connection in accordance with the present invention; FIG.", "24b is a partial perspective view of the connection of FIG.", "24a attaching two iso-truss structures; FIG.", "25a is a perspective view of another connection in accordance with the present invention; FIG.", "26 is a perspective view of an attachment member in accordance with the present invention; FIG.", "27 is a perspective view of an iso-truss structure with an attachment member of FIG.", "26; FIG.", "28a is a perspective view of an iso-truss structure with an exterior shell in accordance with the present invention; FIG.", "28b is a perspective view of the exterior shell of FIG.", "28a; FIG.", "29 is a perspective view of the attachment member of FIG.", "26; FIG.", "30 is a perspective view of an iso-truss structure with attachment members supporting platforms; FIG.", "31 is a perspective view of an iso-truss structure with another configuration of attachment members; FIG.", "32 is a side view of a flat member attached to an iso-truss structure in accordance with the present invention; FIG.", "33 is a perspective view of flat members attached to an iso-truss structure in accordance with the present invention; FIG.", "34a is a side view of a flat member attached to an iso-truss structure in accordance with the present invention; FIG.", "34b is an end view of the flat member of FIG.", "34a; FIG.", "35 is an end view of an attachment of a flat member to an iso-truss structure in accordance with the present invention; FIG.", "36 is an end view of an attachment of a flat member to an iso-truss structure in accordance with the present invention; FIG.", "37a is a top view of an attachment to an iso-truss structure in accordance with the present invention; FIG.", "37b is a perspective view of the attachment of FIG.", "37a; FIG.", "38a is a side view of a tapering iso-truss structure of the present invention; FIG.", "38b is a side view of another tapering iso-truss structure of the present invention; FIG.", "39 is a side view of a flexible iso-truss structure of the present invention shown in a curved configuration; FIG.", "40a is a side view of an angled iso-truss structure in accordance with the present invention; FIG.", "40b is a side view of another angled iso-truss structure in accordance with the present invention; FIG.", "41 is a side view of a curved iso-truss structure in accordance with the present invention; FIG.", "42 is a side view of a circular iso-truss structure in accordance with the present invention; FIG.", "43 is a side view of a curved angular iso-truss structure in accordance with the present invention; FIG.", "44 is a side view of another curved angular iso-truss structure in accordance with the present invention; FIG.", "45 is a side view of another iso-truss structure in accordance with the present invention; FIG.", "46 is a detailed perspective view of a braided sock in accordance with the present invention; FIG.", "47 is a side view of an integral connector in accordance with the present invention; FIG.", "48 is a side view of another integral connector in accordance with the present invention; FIG.", "49 is a side view of another integral connector in accordance with the present invention; FIGS.", "50 and 51 are side views of a union connector in accordance with the present invention; FIG.", "52 is a side view of an elbow connector in accordance with the present invention; FIGS.", "53 and 54 are side views of a tee connector in accordance with the present invention; FIG.", "55 is a side view of a cross connector in accordance with the present invention; FIG.", "56 is a side view of another connector in accordance with the present invention; FIGS.", "57 and 58 are schematic exploded views of other attachments in accordance with the present invention; FIG.", "59 is a side view of a sign utilizing an iso-truss structure in accordance with the present invention; FIG.", "60 is a side view of another sign utilizing an iso-truss structure in accordance with the present invention; FIG.", "61 is a side view of another sign utilizing an iso-truss structure in accordance with the present invention; FIGS.", "62 and 63 are side views of utility poles utilizing an iso-truss structure in accordance with the present invention; FIG.", "64 is a side view of a light pole utilizing an iso-truss structure in accordance with the present invention; FIGS.", "65-74 are side views of bicycles with frames utilizing iso-truss structures in accordance with the present invention; FIG.", "75 is an exploded view of a bicycle frame utilizing iso-truss structures in accordance with the present invention; FIG.", "76 is a perspective view of the bicycle frame of FIG.", "78; FIG.", "77 is a side view of a mandrel for forming an iso-truss structure in accordance with the present invention; FIG.", "78 is a perspective view of a head for a mandrel for forming an iso-truss structure in accordance with the present invention; FIG.", "79 is a perspective view of a collapsible mandrel for forming an iso-truss structure in accordance with the present invention; FIG.", "80 is a support member utilizing an iso-truss structure in accordance with the present invention; FIG.", "81 is a side view of a basketball support utilizing an iso-truss structure in accordance with the present invention; FIG.", "82 is a side view of a backpack utilizing an iso-truss structure in accordance with the present invention; FIG.", "83 is a perspective view of a boat with a mast or support utilizing an iso-truss structure in accordance with the present invention; FIG.", "84 is a side view of a bridge utilizing an iso-truss structure in accordance with the present invention; FIG.", "85 is a side view of an oil platform utilizing iso-truss structures in accordance with the present invention; FIG.", "86 is a side view of an oil platform utilizing iso-truss structures in accordance with the present invention; FIG.", "87 is a cross-sectional end view of a submarine utilizing an iso-truss structure in accordance with the present invention; FIG.", "88 is a perspective view of a missile or rocket utilizing an iso-truss structure in accordance with the present invention; FIG.", "89a is a perspective view of an aircraft utilizing an iso-truss structure in accordance with the present invention; FIG.", "89b is a cross-sectional end view of the aircraft of FIG.", "89a; FIG.", "90 is a perspective view of a satellite utilizing an iso-truss structure in accordance with the present invention; FIG.", "91 is a side view of a water tower utilizing iso-truss structures in accordance with the present invention FIG.", "92 is a partial side view of a roof system utilizing iso-truss structures in accordance with the present invention; FIG.", "93 is a broken away side view of a kayak utilizing iso-truss structures in accordance with the present invention; FIG.", "94 is a partial broken away side view of a rocket utilizing iso-truss structures in accordance with the present invention; FIG.", "95 is a side view of an artificial reef utilizing iso-truss structures in accordance with the present invention; FIG.", "96 is a partial side view of a drive shaft utilizing iso-truss structures in accordance with the present invention; FIG.", "97 is a side view of a shock absorber utilizing iso-truss structures in accordance with the present invention; FIG.", "98 is a side view of a flexible joint utilizing iso-truss structures in accordance with the present invention; FIG.", "99 is a cross-sectional end view of a pressure vessel or tank utilizing iso-truss structures in accordance with the present invention; FIG.", "100 is a side view of a gear system utilizing iso-truss structures in accordance with the present invention; FIGS.", "101a b are side view of impact barriers utilizing iso-truss structures in accordance with the present invention; FIGS.", "102a and b are cross-sectional end views of impact barriers utilizing iso-truss structures in accordance with the present invention; FIGS.", "103a-c are end views of iso-truss structures in accordance with the present invention.", "DETAILED DESCRIPTION For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used to describe the same.", "It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.", "Any alterations and further modifications of the inventive features illustrated herein, and any additional applications of the principles of the invention as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention.", "Improved Iso-Truss Structure Some basic features of an iso-truss structure are described in U.S. Pat.", "No.", "5,921,048, issued Jul.", "13, 1999, which is herein incorporated by reference.", "As illustrated in FIGS.", "1-5, an improved iso-truss structure, indicated generally at 10, in accordance with the present invention is shown.", "The structure and geometry of the preferred embodiment of the iso-truss structure 10 may be described in numerous ways.", "The iso-truss structure 10 includes a plurality of elements or members 12 arranged in a repeating pattern along the length or longitudinal axis 14 of the structure 10.The structure 10 may be conceptualized and described as a plurality of helical components 20 wrapping about the longitudinal axis 14.Each helical component 20 includes a plurality of straight segments 22 connected end-to-end in a helical configuration.", "In one aspect, each helical component 20 advantageously includes at least four straight segments 22 which form a single, substantially complete rotation about the axis 14.Thus, when viewed along the axis 14, the four straight segments 22 form a square, or have a square cross-sectional shape, best seen in FIG.", "4.The helical components 20 may continue indefinitely forming any number of straight segments 22.The straight segments 22 are oriented at an angle with respect to the axis 14.Preferably, the straight segments 22 are rigidly connected at their ends to adjacent or sequential segments.", "In one aspect, the basic structure of the iso-truss structure 10 includes 1) at least two helical components 30 and 32, and 2) at least one reverse helical component 34, all wrapping around the axis 14.In another aspect, the basic structure 10 includes 1) four helical components 30, 32, 36 and 38, and 2) four reverse helical components 34, 40, 42 and 44.The helical components 30 and 32 wrap around the axis 14 in one direction, for example clockwise, while the reverse helical component 34 wraps around the axis 14 in the opposite direction, for example counterclockwise.", "The helical components 30 and 32, and segments 22 thereof, have a common angular orientation and a common axis 14.The reverse helical component 34, and segments thereof, have a similar helical configuration to the helical components 30 and 32, but an opposing angular orientation.", "This basic structure 10, when viewed from the end or axis 14 (FIG.", "4), appears as an imaginary tubular member of square cross section.", "Referring to FIGS.", "5a-5v, the various helical components are shown being individually added to the structure 10 for clarity.", "The first helical component 30 is shown in FIG.", "5a.", "The segments 22 define a square tube 50, shown in phantom lines.", "For purposes of FIG.", "5a, the square tube 50 includes a bottom, top, and left and right sides, or planes.", "The first helical component 30 includes a first segment 52, in the left plane; a second segment 54 in the top plane; a third segment 56 in the right plane; and a fourth segment 58 in the bottom plane.", "The helical component 30 may continue with many more segments.", "The four segments 22 of the helical component 30 form a single, complete rotation about the axis 14.Referring to FIGS.", "5b-5d, the second, third and fourth helical segments 32, 36 and 38 are shown in bold respectively.", "Referring to FIG.", "5e, the first reverse helical segment 34 is shown in bold.", "The first reverse helical component 34 includes a first segment 60, in the left plane; a second segment 62 in the bottom plane; a third segment 64 in the right plane; and a fourth segment 66 in the top plane.", "The reverse helical component 34 may continue with many more segments.", "The four segments 22 of the reverse helical component 34 form a single, complete rotation about the axis 14.Referring to FIGS.", "5f-5h, the second, third and fourth reverse helical components 40, 42 and 44 are shown in bold respectively.", "Referring to FIG.", "5i, all of the helical components 30, 32, 36 and 38 are shown highlighted.", "Similarly, referring to FIG.", "5j, all of the reverse helical components 34, 40, 42 and 44 are shown highlighted.", "Referring to FIG.", "5k, all of the components in the top and right planes are shown highlighted.", "Referring again to FIG.", "51, building on the basic structure of the iso-truss structure 10 described above, the iso-truss structure 10 advantageously includes an enhanced basic structure, additionally including 1) rotated helical components, and 2) reverse rotated helical components.", "The rotated helical components are similar to the helical components, but are rotated with respect to the helical components.", "Similarly, the reverse rotated helical components are similar to the reverse helical components, but rotated with respect to the reverse helical components.", "The rotated helical components and the rotated reverse helical components also form a square when viewed along the axis 14 (FIG.", "4) which is rotated with respect to the square formed by the helical components 30 and 32 and reverse helical component 34.Referring to FIGS.", "51-5u, the various rotated helical components are shown being individually added to the structure 10, with the helical and reverse helical components removed, for clarity.", "The first rotated helical component 80 is shown in FIG.", "51.The segments 22 define a square tube 82, shown in phantom lines.", "For purposes of FIG.", "51, the square tube 52 includes a forward facing, rearward facing, and upper and lower facing sides, or planes.", "The first helical component 80 includes a first segment 84, in the forward facing plane; a second segment 86 in the lower facing plane; a third segment 88 in the rear facing plane; and a fourth segment 90 in the upper facing plane.", "The rotated helical component 80 may continue with many more segments.", "The four segments 22 of the rotated helical component 80 form a single, complete rotation about the axis 14.Referring to FIGS.", "5m-5o, the second, third and fourth rotated helical segments 92, 94 and 96 are shown in bold respectively.", "Referring to FIG.", "5p, the first rotated reverse helical segment 98 is shown in bold.", "The first rotated reverse helical component 98 includes a first segment 100, in the forward facing plane; a second segment 102 in the upper facing plane; a third segment 104 in the rear facing plane; and a fourth segment 106 in the lower facing plane.", "The rotated reverse helical component 98 may continue with many more segments.", "The four segments 22 of the rotated reverse helical component 98 form a single, complete rotation about the axis 14.Referring to FIGS.", "5q-5s, the second, third and fourth rotated reverse helical components 110, 112 and 114 are shown in bold respectively.", "All of the components are shown in FIG.", "5v.", "Referring again to FIGS.", "1-5, the iso-truss structure 10 has a plurality of helical components 20, including: 1) four helical components 30, 32, 36 and 38; 2) four reverse helical components 34, 40, 42 and 44; 3) four rotated helical components 80, 92, 94 and 96; and 4) four rotated reverse helical components 98, 110, 112 and 114.Thus, the structure 10 has a total of sixteen helical components 20.As described above, the straight segments 22 of the helical components 30, 32, 36 and 38 have a common angular orientation, a common axis 14, and are spaced apart from each other at equal distances.", "Similarly, the segments of the reverse helical components 34, 40, 42 and 44 have a common angular orientation, a common axis 14, and are spaced apart from each other at equal distances.", "But the straight segments of the reverse helical components 34, 40, 42 and 44 have an opposing angular orientation to the angular orientation of the segments of the helical components 30, 32, 36 an 38.Again, this structure, when viewed from the end or axis 14, appears as an imaginary tubular member of square cross section, as shown in FIG.", "4.The straight segments of the rotated helical components 80, 92, 94 and 96 have a common angular orientation, a common axis 14, and are spaced apart from each other at equal distances, like the helical components 30, 32, 36 and 38.The segments of the rotated reverse helical components 98, 110, 112 and 114 have a common angular orientation, a common axis 14, and are spaced apart from each other at equal distances, like the reverse helical components 34, 40, 42 and 44.But the straight segments of the rotated reverse helical components 98, 110, 112 and 114 have an opposing angular orientation to the angular orientation of the segments of the rotated helical components 80, 92, 94 and 96.The rotated helical components 80, 92, 94 and 96 and the rotated reverse helical components 98, 110, 112 and 114 are rotated with respect to the helical components 30, 32, 36 and 38 and reverse helical components 34, 40, 42 and 44.In other words, this structure, when viewed from the end or axis 14, appears as an imaginary tubular member of square cross section, but is rotated with respect to the imaginary tubular member created by the helical and reverse helical components, as shown in FIG.", "4.Together, the helical, reverse helical, rotated helical, and rotated reverse helical components appear as an imaginary tubular member having an eight-pointed star cross section when viewed from the axis 14, as shown in FIG.", "4.Two or more single elements 12 connect or intersect at joints 120 (FIG.", "4).", "The elements 12 may be rigidly connected, flexibly connected, or merely intersect at the joints 120.A node is formed where intersecting elements 12 are connected.", "An external node 122 is formed where intersecting elements 12 meet at the perimeter of the structure 10, best seen in FIG.", "4.An internal node 124 is formed where intersecting elements 12 meet at the interior of the structure 10, as seen in FIG.", "4.The iso-truss structure 10 may be referred to as an eight-node configuration, referring to its eight external nodes 122, best seen in FIG.", "4.A bay 128 (FIGS.", "1 and 2) is formed by a repeating unit or pattern measured in the direction of the longitudinal axis 14.A bay 128 contains a single pattern formed by the elements 12.The structure 10 may comprise any number of bays 128.In addition, the length of the bay 128 may be varied.", "An internal angle 130 (FIG.", "3) is formed by a plane created by two corresponding elements 12 of a tetrahedron and a plane created by opposing elements of the same tetrahedron.", "The repeating pattern may be described as a number of triangles or tetrahedrons.", "The triangles and tetrahedrons are of various sizes with smaller triangles and tetrahedrons being interspersed among larger triangles and tetrahedrons.", "The structure 10 may be conceptualized as two, imaginary tubular members of square cross section overlaid to form a single imaginary tube with a cross section like an eight-pointed star, as shown in FIG.", "4.Or, when viewed from the end or longitudinal axis 14, the structure 10 has the appearance of a plurality of triangles spaced from the axis 14 and oriented about a perimeter to form an imaginary tubular member of polyhedral cross section in the interior of the structure 10.In the case of the preferred embodiment, eight triangles are spaced about the longitudinal axis to form an imaginary tubular member of octagonal cross section in the interior of the structure 10.In addition, when viewed from the end or the axis 14, it is possible to define eight planes parallel with the axis 14.The planes extend between specific external nodes 122 in an eight-pointed star configuration.", "The planes are oriented about the axis 14 at 45 degree intervals.", "Furthermore, within a bay 128, a ring of triangular grids is formed which are believed to have strong structural properties.", "This ring of triangular grids circle the interior of the structure 10 in the center of the bay, as shown in FIG.", "4.It is believed that this strength is due to a greater number of connections.", "The helical components 30, 32, 36 and 38 intersect with reverse helical components 34, 40, 42 and 44 at external nodes 122.Similarly, rotated helical components 80, 92, 94 and 96 intersect with rotated reverse helical components 98, 110, 112 and 114 at external nodes 122.The helical components 30, 32, 46 and 38 intersect with rotated reverse helical components 98, 110, 112 and 114 at internal nodes 124.Similarly, the rotated helical components 34, 40, 42 and 44 intersect with reverse helical components 80, 92, 94 and 96 at internal nodes 124.The helical components 30, 32, 36 and 38 and rotated helical components 80, 92, 94 and 96 do not intersect.", "Likewise, the reverse helical components 36, 40, 42 and 44 and rotated reverse helical components 98, 110, 112 and 114 do not intersect.", "In addition to the plurality of helical members, the structure 10 also may have eight internal axial members 132 (FIGS.", "2 and 4) located in the interior of the structure 10 and intersecting the plurality of helical members 20 at internal nodes 120.The axial members 132 are parallel with the longitudinal axis 14.The external and internal nodes 122 and 124 may form rigid connections, or the components may be rigidly connected together.", "In addition, the axial members 132 may be rigidly coupled to the components at the internal nodes 124.The components can be made from a composite material.", "The helical configuration of the structure 10 makes it particularly well suited for composite construction.", "The components are coupled together as the fibers of the various components overlap each other.", "The fibers may be wound in a helical pattern about a mandrel following the helical configuration of the member, as described in greater detail below.", "This provides great strength because the segments of a component are formed by continuous strands of fiber.", "The elements or components may be a fiber, such as fiber glass, carbon, boron, basalt or Kevlar (aramid), in a matrix, such as a thermoset (epoxy, vinyl ester, etc.", "), or even a thermoplastic (polyester, polypropylene, PVC, etc.).", "In addition, an additive may be included in the resin or matrix, such as UV protectors, or chemical repellents.", "Alternatively, the structure 10 may be constructed of any suitable material, such as wood, metal, plastic, or ceramic and the like.", "The elements of the member may consist of prefabricated pieces that are joined together with connecters at the nodes 122.The connector has recesses formed to receive the elements.", "The recesses are oriented to obtain the desired geometry of member 10.It is believed that the multiple symmetric and highly redundant nature of the structure 10 provides an attractive, efficient, and damage tolerant structure, with the three-dimensional configuration of the structure 10 providing substantial resistance to local buckling.", "The structure 10 incorporates stable geometric forms with members that spiral in a piecewise linear fashion in opposing directions around a central cavity.", "The helical and longitudinal members are repeatedly interwoven, yielding a highly redundant and stable configuration.", "In addition, the structure 10 takes advantage of the mechanical properties of continuous fiber in the primary load paths.", "The load is transferred through beam segments to the intersections, where it disperses through other beam segments.", "Each member carries primarily axial loads, taking full advantage of the inherent strength and stiffness of continuous fiber-reinforced composites.", "The helical members primarily carry the torsion and transverse shear loads and stabilize the longitudinal members against buckling when loaded in flexure or axial compression, while the longitudinal members primarily carry the axial and flexural loads and stabilize the helical members against buckling when loaded in torsion or transverse shear.", "Multiple interweaving of the longitudinal and helical members at the joints or nodes provides a strong interlocking mechanism to enable this type of interdependent three-dimensional stabilization.", "Furthermore, the highly redundant nature of the structure 10 makes it very damage tolerant.", "Removal of a single member results in only fractional degradation of the overall structure.", "In fact, removal of a complete node reduces the effective properties by approximately 1/N, where N represents the number of nodes in a single cross-section.", "This damage tolerance capability provides a significant performance advantage over traditional shell structures.", "Failure of composite iso-grid structures typically displays a more ductile overall behavior than is generally observed in advanced composite structures.", "Although the initial response is still linear elastic to the ultimate load, the subsequent behavior after damage initiation is generally nonlinear.", "In compression, this nonlinearity generally includes a roughly 1/N drop in load each time the members through one of the nodes fail.", "In flexure, the failure is less ductile, since the load is concentrated in fewer members.", "Failure initiation under one load type causes only minimal reduction in strength when loaded in another direction, although the stiffness may be more adversely affected.", "Furthermore, failure of the principal load carrying members has little or no effect on the ability of the secondary load carrying members to resist simple loading.", "Failure of one bay in compression has little effect on the torsion capacity of the structure, although the corresponding toughness is reduced.", "In other words, local failure of the primary members has little effect on the capacity of the secondary members.", "From the basic configuration of the structure 10 described above, several alternative configurations are possible with the addition of additional members.", "Referring to FIGS.", "6-8, external axial members 140 may also be located at the perimeter of the structure 10 and intersect the plurality of helical members 20 at the external nodes 122.The axial members 140 are parallel with the longitudinal axis 14.In addition, perimeter members 144 may be located around the perimeter between nodes 122 that lay in a plane perpendicular to the longitudinal axis 14.The perimeter members 144 form a polyhedron when viewed from the axis 14, as shown in FIG.", "8.The perimeter members 144 may be located around the perimeter of the structure 10 between nodes 122 on a diagonal with respect to the longitudinal axis 14.These diagonal perimeter members may be formed by segments of additional helical components wrapped around the perimeter of the plurality of helical components 20.The diagonal perimeter members may extend between adjacent nodes 122, or extend to alternating nodes 122.Such perimeter members may form another iso-truss structure about the first, or a double iso-truss structure.", "Such a configuration creates a relatively smooth outer surface or supporting structure that simplifies application of an outer skin for cosmetic of structural purposes.", "The double iso-truss structure also provides enhanced stiffness per unit weight.", "As stated above, the improved iso-truss structure of the present invention preferably includes sixteen helical components which each include four segments forming a full rotation about the axis 14 to form square cross sections, and may be referred to as an eight node structure.", "A side-by-side comparison of the eight and six node configurations is shown in FIGS.", "10a-f and 11a-11f, respectively.", "The eight node structure 10 is shown in FIGS.", "10a and 10b, while the six node structure is shown in FIGS.", "11a and 11b.", "External axial members 140 and perimeter members 144 have been added to the structures shown in FIGS.", "10c and 10d for the eight node structure, and FIGS.", "11c and 11d for the six node structure.", "As stated above, the external axial members 140 and perimeter members 144 may form another iso-truss structure about the first, or a double iso-truss structure.", "The internal axial members have been removed from the structure shown in FIGS.", "10e and 10f for the eight node structure, and FIGS.", "11e and 11f for the six node structure.", "The eight node configuration results in the structure 10 having parallel sides, which makes the structure more square and better suited for applications which prefer square geometries.", "For example, the eight node configuration fits better in a box, due to its parallel and perpendicular sides, permitting greater suitability for numerous internal stiffening applications where the dimensions of the structure are constrained.", "In addition, the increased number of nodes increases the angle between adjacent segments or members of each helical component.", "It will be appreciated that with a six node configuration, each helical component would have three segments or members forming a complete rotation, or a triangle, with a relatively sharp angle between adjacent segments or members.", "Such sharp angles act as points of stress concentration, and may be subject to failure.", "With an eight node configuration, however, each helical component has four segments or members forming a substantially complete rotation, or a square, with relatively wider angles, which may have reduced stress and failure.", "Furthermore, the nodes may be more rounded to further reduce stress concentration.", "The eight node configuration, with wider angles, facilitates rounded nodes, and thus reduces stress concentrations.", "In addition, the eight node configuration has more unobstructed internal space (free volume) as a percentage of the total cross-sectional area, permitting easier fabrication and yielding more internal volume for non-structural purposes than the six node configuration.", "Performance Characteristics Referring to FIGS.", "9a-9f, the performance of the iso-truss structure 10 of the present invention is shown with respect to other configurations.", "As indicated above, the iso-truss structure 10 of the present invention includes eight external nodes 122, and may be referred to as an eight node structure.", "In addition, the iso-truss structure 10 of the present invention includes sixteen helical components which each include four segments forming a full rotation about the axis 14 to form square cross sections.", "A basic structure disclosed in U.S. Pat.", "No.", "5,921,048 includes only twelve helical components, each with only three straight segments forming triangular cross sections, and thus includes only six external nodes.", "A side-by-side comparison of the eight and six node configurations is shown in FIGS.", "10a-f and 11a-11f, respectively.", "Referring to FIG.", "9a, the bending strength of various configurations of structures are shown.", "In particular, the bending strength of several structures is shown which have six, eight, nine, ten and twelve nodes.", "It can be seen from the figure that an eight-noded structure has the surprising and unexpected result of significantly increasing the bending strength.", "Referring to FIG.", "9b, the torsional strength of various configurations of structures with various numbers of nodes is shown.", "Again, it can be seen from the figure that an eight node structure 10 has the surprising and unexpected result of significantly increasing the torsional strength of the structure 10.While increasing the number of nodes beyond eight causes an increase in both bending and torsional strength, the increase is not nearly as significant as the increase from six to eight nodes.", "Angular Configuration Referring again to FIGS.", "2 and 3, an angle 130 is formed between a helical component 30 and a reverse helical component 34, or the segments thereof.", "Preferably, this angle 130 is greater than or equal to 45 degrees; more preferably greater than 60 degrees; and most preferably greater than or equal to 75 degrees.", "Referring again to FIG.", "9a, it can be seen that the bending strength surprisingly and unexpectedly increases a significant amount as the angle 130 between the helical and reverse helical components 30 and 34 is increased.", "Similarly referring to FIG.", "9b, the torsional strength of the structure 10 also surprisingly and unexpectedly increases a significant amount when the angle 130 is 75 degrees.", "The torsional properties appear to be greatest at an angle 130 of approximately 90 degrees.", "From the figures, it can be seen that the bending and axial (tension) properties of the structure improve as the angle 130 increases.", "Other properties, however, such as buckling and torsion appear to be reduced as the angle increases.", "One problem with tubular composite structures is their poor bending properties, or they bend too easily.", "The structure of the present invention, however, and the increased angle, demonstrates improved, or stiffer, bending properties.", "End Connections Referring to FIGS.", "12a and b, an end plate 120 is shown for attaching the iso-truss structure 10 to other structures and objects, and/or for facilitating manufacture of the iso-truss structure 10.The end plate 120 is attached to an end of the helical components 20 in order to attach the helical components and the structure 10 to another object.", "The end plate 120 includes a plurality of apertures 121 through which bolts or the like may be used to secure the end plate 120, and thus the iso-truss structure 10, to another object.", "In addition, the end plate 120 includes a perimeter 122 with a plurality of indentations 123a.", "The indentations 123a may receive the helical and/or external axial components, or the strands of fiber forming the helical or external axial components.", "For example, strands of fiber may be wound around the end plate through the indentations 123a, such that the end plate 120 is integrally formed with the iso-truss structure 10, thus providing a strong attachment between the end plate 120 and the iso-truss structure 10.A strand of fiber may pass through one indentation 123a, wrap around the end plate 120, and pass back through another indentation 123a.", "Furthermore, the end plate 120 may include a center aperture 124 through which a mandrel is received during the manufacturing process, as discussed in greater detail below.", "Further indentations 123b may also be provided for receiving the internal axial members 132, or the strands of fiber comprising the internal axial members.", "Referring to FIG.", "13, an angled end plate 125 may be provided for attaching to the iso-truss structure 10 at an angle with respect to the longitudinal axis 14.The angled end plate 125 is similar in many respects to the end plate 120 except that the angled end plate 125 is elongated in one direction to accommodate its attachment at an angle.", "Such an angled end plate 125 may be used to attach two iso-truss structures together at an angle.", "For example, the angled end plate 125 may be configured to attach to an iso-truss structure at a 45 degree angle.", "Thus, two iso-truss structures may be connected by angled end plates 125 to form a 90 degree angle there between.", "Referring to FIGS.", "14a-c, another end plate 126 is shown for attaching to the structure 10 in order to attach the structure to another object.", "The end plate 126 includes a groove or slot 127 for receiving the structure.", "Preferably, the groove 127 is octagonal for receiving the inner portion of the structure.", "The groove or slot 127 can be formed about the plate 126 near the edge or perimeter creating a perimeter wall 128.The perimeter wall 128 can be slotted 129 to form a plurality of flaps or fingers 130, which may be flexible to bend outwardly to receive the structure, and resilient to bend back inwardly once the structure is received, such that the structure “snaps” into the groove 127 between the plate 126 and fingers 130.Other grooves or indentations 131 may be formed in the plate 126 or fingers 130 and located and oriented to receive the various segments of the structure therein, such that the fingers 130 “snap” around the various segments to hold the structure to the plate 126.Holes 132 can be formed through the fingers 130, the groove 127, and into the plate 126 to receive bolts or screws to further secure the structure in the groove 127.The holes 132 are located such that the bolts or screws pass through the structure around various segments thereof.", "Such a configuration has the advantage that the structure can be snapped into the plate.", "Referring to FIGS.", "15 and 16, another end plate 136 is shown for attachment to the structure 10.A plurality of U-shaped bolts or members 137 extend around various segments or nodes of the structure 10 and are secured to the plate 136 to secure the structure to the plate.", "The U-shaped bolts or members 137 may be angled such that bolts or members 137 extend radially through the structure 10 and then angle longitudinally or axially towards the end plate 136.Holes may be formed in the plate 136 for receiving the bolts or members 137, which may be secured by nuts threaded onto the ends thereof.", "The bolts or members 137 may be located outside the structure 10, as shown, or may be located inside.", "In addition, the bolts or members 137 may engage the structure 10 at external nodes 122, and engage both helical members, and external axial member 140.Such a configuration may be less expensive to fabricate.", "Referring to FIGS.", "17a and b, another end connector 140 is shown which includes a base 141 with a plurality of fingers 142 which extend into the structure 10, and are received within the openings formed between the various segments of the structure 10.The base 141 may be annular, with the fingers 142 disposed around the annular base 141 and extending longitudinally, or axially.", "Preferably, the base 141 is sized to fit within the central cavity or space between the segments or helical members.", "In addition, the connection 140 preferably includes eight fingers 142, to extend into the eight triangular openings or voids formed between the segments of the structure 10.A center ring 143 is disposed in the central cavity or space, and is attached to the fingers 142 by fasteners 144, such as bolts.", "Thus, the center ring 143 and fasteners 144 secure the fingers 142 and base 141 to the structure 10.Other objects may be secured to the base 141 to attach such objects to the structure 10.The configuration of the connection 140 allows the base 141 and fingers 142 to be easily slid into the end of the structure, and attached to the center ring 143 by the fasteners 144.In addition, the connection 140 is entirely disposed within the circumference or perimeter of the structure 10 such that the connection 140 does not protrude therefrom.", "The fingers may be flexible and resilient to be bent inwardly as the fasteners are tightened, gripping the structure.", "Referring to FIGS.", "18a and b, a similar connection 146 is shown in which a C-clamp type fastener 147 is utilized in place of the center ring 143 described above.", "In addition, L-shaped members 148 are secured to or protrude from the fingers 142 and extend into the central cavity or space.", "The C-clamp 147 surrounds the angled portion of the L-shaped members 148, securing them together, and thus securing the fingers 142 and base 141 to the structure 10.Such a configuration of the connection 146 allows the fingers 142 to bend inwardly towards the center as the C-clamp 147 is tightened.", "Thus, the fingers 142 may grip the structure 10.Referring to FIGS.", "19a and b, a similar connection 150 is shown in which the fingers 142 are paired together, or connected in pairs.", "An L-shaped member 151 is attached to, or extends from, each pair towards the middle of the central cavity or space in the structure 10.Opposing L-shaped members 151 are coupled together by fasteners 152, such as bolts.", "The fasteners 152 may be tightened, drawing the L-shaped members 151, and thus the fingers 142, inwardly.", "Thus, the fingers 142 may grip the structure 10.Referring to FIGS.", "20a and b, another end connection has an end plate 154 with a plurality of fingers 155 extending therefrom in the axial or longitudinal direction.", "Preferably, the fingers 155 are sized, shaped and located to extend into the openings between the segments of the structure 10.Thus, the connection preferably includes eight fingers 155 with triangular cross-sectional shapes to fit snugly or completely in the openings between the segments.", "In addition, the connection includes a ring member 156 disposed about the exterior of the structure 10, preferably about a narrow portion or the inner nodes.", "The fingers 155 and ring member 156 are attached, such as by fasteners, to secure the base plate 154 to the structure 10.The fingers 155 may have slots or indentations for receiving the ring member 156.In addition, the ring member 156 may be segmented, or formed of more than one piece, in order to dispose the ring member 156 about the exterior of the structure 10 at a narrow portion.", "Referring to FIGS.", "21a and b, a similar connection is shown in which a plurality of retaining members 157 are attached to the fingers 155 to retain the structure 10 on the fingers 155 and base plate 154.The fingers 155 can include slots, holes, or the like, for receiving the retaining members 157 therethrough.", "The retaining members 157 can extend through the fingers, and the segments of the structure 10.Thus, the fingers 155 and base plate 154 may be slid onto the end of the structure 10, and the retaining members 157 disposed through the fingers 155 and structure 10, to secure the base plate 154 to the structure.", "Referring to FIGS.", "22a and b, another end connection 160 is shown with a base 161 and a plurality of fingers 162.The base 161 may be annular, and sized to extend around the exterior of the structure 10.The fingers 162 may extend inwardly from the annular base 161 to be received in the spaces between the segments of the structure 10.The base 161 preferably is octagonal to receive the structure therein, and to extend completely around the circumference or perimeter of the structure 10.Other objects may be secured to the exterior of the structure 10 by attaching such objects to the base 161.Referring to FIG.", "23, a similar end connection 164 is shown 164 with a base 165 which extend only partially about the circumference or perimeter of the structure 10.Again, other objects may be secured to the structure 10 by attaching such objects to the base 165.Referring to FIGS.", "24a and b, another type of end connection 170 is shown for attaching two structures 10 and 171 together, preferably in an end-to-end configuration.", "Such a connection 170 may be useful in assembling multiple structures 10 and 171 together to form a larger structure.", "The connection 170 includes opposite first and second ends 172 and 173 configured to engage and couple to the first and second structures 10 and 171, respectively.", "The connection 170 includes an elongated, axial member 174 configured to extend along the axis or longitude of the structures 10 and 171.The axial member 174 preferably is segmented into first and second portions adjustably attached together by an adjustable attachment member 175.The proximal ends of the first and second portions can be threaded, while the attachment member 175 can have opposite threaded openings receiving the proximal ends.", "Thus, turning the attachment member 175 either draws the first and second portions together, or further separates them.", "The ends 172 and 173 are configured to engage and attach to the structures 10 and 171, respectively.", "Each end 172 and 173 preferably is formed into a hook-like configuration for securing to the segments of the structures.", "The ends 172 and 173 can include an angled, U-shaped member 176 for engaging the segments of the structures.", "Thus, members 176 extend from the ends inwardly towards the structures, and then angle longitudinally or axially, to form a hook.", "In addition, the U-shaped members 176 may extend along either side of an axial member.", "Thus, the U-shaped members 176 can be hooked to the structures, and the first and second portions of the axial member 174 drawn together by rotating the attachment member 175, in order to draw the first and second structures 10 and 171 together in a secure or attached relationship.", "Referring to FIG.", "25, a similar attachment 178 is shown in which hoops or loops 179 are formed at the ends 172 and 173 for surrounding segments of the structures 10 and 171.The hoops or loops 179 can be formed by angled U-shaped members with ends received in brackets at the ends 172 and 173 of the axial member 174.Intermediate Connections In addition to connecting the iso-truss structure 10 at its ends, it may be necessary or desirable to attach other objects at an intermediate point of the iso-truss structure.", "Referring to FIG.", "26, an attachment member 180 may be provided for attaching to the iso-truss structure 10 at an intermediate location.", "The attachment member 180 may have a triangular cross section, or a portion with a triangular-like cross section.", "Thus, the triangular cross section of the attachment member 180 may be received through a triangular opening in the iso-truss structure 10, as shown in FIG.", "27.Preferably, the triangular shape of the attachment member 180 matches the size and triangular shape of the openings through the structure 10, to form a snug, or firm fit.", "A plurality of grooves 182 may be formed in the attachment member for receiving the helical components.", "Therefore, other objects may be attached to the attachment member 180 in order to attach the objects to the iso-truss structure 10.For example, a pair of attachment members 180 may extend through the structure 10, to support other objects, such as cross members of utility poles to support utility lines, etc.", "Referring to FIG.", "27, the ends of the attachment member 180 may have indentations 184 formed in the triangular cross section to receive and facilitate the use of fasteners 185, such as bolts.", "The indentations 184 create a flat flange 186 for the fasteners 185.As stated above, various other objects may be attached to the structure 10, or the attachment members 180.Referring to FIGS.", "28a and b, an exterior shell 190 may be attached to the structure 10.The shell 190 may be utilized to protect the structure 10 or as a platform for attaching other objects to the shell 190, and thus to the structure 10.The shell 190 can have any appropriate shape.", "The shell 190 may be octagonal, or have an octagonal cross-section, to match the exterior or perimeter of the structure 10.Attachment members 191, similar to those described above, extend through the structure 10, and can have triangular cross-sections.", "The shell 190 may be provided in lateral or radial portions, such as first and second halves which each extend longitudinally or axially along the length of the structure 10.Each half of the shell 190 can be attached to the ends of the attachment members 191.For example, apertures may be formed in the shell 190, and bores formed axially in the ends of the attachment members 191, to receive fasteners, such as bolts, which extend through the apertures an bores to secure the shell 190 to the attachment members 191.The shell 190 may prevent climbing on the structure 10, protect the structure 10, or have various other objects attached thereto.", "Referring to FIG.", "29, it will be noted that the attachment members 180, as described above, may be configured in groups or pairs.", "In addition, the pairs of attachment members 180 may be oriented to point towards one another, forming an hour-glass profile, or away from one another, forming a diamond shaped profile.", "Furthermore, the attachment members 180 may be grouped and oriented to extend from opposite sides, and/or radiate outwardly on more than one or two sides, such as four orthogonal sides, as shown.", "Brackets 193 can be configured to surround the ends of the pair of attachment members 180.Various objects may be attached to the brackets 193, such as eyes for suspending other objects, as shown.", "Referring to FIG.", "30, platforms 195 may be attached to the pairs of attachment members 180.Referring to FIG.", "31, multiple attachment members 180 can be configured to extend through the structure 10 in a square configuration, allowing attachment from multiple sides.", "Each attachment member 180 can include an elongated protrusion 194, and be attached to adjacent members.", "The attachment members described above preferably are triangular to match the openings extending through the structure 10.Referring to FIG.", "32, flat attachment members 200 may extend through the openings in the structure.", "The flat attachment members 200 can include indicia and can be utilized as signs, or can be utilized as platforms.", "U-bolts 201 can be used to attach the flat attachment member 200 to segments, such as the exterior axial members.", "Referring to FIG.", "33, other flat members 206 can be attached to the exterior of the structure 10.Hooks 207 can be formed on one side of the flat members 206 for engaging or hooking to the segments of the structure 10.Other object can be attached to the other side of the flat member 206, or indicia may be provided on the other side.", "Referring to FIGS.", "34a and b, flat members 210 can be attached to the exterior of the structure 10 utilizing attachment members 211, similar to those described above.", "One or more attachment members 211 may extend through the structure 10 near the exterior.", "Fasteners 212, such as U-bolts, can extend around the attachment members 211 and attach to the flat member 210, such as by extending through apertures therein.", "Referring to FIG.", "35, attachment members 216 can extend through the structure 10 and attach directly to a flat member 217.The attachment members 216 may be configured in a block U-shaped configuration to engage more of the structure 10.Alternatively, rounded U-shaped attachment members 218 may extend through the structure 10, as shown in FIG.", "36.Many of the attachment members described above have been described as extending through the structure 10.Referring to FIGS.", "37a and b, attachment members 220 may extend into the structure 10, and be coupled in the central cavity or space, without extending entirely through the structure 10.The members 220 may be provided with flanges that are attached with a fastener.", "In addition, the attachment member may have other cross sectional shapes and be configured to extend through other cross sectional openings in the structure.", "For example, the attachment member may have a quadrilateral cross sectional shape and extend through a quadrilateral opening in the structure.", "One or more nodes may be removed or left out to facilitate attachment of an object to the structure.", "For example, leaving out one node presents a flatter side.", "In addition, opposite nodes can be left out for flatter, opposite sides, for an attachment through the structure.", "Tapering Iso-Truss Structure Referring to FIGS.", "38a and 38b, iso-truss structures are shown which are similar to the iso-truss structure 10 described above, but taper in one or more directions.", "Referring to FIG.", "38a, an iso-truss structure 230 tapers from a wider first end 231 to a narrower second end 232.The individual segments 12 which form the helical components of the structure 230 vary in length from being longer at the first end 231 to shorter at the second end 232, such that the entire structure 230 tapers.", "The helical components may continue to wrap around the longitudinal axis with the same angular orientation.", "The structure 230 may also include axial members 233 which are not parallel with the longitudinal axis 14 of the structure 230.Referring to FIG.", "38b, another iso-truss structure 234 may have narrow ends 235 and 236 and a wider middle 237.Again, the individual segments 12 forming the helical components may vary in length from longer at the middle 237 to shorter at the ends 235 and 236.It is of course understood that the structure may taper in the middle, and thus have wider ends and a narrower middle.", "Flexible or Bendable Iso-Truss Structure Referring to FIG.", "39, a flexible or bendable iso-truss structure 240 is shown which is similar in many respects to the iso-truss structure 10 described above, but does not include any axial members.", "Again, the individual components 12 and the helical members may be rigidly interconnected, but the segments 22 can include a degree of flexibility.", "Thus, the iso-truss structure 240 may bend laterally between a first straight configuration, similar to FIG.", "5t, and a second curved configuration as shown in FIG.", "39.In the straight position, the structure 240 includes a straight longitudinal axis 14, as in FIG.", "5t.", "Referring to FIG.", "39 and the curved position, the segments and helical components bend and flex such that the entire structure 240 bends laterally about an arcuate or curved axis 242.The lack of the longitudinal components allows the structure 240 to bend or flex in a lateral direction.", "It has been discovered, however, that although the structure 240 is capable of bending in a lateral direction, the structure 240 continues to maintain its torsional stiffness, or resist rotation about the longitudinal axis 14.In addition, a similar structure also can compress and/or expand axially or longitudinally.", "Thus, the structure may expand and/or compress, preferably storing energy, so that the structure can function as a spring member.", "Angled Iso-Truss Structures Referring to FIG.", "40a, a structural member 250 is shown which is similar in many respects to the structural member 10 described above, but includes two sections 252 and 254 which form an angle with respect to one another.", "For example, the two sections 252 and 254 may form a right angle.", "In addition, the two sections 252 and 254 can be integrally formed, or the helical components of one section 252 continue to form the helical components of the second section 254.Thus, the structure 250 forms a continuous angled structure which may be stronger than a separate structure formed with some type of connection.", "Such an arrangement or configuration may be utilized in constructing more complicated structures.", "The structure 250 may have exterior axial members 256 attached to the external nodes 122.Alternatively, a structure 258 may be angled, but without exterior axial members, as shown in FIG.", "40b.", "Curved Iso-Truss Structures Referring to FIG.", "41, a curved iso-truss structure 270 is shown which is similar to the iso-truss structure 10 described above, but has a curved or arcuate longitudinal axis 272.The helical components forming the arcuate structure 270 have segments of different lengths.", "For example, the inside segments 274 on the inside of the curve can be shorter than the outside segments 276 on the outside of the curve.", "In addition, the axial members 278 are also curved and parallel with the curved longitudinal axis 272.Such curved structures 270 may produce less stress than sharp angles.", "Referring to FIG.", "42, a circular iso-truss structure 280 may be formed.", "The circular structure 280 may be continuous as shown.", "The circular structure may have exterior axial members.", "The curved or circular configurations of the iso-truss structure are believed to impart the same structural advantages of the straight iso-truss structures to the curved and circular structures.", "Referring to FIG.", "43, an iso-truss structure 300 may include a curved portion 302 joining to other portions 304 and 306 which may be straight.", "Such a configuration is similar to the sharp angular configuration shown in FIG.", "40b, but provides curvature at the connection of the sections 304 and 306.The curved section 302 is similar to the curved structure 270 described above.", "Such a configuration can be utilized for more complex structures as described in further detail below.", "Such curved portions may be stronger and prevent stress concentrations of sharper angles.", "The structure may have a broad curved section as shown in FIG.", "43, or may have a sharper curved section as shown in FIG.", "44.Referring to FIG.", "44, a structural member 320 is shown in which the structure 320 forms a right angle bend around a external node 324.Thus, a number of helical components may pass through the node 324.The helical components may be continuously formed through the curve.", "The structure may include external axial components 326.Referring to FIG.", "45, an iso-truss structure 330 may be formed with multiple bends or curvatures 332, and/or with more complicated or sharp curvatures.", "For example, a structure may be formed with multiple right angle curvatures.", "As another example, a structure may be formed with sharp curvatures, broad curves, or with multiple different curvatures, like an S-shape.", "Braided Pre-Form As stated above, many of the above-described structures may be formed by resin impregnated fibers, to form rigid structures.", "Many of the above-described structures may also be provided in a braided pre-form configuration.", "The structures may be formed by winding strands of fiber together.", "In addition, additional strands of fiber may be wrapped around segments to hold the fibers together.", "The strands of fiber, however, without their resin, remain flexible, and may be collapsed and expanded as desired.", "Thus, such a braided pre-form may be collapsed or substantially compacted into a small area for transportation, etc.", "The braided pre-form may then be expanded and impregnated with resin to form the desired structure.", "Referring to FIG.", "46, the long fibers forming the segments or helical members, may be sheathed in a braided sock 348 disposed around the fibers.", "Such a sock 348 maintains the internal long fibers together, to prevent tangling, etc.", "In addition, the fibers or segments can be twisted to compact the fibers.", "Furthermore, the segments, or fibers thereof, can be wrapped, such as in a spiral, with other fibers for compaction.", "Integral Connectors Referring to FIG.", "47, the structure 10 can be provided at its ends with connectors 350.Such connectors 350 can be integrally formed with the structure 10, such as by fiber reinforced resin extending continuously between the structure 10 and the connectors 350.The connectors 350 are configured to attach or couple the structure 10 to mating connectors or structures.", "Thus, the connectors 350 may be formed as protrusions or indentations, such as male and female connectors, for mating with opposite indentations or protrusions, respectively, or female and male connectors.", "The connectors 350 can have a circular cross-sectional shape, similar to cylindrical composite tubes, and be received within a circular opening in a receiving connector, as described below.", "The connector 350 may be threaded 353, or have external threads, as shown in FIG.", "49, and threadedly mate with internal threads of a receiving connector, described below.", "The connectors 350 may be protrusions, or male connectors, as shown, or may be indentations, or female connectors.", "Alternatively, the connectors 350 can have a hexagonal cross-sectional shape 356, or an octagonal cross-sectional shape, for mating with a similar shaped connector 357, as shown in FIG.", "48.It is of course understood that the connectors can have any appropriate shape, including for example, square or triangular.", "Various shaped members may be provided for connecting structures.", "For example, union 360 or 361 can have opposing openings for receiving connectors 352 or 356 from two structures, to couple the structures together in an end-to-end configuration, as shown in FIGS.", "50 and 51.An elbow 362 can have an angled configuration, such as a 90 degree angle, to coupled two structures together at an angle, as shown in FIG.", "52.It is of course understood that any appropriate angle can be provided.", "A tee 364 or 357 can have a T-shaped body for coupling a structure at an angle, as shown in FIGS.", "53 and 54.A cross 366 can have four openings, as shown in FIG.", "55.Other connectors may connect the structures to a base 354, as shown in FIG.", "56.Other Attachments Other attachments also are possible.", "Referring to FIGS.", "57 and 58, for example, a plurality of members 380 or 381 extend through the structure transverse to one another.", "The members 380 and 381 can include grooves 382 for mating with one another in an overlapping relationship.", "For example, for a six-node structure, three members 380 can extend through the structure and mate as they overlap one another.", "Holes 384 may be formed in the members 380 for receiving fasteners, such as bolts, which extend through the members 380 and into a base 386 or 387.Thus, the members 380 extend through the structure, attaching the structure to the plate 386.Signs Referring to FIGS.", "59-61, such iso-truss structures as described above may be used to hold signs.", "Referring to FIG.", "59, a straight iso-truss structure 400 may be vertically oriented and have a first end 402 secured to a support surface, such as the ground, and an opposite second end elevated above the first end 402.A sign 406 may be attached to the upper-end 404 of the iso-truss structure 400.The sign 406 may include various indicia.", "Referring to FIG.", "60, an iso-truss structure 410 may include a vertical component 412, the horizontal component 414, and a curved component 416 joining the vertical and horizontal components 412 and 414.The vertical component 412 may be vertically oriented and secured to a support surface, such as a road side.", "The horizontal section 414 may be secured to the vertical section 412, such as through a curved or acuate section 416, as described above.", "A sign member 416 may be secured to the horizontal member 414.Thus, a sign 416 may be suspended or elevated above a road way.", "Referring to FIG.", "61, an iso-truss structure 420 may include a pair of vertical members 422 and 424 disposed on opposite sides of a roadway.", "A horizontal component 426 may be suspended between the two vertical sections 422 and 424.A sign member 428 may be secured to the horizontal member.", "Utility Poles Referring to FIG.", "62, an iso-truss structure 440 may be vertically oriented and attached to a support surface, such as the ground.", "One or more arms 442 may be secured or attached to the iso-truss structure 440 at a location above the ground, and extend generally horizontally outwardly.", "Such arms 442 may be similar to the attachment member described above.", "Utilities lines 444, such as phone, cable, or electrical lines, may be suspended from the arms 442.Referring to FIG.", "63, the structural member 440 may include non-conductive attachment members 446 for attaching the utility lines 444 to the structure.", "The utility lines 444 may extend along a portion of the lengths of the iso-truss structure 440.Referring to FIG.", "64, an iso-truss structure 450 may be vertically oriented and provided at its top end 452 with light structures or light sources 454 for providing illumination.", "Such light sources 454 may be secured to the top end 452, such as with an end plate as described above.", "Bicycle Frames Referring to FIGS.", "65-74, the iso-truss structures described above may be utilized for bike frames, and thus advantageously provide the advantages of strength and light weight.", "The bike frame includes a handle bar location 500 attached to a handle bar 502 and/or front fork 504; a seat location 506 for attachment to a seat stem 508; a pedal location 510 attached to a pedal assembly 512; a rear wheel location 514 attached to a rear wheel 516.The frame 520 includes a plurality of members extending to and between the handle bar, seat, pedal and rear wheel locations 500, 506, 510, and 514.For example, the frame 520 includes a vertical member 522 extending between the pedal location 510 and the seat location 506.In addition, the frame 520 includes a horizontal member 524 extending between the handle bar location 500 and the seat location 506.Finally, the frame member 520 includes a diagonal member 526 extending between the handle bar location 500 and the pedal location 510.The various components or sections 522, 524, and 526, are similar to the iso-truss structures described above, and are assembled to form a triangular frame 520.The frame 520 provides strength and reduced weight.", "Referring to FIG.", "66, only a single diagonal member 532 extends from the handle bar location 500 to the vertical member 522.The frame 530 forms something of a T-shape and eliminates a component for reducing weigh.", "Referring to FIG.", "67, another bike frame 540 may include an arcuate member 542 extending from the seat location 506 to the pedal location 510, and a diagonal member 532 extending from the handle bar location 500 to the arcuate member 542.The arcuate member 542 may more closely match the curvature of the rear wheel 516 and provide additional bending strength.", "Referring to FIG.", "68, another bike frame 550 may include members 552 extending from the seat location 506 to the rear wheel location 514, and another member 554 extending from the pedal location 510 to the rear wheel location 514, or a triangle formed of the iso-truss structure.", "Thus, more of the frame may be formed of lighter weight iso-truss structure.", "Referring to FIG.", "69, another bike frame 560 may include a plurality of members which extend inwardly towards a central location 562.A diagonal member 564 may extend from the handle bar location 500 to the cental location 562.Similarly, a lower member 566 may extend from the pedal location to the central section 562.Finally, an upper member 512 may extend from the seat location 560 to the central location 562.Such a configuration utilizes straight structures which may be easier to manufacture.", "Referring to FIG.", "70, another bike frame 570 may utilize curved or arcuate members.", "For example, an upper member 572 may curve broadly from the handle bar location 500, past the seat location 506, and to the rear wheel location 514.A lower member 574 may extend in a broad arc from the handle bar location 500 to the pedal location 510.The curvature of the member 572 and 574 may provide additional strength.", "Referring to FIG.", "71, another frame 580 may include a broad arcuate member 582 extending from the handle bar location 500 to the pedal location 510, while an additional member 584 extends from the arcuate member 582 past the seat location 506 and towards the rear wheel location 514.Referring to FIG.", "72, another bike frame 590 may include an upwardly curving member 592 extending from the handle bar location 500 pass the seat location to the rear wheel location 514, while a lower member 594 extends from the handle bar location past the pedal location 510 and towards the rear wheel location 514.Thus, the entire frame 590 is formed of the iso-truss structure.", "Referring to FIG.", "73, another bike frame 600 may have an S-shaped member 602 extending in a first arc from the handle bar location 500 and bending into a second arc extending towards the pedal location 510.An upper member 604 extends from the seat location 506 in an arcuate fashion towards the S-shaped members 602.Referring to FIG.", "74, another bike frame 610 forms an S-shape member 612 extending from the handle bar location 500 to the pedal location 510.A vertical member 614 extends upwardly from the pedal location 510 towards the seat location 506.Finally, a rear member 616 extends from the vertical member 614 towards the rear wheel location 514.Referring to FIGS.", "75 and 76, another bike frame 620 is shown in which iso-truss structures are disposed between connectors.", "A handle bar connector 622 may be disposed at the handle bar location 500 and configured to receive an upper horizontal member 624 and a lower diagonal member 626.An upper horizontal member 624 and a lower diagonal member 626 may be received on extensions of the handle bar connector 622.A seat connector 628 may be disposed at the seat location 506 and have extensions to receive the upper horizontal member 624 and a vertical member 630.A lower member 632 is attached at the pedal location 510 and has extensions to receive the lower diagonal member 626 and the vertical member 630.Thus, relatively straight iso-truss structures 624, 626, and 630 may be utilized and attached to the connectors 622, 628, and 632.Method of Manufacturing As discussed above, the iso-truss structures preferably are formed by fibers impregnated with resin.", "In addition, the iso-truss structures or helical components preferably are formed by continuous strands of fiber wrapping around the longitudinal axis and along the length of the iso-truss structure.", "Such a composite iso-truss structure may be formed using a mandrel.", "It will be appreciated that the complicated geometry of the iso-truss structure presents a manufacturing challenge.", "Referring to FIG.", "77, a mandrel 700 is shown with fibers 702 disposed thereon forming the iso-truss structures described above.", "The mandrel 700 can be elongated and shaped to match the desired shape of the iso-truss structure.", "For example, as shown in FIG.", "77, the mandrel 700 is elongated and straight to form an elongated and straight iso-truss structure.", "It is, of course, understood, that the mandrel 700 may be curved or arcuate, or form other angles in accordance with the desired shape of the iso-truss structure.", "In addition, the mandrel 700 can be rotationally disposed such that the mandrel 700 may be rotated as the fibers 702 are wrapped thereon.", "The mandrel 700 may include an elongated core or body 704, and a plurality of heads 706 disposed thereon.", "The core or body 704 preferably has a reduced or smaller diameter with respect to the iso-truss structure, such that the core or body 704 may reside within the iso-truss structure without interfering with any of the segments or helical components.", "The heads 706 preferably are spaced apart from the core or body 704.The heads 706 extend radially from the core or body 704 and towards the exterior nodes 122 of the iso-truss structure.", "The heads 706 are configured to receive the strands of fiber 702 as they are wrapped about the mandrel 700.Therefore, for an eight node iso-truss structure, eight heads 706 extend radially around the circumference of the core or body 704.In addition, a number of heads 706 extend along the length of the core or body 704 in accordance with the length of the desired iso-truss structure.", "Referring to FIG.", "78, the heads 706 are shown in greater detail.", "Each head 706 preferably includes a plurality of indentations 710 for receiving strands of fibers 702.The indentations 710 preferably include two sets of deep indentations 712 and 714 for receiving the strands of fiber 702 forming a pair of opposing helical components.", "Thus, the set of deep indentations 712 and 714 preferably extend downwardly at an angle to match the angle of the segments.", "Each set of deep indentations 712 and 714 preferably include two aligned indentations formed at an angle with respect to one another such that each indentation of the set is performing a different segment of the same helical component.", "The inner section of the indentations 716 is located at the exterior node 122 of the iso-truss structure.", "In addition, the indentations 710 preferably include one or more sets of shallow indentations 718 and 720.One set of shallow indentations 718 may be utilized to form longitudinal components of the iso-truss structure, while the other shallow indentations 720 may be utilized to form radial or lateral components of the iso-truss structure.", "In order to form an iso-truss structure as described above, strands of fiber can be wrapped around the mandrel in order to create the helical components and segments thereof.", "The strands of fiber 702 may be wrapped about the mandrel as described above with respect to the helical components, placing the strands of fiber in the indentations of the head.", "In addition, the strands of fiber may be impregnated with resin as they are wrapped around the mandrel 700.Alternatively, the strands of fiber may be wrapped around the mandrel without impregnating them with resin as discussed above to form a braided pre-form.", "The resin is then cured and the mandrel may then be removed from the iso-truss structure.", "Alternatively, the iso-truss structure may be integrally formed with a mandrel and the mandrel may remain therein.", "It will be appreciated that the complex geometry of the iso-truss structure, and the extension of the heads from the mandrel, create a challenge in removing the mandrel from the iso-truss structure.", "Various types of mandrels may be utilized in order to form the iso-truss structure.", "For example, a dissolvable mandrel may be formed by salt, or sand with a binder, which is dissolved to remove the mandrel from the iso-truss structure.", "As another example, eutectic metals may be used which can be melted away from the iso-truss structure.", "As another example, a balloon mandrel may be utilized which includes a sand-filled bladder which is packed with sand and vacuum sealed to form the mandrel, and then the vacuum is released and the bladder emptied of sand to remove the mandrel from the iso-truss structure.", "In addition, the iso-truss structure may be formed by wet or dry wrapping fibers around an internal mold, and then enclosed by an external mold, similar to injection molding.", "Such a molding process can provide good consolidation, good shape definition, and good surface finish.", "Referring to FIG.", "79, a collapsible mandrel 720 is shown which advantageously may be removed from an iso-truss structure and reused.", "The collapsible mandrel 720 is similar to the mandrel 700 described above and can include an elongated tubular body 722 and a plurality of beads 724.The hollow tubular body 722 can include a plurality of holes or apertures 726 for receiving a plurality of pins 728 therein.", "The pins 728 may be inserted through the holes or apertures 726 of the tubular body 722, and the heads 724 disposed on a pin 728.Thus, the heads 724 extend from the tubular body 722 on the pin 728.An elongated core 730 is removably disposed within the tubular body 722.In addition, a plurality of inserts 732 are also removably disposed in a tubular body 722 between the core 730 and the tubular body 722.The insert 732 also includes a plurality of holes or apertures 734 for receiving the pin 728.Thus, the pin 728 extends through the tubular body 722 and the insert 734 to abut the core 730.Thus, the core 730 maintains the heads extending from the tubular body 722 on the pin 728.After the iso-truss structure has been formed on the mandrel 720, the core 730 may be removed from the tubular body 722 by sliding the core 730 outwardly from the tubular body 722.Removal of the core 730 allows the insert 732 to be removed from the tubular body 722, and the pins 728 to move inwardly into the tubular body 722.Thus, the pins may be removed and the tubular body 722 removed from the iso-truss structure.", "In addition, the heads 724 may be removed.", "Referring again to FIG.", "77, an end plate 120 may be disposed on the mandrel 700 at one or both ends thereof.", "As discussed above with respect to the FIGS.", "12a and b, the end plate 120 has a hole or aperture 124 through which the core or body 704 of the mandrel 700 may be received.", "The strands of fiber 702 may then be wrapped around the indentations 123 through the end plate 120 to integrally form the end plate 120 with the iso-truss structure.", "The core or body 704 of the mandrel 700 may then be removed through the aperture 124 of the end plate 120.The mandrel 740 may be assembled by inserting the pins 728 into apertures in the core or tube 722.Collars also may be disposed at the ends of the tube 722 to form the integral connectors, as described above.", "The heads 724 are disposed on the pins 728.The fibers are wrapped about the heads 724 to form the helical members and axial members.", "In addition, the fibers are wrapped around the collars to form the integral connectors.", "The mandrel is removed to leave the structure.", "Additional Applications Referring to FIG.", "80, a support member 750 may utilize an elongated iso-truss structure 752 as discussed above to hold and secure precast concrete forms 754.The support member 750 may include an iso-truss structure 752 with end plates 120 on the ends thereof to receive connection members 754 and 756 for engaging the ground and the concrete form 754.The strength of the iso-truss structure 752 provides strength for holding up the precast concrete form 754, while the light weight of the iso-truss structure 752 allows the support members 750 to be easily manipulated and handled.", "Referring to FIG.", "81, basketball support 760 is shown for supporting a basketball standard 762.The basketball support 760 may include an iso-truss structure 764 as described above.", "The basketball support 760 may include a vertical iso-truss section 766 and a horizontal iso-truss structure 768 connected to the vertical section 766 for extending the basketball standard 762 over the court.", "Referring to FIG.", "82, a backpack 770 may include a frame 772 which utilizes iso-truss structure 774 as described above.", "The frame 772 may include a perimeter formed of iso-truss structures including a pair of spaced apart vertical members and interconnecting horizontal members.", "The iso-truss structure 774 provides strength and light weight to the backpack 770.Referring to FIG.", "83, iso-truss structure 790 may be utilized to form a mast or other support structures 792 on a boat 794 or other marine structure.", "The iso-truss structure 790 may be formed with composite material, and thus resist corrosion.", "Referring to FIG.", "84, a bridge 800 is shown utilizing an iso-truss structure 802.The iso-truss structure 802 may be arcuate and various bridge components may be suspended therefrom.", "Referring to FIGS.", "85 and 86, an oil platform 810 is shown utilizing iso-truss structures 812 as support columns for supporting the oil platform 810.Again, these iso-truss structures 812 preferably are formed with composite material to resist corrosion.", "In addition, the open structure of the iso-truss structures 812 provide lower drag forces on a structure.", "Referring to FIG.", "87, an iso-truss structure 830 is shown utilized with a submarine 832.The iso-truss structure 830 provides the internal structure supporting the hull 834 of the submarine 832.Thus, the hull 834 is formed around the iso-truss structure 830, while the interior of the iso-truss structure 830 may be utilized for the crew, and interior wall support.", "In addition, the hollow or open structure between the segments or helical components of the iso-truss structure 830 may also be utilized for equipment, piping, etc.", "It is, of course, understood that the iso-truss structures may be utilized for other structures, vehicles, and vessels.", "Referring to FIG.", "88, an iso-truss structure 840 may be utilized for aircraft or airborne devices such as artillery or missiles 842.Again, the iso-truss structure 840 provides an exterior shell or exoskeleton for supporting an outer skin, and an interior for containing other items.", "Thus, the iso-truss structure 840 provides strength and light weight, which is particularly useful in aircraft or airborne applications.", "Referring to FIGS.", "89a and b, an iso-truss structure 842 may be included as part of the fuselage of an aircraft.", "Passenger seats may be located in the central void or space of the structure 842, while other components, such as wiring, hydraulics, fuel lines, etc., may be disposed within the structure 842 itself, or between the segments.", "Such iso-truss structures also may be utilized for wing structures, and other components of the aircraft.", "Referring to FIG.", "90, the structures described above also may be utilized in aerospace applications, such as with satellites or other orbiting structures 844.The structures may be collapsible/expandable to optimize limited cargo space.", "The structures also may be partially formed, such as a braided preform described above, and finally formed in space.", "Referring to FIG.", "91, iso-truss structures 846 also may be utilized in water tower applications.", "The iso-truss structures can be used in buildings and construction.", "Referring to FIG.", "92, a roofing system 900 can utilize iso-truss structures similar to those described above.", "Inclined or horizontal iso-truss structures 902 can form beams to support a roof 904.Vertical iso-truss structures 906 can be used as columns to support the roof inclined iso-truss structures 902.The iso-truss provides structural strength, and is light weight.", "The iso-truss structures can be used in vessels, boats and ships.", "Referring to FIG.", "93, a kayak 910 can utilize tapping iso-truss structures as described above.", "A frame 912 can be formed by an iso-truss structure that tappers at each end.", "A skin or shell 914 can be formed over the frame 912.A portion or side 916 of a bay of the iso-truss can be removed to allow access into the kayak 910, and allow the user's body to extend through the frame 912, and into the hollow of the frame.", "The iso-truss provides structural strength to the kayak, while providing space on the interior for the passenger.", "Referring to FIG.", "94, a solid fuel rocket 917 is shown with an iso-truss structure 918.The solid rocket fuel can be disposed about the members of the iso-truss.", "The iso-truss can burn as the rocket fuel burns, thus eliminating falling rocket casings.", "The nozzle 919 can be configured to travel along the rocket 917 as the fuel and iso-truss burn.", "Referring to FIG.", "95, an artificial reef 920 can be created using a plurality of iso-truss structures 922.The iso-truss can be weighted so that it sinks to the bottom of the sea floor.", "For example, a weight 924, such as concrete, can be attached to one end of the iso-truss.", "The other end can be free to extend upwardly from the sea floor.", "Thus, the iso-truss can be transported to the desired location, and dropped overboard.", "Several iso-truss structures can be attached together.", "The iso-truss structures can be formed with an environmentally friendly epoxy to promote growth on the iso-truss.", "The iso-truss structures also can be used to transmit torque or rotational movement.", "Referring to FIG.", "96, a drive shaft 930 can be formed with an iso-truss structure 932 similar to those described above.", "The drive shaft or iso-truss can be rigid or flexible.", "One end of the drive shaft 930 can be coupled to an engine or transmission 934, while the other end can be coupled to a transfer case or wheel 936.Such a configuration can be useful for vehicles.", "It will be appreciated that such a drive shaft can be used in other applications as well.", "In addition, the iso-truss can be used for drills, such as oil, water and mining drills.", "In such a configuration, one end can be coupled to a driver, while the other end is coupled to a drill bit or cutter.", "Referring to FIG.", "97, a shock absorber 940 utilizes an iso-truss structure 942 with no axial members.", "Thus, the iso-truss structure can compress in a longitudinal or axial direction to absorb shock.", "In addition, a bladder 944, such as a gas filled bladder, can be disposed in the iso-truss.", "Referring to FIG.", "98, an iso-truss structure 950 can be configured with both rigid sections 952, and a flexible section 954 to form a joint.", "The rigid sections 952 can be formed with axial members for stiffness or rigidity, while the flexible section 954 can be formed without the axial members for flexibility.", "Referring to FIG.", "99, a tank or pressure vessel 960 can include an iso-truss structure 962 in which an continuous interior wall 964 is formed.", "The tank or pressure vessel 960 can contain fluids, such as liquids or gases.", "Referring to FIG.", "100, a gear system 970 includes a plurality of gears 972 formed of iso-truss structures which rotate and engage one another.", "The exterior nodes of the gears 972 or iso-truss structures intermesh.", "Referring to FIGS.", "101a and 101b, impact barriers 974 and 976 can include iso-truss structures.", "The iso-truss structure can be oriented to be impacted axially or longitudinally, as shown in FIG.", "101a, or laterally, as shown in FIG.", "101b.", "Referring to FIGS.", "102a and 102b, the impact barriers can include a compressible material, such as foam, disposed in and/or around the iso-truss structure.", "In one aspect, the foam material 980 can form a shell around all or some of the iso-truss structure, and between the internal and external nodes.", "In another aspect, the foam material 982 can be disposed in the interior of the iso-truss structure.", "Referring to FIGS.", "103a-c, the iso-truss structure can be elongated on one side, or in one direction, to create an elongated cross-section.", "Such configuration can be better suited or more efficient in applications where one direction has preferential loading, such as a floor joist.", "The configuration can have different structural properties in different directions, so that the iso-truss can be configured for the loads of a particular application.", "The configurations shown in FIGS.", "103a-c are similar in many respects to the iso-truss structures described above and illustrated herein.", "Some of the segments of the helical components have been elongated with respect to the others, or have a greater length, to create the elongated cross section.", "In addition, the angular orientation between some adjacent or sequential segments is greater.", "Referring to FIG.", "103a, an eight node iso-truss structure 1000 is shown.", "Some of the helical components include longer segments 1002 and shorter segments 1004 to create a rectangular cross sectional shape.", "For example, the helical and reverse helical components can form the rectangular cross sectional shape.", "Other of the helical components include larger angles 1006 between some adjacent segments, and smaller angles 1008 between other adjacent segments, to create a diamond shaped cross section.", "For example, the rotated and rotated reverse helical components can form the diamond shaped cross section.", "Referring to FIG.", "103b, a ten node iso-truss structure 1010 is shown which is elongated to have a more elliptical shape.", "The helical components can have both 1) segments of different lengths, and 2) different angles between adjacent segments.", "For example, the helical and reverse helical components can form a first, elongated pentagon 1012, while the rotated and rotated reverse helical components form a second, elongated pentagon 1014, which together form the elliptical shape.", "In addition, the helical components have five segments forming a single, substantially complete rotation.", "Referring to FIG.", "103c, another iso-truss structure 1020 is shown which has multiple cross sectional shapes.", "The structure 1020 can include both rectangular cross sectional shapes, and elongated diamond cross sectional shapes.", "The helical components have four segments per rotation, but utilizes three helical components for every two helical components in the typical structure.", "The iso-truss structures described above can be utilized in other applications as well.", "For example, the iso-truss structure can be included in the mast of a boat with a sail coupled thereto.", "The iso-truss structure can be included in a flag post with a flag coupled thereto.", "The iso-truss structure can be included in a fence post with fence members attached thereto.", "In addition, a skin, covering or wrap may be disposed around the structure.", "Such a skin may strengthen the structure, prevent climbing, and/or be aesthetic.", "The iso-truss structures described above also may be utilized to reinforce concrete.", "For example, concrete may be poured or otherwise formed about the structures, and may fill the interior of the structures.", "The iso-truss structures have been described above with particular reference to an eight node structure in which the helical components have four straight segments forming a single, complete rotation about the axis.", "It is of course understood that other configuration can be useful, including for example, structure with five, six, seven, nine, twelve, etc.", "nodes.", "It is to be understood that the above-described arrangements are only illustrative of the application of the principles of the present invention.", "Numerous modifications and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of the present invention and the appended claims are intended to cover such modifications and arrangements.", "Thus, while the present invention has been shown in the drawings and fully described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the invention, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, variations in size, materials, shape, form, function and manner of operation, assembly and use may be made, without departing from the principles and concepts of the invention as set forth in the claims." ] ]

[ [ "Dishwashing compositions comprising floating particles", "A machine dishwashing detergent composition is disclosed.", "The composition includes delayed-release solid particles comprising at least one ingredient that is intended to perform its function during the rinse cycle of an automatic dishwashing cycle wherein the particles float in water and have no more than one dimension bigger than 1 cm." ], [ "1.Machine dishwashing detergent composition comprising delayed-release solid particles which particles have a composition comprising at least one ingredient that is intended to perform its function during the rinse cycle characterised in that said particles float in water and have no more than one dimension bigger than 1 cm.", "2.Composition according to claim 1 characterised in that the floating particles have not more than one dimension bigger than 0,5 cm preferably having no more than one dimension bigger than 0,2 cm.", "3.Composition according to claims 1 or 2 characterised in that the surface of the particles is made sticky to metallic or plastic surfaces.", "4.Composition according to any of preceding claims characterised in that the particle has a bulk density lower than 1 g/cm3.5.Composition according to any of preceding claims characterised in that the solid particles remain substantially undissolved in an amount of at least 30% at the end of the last washing cycle when the particles are dosed to a MIELE dishwasher together with 30 g. of standard ICE B dishwashing detergent and the dishwasher is run at its 55° C. mild program.", "6.Composition according to any of preceding claims characterised in that the particle is substantially water-insoluble at low temperatures (<55° C.) and it is soluble or at least dispersible at the higher temperatures of the rinse cycle.", "7.Composition according to any of preceding claims characterised in that the particle is substantially insoluble at relatively high ionic strength and/or high pH and it is solubilised upon decrease of pH or ionic strength.", "8.Composition according to claims 1 to 6 characterised in that the particle comprises a rinse additive.", "9.Composition according to claims 1 to 6 characterised in that the particle comprises a fragrance.", "10.Composition according to the invention substantially as hereinbefore described with reference to any one of Examples 1 and 2." ], [ "The present application relates to detergent compositions particularly for use in a domestic dishwashing machine.", "Although modern dishwashing machines have in most cases a number of different cleaning programs working at different temperatures and having different duration, all the cleaning processes consist basically of the following cycles: pre-wash cycle, main wash cycle, rinse cycle and drying cycle.", "Between the pre-washing and the washing cycle and between the washing and the rinsing cycle the liquor in the machine is pumped off and new fresh water is fed.", "Different products are used in dishwashers to achieve optimum results, these products being normally delivered at different moments during the overall machine cleaning process: a detergent is normally needed for the washing cycle and a rinse aid is normally desirable for the rinse cycle.", "The function of the rinse aid is to avoid that, during rinsing, water droplets are left particularly onto the glassware which droplets upon drying leave residues coming from the substances dissolved in water.", "Although dishwashing machines are equipped with separate dispensing systems to dispense detergent at the wash cycle and rinse aid at the rinse cycle, products in tablet form have recently appeared in the marketplace which products, when dosed at the wash cycle, combine the functionality of a dishwashing detergent and a rinse aid.", "These products consist of a tablet with a first detergent portion and a second solid portion comprising rinse aid additives, whereby means are used to prevent the second rinse aid containing portion from completely solubilising during the washing cycles.", "The rinse aid contained in said second portion is then mainly released at the rinse cycle where it can perform its function.", "The delayed release of the rinse composition has been achieved using two different approaches.", "In a first approach the solid rinse aid containing portion is formulated to be substantially water-insoluble at low temperatures (<55° C.) which are encountered during the washing cycle while it is soluble or at least dispersible at the higher temperatures of the rinse cycle.", "The second approach uses chemical means to control the solubilisation of the rinse aid containing composition.", "During the wash cycle the washing liquor containing the detergent has normally a high ionic strength and/or a high pH.", "After the washing cycle the washing liquor is pumped off and fresh water comes in, causing a substantial decrease of the ionic strength and/or the pH of the washing liquor.", "It has been proposed to formulate the rinse aid composition including ionic strength- or pH-sensitive materials to control the solubility of the composition as a function of these two parameters.", "Both approaches do require that the solid rinse-aid-containing portion survives the washing cycle and reaches the rinse cycle in a substantially unaltered form and that it is not removed from the machine by the pumping steps occurring between cycles as described above.", "The products present on the market have guaranteed that the solid rinse aid containing portions are not pumped off from the machine by making them sufficiently big to prevent them from being pumped away in their undissolved state.", "The requirement of a big particle size for the solid rinse aid portion is not a problem when this portion is part of a unit dose product (i.e.", "a tablet) because then the correct proportion of rinse aid to detergent is pre-established by the manufacturer.", "Conversely the need to work with big particles is a strong limitation when wishing to formulate non-unit-dose products (i.e.", "powders, liquids, gels, very small tablets) Big particles will tend to settle in liquid or gel compositions and will segregate in powder compositions.", "Additionally it will be very difficult for the consumer to guarantee an accurate dosage of the rinse aid composition when big particles of rinse aid are used.", "In PCT patent application number WO 95/29982 A1 it has been proposed to incorporate into a dishwashing detergent composition small (pref.", "100-2,500μ) delayed-release composite particles with a core comprising a rinse aid material and a waxy coating encapsulating said core.", "Although the application claims that these particles provide an effective reduction of residual water left on dishes, the applicant has tried to use the products and has found that very poor results in terms of actual spotting on glassware can be obtained.", "This is a consequence of the pumping off of most of the coated particles before they can actually reach the rinse cycle.", "The applicant has now surprisingly found that when compositions comprising ingredients that are intended to perform its function during the rinse cycle (i.e.", "rinse aids) are formulated in the form of delayed-release small particles which float in water, these particles can be successfully incorporated into dishwashing formulations without being substantially pumped off thereby providing effective rinse performance.", "By particles it is meant here solid material of whatever shape having no more than one dimension bigger than 1 cm more preferably having no more than one dimension bigger than 0,5 cm even more preferably having no more than one dimension bigger than 0,2 cm.", "The term particle in this specification includes granules, beads, flakes, noodles and the like.", "It is preferred that the particles of the invention have no dimension bigger 1 cm more preferably bigger than 0,5 cm even more preferably bigger than 0,2 cm.", "When rinse particles according to the invention are to be incorporated into powder formulations it is preferred that they do not have any dimension bigger than 3 mm.", "When rinse particles according to the invention are to be incorporated into liquid formulations it is preferred that they do not have any dimension bigger than 3 mm.", "If the particles according to the invention are to be incorporated into formulations made in the form of small tablets, it is preferred that they do have a volume not smaller than half and not bigger than double than the dimension of the tablets with which they are mixed.", "There are a variety of factors influencing the floatability of the particles: bulk density of the particles, presence of effervescent systems, surface tension between particles and water among others.", "It is however advantageous to use particles having a bulk density lower than 1 g/cm3.Particles with density lower than 1 g/cm3 can be obtained by using, in the formulation of the tablets, ingredients having low density but they can also be obtained by entrapping air within the particles.", "It has also been surprisingly found by the applicants that floating particles show an increase tendency to migrate and then stick to the walls of the dishwashing machine which further contributes to guarantee their survival during the pumping off cycles.", "The natural tendency of floating particles to stick to the walls of the machine can be further increased by having the particle surface comprise a material which tends to adhere to metal or plastic surfaces normally found in a dishwasher.", "This material can be either incorporated into the particle's composition when the particle is uniform or can also be applied as a coating onto the surface of the tablet.", "Although the expert in the field may well be aware of testing methods to assess the degree of stickiness of particles to specific surfaces, the following process has been found useful for this evaluation: A 2-litre recipient equipped with a stirrer and having controllable draining means at its bottom, is filled with 1 litre water and 0,5 g. of the particles to be tested are added thereto.", "The liquid is agitated during 5 minutes at 200 rpm.", "The stirrer is then stopped and the liquid is left to stand for 5 minutes.", "After this period the liquid is drained out of the recipient.", "The particles remaining in the wall of the recipient are collected and weighted.", "The weight ratio of remaining particles to the weight of particles initially added is a measure of the tendency of the particles to stick to the recipient's walls.", "The tests may be repeated with recipients made of different materials (i.e.", "plastic stainless steel, aluminium .", ".", ". )", "to simulate the different surfaces present in dishwashing machines.", "As possible means of controlling the release of the ingredients that are intended to perform its function during the rinse cycle so that it acts mainly after the washing cycle any of the two approaches that have been explained above and have been used in dishwashing tablets with incorporated rinse aids may be used.", "The applicant also wishes to explicitly incorporate here by reference the means of controlling release disclosed in its PCT patent application number WO 00/06688 A1.By particles showing a delayed release profile it is meant here, particles more than 30% wt.", "of which will remain substantially undissolved at the end of the last washing cycle when the particles are dosed to a MIELE dishwasher together with 30 g. of standard ICE B dishwashing detergent and the dishwasher is run at its 55° C. mild program.", "According to a further aspect of the present invention there is provided a detergent composition for use in automated washing cycles including a rinse cycle, the composition including particles comprising at least one ingredient for use in the rinse cycle, wherein the particles are adapted to float in water.", "Preferably, the particles have no more than one dimension bigger than 1 cm.", "EXAMPLES Example 1 Particles (Floating particle A) as used in example 1 were prepared according to the following instructions: 80 g of paraffin wax with a melting point at 60-62° C. (Wintershall 60/62 DAB) were smoothly heated to a temperature of 80° C. Then while vigorously stirring 80 g of Synperonic LF/RA 30 (Uniqema) were added to the wax while maintaining a temperature of 70° C. Then the mixture was passed through a nozzle with a nozzle size of 0.4 mm.", "droplets were formed which were allowed to chill while falling through a cooling channel to result in solid beads with a size of 0.5-1.5 mm.", "The particles float when added to water.", "They also tend to move to the side walls of a beaker when dosed to the centre of the water surface in such beaker.", "These particles were then added to automatic dishwashing compositions as shown in table 1: TABLE 1 Comparative Formulation Formulation Ingredient Formulation 1 2 3 Disilicate 2.70 2.70 2.70 Sodiumtripolyphosphate 32.00 32.00 32.00 Sodiumcarbonate 37.50 37.50 37.50 Polymer 4.00 4.00 4.00 Floating particle B 0.00 7.50 15.00 Sodiumsulfate 16.65 9.15 1.65 Sodium percarbonate 4.00 4.00 4.00 Enzyme 2.00 2.00 2.00 Surfactant 0.85 0.85 0.85 Corrosion inhibitor 0.15 0.15 0.15 Fragrances 0.15 0.15 0.15 100.00 100.00 100.00 Formulations 1-3 were then investigated in a domestic dishwashing appliance according to their rinse aid performance.", "All formulations were dosed at a level of 30 g per wash.", "The dishwasher used was a BOSCH SMS 5062 and the cleaning program Universal 50° C. was chosen.", "Water hardness was 2° dH.", "Test Procedure A soil in a closed basket is placed in the lower rack of the dishwasher.", "The dishwasher is equipped with a number of tableware specimens.", "After the detergent is dosed into the dosage chamber the front door is closed and the program is started.", "After the program has finished, the front door is kept closed for additional 10 minutes.", "Then the door is fully opened.", "The evaluation can be started when the tableware is dry.", "Rating of the items is according to the instructions below.", "The grading presented in table 2 is generated by calculating the mean over all tableware items of similar kind.", "All dishes are rated separately under a lamp (suggestion: 500-1000 W-halogen light) in terms of spotting.", "The Machine Load: 20 glass tumblers 20 porcelain plates 20 pieces of cutlery Rating for Spotting: 4=no spots on specimen surface 3=1-4 spots 2=more than 4 spots up to 25% of surface covered with spots 1=25% up to 50% of surface covered with spots 0=more than 50% of surface covered with spots TABLE 2 Rinse aid performance of formulations 1-3 Glass tumblers Plates cutlery Over all Formulation 1 2.0 2.0 2.1 2.0 Formulation 2 2.5 2.4 2.6 2.5 Formulation 3 2.9 2.9 3.3 3.0 The results show that an increasing amount of “floating particle A” yields in a significant better rinse aid performance of the detergent formulation.", "It should be noted that if the dishwashing machine cycle is interrupted after the wash cycle or before the rinse cycle a substantial amount of “floating particle A” can be detected in the machine.", "Example 2 Particles (Floating particle B) as used in example 2 were prepared according to the following instructions: 80 g of paraffin wax with a melting point at 46-48° C. were smoothly heated to a temperature of 55° C. Then while vigorously stirring 30 g of a fragrance oil commonly used in house hold cleaning formulations was added to the wax while maintaining a temperature of 50-55° C. Then the mixture was passed through a nozzle with a nozzle size of 0.4 mm.", "droplets were formed which were allowed to chill while falling through a cooling channel to result in solid beads with a size of 0.5-1.5 mm.", "The beads were then placed in a standard fluid bed coater and were coated with a solution comprised of: Hydroxypropylmethylcellulose 3% Polymer I 7% HCl <1% Water 89-90% (Polymer I: Terpolymer comprised of the monomers methacrylmethylester (l), dimethylaminopropyl-methacrylat (m), dimethylaminopropyl-methacrylamid (n) with l/(l+m+n)=0,35; m/(l+m+n)=0,45; l+m+n=1500-1800) The coating process was conducted until the particles gained about 4% in weight.", "The particles float when added to water.", "They also tend to move to the side walls of a beaker when dosed to the centre of the water surface in such beaker.", "There they stick to the walls due to a swelling of the coating layer.", "These particles were then added to automatic dishwashing compositions as shown in table 3: TABLE 3 Comparative Formulation Formulation Ingredient Formulation 4 5 6 Disilicate 2.70 2.70 2.70 Sodiumtripolyphosphate 32.00 32.00 32.00 Sodiumcarbonate 37.50 37.50 37.50 Polymer 4.00 4.00 4.00 Sodiumsulfate 15.80 14.80 12.80 Sodium percarbonate 4.00 4.00 4.00 Enzyme 2.00 2.00 2.00 Surfactant 0.85 0.85 0.85 Corrosion inhibitor 0.15 0.15 0.15 Floating particle B 0.00 2.00 4.00 Fragrances 1.00 0.00 0.00 100.00 100.00 100.00 Formulation 4 and 6 contain almost the same amount of fragrance.", "Formulation 5 has only half the amount of fragrance.", "Formulations 4-6 were then investigated in a domestic dishwashing appliance according to their fragrance release profile.", "All formulations were dosed at a level of 30 g per wash.", "The dishwasher used was a BOSCH SMS 5062 and the cleaning program Universal 50° C. was chosen.", "Water hardness was 2° dH.", "Test Procedure A dishwasher is equipped with a full load of tableware.", "A frozen soil cube as described in the IKW Method (IKW-Working Group Machine Dishwashing Detergents, “Method of Determination of the Cleaning Performance of Machine Dishwashing Detergent (Parts A and B)”) is placed in the bottom of the machine.", "After the detergent is dosed into the dosage chamber the front door is closed and the program is started.", "After the program has finished, the front door is kept closed for additional 5 minutes.", "Then the door is fully opened.", "The evaluation is started directly when the door is opened.", "Rating of the fragrance smell intensity is done by a panel of 5 people according to the instructions below.", "The grading presented in table 4 is generated by calculating the mean over all grades given by the panellists and over all trials.", "Rating of Fragrance Smell Intensity: 4=very intense and fresh smell 3=intense and fresh smell 2=fresh smell 1=traces of smell noticeable 0=smell was not noticed TABLE 4 fragrance smell intensity of formulations 4-6 Trial 1 Trial 2 Trial 3 Over all Formulation 1 1.0 1.2 1.1 1.1 Formulation 2 1.6 1.7 2.1 2.5 Formulation 3 3.3 3.1 2.9 3.0 The results show that an increasing amount of “floating particle B” yields in a significant better fragrance smell intensity grading by the panellists.", "Important is that by only using half amount of fragrance the fragrance smell intensity grading is superior when using “floating particle B” compared to a formulation with free fragrance.", "It should be noted that if the dishwashing machine cycle is interrupted after the wash cycle or before the rinse cycle a substantial amount of “floating particle B” can be detected in the machine." ] ]

[ [ "Providing shorter uniform frame lengths in dynamic time warping for voice conversion", "A method and apparatus for frame matching is disclosed.", "The frame matching includes receiving numbers of frames in first and second input signals within a voice unit.", "A uniform frame length of the first input signal is then updated to a time sample period of the first input signal divided by the number of frames in the second input signal, when the number of frames in the second input signal is greater than or equal to the number of frames in the first input signal.", "Otherwise, a uniform frame length of the second input signal is updated to a time sample period of the second input signal divided by the number of frames in the first input signal." ], [ "1.A method for frame matching, comprising: receiving numbers of frames in first and second input signals within a voice unit; and updating a uniform frame length of said first input signal to a time sample period of said first input signal divided by the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal.", "2.The method of claim 1, further comprising: second updating a uniform frame length of said second input signal to a time sample period of said second input signal divided by the number of frames in said first input signal, when the number of frames in said second input signal is less than the number of frames in said first input signal.", "3.The method of claim 2, further comprising: third updating the number of frames in said first input signal to the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal; and fourth updating the number of frames in said second input signal to the number of frames in said first input signal, when the number of frames in said second input signal is less than the number of frames in said first input signal.", "4.The method of claim 3, further comprising: training said first and second input signals with the updated numbers of frames and the updated uniform frame lengths 5.The method of claim 1, wherein said first input signal is a target voice signal, and said second input signal is a source voice signal.", "6.The method of claim 1, wherein said voice unit is a syllable.", "7.The method of claim 6, wherein said number of frames is a number of pitch marks within a syllable.", "8.The method of claim 1, further comprising: parsing a voice stream of each of said first and second input signals into at least one voice unit.", "9.The method of claim 8, further comprising: segregating each voice unit into voiced and unvoiced sections.", "10.The method of claim 9, further comprising: determining the number of frames in said first and second input signals within the voice unit.", "11.A method for frame matching, comprising: receiving numbers of frames in first and second input signals within a voice unit; and first updating a uniform frame length of said first input signal to a time sample period of said first input signal divided by the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal, and otherwise second updating a uniform frame length of said second input signal to a time sample period of said second input signal divided by the number of frames in said first input signal.", "12.The method of claim 11, further comprising: third updating the number of frames in said first input signal to the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal; and fourth updating the number of frames in said second input signal to the number of frames in said first input signal, when the number of frames in said second input signal is less than the number of frames in said first input signal.", "13.A computer readable medium containing executable instructions which, when executed in a processing system, causes the system to perform frame matching, comprising: receiving numbers of frames in first and second input signals within a voice unit; and updating a uniform frame length of said first input signal to a time sample period of said first input signal divided by the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal.", "14.The computer readable medium of claim 13, further comprising: second updating a uniform frame length of said second input signal to a time sample period of said second input signal divided by the number of frames in said first input signal, when the number of frames in said second input signal is less than the number of frames in said first input signal.", "15.The medium of claim 14, further comprising: third updating the number of frames in said first input signal to the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal; and fourth updating the number of frames in said second input signal to the number of frames in said first input signal, when the number of frames in said second input signal is less than the number of frames in said first input signal.", "16.A frame matching system, comprising: a storage element to receive and store numbers of frames in first and second input signals within a voice unit; and a processor to update a uniform frame length of said first input signal to a time sample period of said first input signal divided by the number of frames in said second input signal, when the number of frames in said second input signal is greater than or equal to the number of frames in said first input signal, and otherwise to update a uniform frame length of said second input signal to a time sample period of said second input signal divided by the number of frames in said first input signal.", "17.The system of claim 16, further comprising: a voice unit detector to parse a voice stream of each of said first and second input signals into at least one voice unit.", "18.The system of claim 17, further comprising: a voice/unvoice detector to segregate each voice unit into voiced and unvoiced sections.", "19.The system of claim 18, further comprising: a voice frame mark generator to determine the number of frames in said first and second input signals within the voice unit.", "20.A system, comprising: a receiving element to receive and store source and target training feature vectors; and a processor to compute mean square error between converted voice of said source training feature vector and said target training feature vector, where said mean square error provides a quality measure of the converted voice.", "21.The system of claim 20, wherein said processor includes: a conversion operation element to receive said source training feature vector, and to generate a conversion operation of said source vector; a first adder to subtract the conversion operation from said target training feature vector, and to generate a first vector; a mean operation generator to generate a mean of the target training feature vector; a second adder to subtract the mean from the target training feature vector, and to generate a second vector; a first distance calculator to compute a square distance of the first vector, and to generate a third vector; a second distance calculator to compute a square distance of the second vector, and to generate a fourth vector; a first summing element to sum elements in the third vector, and to generate a fifth vector; a second summing element to sum elements in the fourth vector, and to generate a sixth vector; and a divider to divide the fifth vector by the sixth vector, and to generate the mean square error.", "22.The system of claim 21, further comprising: a first normalizing element to divide the fifth vector by a first normalizing value; and a second normalizing element to divide the sixth vector by a second normalizing value.", "23.A method, comprising: computing mean square error between converted voice of a source training feature vector and a target training feature vector, where the mean square error provides a quality measure of the converted voice.", "24.The method of claim 23, wherein said mean square error is computed as ɛ MSE = 1 N ⁢ ∑ n = 1 N ⁢  y n - F ⁡ ( x n )  2 1 N ⁢ ∑ n = 1 N ⁢  y n - μ y  2 , where xn and yn are the source and target training feature vectors, μy is a mean of the target training feature vector, and F(.)", "is a conversion operation." ], [ "<SOH> BACKGROUND <EOH>The present disclosure relates to voice conversion using dynamic time warping, and more particularly, to using shorter uniform frame lengths in dynamic time warping.", "Consequently for purposes of illustration and not for purposes of limitation, the exemplary embodiments of the invention are described in a manner consistent with such use, though clearly the invention is not so limited.", "In voice conversion, an acoustic feature of speech, such as, for example, its spectral profile or average pitch, may be analyzed to represent it as a sequence of numbers.", "The feature may then be modified from the source speaker's voice in accordance with statistical properties of a target speaker's voice.", "A typical voice converter may have a reference vocabulary stored as acoustic patterns called templates.", "An input utterance may be converted to digital form and compared to the reference templates.", "The most similar template is selected as the identity of the input.", "In order to compare an input pattern, e.g.", "a spoken word, with a reference, each word is divided into a sequence of time frames.", "In each time frame, signals representative of acoustic features of the speech pattern are obtained.", "For each frame of the input word, a frame of the reference word is selected.", "Signals representative of the similarity or correspondence between each selected pair of frames are obtained responsive to the acoustic feature signals.", "The correspondence signals for the sequence of input and reference word frame pairs are used to obtain a signal representative of the global or overall similarity between the input word and a reference word template.", "Since there are many different ways of pronouncing the same word, the displacement in time of the acoustic features comprising the word is variable.", "Different utterances of the same word, even by the same individual, may be widely out of time alignment.", "The selection of frame pairs is therefore not necessarily linear.", "Matching, for example, the fourth, fifth and sixth frames of the input utterance with the fourth, fifth and sixth frames respectively of the reference word may distort the similarity measure and produce unacceptable errors.", "Dynamic time warping (DTW) techniques may be used to align the frames of a test and reference pattern in an efficient manner.", "The DTW technique is used to cope with a difference in length of utterance according to the individual personalities of the unspecified person.", "The alignment is efficient in that the global similarity measure assumes an extremum.", "It may be, for example, that the fifth frame of the test word should be paired with the sixth frame of the reference word to obtain the best similarity measure.", "Since the acoustic feature vector is based on short-term quasi-stationary speech signal analysis, the vector needs to be extracted from the speech waveform frame-by-frame.", "To make sure that the corresponding frames of the source and target speakers' voices contain substantially similar content, the two speakers need to input speech read from substantially similar text.", "However, experiments have revealed that the DTW technique has difficulty in matching frames when the two voices are substantially different than when they are substantially similar." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 illustrates an example process of frame matching using the conventional dynamic time warping (DTW) technique.", "FIG.", "2 is a block diagram of a speech conversion system in accordance with an embodiment of the present disclosure.", "FIG.", "3 is a flowchart illustrating a process of generating shorter uniform frame lengths according to an embodiment.", "FIG.", "4 illustrates an example process of frame matching according to an embodiment.", "FIG.", "5 is a block diagram of the normalized mean square error (MSE) generator according to an embodiment.", "FIG.", "6 shows a comparison plot of the normalized mean square errors measured for a Mandarin speech conversion experiment.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND The present disclosure relates to voice conversion using dynamic time warping, and more particularly, to using shorter uniform frame lengths in dynamic time warping.", "Consequently for purposes of illustration and not for purposes of limitation, the exemplary embodiments of the invention are described in a manner consistent with such use, though clearly the invention is not so limited.", "In voice conversion, an acoustic feature of speech, such as, for example, its spectral profile or average pitch, may be analyzed to represent it as a sequence of numbers.", "The feature may then be modified from the source speaker's voice in accordance with statistical properties of a target speaker's voice.", "A typical voice converter may have a reference vocabulary stored as acoustic patterns called templates.", "An input utterance may be converted to digital form and compared to the reference templates.", "The most similar template is selected as the identity of the input.", "In order to compare an input pattern, e.g.", "a spoken word, with a reference, each word is divided into a sequence of time frames.", "In each time frame, signals representative of acoustic features of the speech pattern are obtained.", "For each frame of the input word, a frame of the reference word is selected.", "Signals representative of the similarity or correspondence between each selected pair of frames are obtained responsive to the acoustic feature signals.", "The correspondence signals for the sequence of input and reference word frame pairs are used to obtain a signal representative of the global or overall similarity between the input word and a reference word template.", "Since there are many different ways of pronouncing the same word, the displacement in time of the acoustic features comprising the word is variable.", "Different utterances of the same word, even by the same individual, may be widely out of time alignment.", "The selection of frame pairs is therefore not necessarily linear.", "Matching, for example, the fourth, fifth and sixth frames of the input utterance with the fourth, fifth and sixth frames respectively of the reference word may distort the similarity measure and produce unacceptable errors.", "Dynamic time warping (DTW) techniques may be used to align the frames of a test and reference pattern in an efficient manner.", "The DTW technique is used to cope with a difference in length of utterance according to the individual personalities of the unspecified person.", "The alignment is efficient in that the global similarity measure assumes an extremum.", "It may be, for example, that the fifth frame of the test word should be paired with the sixth frame of the reference word to obtain the best similarity measure.", "Since the acoustic feature vector is based on short-term quasi-stationary speech signal analysis, the vector needs to be extracted from the speech waveform frame-by-frame.", "To make sure that the corresponding frames of the source and target speakers' voices contain substantially similar content, the two speakers need to input speech read from substantially similar text.", "However, experiments have revealed that the DTW technique has difficulty in matching frames when the two voices are substantially different than when they are substantially similar.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 illustrates an example process of frame matching using the conventional dynamic time warping (DTW) technique.", "FIG.", "2 is a block diagram of a speech conversion system in accordance with an embodiment of the present disclosure.", "FIG.", "3 is a flowchart illustrating a process of generating shorter uniform frame lengths according to an embodiment.", "FIG.", "4 illustrates an example process of frame matching according to an embodiment.", "FIG.", "5 is a block diagram of the normalized mean square error (MSE) generator according to an embodiment.", "FIG.", "6 shows a comparison plot of the normalized mean square errors measured for a Mandarin speech conversion experiment.", "DETAILED DESCRIPTION In recognition of the above-described difficulties in using the conventional dynamic time warping (DTW) technique, the present disclosure describes a system and method for providing shorter uniform frame lengths for the DTW technique in voice conversion.", "Thus, the present system of providing shorter frame lengths reinforces the DTW technique when the two voice signals are significantly different.", "However, the present system should work well for all cases.", "FIG.", "1 illustrates an example process of frame matching using the conventional DTW technique.", "The illustrated process shows pitch marks of both source 100 and target 102 voice signals with uniform frame lengths.", "The process also shows syllable boundary marks 104, 106 for the source 100 and target 102 signals, respectively.", "The source signal 100 is represented as i frames, while the target signal 102 is represented as j frames.", "For the case where the source voice is substantially similar to the target voice, the i-th frame of the source signal 100 should correspond to the j-th frame of the target signal 102.However, in FIG.", "1, the source voice is shown to be substantially different from the target voice.", "Thus, the DTW technique may erroneously match the i-th frame of the source signal 100 with the (j+1)-th frame of the target signal 102, or even with the (j+2)-th frame.", "This situation may be comparable to correlating the pronunciation of a letter ‘o’ by a source voice to the pronunciation of a Letter ‘e’ by a target voice.", "This erroneous correspondence may result in relatively long uniform frame length, as shown in 108.Furthermore, this erroneous correspondence may change the mapping operation, and therefore, introduce artifacts or noise in the converted voice.", "A block diagram of a speech conversion system 200 in accordance with an embodiment of the present disclosure is shown in FIG.", "2.The speech conversion system 200 receives source and target voice signals.", "The system 200 is arranged to convert the source voice signal into a corresponding target voice signal.", "Therefore, the corresponding target voice signal includes substantially similar speech/text of the source voice signal in target voice.", "The speech conversion system 200 includes a frame length generator 220 adapted to provide shorter uniform frame lengths than the frame lengths provided by the conventional DTW technique.", "The system 200 also includes voice unit boundary detectors 202, 212, voice/unvoice detectors 204, 214, and voice frame mark generators 206, 216.The system 200 further includes a training model 222, which receives the frame number and the uniform frame length for each frame number, and generates a conversion operation.", "In the illustrated embodiment of FIG.", "2, the voice unit boundary detectors 202, 212 are syllable boundary detectors 202, 212 that receive source or target voice signal, and parse sentences or words into recognizable syllables.", "Thus in this particular embodiment, the voice unit boundary represents a syllable.", "However in other embodiments, the voice unit boundary may represent different segment or part of speech.", "In an alternative embodiment, the system 200 may include only one each of the voice unit boundary detector 202 voice/unvoice detector 204 and the voice frame mark generator 206.In this particular embodiment, the source and target signals may then be routed or multiplexed through the detectors 202, 204 and the generator 206, sequentially or in parallel.", "The voice/unvoice detector 204, 214 segregates the parsed voice unit or syllable into voiced and unvoiced sections.", "The voice/unvoice segregation is applied to the voice unit to allow the generation of pitch marks or frame marks.", "The voice frame mark generator 206, 216 generates these pitch marks or frame marks only on the voiced section of the voice unit.", "The generator 206, 216, however, may generate any other marks to indicate the voice unit.", "Typically, the correlated processing duration for the voiced section of the voice unit is approximately between 200 and 400 milliseconds.", "FIG.", "3 is a flowchart illustrating a process of generating shorter uniform frame lengths in accordance with an embodiment of the present disclosure.", "The process may be implemented in the frame length generator 220 of FIG.", "2.This process may be programmed as computer software.", "The process may also be hard-coded in a read-only memory (ROM) or in a logic array.", "The illustrated process includes receiving the number of frames in source (Ns) and target (Nt) signals within a parsed voice unit such as a syllable, at 300.Only the voiced section of the syllable may be processed.", "At 302, the number of frames in the source signal (Ns) is compared to the number of frames in the target signal (Nt).", "If the number of frames (Ns) in the source signal is greater than or equal to the number of frames (Nt) in the target signal, the number of frames (Ns) and the uniform frame length (Ls) of the source signal are unchanged.", "However, the number of frames (Nt) and the uniform frame length (Lt) of the target signal are modified, at 306.In the illustrated embodiment, the number of frames (Nt) of the target signal is set to the number of frames (Ns) in the source signal.", "Moreover, the uniform frame length (Lt) of the target signal is set to the time sample period (nt) of the target signal divided by the number of frames (Nt) of the target signal.", "Otherwise if the number of frames (Ns) in the source signal is less than the number of frames (Nt) in the target signal, the number of frames (Nt) and the uniform frame length (Lt) of the target signal are unchanged.", "However, the number of frames (Ns) and the uniform frame length (Ls) of the source signal are modified, at 304.In the illustrated embodiment, the number of frames (Ns) of the source signal is set to the number of frames (Nt) in the target signal.", "Moreover, the uniform frame length (Ls) of the source signal is set to the time sample period (ns) of the source signal divided by the number of frames (Ns) of the source signal.", "Therefore, the above-described process operates to use the larger number for the number of frames in the input signal to obtain the shorter uniform frame length.", "FIG.", "4 illustrates an example process of frame matching according to an embodiment of the present disclosure.", "The process includes using the DTW technique, but enhanced with shorter uniform frame length generation (see FIG.", "3).", "The illustrated process shows voice frame marks of both source 400 and target 402 voice signals with uniform frame lengths, similar to those in FIG.", "1.However, the new process illustrates the benefit of using the frame length generator 220.The illustrated frame matching process shows that by using the shorter uniform frame length generation process of FIG.", "3, the uniform frame lengths 404 may be significantly shortened.", "The effectiveness of the new process, illustrated in FIG.", "4, may be measured by comparing the normalized mean square errors (MSE).", "The MSE of the conventional DTW technique may be compared to the MSE of the new process, after training.", "The normalized MSE between the converted training voice and the target training voice may be computed as follows: ɛ MSE = 1 N ⁢ ∑ n = 1 N ⁢  y n - F ⁡ ( x n )  2 1 N ⁢ ∑ n = 1 N ⁢  y n - μ y  2 , ( 1 ) where xn and yn are the source and target training feature vectors, μy is the mean of the target training feature vector, and F(.)", "is the conversion operation.", "The conversion operation may be chosen so that it corresponds to the mean of the minimum normalized MSE.", "FIG.", "5 is a block diagram of the normalized mean square error (MSE) generator 500 according to an embodiment of the present disclosure.", "The generator 500 includes a conversion operation 502 and a mean operation 504.The generator 500 also includes adders 506, 508, distance calculators 510, 512, summing elements 514, 516, and normalizing elements 518, 520.The generator 500 further includes a divider 522.The generator 500 receives the source and target training feature vectors, Xn and Yn, respectively.", "The feature vectors are then processed, summed, and normalized to produce a mean square error according to equation (1) above.", "As mentioned above, the normalized MSE generator 500 may be used to measure the effectiveness of the new process illustrated in FIGS.", "3 and 4.In an alternative embodiment, the normalized MSE generator 500 may be used to determine which source signal includes “substantially different” voice from the target signal.", "If the normalized MSE is large, then the two signals may have substantially different voice.", "Otherwise if the normalized MSE is small, then the two signals may have substantially similar voice.", "Therefore in the alternative embodiment, the determination may be used to apply the shorter uniform frame length generation only when the two signals have substantially different voice.", "Advantages of the present disclosure may be evaluated both objectively and subjectively.", "The subjective evaluation may be made by listening to the converted voice, which has noise and other artifacts removed.", "The shorter uniform frame length generation process, illustrated in FIGS.", "3 and 4, provides removal of noise and artifacts.", "Furthermore, the objective evaluation may be made by measuring the normalized MSE according to equation (1), and as illustrated in FIG.", "5.FIG.", "6 shows a comparison plot of the normalized mean square errors measured for a Mandarin speech conversion experiment.", "The experiment was performed to convert female Mandarin voice to male voice.", "Curve 600 illustrates the measured MSE using the conventional DTW technique.", "Curve 602 illustrates the measured MSE using the DTW technique enhanced with the shorter uniform frame length generation process.", "The plot shows that the DTW technique enhanced with the shorter uniform frame length generation process produces consistently smaller mean square error.", "While specific embodiments of the invention have been illustrated and described, such descriptions have been for purposes of illustration only and not by way of limitation.", "Accordingly, throughout this detailed description, for the purposes of explanation, numerous specific details were set forth in order to provide a thorough understanding of the present invention.", "It will be apparent, however, to one skilled in the art that the system and method may be practiced without some of these specific details.", "In other instances, well-known structures and functions were not described in elaborate detail in order to avoid obscuring the subject matter of the present invention.", "Accordingly, the scope and spirit of the invention should be judged in terms of the claims which follow." ] ]

[ [ "Apparatus and method for producing porous polymer particles", "An apparatus and method for producing porous polymer particles is disclosed.", "The apparatus includes a rotating atomizer wheel (39) onto which a uniform thin layer of a polymer may be applied via a distributor (40), followed by the movement of the polymer to the periphery of the wheel due to centrifugal force and the subsequent release of free flying particles at the periphery of the wheel.", "The apparatus further includes a catch tray (14) to collect the porous polymer particles produced and an enclosure defining a partition between an interior environment and an exterior environment of the apparatus.", "The enclosure includes an aperture allowing a gaseous exchange between the interior and exterior environments." ], [ "1.An atomizer machine for the production of porous polymer particles, comprising: a) an atomizer wheel having an edge, wherein said wheel is rotatable about an axis; b) a distributor for depositing polymer in fluid state to said wheel; c) a catch tray disposed under the atomizer wheel to collect the polymer particles formed as a result of ejection of the polymer from the edge as the atomizer wheel rotates; d) an enclosure, enclosing said atomizer wheel, said distributor and said catch tray, said enclosure defining a partition between an interior environment of said atomizer machine and an exterior environment of said atomizer machine; e) an aperture on said enclosure allowing a gaseous exchange between the interior environment of said atomizer machine and the exterior environment of said atomizer machine.", "2.An atomizer machine as defined in claim 1, wherein said aperture is of variable size.", "3.An atomizer machine as defined in claim 2, wherein said enclosure includes a peripheral wall surrounding said atomizer wheel, said distributor and said catch tray and a roof portion covering said peripheral wall.", "4.An atomizer machine as defined in claim 3 wherein said peripheral wall is generally circular.", "5.An atomizer machine as defined in claim 4, wherein said aperture extends circumferentially along said peripheral wall.", "6.An atomizer machine as defined in claim 4, wherein said peripheral wall includes an upper portion and a lower portion, said aperture being defined between said upper portion and between said lower portion.", "7.An atomizer machine as defined in claim 6, further comprising an actuator to displace said upper portion and said lower portion with relation to one another to vary the size of said aperture.", "8.An atomizer machine as defined in claim 7, wherein said actuator is operative to displace said upper portion along said axis to vary the size of said aperture.", "9.An atomizer machine as defined in claim 2, including a temperature control unit to regulate a temperature in said interior environment.", "10.An atomizer machine as defined in claim 2, including a humidity control unit to regulate a level of humidity in said interior environment.", "11.An atomizer machine as defined in claim 9, further comprising a monitor capable of indicating a level of temperature in said interior environment.", "12.An atomizer machine as defined in claim 10, further comprising a monitor capable of indicating a level of humidity in said interior environment.", "13.An atomizer machine according to claim 9 wherein the temperature control unit comprises at least one of: a unit for controlling a size of said aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of said atomizer wheel, over the wheel and in the enclosure, and at least one steam trap for de-misting air in said interior environment and preventing water droplets from falling on said atomizer wheel.", "14.An atomizer machine according to claim 10 wherein said humidity control unit comprises at least one of: a unit for controlling a size of said aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of said atomizer wheel periphery, over the wheel and in the enclosure, and at least one steam trap for de-misting the air in the interior environment and preventing water droplets from falling on said atomizer wheel.", "15.An atomizer machine as defined in claim 13, further comprising a monitor capable of indicating a level of temperature in said interior environment.", "16.An atomizer machine as defined in claim 14, further comprising a monitor capable of indicating a level of humidity in said interior environment.", "17.An atomizer machine according to claim 1 further comprising a trajectory control means to control a trajectory of the particles from a periphery of said atomizer wheel to said catch tray.", "18.An atomizer machine according to claim 17 wherein the trajectory control means comprises a unit for controlling a size of said aperture, disposing steam valves at the periphery of said atomizer wheel, over said atomizer wheel and directly into said enclosure, and controlling airflow patterns at the periphery of said atomizer wheel.", "19.An atomizer machine according to claim 1 further comprising a reactor for producing the polymer and at least one temperature controlled conduit for feeding the polymer to the distributor.", "20.An atomizer machine according to claim 19 wherein the at least one conduit consists of a double jacket tube defining an inner passage for feeding the polymer to the distributor and an outer envelope surrounding the inner passage, through which outer envelope a temperature liquid is flowed to control a level of temperature of the polymer.", "21.An atomizer machine according to claim 1, wherein said distributor rotates in the same direction as said atomizer wheel.", "22.An atomizer machine according to claim 1 wherein the distributor comprises a plurality of holes.", "23.An atomizer machine according to claim 22 wherein the plurality of holes are disposed in a circle.", "24.An atomizer machine according to claim 22 wherein the distributor has 24 holes.", "25.An atomizer machine according to claim 1 wherein said atomizer wheel has a flat surface.", "26.An atomizer machine according to claim 1, further comprising a shaft for receiving said atomizer wheel, said shaft being conical and tapered so as to reduce vibrations during rotation of said atomizer wheel.", "27.An atomizer machine according to claim 1, further comprising a shaft for receiving said atomizer wheel, said shaft having a threaded section for securing said atomizer wheel to said shaft.", "28.An atomizer machine according to claim 1, further comprising a sorting bin for receiving and sorting the particles from said catch tray.", "29.An atomizer machine according to claim 1, wherein said atomizer wheel has a perimeter, and wherein said atomizer wheel has at said perimeter radially projecting teeth.", "30.An atomizer machine according to claim 1, further comprising at least one baffle disposed within the enclosure for regulating air flow within said internal environment.", "31.The atomizer machine according to claim 30 wherein the at least one baffle is a plurality of baffles.", "32.The atomizer machine according to claim 31 wherein the plurality of baffles comprises 4 baffles.", "33.A method for producing polymer particles, said method comprising: a) providing an atomizer wheel, distributor and a catch tray enclosed by an enclosure defining a partition between an interior and an exterior environment and having an aperture for allowing gaseous exchange between said interior and said exterior environment; and b) allowing gaseous exchange through said aperture thereby to regulate at least one condition of temperature, humidity or air flow within said interior environment.", "34.The method of claim 33 further comprising varying a size of said aperture to vary a rate of gaseous exchange." ], [ "<SOH> BACKGROUND <EOH>The capacity of certain porous support particles to cause selective retardation based on either size or shape is well known.", "Such particles are used in chromatographic separation techniques, for example gel filtration, to separate biological macromolecules, e.g.", "proteins, DNA, RNA polysaccharides and the like.", "The sieving particles are characterized by the presence of a microporous structure that exerts a selective action on the migrating solute macromolecules, restricting passage of larger particles more than that of the smaller particles.", "Thus, the utility of sieving lies in the capacity of the particles to distinguish between molecules of different sizes and shapes.", "Affinity chromatography is a chromatographic method used for the isolation of proteins and other biological compounds.", "This technique is performed using an affinity ligand attached to a support particle and the resulting adsorbent packed into a chromatography column.", "The target protein is captured from solution by selective binding to the immobilized ligand.", "The bound protein may be washed to remove unwanted contaminants and subsequently eluted in a highly purified form.", "Good separation using chromatography techniques depends on the size of particles, the size distribution of particles and the porosity of the particles.", "The beads, once packed into a column, should be of a high strength in order to support the liquid flow rates observed during purification and column regeneration.", "The effect of polymer concentration and other preparation parameters on agarose particle porosity and strength are presented in S. Hjertén and K. O. Eriksson, Analytical Biochemistry, 137, 313-317 (1984), herein incorporated by reference.", "Additional fundamental information is presented in Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995, herein incorporated by reference.", "The description of chemical additives that help to improve the agarose particle porosity are found in M. Letherby and D. A.", "Young, J. Chem.", "Soc., Faraday Trans.", "1, 77, 1953-1966 (1981) and in M. Tako and S. Nakamura, Carbohydrate Research, 180, 277-284 (1988), both herein incorporated by reference.", "Many particle formation methods and apparatus have been developed using centrifugal action to divide a liquid or into droplets or particles.", "Rotary atomizer machines in general are discussed in the text Spray Drying Handbook, K. Masters, Fifth edition, Longman Scientific & Technical, Longman Group UK Limited, herein incorporated by reference.", "Other relevant references related to atomization are Atomization and Sprays, A. Lefebvre, Hemisphere Publications, 1989 and Liquid Atomization, L. Bayvel and Z. Orzechowski, Taylor and Francis, 1993, both herein incorporated by reference.", "A fundamental theory used in the present invention is known as “spray congealing”, based on spray drying principles with the exception that solidification is the objective instead of drying.", "Traditional emulsion based methods for agarose bead preparation are described in, for example, Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995 and in “The Preparation of Agarose Spheres for Chromatography of Molecules and Particles”, Biochimica et Biophysica Acta, 79, 393-398 (1964).", "The particle size distribution produced by known apparatus and methods require further sorting steps or procedures in order to select particles of uniform size required for chromatography.", "The additional sorting steps introduce further costs that could be avoided if the factors determining size distribution of the particles and operating variables are closely controlled.", "Without additional sorting steps, the products manufactured by conventional rotary atomization or emulsion techniques cannot be used in applications where the size distribution of the particles must be very narrow.", "For example, when using particles in blood purification applications, small particles must be avoided as small particles could be caught by the carrier fluid and would result in contamination of the purified material.", "Of course a narrow particle size distribution improves performances of particles in many applications, including chromatographic applications.", "Operating variables that influence droplet size produced from atomizer wheels and hence particle size include speed of rotation, wheel diameter, wheel design, feed rate, viscosity of feed and air, density of feed and air and surface tension of feed.", "The atmosphere within which a particle passes is important in order to avoid reduction of pore size.", "In particular, humidity and temperature control avoids particle desiccation during polymerization and gelling stages.", "Particle desiccation reduces pore size.", "It is desirable to have a machine and process to produce particles using centrifugal action in such a manner as that the particles have a narrow particle size distribution with both high porosity and flow.", "Lengthy consideration of prior art devices and processes has identified a number of factors that may be responsible for the wider size distribution of particles.", "Such factors include interruptions on the wheel surface that may impede radial acceleration of the particle solution and adhesion to the surface of the wheel; lack of adequate temperature control on the atomizer wheel that may result in changes in feed viscosity and particle structure; and uncontrolled airflow patterns at the perimeter of the atomizer wheel that may result in particle twinning due to collisions between particles prior to gelation and in undesired drying of the particles due to a modification in their path down from the wheel to the collecting liquid." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Applicants have recognized that control of humidity and temperature within specific parameters in the immediate area of the atomizer wheel will yield particles of a narrower size distribution than previously possible with both good porosity and rigidity.", "Specifically, It has been discovered that the air flow rate, temperature and humidity may be controlled in the immediate area of the atomizer wheel with sufficient accuracy to produce particles of a narrow size distribution.", "Control of temperature and humidity is achieved by the combination of temperature and humidity control means and an enclosure comprising an aperture, thus partially enclosing the atomizer machine.", "The apparatus and method of the present invention produce particles having improved properties including very good bead shape and a narrower size distribution than possible with conventional production apparatus and methods.", "The apparatus and method are particularly well suited for the production of agarose beads for use in chromatography.", "According to a first broad aspect, the invention provides an atomizer machine for the production of porous polymer particles, comprising: a) an atomizer wheel having an edge, wherein the wheel is rotatable about an axis; b) a distributor for depositing polymer in fluid state to the wheel; c) a catch tray disposed under the atomizer wheel to collect the polymer particles formed as a result of ejection of the polymer from the edge as the atomizer wheel rotates; d) an enclosure, enclosing the atomizer wheel, the distributor and the catch tray, the enclosure defining a partition between an interior environment of the atomizer machine and an exterior environment of the atomizer machine; e) an aperture on the enclosure allowing a gaseous exchange between the interior environment of the atomizer machine and the exterior environment of the atomizer machine.", "In an embodiment, the above-mentioned aperture is of variable size.", "In an embodiment, the above-mentioned enclosure includes a peripheral wall surrounding the atomizer wheel, the distributor and the catch tray and a roof portion covering the peripheral wall.", "In an embodiment, the above-mentioned peripheral wall is generally circular.", "In an embodiment, the above-mentioned aperture extends circumferentially along the peripheral wall.", "In an embodiment, the above-mentioned peripheral wall includes an upper portion and a lower portion, the aperture being defined between the upper portion and between the lower portion.", "In an embodiment, the above-mentioned atomizer machine further comprises an actuator to displace the upper portion and the lower portion with relation to one another to vary the size of the aperture.", "In an embodiment, the above-mentioned actuator is operative to displace the upper portion along the axis to vary the size of the aperture.", "In an embodiment, the above-mentioned atomizer machine includes a temperature control unit to regulate a level of temperature in the interior environment.", "In an embodiment, the above-mentioned atomizer machine includes a humidity control unit to regulate a level of humidity in the interior environment.", "In an embodiment, the above-mentioned temperature control unit comprises at least one of: a unit for controlling a size of the aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of the atomizer wheel, over the wheel and in the enclosure, and at least one steam trap for de-misting air in the interior environment and preventing water droplets from falling on the atomizer wheel.", "In an embodiment, the above-mentioned humidity control unit comprises at least one of: a unit for controlling a size of the aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of the atomizer wheel, over the wheel and in the enclosure, and at least one steam trap for de-misting the air in the interior environment and preventing water droplets from falling on the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a monitor capable of indicating a level of temperature in the interior environment.", "In an embodiment, the above-mentioned atomizer machine further comprises a monitor capable of indicating a level of humidity in the interior environment.", "In an embodiment, the above-mentioned atomizer machine further comprises a trajectory control means to control a trajectory of the particles from a periphery of the atomizer wheel to the catch tray.", "In an embodiment, the above-mentioned trajectory control means comprises a unit for controlling a size of the aperture, disposing steam valves at the periphery of the atomizer wheel, over the atomizer wheel and directly into the enclosure, and controlling airflow patterns at the periphery of the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a reactor for producing the polymer and at least one temperature controlled conduit for feeding the polymer to the distributor.", "In an embodiment, the above-mentioned at least one conduit consists of a double jacket tube defining an inner passage for feeding the polymer to the distributor and an outer envelope surrounding the inner passage, through which outer envelope a temperature liquid is flowed to control a level of temperature of the polymer.", "In an embodiment, the above-mentioned distributor rotates in the same direction as the atomizer wheel.", "In an embodiment, the above-mentioned distributor comprises a plurality of holes.", "In an embodiment, the above-mentioned plurality of holes are disposed in a circle.", "In an embodiment, the above-mentioned distributor has 24 holes.", "In an embodiment, the above-mentioned atomizer wheel has a flat surface.", "In an embodiment, the above-mentioned atomizer machine further comprises a shaft for receiving the atomizer wheel, the shaft being conical and tapered so as to reduce vibrations during rotation of the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a shaft for receiving the atomizer wheel, the shaft having a threaded section for securing the atomizer wheel to the shaft.", "In an embodiment, the above-mentioned atomizer machine further comprises a sorting bin for receiving and sorting the particles from the catch tray.", "In an embodiment, the above-mentioned atomizer wheel has a perimeter, the perimeter having radially projecting teeth.", "In an embodiment, the above-mentioned atomizer machine further comprises at least one baffle disposed within the enclosure for regulating air flow within the internal environment.", "In an embodiment, the above-mentioned at least one baffle is a plurality of baffles.", "In an embodiment, the above-mentioned plurality of baffles is 4 baffles.", "According to a second broad aspect, the invention provides a method for producing polymer particles, the method comprising: a) providing an atomizer wheel, distributor and a catch tray enclosed by an enclosure defining a partition between an interior and an exterior environment and having an aperture for allowing gaseous exchange between the interior and the exterior environment; and b) allowing gaseous exchange through the aperture thereby to regulate at least one condition of temperature, humidity or air flow within the interior environment.", "In an embodiment, the above-mentioned method further comprises varying a size of the aperture to vary a rate of gaseous exchange.", "In a further embodiment, the above mentioned enclosure comprises a dome.", "The dome partially enclosing the atomizer machine at once creates an open-system and creates a zone surrounding the machine.", "The open system is necessary to obtain an air flow current from within the zone to the exterior of the zone.", "The air flow current contributes to the control of temperature and humidity by preventing build-up of heat within the immediate vicinity of the wheel as a result of rapid rotation of the wheel.", "The creation of a zone surrounding the machine is necessary to define an area within which a desired temperature and humidity profile may be maintained.", "It has been found that accurate control of the factors that determine the temperature and humidity surrounding the machine is not possible in absence of a structure that defines a zone within which the temperature and humidity control means operate to maintain the desired temperature and humidity profile.", "Advantageously, the dome is adjustable, thus providing adjustment of the aperture size, to compensate for variations in the factors that affect the temperature and humidity in the immediate vicinity of the gel and particles.", "Temperature, humidity and turbulence of the air surrounding the apparatus and inside the apparatus affect the properties of beads: porosity, flow, average particle size, particle size distribution, bead shape and non-specific binding.", "In a further embodiment, the invention further provides an atomizer machine for the production of porous polymer particles having a narrow size distribution comprising: a) an atomizer wheel rotating about an axis; b) a distributor for providing a uniform thin layer of a gelatinous polymer on the wheel; c) a shaft connecting the wheel to a rotor; d) a catch tray disposed under the wheel to collect the particles; e) a dome partially enclosing the atomizer wheel and catch tray so as to maintain an open system and defining a zone surrounding the wheel and catch tray; f) a means for temperature and humidity control for creating and maintaining a temperature and humidity gradient within the zone; wherein the gelatinous polymer deposited on the rotating wheel moves to the periphery of the wheel under action of centrifugal force, the film being broken into free flying particles at the edge of the wheel.", "These and other aspects of the invention shall become apparent to those of ordinary skill in the art upon consideration of the following description of specific embodiments in conjunction with the accompanying drawings." ], [ "FIELD OF THE INVENTION The present invention relates to an apparatus and process for the formation of porous polymer particles for use in chromatography techniques.", "BACKGROUND The capacity of certain porous support particles to cause selective retardation based on either size or shape is well known.", "Such particles are used in chromatographic separation techniques, for example gel filtration, to separate biological macromolecules, e.g.", "proteins, DNA, RNA polysaccharides and the like.", "The sieving particles are characterized by the presence of a microporous structure that exerts a selective action on the migrating solute macromolecules, restricting passage of larger particles more than that of the smaller particles.", "Thus, the utility of sieving lies in the capacity of the particles to distinguish between molecules of different sizes and shapes.", "Affinity chromatography is a chromatographic method used for the isolation of proteins and other biological compounds.", "This technique is performed using an affinity ligand attached to a support particle and the resulting adsorbent packed into a chromatography column.", "The target protein is captured from solution by selective binding to the immobilized ligand.", "The bound protein may be washed to remove unwanted contaminants and subsequently eluted in a highly purified form.", "Good separation using chromatography techniques depends on the size of particles, the size distribution of particles and the porosity of the particles.", "The beads, once packed into a column, should be of a high strength in order to support the liquid flow rates observed during purification and column regeneration.", "The effect of polymer concentration and other preparation parameters on agarose particle porosity and strength are presented in S. Hjertén and K. O. Eriksson, Analytical Biochemistry, 137, 313-317 (1984), herein incorporated by reference.", "Additional fundamental information is presented in Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995, herein incorporated by reference.", "The description of chemical additives that help to improve the agarose particle porosity are found in M. Letherby and D. A.", "Young, J. Chem.", "Soc., Faraday Trans.", "1, 77, 1953-1966 (1981) and in M. Tako and S. Nakamura, Carbohydrate Research, 180, 277-284 (1988), both herein incorporated by reference.", "Many particle formation methods and apparatus have been developed using centrifugal action to divide a liquid or into droplets or particles.", "Rotary atomizer machines in general are discussed in the text Spray Drying Handbook, K. Masters, Fifth edition, Longman Scientific & Technical, Longman Group UK Limited, herein incorporated by reference.", "Other relevant references related to atomization are Atomization and Sprays, A. Lefebvre, Hemisphere Publications, 1989 and Liquid Atomization, L. Bayvel and Z. Orzechowski, Taylor and Francis, 1993, both herein incorporated by reference.", "A fundamental theory used in the present invention is known as “spray congealing”, based on spray drying principles with the exception that solidification is the objective instead of drying.", "Traditional emulsion based methods for agarose bead preparation are described in, for example, Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995 and in “The Preparation of Agarose Spheres for Chromatography of Molecules and Particles”, Biochimica et Biophysica Acta, 79, 393-398 (1964).", "The particle size distribution produced by known apparatus and methods require further sorting steps or procedures in order to select particles of uniform size required for chromatography.", "The additional sorting steps introduce further costs that could be avoided if the factors determining size distribution of the particles and operating variables are closely controlled.", "Without additional sorting steps, the products manufactured by conventional rotary atomization or emulsion techniques cannot be used in applications where the size distribution of the particles must be very narrow.", "For example, when using particles in blood purification applications, small particles must be avoided as small particles could be caught by the carrier fluid and would result in contamination of the purified material.", "Of course a narrow particle size distribution improves performances of particles in many applications, including chromatographic applications.", "Operating variables that influence droplet size produced from atomizer wheels and hence particle size include speed of rotation, wheel diameter, wheel design, feed rate, viscosity of feed and air, density of feed and air and surface tension of feed.", "The atmosphere within which a particle passes is important in order to avoid reduction of pore size.", "In particular, humidity and temperature control avoids particle desiccation during polymerization and gelling stages.", "Particle desiccation reduces pore size.", "It is desirable to have a machine and process to produce particles using centrifugal action in such a manner as that the particles have a narrow particle size distribution with both high porosity and flow.", "Lengthy consideration of prior art devices and processes has identified a number of factors that may be responsible for the wider size distribution of particles.", "Such factors include interruptions on the wheel surface that may impede radial acceleration of the particle solution and adhesion to the surface of the wheel; lack of adequate temperature control on the atomizer wheel that may result in changes in feed viscosity and particle structure; and uncontrolled airflow patterns at the perimeter of the atomizer wheel that may result in particle twinning due to collisions between particles prior to gelation and in undesired drying of the particles due to a modification in their path down from the wheel to the collecting liquid.", "SUMMARY OF THE INVENTION Applicants have recognized that control of humidity and temperature within specific parameters in the immediate area of the atomizer wheel will yield particles of a narrower size distribution than previously possible with both good porosity and rigidity.", "Specifically, It has been discovered that the air flow rate, temperature and humidity may be controlled in the immediate area of the atomizer wheel with sufficient accuracy to produce particles of a narrow size distribution.", "Control of temperature and humidity is achieved by the combination of temperature and humidity control means and an enclosure comprising an aperture, thus partially enclosing the atomizer machine.", "The apparatus and method of the present invention produce particles having improved properties including very good bead shape and a narrower size distribution than possible with conventional production apparatus and methods.", "The apparatus and method are particularly well suited for the production of agarose beads for use in chromatography.", "According to a first broad aspect, the invention provides an atomizer machine for the production of porous polymer particles, comprising: a) an atomizer wheel having an edge, wherein the wheel is rotatable about an axis; b) a distributor for depositing polymer in fluid state to the wheel; c) a catch tray disposed under the atomizer wheel to collect the polymer particles formed as a result of ejection of the polymer from the edge as the atomizer wheel rotates; d) an enclosure, enclosing the atomizer wheel, the distributor and the catch tray, the enclosure defining a partition between an interior environment of the atomizer machine and an exterior environment of the atomizer machine; e) an aperture on the enclosure allowing a gaseous exchange between the interior environment of the atomizer machine and the exterior environment of the atomizer machine.", "In an embodiment, the above-mentioned aperture is of variable size.", "In an embodiment, the above-mentioned enclosure includes a peripheral wall surrounding the atomizer wheel, the distributor and the catch tray and a roof portion covering the peripheral wall.", "In an embodiment, the above-mentioned peripheral wall is generally circular.", "In an embodiment, the above-mentioned aperture extends circumferentially along the peripheral wall.", "In an embodiment, the above-mentioned peripheral wall includes an upper portion and a lower portion, the aperture being defined between the upper portion and between the lower portion.", "In an embodiment, the above-mentioned atomizer machine further comprises an actuator to displace the upper portion and the lower portion with relation to one another to vary the size of the aperture.", "In an embodiment, the above-mentioned actuator is operative to displace the upper portion along the axis to vary the size of the aperture.", "In an embodiment, the above-mentioned atomizer machine includes a temperature control unit to regulate a level of temperature in the interior environment.", "In an embodiment, the above-mentioned atomizer machine includes a humidity control unit to regulate a level of humidity in the interior environment.", "In an embodiment, the above-mentioned temperature control unit comprises at least one of: a unit for controlling a size of the aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of the atomizer wheel, over the wheel and in the enclosure, and at least one steam trap for de-misting air in the interior environment and preventing water droplets from falling on the atomizer wheel.", "In an embodiment, the above-mentioned humidity control unit comprises at least one of: a unit for controlling a size of the aperture, a unit for controlling a level of temperature of the distributor and wheel, a unit for controlling a level of temperature and a flow rate of water in the catch tray, at least one valve providing at least one respective vapor stream at a periphery of the atomizer wheel, over the wheel and in the enclosure, and at least one steam trap for de-misting the air in the interior environment and preventing water droplets from falling on the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a monitor capable of indicating a level of temperature in the interior environment.", "In an embodiment, the above-mentioned atomizer machine further comprises a monitor capable of indicating a level of humidity in the interior environment.", "In an embodiment, the above-mentioned atomizer machine further comprises a trajectory control means to control a trajectory of the particles from a periphery of the atomizer wheel to the catch tray.", "In an embodiment, the above-mentioned trajectory control means comprises a unit for controlling a size of the aperture, disposing steam valves at the periphery of the atomizer wheel, over the atomizer wheel and directly into the enclosure, and controlling airflow patterns at the periphery of the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a reactor for producing the polymer and at least one temperature controlled conduit for feeding the polymer to the distributor.", "In an embodiment, the above-mentioned at least one conduit consists of a double jacket tube defining an inner passage for feeding the polymer to the distributor and an outer envelope surrounding the inner passage, through which outer envelope a temperature liquid is flowed to control a level of temperature of the polymer.", "In an embodiment, the above-mentioned distributor rotates in the same direction as the atomizer wheel.", "In an embodiment, the above-mentioned distributor comprises a plurality of holes.", "In an embodiment, the above-mentioned plurality of holes are disposed in a circle.", "In an embodiment, the above-mentioned distributor has 24 holes.", "In an embodiment, the above-mentioned atomizer wheel has a flat surface.", "In an embodiment, the above-mentioned atomizer machine further comprises a shaft for receiving the atomizer wheel, the shaft being conical and tapered so as to reduce vibrations during rotation of the atomizer wheel.", "In an embodiment, the above-mentioned atomizer machine further comprises a shaft for receiving the atomizer wheel, the shaft having a threaded section for securing the atomizer wheel to the shaft.", "In an embodiment, the above-mentioned atomizer machine further comprises a sorting bin for receiving and sorting the particles from the catch tray.", "In an embodiment, the above-mentioned atomizer wheel has a perimeter, the perimeter having radially projecting teeth.", "In an embodiment, the above-mentioned atomizer machine further comprises at least one baffle disposed within the enclosure for regulating air flow within the internal environment.", "In an embodiment, the above-mentioned at least one baffle is a plurality of baffles.", "In an embodiment, the above-mentioned plurality of baffles is 4 baffles.", "According to a second broad aspect, the invention provides a method for producing polymer particles, the method comprising: a) providing an atomizer wheel, distributor and a catch tray enclosed by an enclosure defining a partition between an interior and an exterior environment and having an aperture for allowing gaseous exchange between the interior and the exterior environment; and b) allowing gaseous exchange through the aperture thereby to regulate at least one condition of temperature, humidity or air flow within the interior environment.", "In an embodiment, the above-mentioned method further comprises varying a size of the aperture to vary a rate of gaseous exchange.", "In a further embodiment, the above mentioned enclosure comprises a dome.", "The dome partially enclosing the atomizer machine at once creates an open-system and creates a zone surrounding the machine.", "The open system is necessary to obtain an air flow current from within the zone to the exterior of the zone.", "The air flow current contributes to the control of temperature and humidity by preventing build-up of heat within the immediate vicinity of the wheel as a result of rapid rotation of the wheel.", "The creation of a zone surrounding the machine is necessary to define an area within which a desired temperature and humidity profile may be maintained.", "It has been found that accurate control of the factors that determine the temperature and humidity surrounding the machine is not possible in absence of a structure that defines a zone within which the temperature and humidity control means operate to maintain the desired temperature and humidity profile.", "Advantageously, the dome is adjustable, thus providing adjustment of the aperture size, to compensate for variations in the factors that affect the temperature and humidity in the immediate vicinity of the gel and particles.", "Temperature, humidity and turbulence of the air surrounding the apparatus and inside the apparatus affect the properties of beads: porosity, flow, average particle size, particle size distribution, bead shape and non-specific binding.", "In a further embodiment, the invention further provides an atomizer machine for the production of porous polymer particles having a narrow size distribution comprising: a) an atomizer wheel rotating about an axis; b) a distributor for providing a uniform thin layer of a gelatinous polymer on the wheel; c) a shaft connecting the wheel to a rotor; d) a catch tray disposed under the wheel to collect the particles; e) a dome partially enclosing the atomizer wheel and catch tray so as to maintain an open system and defining a zone surrounding the wheel and catch tray; f) a means for temperature and humidity control for creating and maintaining a temperature and humidity gradient within the zone; wherein the gelatinous polymer deposited on the rotating wheel moves to the periphery of the wheel under action of centrifugal force, the film being broken into free flying particles at the edge of the wheel.", "These and other aspects of the invention shall become apparent to those of ordinary skill in the art upon consideration of the following description of specific embodiments in conjunction with the accompanying drawings.", "BRIEF DESCRIPTION OF THE DRAWINGS In the drawings: FIG.", "1 illustrates an apparatus for producing porous particles according to an embodiment of the invention; FIG.", "2 illustrates the central column of the apparatus of FIG.", "1 showing bottom and top steam diffusers and shaft-wheel-distributor assembly; FIG.", "3 illustrates the shaft-wheel-distributor assembly of FIG.", "2; FIG.", "4 is a bottom view of the bottom steam diffuser of FIG.", "2 that distributes steam to the edge of the wheel; FIG.", "5 is a side view of the bottom steam diffuser of FIG.", "2; FIG.", "6 is a top view of the top steam diffuser of FIG.", "2 and a partial side view of the top steam diffuser of FIG.", "2; and FIG.", "7 is a bottom view of the top steam diffuser of FIG.", "2 and a partial side view of the top steam diffuser of FIG.", "2.DETAILED DESCRIPTION OF THE INVENTION One embodiment of the liquid atomization apparatus of the invention is illustrated in FIG.", "1.A solution comprising a polymer is prepared in reactor (1).", "Solid particles are formed from the solution in a beader (2).", "A heated tube (12) connects the reactor (1) to the beader (2).", "The apparatus and method of the invention will be described in conjunction with the production of agarose beads.", "However, the apparatus may be used to produce particles of any other polymer.", "The polymer is first slowly poured into a solvent, in an embodiment, at room temperature, under vigorous stirring in a sealed stainless steel reactor (1), yielding a mixture.", "Suitable solvents include, but are not limited to, water, aqueous salt solutions, and organic solvents.", "The mixture is heated up to over 90° C. to allow a complete dissolution of the agarose, forming a solution.", "The solution is quickly cooled down to an intermediate temperature between dissolution and gelling temperatures, where a special additive can be added to the solution in order to improve bead porosity.", "This additive or chemical can be any chemical that helps obtain improved porosity, such as a salt (for example, ammonium sulfate) or a surfactant, in an embodiment, ammonium sulfate.", "Then, the solution is slowly cooled down to the process temperature, close enough to the gelling temperature, at a rate, for example, of up to about 0.5° C./min, in an embodiment, not more than 0.1° C./min.", "Once the gel has reached its process temperature, it is pumped through a heated tube (12) maintained at the gel process temperature, from the reactor (1) to a nozzle (42), using a gear pump (11) also maintained at the gel process temperature by means of a pump head heater (not illustrated).", "The gel is supplied to a distributor (40) by the nozzle (42), and evenly distributed on the atomization wheel (39) by means of a distributor (40).", "A thin uniform layer is formed by both centrifugal force and the use of the distributor (40), and split by teeth (43) into filaments, which are broken in uniform sized spheres by the air flowing at the atomization wheel (39) edge.", "The beads travel through the surrounding air in the dome (13), where relative humidity and temperature are accurately controlled (from hot and humid at the atomization wheel (39) edge to less hot and less humid air at the catch tray (14) level) before they fall into the catch tray (14).", "The temperature and humidity profiles in the dome (13), between the atomization wheel (39) and surface of the catch tray (45), are accurately controlled in order to make sure that the bead formed turns into solid phase prior to reaching the catch tray surface.", "A liquid, for example water, is continuously recirculated at a flow rate in a closed loop from the catch tray (14) to a siever (20) and back to the catch tray via a recirculation pump (23).", "The flow rate is adjusted in such way that the surface of the catch tray (45) is always covered with a thin continuous layer of liquid.", "A heat exchanger (21) is installed in the inlet reservoir (22) of the recirculation pump (23) to control the catch tray (14) temperature.", "The beads can be collected at the outlet of the siever machine (20) in a sealed bucket (24) for packaging.", "The beader (2) thus contains a dome (13) and a catch tray (14).", "The dome (13) is not attached to the catch tray (14), leaving the beader (2) open for air exchanges with the production room.", "The dome skirt (15) controls the gap between the dome (13) and the catch tray (14), which is responsible for the fresh air inlet into the process.", "Therefore the dome (13) defines a zone surrounding the apparatus and partially encloses the atomizer wheel in an open system.", "In certain embodiments, the temperature and humidity of the ambient air in the production room should be accurately controlled between 20-23° C. and 25-75% humidity, respectively, in order to get an adequate temperature and humidity profile in the dome (13).", "Deviations from these recommended adjustments could be compensated by variations in other process parameters such as the gap between the dome (13) and the catch tray (14) as an example.", "The catch tray (14), which has a slope from the center to the edge, collects the beads off the atomization wheel in a liquid that is in continuous recirculation.", "In order to allow the dome to move up and down for maintenance, cleaning and atomization wheel (39) installation, a system of, for example, at least one rod with an air cylinder, for example, three rods (18) with three air cylinders (19), may be used.", "A rigid structure (44) stabilizes the dome and avoids any instability that could result in vibration or movements of the dome (13).", "Two columns are included in the beader: a top column (4), which is attached to the dome (13), and a bottom column (3), which is attached to the catch tray (14).", "These two columns are clearly illustrated in drawings 2 to 7.The bottom column (3) holds the atomization wheel and helps in the control of temperature and humidity profiles in the dome (39), while the top column (4) controls the environment over the atomization wheel and helps in the control of the temperature and humidity in the dome (13).", "For maximum advantage, these two columns should be centered at all times.", "Both the rods (18) and the rigid structure (44) of the dome (13) guarantee centering of the two columns (3) and (4).", "A main steam supply (5) is split in two steam lines (6) and (7), that are required for the control of both temperature and humidity in the dome (13).", "A collecting liquid (water in the illustrated example) is distributed from the inlet reservoir (22) to the catch tray (14) through a splitter (16) which can be located at the center of the catch tray (14).", "The liquid forms a uniform and evenly distributed thin film on the catch tray surface (45) and ends in tubes (17) that are connected to the siever (20).", "For maximum advantage, the catch tray surface (45) should be continuously covered by a thin layer of liquid in order to prevent drying of the beads as they fall on the catch tray (14).", "The liquid flow rate in the catch tray (14) and its temperature affect the control of humidity and temperature in the dome (13).", "The center part of the dome is illustrated in FIG.", "2.A flat atomization wheel (39) that, in certain embodiments, can have radially projecting teeth (43) at an edge thereof, is covered by a distributor (40), which is centered with the atomization wheel (39) and, in certain embodiments, rotates at the same speed as the atomization wheel.", "A plate (58) is screwed to the tapered shaft (29), keeping the atomization wheel-distributor assembly in place.", "The distributor (40) is the recipient for the gel that comes out from a nozzle (42).", "The gel falls on a distributor lip (67), which is filled with holes (66), allowing the gel to be evenly distributed at the bottom of the distributor (40).", "These holes should occupy almost all the distributor lip (67) surface and be spaced in such a way that sufficient strength of the distributor (40) is maintained.", "An inside cylinder (60) of the distributor (40) is longer than an outside cylinder (59) giving a constant and reproducible gap between the atomization wheel (39) and the distributor (40).", "This design avoids the use of spacers, which would unbalance the atomization wheel-distributor assembly and increase particle size distribution.", "In mounting high speed rotary bodies it is advantageous that the rotating mass be balanced.", "A suitable mounting arrangement for securely positioning the wheel on the rotatable shaft is to use a tapered shaft (29) in order to make atomization wheel-shaft alignment easy and reproducible and to maintain balancing.", "The inside of the atomization wheel (39) is machined with the same slope as the tapered section (61) of the tapered shaft (29).", "In an embodiment, the atomization wheel (39) does not touch the bottom of the tapered section (61) but is supported by the tapered section (61) itself.", "This has been designed to avoid any screwed part that would make the alignment difficult to reproduce.", "For maximum advantage, the tapered shaft-atomization wheel-distributor assembly should be balanced, advantageously, at all speeds within the rotation speed range utilized, to eliminate vibration.", "The atomization wheel (39) stands over a bottom steam diffuser (31), which helps the regulation of temperature and humidity in the dome (13) and in the area close to the atomization wheel (39).", "The bottom steam diffuser (31) is connected to a bottom steam line (7) where a steam trap (48) removes any steam condensate located in the bottom steam line (7).", "A needle valve (65), located as close as possible to the steam trap (48), accurately controls the steam flow rate to the bottom steam diffuser (31).", "Steam is distributed into the dome (13) via slots (50) located on the side of the bottom steam diffuser (31).", "A bottom plate (46) and a top plate (38) are part of the bottom steam diffuser (31) and can be affixed to it using screws (49), for example.", "A drain (47) allows the evacuation of any condensation that could occur in the bottom steam diffuser (31) and avoids water accumulation that would result in steam bubbling and result in a change in humidity and temperature conditions in the dome (13).", "An annular plate (30) having holes, for example, very small holes, covers the side of the bottom steam diffuser (31).", "The small holes of the annular plate (30) cover a limited sector of the annular plate (30).", "For example, the sector may be defined by the first 60° starting at the bottom of the annular plate (30), in order to guide the steam in the dome (13) and not under the atomization wheel (39) or at the atomization wheel (39) edge, close to the teeth (43).", "The bottom column (3) also holds a motor (not illustrated) that controls atomization wheel (39) RPMs (revolutions per minute).", "The top steam diffuser (26) is connected to the top column (4) using flange (28), spacer (25) and connection (27).", "The spacer (25) and connection (27) avoid any chimney effect in the top column (4) that could result from the high spinning rate of the atomization wheel and thus affect the temperature and humidity conditions in the dome (13) and in the area close to and above the atomization wheel (39).", "The steam in the top steam line (6) goes through a demister (69) where most water drops resulting from steam condensation are removed.", "A steam trap (68) completes condensate removal from the top steam line (6).", "The top steam line (6) is then split into three steam lines (8), (9) and (10), where needle valves (62), (63) and (64) respectively, accurately control the steam flow rate in the areas of the top steam diffuser (26).", "The first steam line (10) is split into a group of holes (55) located immediately above the distributor (40), and keeps the air above the distributor (40) fully saturated in order to prevent the liquid sprayed from drying under the effect of the fast air flow rate generated by pumping caused by the rotation of the atomization wheel (39).", "The second steam line (9) is split into a second group of holes (54) forming a circle located outside the distributor (40) but still above the atomization wheel (39).", "This second steam line (9) is also required to avoid drying of the liquid on the atomization wheel (39) but also to maintain the required temperature profile above the atomization wheel (39).", "A ring (57) restricts exchanges between the dome (13) and the area above the atomization wheel (39) and helps to control temperature and humidity conditions above the atomization wheel (39).", "The third steam line (8) supplies steam to a group of holes (53) located above the atomization wheel (39) but outside the ring (57), directing steam in the dome (13), close to the atomization wheel (39) edge.", "The combination of appropriate adjustments to the following process parameters combined with the presence of a demister (69), steam traps (48) and (68), bottom steam diffuser (31) and top steam diffuser (26) controls the temperature and humidity profiles in the dome (13), in the area above the atomization wheel and at the atomization wheel edge: distance between the dome (13) and the catch tray (14), steam pressure, temperature and flow rate of the liquid in the catch tray, humidity and temperature of the air surrounding the apparatus (production room), needle valves (62), (63), (64), (65) adjustments, distance between the atomization wheel (39) and the ring (57) of the top steam diffuser (51), atomization wheel (39) spinning rate, distance between the atomization wheel (39) and the surface of the catch tray (45).", "These parameters control temperature and humidity profiles and are adjusted according to the product manufactured and desired properties.", "Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art.", "Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve substantially the same result in substantially the same way.", "Numeric ranges are inclusive of the numbers defining the range.", "In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”.", "The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.", "EXAMPLES Particles produced by the apparatus and method of the present invention have a very narrow size distribution as illustrated by the following examples describing agarose bead manufacture.", "The polymer preparation steps and temperatures and most process parameters are specific to agarose preparation and could differ depending on the polymer used for particle formation.", "Example 1 Preparation of 4% 100 μm Agarose Beads 300 g of agarose was slowly poured in 4.25 L of purified water under vigorous mixing.", "This solution was heated up to 97-99° C. for 30 minutes and cooled down to 70° C. A heating/cooling fluid was used in the jacket of the reactor to control the temperature accurately.", "750 mL of a 0.75M ammonium sulfate solution, maintained at 70° C., was added very slowly and under vigorous stirring to the previous agarose solution in order to prevent local salting out, which would result in the formation of lumps.", "The final solution was cooled to 56-57° C. at a rate not more than 0.1° C./min.", "In the meantime, the beader was started for stabilization.", "Atomization wheel-column centering was checked, and the distance between the atomization wheel and the top column was adjusted to 15 mm.", "The dome opening (distance between the dome and the catch tray) was adjusted to 7 cm.", "The atomization wheel speed was adjusted to 4900-5100 RPM , needle valves were all adjusted at 7 and steam pressure was set at 5 psig at the boiler outlet.", "These steam adjustments allowed the control of dome temperature at 36-39° C. close to the edge of the atomization wheel and were adequate for the product manufactured and the size of the dome.", "Approximately 60 L/minute of purified water maintained at 16-19° C. were recirculated in the catch tray to ensure that the catch tray surface is continuously covered with a thin film of water.", "This water flow rate is also appropriated for the control of temperature and humidity in the dome.", "Resulting stabilized beader temperatures were the following: Atomization wheel temperature: 56-60° C. Catch tray temperature: 16-19° C. Dome temperature close to the atomization wheel edge: 37-39° C. Temperature at area above atomization wheel: 71-73° C. Once the beader was stabilized and the gel at the right temperature, the gear pump was turned on, feeding 1.6 L of gel/hr to the atomization wheel.", "The following properties were recorded: Speci- Lot Lot Lot Properties/Lot fications 000612381 001016434 001017435 Porosity Thyroglobulin 0.35-0.53 0.44 0.48 0.48 Apoferritin 0.50-0.76 0.60 0.63 0.64 β-Amylase 0.54-0.80 0.64 0.67 0.68 Alcohol 0.58-0.86 0.69 0.72 0.72 Dehydrogenase Albumin 0.61-0.91 0.72 0.74 0.76 (Bovine Serum) Carbonic 0.68-1.00 0.85 0.85 0.87 Anhydrase Pressure vs Flow >20 cm/hr 35 22 22 Particle Size Analysis Average N/A 106 103 101 Size (μm) % Between N/A 98% 97.4% 90% 76-140 microns before sieving % Between Greater 99.7% 99% 99% 76-140 microns than 95% after sieving Non-Specific Less than 3.1 2.5 2.2 Binding 8 μg cyt./ml gel Microscopy Less 0.89% 0.7% 0.9% than 3% broken, fused, damaged beads Process reproducibility has been demonstrated and is clearly documented.", "The particle size distribution before sieving is very narrow, a lot more than any equivalent product available on the market at the moment.", "The distribution can be significantly improved by sieving, without significantly reducing the global yield.", "As an example, a 5 L batch prepared as above gave reproducibly 6.5 to 6.8 L of beads.", "Example 2 Preparation of 4-100 μm Agarose Beads Using Apparatus Further Comprising Baffles A plurality of 3 inch or 6 inch baffles were inserted into the dome in order to get a more homogeneous temperature profile in the dome.", "Four baffles were equally distributed on the inside of the dome, vertically, to inhibit the effect of room conditions in the dome.", "With the baffles in place, the dome is less sensitive to ambient conditions, and dome conditions are more easily reproduced.", "In addition, a wider range of particle size can be produced in the same apparatus when the baffles are used.", "The increase in disk RPM required to produce smaller particles affects the air pattern and hydrodynamics in the dome.", "The presence of baffles makes the temperature profiles less dependant on disk RPM.", "Also, the production of large particles (above 200 microns) resulted in projection of particles on the walls of the dome, due to the inertia of the particles produced.", "The presence of baffles affects the air pattern in such a way that the particles formed fall closer to the bottom column, making possible the manufacture of large particles without changing the design of the equipment.", "4% 100 microns agarose beads have been manufactured using conditions in the example above to demonstrate that the presence of baffles do not affect particle properties.", "Lot 001103443 was manufactured using 6″ baffles, while lot 001113447 was manufactured using 3″ baffles.", "Lot 000612381 is reproduced in the table for comparison purposes.", "Speci- Lot Lot Lot Properties/Lot fications 000612381 001113447 001103443 Porosity Thyroglobulin 0.35-0.53 0.44 0.45 0.43 Apoferritin 0.50-0.76 0.60 N/A N/A β-Amylase 0.54-0.80 0.64 N/A N/A Alcohol 0.58-0.86 0.69 N/A N/A Dehydrogenase Albumin 0.61-0.91 0.72 N/A N/A (Bovine Serum) Carbonic 0.68-1.00 0.85 N/A N/A Anhydrase Pressure vs Flow >20 cm/hr 35 28 24 Particle Size Analysis Average N/A 106 101 100 Size (μm) % Between N/A 98% 97.0% 97.2% 76-140 microns before sieving % Between Greater 99.7% 98.9% 98.6 76-140 microns than 95% after sieving Non-Specific Less than 3.1 N/A N/A Binding 8 μg cyt./ml gel Microscopy Less 0.89% 1.9% 1.5% than 3% broken, fused, damaged beads Example 3 Preparation of 5% 200μm Agarose Beads A procedure similar to the one described for the preparation of 4% 100 microns agarose beads has been applied for the manufacturing of 200 microns agarose beads at the 2 L scale.", "The differences are set forth in the present description.", "110 g of agarose was slowly poured in 1.7 L of purified water under vigorous mixing.", "This solution was heated up to 97-99° C. for 30 minutes and cooled down to 70° C. 300 mL of a 0.75M ammonium sulfate solution, maintained at 70° C., was added very slowly and under vigorous stirring to the previous agarose solution.", "The final solution was cooled to 55-57° C. at a rate not more than 0.1° C./min.", "In the meantime, the beader was started for stabilization.", "The distance between the atomization wheel and the top column was adjusted to 15 mm.", "The dome opening was adjusted to 4 cm.", "The atomization wheel speed was adjusted to about 2000 RPM, needle valves were all adjusted at 3 and steam pressure was set at 5 psig at the boiler outlet.", "These steam adjustments allowed the control of dome temperature at 47-50° C. close to the edge of the atomization wheel and were adequate for the product manufactured and the size of the dome.", "Approximately 60 L/minute of purified water maintained at 14-16° C. was recirculated in the catch tray.", "Resulting stabilized beader temperatures were the following: Atomization wheel temperature: Not available due to the dome opening Catch tray temperature: 14-16° C. Dome temperature close to the atomization wheel edge: 47-50° C. Temperature at area above atomization wheel: 78-80° C. Once the beader was stabilized and the gel at the right temperature, the gear pump was turned on, feeding about 2.8 L of gel/hr to the atomization wheel.", "The following properties were recorded: Lot Lot Properties/Lot Specifications 001116450 001123453 Porosity Thyroglobulin N/A 0.02 0.03 Apoferritin N/A 0.46 0.11 β-Amylase N/A 0.63 0.64 Alcohol N/A 0.70 0.73 Dehydrogenase Albumin N/A 0.71 0.74 (Bovine Serum) Carbonic N/A 0.85 0.86 Anhydrase Pressure vs Flow N/A 114 106 Particle Size Analysis Average N/A 200 199 Size (μm) % Between N/A N/A N/A 150-300 microns before sieving % Between N/A 96% 96% 150-300 microns after sieving Non-Specific N/A N/A N/A Binding Microscopy N/A 2.5 3.9 Example 4 Preparation of 4% 125 μm Agarose Beads The procedure described for the production of 4% 100 microns agarose beads has been applied for the production of 4% 125 microns agarose beads at the 3 L scale.", "The differences are set forth in the present description.", "180 g of agarose was slowly poured in 2.55 L of purified water under vigorous mixing.", "This solution was heated up to 97-99° C. for 30 minutes and cooled down to 70° C. 450 mL of a 0.75M ammonium sulfate solution, maintained at 70° C., was added very slowly and under vigorous stirring to the previous agarose solution.", "The final solution was cooled to 55-57° C. at a rate not more than 0.1° C./min.", "In the meantime, the beader was started for stabilization.", "The distance between the atomization wheel and the top column was adjusted to 15 mm.", "The dome opening was adjusted to 7 cm.", "The atomization wheel speed was adjusted to 3700-3800 RPM, needle valves were all adjusted at 4-5 and steam pressure was set at 5 psig at the boiler outlet.", "These steam adjustments allowed the control of dome temperature at 35-37° C. close to the edge of the atomization wheel and were adequate for the product manufactured and the size of the dome.", "Approximately 60 L/minute of purified water maintained at 14-16° C. were recirculated in the catch tray.", "Resulting stabilized beader temperatures were the following: Atomization wheel temperature: 56-58° C. Catch tray temperature: 14-16° C. Dome temperature close to the atomization wheel edge: 35-37° C. Temperature at area above atomization wheel: 71-75° C. Once the beader was stabilized and the gel at the right temperature, the gear pump was turned on, feeding about 1.9 L of gel/hr to the atomization wheel.", "The following properties were recorded: Speci- Lot Lot Lot Properties/Lot fications 010118464 010124465 010125466 Porosity Thyroglobulin 0.35-0.53 0.45 0.47 0.48 Apoferritin 0.50-0.76 N/A N/A N/A β-Amylase 0.54-0.80 N/A N/A N/A Alcohol 0.58-0.86 N/A N/A N/A Dehydrogenase Albumin 0.61-0.91 N/A N/A N/A (Bovine Serum) Carbonic 0.68-1.00 N/A N/A N/A Anhydrase Pressure vs Flow >30 cm/hr 44 53 45 Particle Size Analysis Average N/A 124 120 123 Size (μm) % Between N/A N/A N/A N/A 95-165 microns before sieving % Between Greater 99.5% 98.5% 99.5% 95-165 microns than 95% after sieving Non-Specific Less than N/A N/A N/A Binding 8 μg cyt./ml gel Microscopy Less 1.3 1.3 0.6 than 3% broken, fused, damaged beads Example 5 Preparation of 4% 60 μm Agarose Beads The procedure described for the production of 4% 100 microns agarose beads has been applied for the production of 4% 60 microns agarose beads at the 2 L scale.", "The differences are set forth in the present description.", "120 g of agarose was slowly poured in 1.7 L of purified water under vigorous mixing.", "This solution was heated up to 97-99° C. for 30 minutes and cooled down to 70° C. 300 mL of a 0.75M ammonium sulfate solution, maintained at 70° C., was added very slowly and under vigorous stirring to the previous agarose solution.", "The final solution was cooled to 56-58° C. at a rate not more than 0.1° C./min.", "In the meantime, the beader was started for stabilization.", "The distance between the atomization wheel and the top column was adjusted to 15 mm.", "The dome opening was adjusted to 7 cm.", "The atomization wheel speed was adjusted to 7200 RPM , needle valves were all adjusted at 7 and steam pressure was set at 5 psig at the boiler outlet.", "Those steam adjustments allowed the control of dome temperature at 33-35° C. close to the edge of the atomization wheel and were adequate for the product manufactured and the size of the dome.", "Approximately 60 L/minute of purified water maintained at 16-19° C. were recirculated in the catch tray.", "Resulting stabilized beader temperatures were the following: Atomization wheel temperature: 56-58° C. Catch tray temperature: 16-19° C. Dome temperature close to the atomization wheel edge: 33-35° C. Temperature at area above atomization wheel: 68-70° C. Once the beader was stabilized and the gel at the right temperature, the gear pump was turned on, feeding about 0.6 L of gel/hr to the atomization wheel.", "The following properties were recorded: Speci- Lot Lot Lot Properties/Lot fications 001018436 001019437 001108446 Porosity Thyroglobulin >0.20 0.56 0.47 0.45 Apoferritin N/A N/A N/A N/A β-Amylase N/A N/A N/A N/A Alcohol N/A N/A N/A N/A Dehydrogenase Albumin N/A N/A N/A N/A (Bovine Serum) Carbonic N/A N/A N/A N/A Anhydrase Pressure vs Flow >5 cm/hr 10 7 7 Particle Size Analysis Average N/A 59 61 59 Size (μm) % Between N/A 92.3% 90.5% 86.7% 30-95 microns before sieving % Between N/A 91.3% 90.5% 90.5% 30-95 microns after sieving Non-Specific N/A N/A N/A N/A Binding Microscopy Less 0% 1.3% 0.8% than 3% broken, fused, damaged beads Again lot 001108446 was manufactured using the 6″ baffles as described in the example above and compared to the standard material to demonstrate that the presence of baffles do not affect the particle properties.", "Example 6 Preparation of 6% 100 μm Agarose Beads The procedure described for the production of 4% 100 microns agarose beads has been applied for the production of 6% 100 microns agarose beads at the 5 L scale.", "The differences are set forth in the present description.", "380 g of agarose was slowly poured in 4.25 L of purified water under vigorous mixing.", "This solution was heated up to 97-99° C. for 30 minutes and cooled down to 70° C. 750 mL of a 0.75M ammonium sulfate solution, maintained at 70° C., was added very slowly and under vigorous stirring to the previous agarose solution.", "The final solution was cooled to 59-61° C. at a rate not more than 0.1° C./min.", "In the meantime, the beader was started for stabilization.", "The distance between the atomization wheel and the top column was adjusted to 15 mm.", "The dome opening was adjusted to 7 cm.", "The atomization wheel speed was adjusted to 4900-5100 RPM , needle valves were all adjusted at 7-9 and steam pressure was set at 5 psig at the boiler outlet.", "Those steam adjustments allowed the control of dome temperature at 37-39° C. close to the edge of the atomization wheel and were adequate for the product manufactured and the size of the dome.", "Approximately 60 L/minute of purified water maintained at 16-20° C. were recirculated in the catch tray.", "Resulting stabilized beader temperatures were the following: Atomization wheel temperature: 59-63 Catch tray temperature: 16-20° C. Dome temperature close to the atomization wheel edge: 37-39° C. Temperature at area above atomization wheel: 71-74° C. Once the beader was stabilized and the gel at the right temperature, the gear pump was turned on, feeding about 1.7 L of gel/hr to the atomization wheel.", "The following properties were recorded: Lot Lot Properties/Lot Specifications Lot B28902 B32904 001026439 Porosity Thyroglobulin 0.25-0.44 0.28 0.31 0.27 Apoferritin 0.39-0.59 0.44 0.47 N/A β-Amylase 0.48-0.72 0.50 0.52 N/A Alcohol 0.49-0.73 0.56 0.57 N/A Dehydrogenase Albumin 0.52-0.78 0.59 0.62 N/A (Bovine Serum) Carbonic 0.66-0.98 0.73 0.77 N/A Anhydrase Pressure vs Flow >45 cm/hr 60 64 52 Particle Size Analysis Average N/A 101 101 102 Size (μm) % Between N/A 80.3 80.1 68% 76-140 microns before sieving % Between >95% 99% 99% 99.4% 76-140 microns after sieving Non-Specific Less than 8 μg 1.7 2.9 N/A Binding cyt./ml gel Microscopy Less than 3% 0% 0.3% 1.6 broken, fused, damaged beads Batch 001026439 was produced using a higher pump flow rate.", "According to the theory, the pump flow rate could be significantly increased without affecting the product quality.", "This has been confirmed with lot 001026439, where the pump was increased to its limit and delivering about 3.5 L/hr of gel on the atomization wheel without affecting the properties only the particle size distribution before sieving was slightly broader when the pump flow rate is increased, resulting in a lower product yield.", "Therefore the gel flow rate fed to the atomization disk is not limited to the examples above, higher and lower feed rates can result in the same product.", "In an embodiment, the atomizer machine may further comprise at least one baffle, in a further embodiment, a plurality of baffles, in a further embodiment, 4 baffles, which is/are disposed within the enclosure and can affect/regulate the air pattern in the interior environment.", "The particles produced by the apparatus and process of present invention may be used in all the chromatographic and electrophoretic methodologies for industrial purification purposes including affinity chromatography, gel filtration, ion-exchange chromatography, as support for grafting different types of ligands; and coating rigid spheres of glass or plastic for types of chromatographic applications." ] ]

[ [ "Engine piston and manufacture", "An engine piston is manufactured by assembling an outer shell, comprising a crown and tubular side wall in which a ring groove region and skirt are defined, with a plate-like mounting member and bonding them together by brazing or welding.", "The mounting member is located within the tubular side wall displaced axially from the crown and is bonded near, but displaced radially from, its centre to the crown and at its periphery to the side wall at the end of the ring groove region.", "The mounting member carries gudgeon pin boss means facing away from the crown.", "The outer shell is formed by extrusion or the like that permits minimal wall thickness and the bonded structure is of light weight but great strength and stiffness, particularly in the ring groove region.", "A combustion bowl formed in the crown facilitates bonding to the mounting member and defines with the mounting member an annular cooling chamber adjacent the ring groove region and crown." ], [ "1-72.", "(canceled) 73.An engine piston comprising a prefabricated outer shell, said shell including a crown centred on a longitudinal piston axis and a tubular side wall extending axially with respect to the periphery of the crown to an open end; and a prefabricated mounting member within the tubular side wall of the shell, said mounting member carrying gudgeon pin bosses and extending transversely to the longitudinal piston axis, said mounting member also having an upper member part interfacing with the crown at a radially extending crown location interface between them serving as a mutual axial location reference and interfacing with the side wall spaced from the crown, defining a transversely extending closure plate-like member closing partly from below a peripheral chamber in the crown, the mounting member being bonded permanently to the crown at a crown bonding interface and bonded permanently to the side wall by at least one axially extending peripheral interface with the side wall.", "74.A piston according to claim 73 wherein the bond between the mounting member and the tubular side wall is at substantially the same longitudinal position as the bond between the mounting member and the crown.", "75.A piston according to claim 73 wherein the tubular side wall includes, adjacent the crown, a region of axially spaced, circumferentially extending ring grooves and the mounting member is bonded to the side wall at the end of the ring groove region remote from the crown.", "76.A piston according to claim 73 wherein the crown has a central region surrounded by a peripheral region, the peripheral region being of integral formation with the tubular side wall, and the central region comprises a combustion bowl having a bowl wall and a bowl floor displaced axially from the crown peripheral region by said bowl wall and the bowl floor being formed by the mounting member bonded to the bowl wall.", "77.A piston according to claim 76 wherein the combustion bowl floor and bowl wall are bonded together at a bonding interface between them extending in a substantially axial direction.", "78.A piston according to claim 77 wherein the bonding interface is accessible from externally of the combustion bowl.", "79.A piston according to claim 76 wherein said radially extending interface between the mounting member and crown serving as a mutual axial location reference is disposed between the mounting member and an end region of the bowl wall.", "80.A piston according to claim 76 wherein the mounting member includes at least one fluid channel between the peripheral chamber and shell open end, at least one said channel extending through the mounting member from a said upper part defining the peripheral chamber to below the combustion bowl floor defined by the mounting member.", "81.A piston as claimed in claim 76 wherein the gudgeon pin bosses are defined integrally with, and provided solely by, the mounting member on a face of the mounting member facing the open end of the tubular wall member and disposed axially between the open end of the shell and the bond between the mounting member and side wall, said bosses including a bore for the passage of a gudgeon pin extending transversely to the longitudinal piston axis and being spaced apart along the axis of said bore the gudgeon pin bosses.", "82.A piston according to claim 73 in which the crown has a central region surrounded by a peripheral region, the peripheral region being of integral formation with the tubular side wall and the central region comprises a combustion bowl having a bowl wall and a bowl floor displaced axially from the crown peripheral region by said bowl wall, the combustion bowl floor being formed integrally with the bowl wall and said mounting member being bonded to the crown at the combustion bowl.", "83.A piston according to claim 82 wherein said radially extending interface between the mounting member and crown serving as a mutual axial location reference is defined between the mounting member and the crown at the junction between the combustion bowl wall and bowl floor.", "84.A piston according to claim 82 wherein said radially extending interface is defined by an axial extension to the combustion bowl wall.", "85.A piston according to claim 82 wherein the mounting member is bonded to the crown at said radially extending crown location interface between the mounting member and crown serving as a mutual axial location reference.", "86.A piston as claimed in claim 82 wherein the gudgeon pin bosses are defined integrally with, and provided solely by, the mounting member on a face of the mounting member facing the open end of the tubular wall member and axially between the open end of the shell and the bond between the mounting member and side wall, said bosses including a bore for the passage of a gudgeon pin extending transversely to the longitudinal piston axis and being spaced apart along the axis of said bore the gudgeon pin bosses.", "87.A piston as claimed in claim 86 wherein the mounting member comprises an axially thin plate having increased axial thickness at the periphery and to effect the piston bosses.", "88.A piston as claimed in claim 86 wherein the mounting member includes a connecting rod aperture extending through the mounting member along the longitudinal piston axis between said gudgeon pin bosses and exposing the central region of the crown to the open end of the tubular side wall.", "89.A piston as claimed in claim 88 wherein the mounting member is of substantially uniform cross-section in said direction of the gudgeon pin bore, except for the connecting rod aperture.", "90.A piston as claimed in claim 88 wherein the mounting member upper part has an upper surface and includes at least one fluid channel between the peripheral chamber and shell open end, at least one said fluid channel extending in the upper surface of the mounting plate from said peripheral chamber to the connecting rod aperture.", "91.A piston as claimed in claim 73 wherein the junction between the periphery of the mounting member and the shoulder means is in line with the open end of the tubular side wall.", "92.A piston as claimed in claim 73 wherein the gudgeon pin boss means includes a bore for the passage of a gudgeon pin transversely to the longitudinal piston axis and the side wall includes cooperating apertures in the tubular side wall and the boss means is bonded with the tubular side wall at said apertures.", "93.A piston as claimed in claim 73 wherein the tubular side wall has therein shoulder means provided by a change in internal diameter of the wall and facing towards the open end of the side wall, and the periphery of the mounting member is bonded to the side wall at the shoulder means.", "94.A piston as claimed in claim 88 wherein the tubular side wall has between the end of the ring groove section and said open end a reduction in thickness defined principally by the shoulder means, the shoulder means comprises a first, smaller thickness of the ring groove region and a second, larger reduction in thickness between the ring groove region and open end.", "95.A piston as claimed in claim 73 wherein the outer shell is made from a metal alloy deformable in the solid state.", "96.A piston as claimed in claim 95 wherein the material is steel.", "97.A piston as claimed in claim 95 wherein the mounting member and outer shell are made from the same material.", "98.A piston as claimed in claim 95 wherein the mounting member and outer shell are bonded together by at least one welded joint.", "99.A method of making an engine piston comprising forming an outer shell part comprising a crown, centered on a longitudinal axis, and a tubular side wall extending axially with respect to the periphery of the crown to an open end, said crown having at least a peripheral region integral with the side wall; forming a mounting member having an upper member part and opposite thereto gudgeon pin bosses and a periphery dimensioned to fit within and interface with the tubular side wall; disposing the mounting member within the tubular side wall such that the gudgeon pin bosses face the open end, the upper part interfaces with the crown at least a radially extending crown interface defining mutual axial location reference, and the periphery interfaces with the side wall at a wall interface spaced axially from the crown periphery and radially from said radially extending crown interface, defining thereby a transversely extending closure plate closing partly from below a peripheral chamber in the crown; and permanently bonding the mounting member to the shell part at said wall interface and to the crown at a crown bonding interface radially inwardly of said wall interface.", "100.The method of claim 99 comprising bonding the mounting member to said crown and wall at interfaces at substantially the same axial position.", "101.The method of claim 99 comprising forming in the tubular side wall adjacent the crown a region of axially spaced circumferentially extending ring grooves and bonding the periphery of the mounting member to the side wall at the end of the ring groove region remote from the crown.", "102.The method of claim 99 comprising forming the tubular side wall and at least a peripheral region of the crown, bounding a central crown region, as an integral shell body and by forming at least part of the central crown region as a combustion bowl having a bowl wall and a bowl floor displaced axially of the crown peripheral region by said bowl wall, and by bonding the mounting member to the crown at the combustion bowl.", "103.The method of claim 102 comprising defining said radially extending crown location interface between the mounting member and crown, serving as a mutual axial location reference between the mounting member and the crown, at the junction between the combustion bowl wall and bowl floor.", "104.The method according to claim 103 comprising defining said radially extending interface by an axial extension to the combustion bowl wall.", "105.The method according to claim 102 comprising bonding the mounting member to the crown at the radially extending crown location interface between the mounting member and crown serving as a mutual axial location reference.", "106.The method of claim 99 comprising forming the tubular side wall and at least a peripheral region of the crown, bounding a central crown region, as an integral shell body and by forming at least part of the central crown region as a combustion bowl having a bowl wall and defining a bowl floor displaced axially of the crown peripheral region by said bowl wall by the mounting member.", "107.The method of claim 106 comprising defining said radially extending crown location interface, serving as a mutual axial location reference, between the mounting member and the combustion bowl wall.", "108.The method of claim 106 comprising bonding the mounting member to the crown by way of a said crown bonding interface between the combustion bowl floor defined by the mounting member and the combustion bowl wall defined by the crown.", "109.The method of claim 108 comprising defining said crown bonding interface extending in a substantially axial direction accessible from outside of the combustion bowl and bonding the combustion bowl floor to the combustion bowl wall by access through the combustion bowl.", "110.The method of claim 109 comprising bonding the combustion bowl floor to the combustion bowl wall metallurgically by applying heat from a source externally of the piston by access through the combustion bowl.", "111.The method of claim 110 comprising bonding the interface by welding.", "112.The method of claim 99 comprising defining the wall interface accessible from externally of the piston and bonding metallurgically by applying heat from a source externally of the piston.", "113.The method of claim 112 comprising bonding the interface by welding.", "114.The method of claim 99 comprising bonding the mounting member to the side wall substantially about the whole of its periphery.", "115.The method of claim 99 comprising casting the mounting member.", "116.The method of claim 99 comprising extruding the mounting member.", "117.The method of claim 99 comprising forming the outer shell part by flow forming about a rotating mandrel shaped to define the interior of the tubular side wall.", "118.The method of claim 99 comprising forming the outer shell part by back extrusion about a mandrel shaped to define the interior of the tubular side wall.", "119.The method of claim 118 comprising forming at least one valve pocket in the crown during extrusion.", "120.The method of claim 99 comprising forming the body shell and mounting member from the same material.", "121.The method of claim 99 comprising forming the outer shell part from steel.", "122.A method of making an engine piston having a crown, centered on a longitudinal axis, and a tubular side wall, extending axially with respect to the periphery of the crown to an open end and defining a region for a belt of ring grooves and a skirt, the method being characterized by rotating a unitary body of metal and by flow forming thereof defining at least a peripheral portion of piston crown and the side wall extending therefrom.", "123.A method of making an engine piston having a crown, centered on a longitudinal axis, and a tubular side wall, extending axially with respect to the periphery of the crown to an open end and defining a region for a belt of ring grooves and a skirt, the method being characterized by back extruding a unitary body of metal to define at least a peripheral portion of piston crown and the side wall extending therefrom." ], [ "This invention relates to pistons for internal combustion engines and to the manufacture thereof.", "The invention is particularly concerned with the manufacture of a strong piston that is also light in weight and suitable for cost-effective mass production for small capacity, high compression engines.", "Pistons for internal combustion engines for mass market automobiles are manufactured in large numbers and subjected to cost constraints, which in turn place limits on manufacturing processes.", "Such pistons are usually, but not necessarily, cast from a light metal alloy, typically aluminium based, and then subjected to a series of machining steps that culminate in a precision component.", "For heavy duty use, for example in compression ignition engines, it is known to manufacture pistons of steel, usually forged, but such pistons have tended to have a weight penalty, notwithstanding extensive machining operations to remove extraneous metal, and have thus far been restricted to large capacity, low-revving engines found in trucks and the like.", "In recent times there has been a need to provide such compression ignition engines for use in smaller, automobile engines, where it is necessary to run at higher speeds and with such high compression pressures.", "Although steel is a material having suitable properties, and has such strength that it could be used in relatively thin sections that mitigate most if not all of the weight penalty, there is difficulty in manufacturing a small one-piece piston that is capable of fulfilling such potential.", "In general such a piston has to be manufactured in steel by forging, with attendant limits to wall thicknesses and shapes that limit weight reduction.", "It has been proposed to assemble or construct a steel piston from separately manufactured parts, as in U.S. Pat.", "No.", "1,667,202 and U.S. Pat.", "No.", "2,244,008.However the crown forms of the pistons shown therein are relatively simple in structure and without an in-crown combustion bowl often required by modern engines.", "Even without such added complexity, it is believed that the number of separate parts and assembly operations required are not conducive to providing a small piston capable of operating within a modern small engine in a cost-effective manner.", "Notwithstanding that a small piston for mass production is subjected principally to constraints of cost, a larger piston for heavy duty application is subjected principally to constraints resulting from weight, so that the ability to produce a light weight piston cost effectively is not restricted in applicability.", "With this in mind, it is an object of the present invention to provide an engine piston of assembled form that is capable of providing strength and light weight in simple form and a method of producing such a piston that is capable of implementation more cost-effectively than hitherto.", "According to a first aspect of the present invention an engine piston comprises an outer shell, including a crown centred on a longitudinal piston axis and a tubular side wall extending axially with respect to the periphery of the crown to an open end and, within the tubular side wall of the shell, a mounting member arranged to extend transversely to the longitudinal axis and bonded both to the crown and to the side wall spaced from the crown, and gudgeon pin boss means carried by the mounting member.", "The term “longitudinal axis” is employed in relation to defining the piston with respect to the geometric centre of the crown, and notwithstanding that the cross section of the piston is other than circular, for example, is to a small extent elliptical or oval.", "Preferably, the tubular side wall includes, adjacent the crown, a region of axially spaced, circumferentially extending ring grooves and the mounting member is bonded to the side wall at the end of the ring groove region remote from the crown and the periphery of the mounting member is bonded to the peripheral side wall substantially at the same axial position as at least one ring groove.", "More preferably, the peripheral region the crown, the tubular side wall and the bonded mounting member define therebetween an annular cooling chamber.", "Preferably, the gudgeon pin boss means is provided integrally with the mounting member, but notwithstanding this the bonding of the mounting member to the crown and to the side wall displaced from the crown create a monocoque type of structure which includes the ring groove region and provides great pressure resistance therefor without need for substantial wall thickness.", "According to a second aspect of the present invention a method of manufacturing an engine piston comprises forming an outer shell part comprising a crown, centred on a longitudinal axis, and a tubular side wall, extending axially with respect to the periphery of the crown to an open end, forming a mounting member, carrying gudgeon pin boss means thereon, with a periphery dimensioned to fit within and interface with the tubular side wall, disposing the mounting member within the tubular side wall such that it interfaces with the crown at a crown interface and interfaces with the side wall at a wall interface and bonding the mounting member to the shell at said crown and wall interfaces.", "Preferably, the method comprises forming the tubular side wall and at least the peripheral part of the crown, bounding a central crown region, as a integral shell body.", "Preferably the outer shell and mounting member are provided separately, as unitary or pre-assembled bodies and are then bonded metallurgically to form the piston.", "In this specification references to bonding metallurgically are intended to mean all known techniques employed in joining metal bodies directly to each other or by way of an intervening metal, and includes brazing and various forms of welding, such as friction welding and laser or other beam or jet welding.", "The outer shell body may be formed by back extrusion or forging.", "Alternatively, the outer shell body may be formed by flow forming.", "If, as may be considered the norm, combustion bowl means is required in the crown this may be formed integrally with the shell body, subject to shape constraints, or may be formed separately and metallurgically bonded.", "The outer shell part and/or mounting member may be made from steel which is suitably ductile, but the method is equally applicable to ductile alloys of non-ferrous materials.", "Embodiments of the invention will now be described by way of example with reference to the accompanying drawings, in which:— FIG.", "1(a) is a sectional elevation through a first embodiment of an engine piston in accordance with the present invention, taken in the direction 1a-1a of FIG.", "1(b), comprising an assembly of outer shell part and mounting member bonded together, FIG.", "1(b) is a sectional elevation through the piston of FIG.", "1(a) at right angles to the plane of that figure in the direction 1b-1b thereof, FIGS.", "1(c) and 1(d) are sectional half elevations through the outer shell of FIGS.", "1(a) and 1(b) respectively, FIG.", "2(a) is a sectional elevation through a second embodiment of an engine piston in accordance with the present invention, taken in the direction 2a-2a of FIG.", "2(b), comprising an assembly of outer shell part and mounting member bonded together, FIG.", "2(b) is a sectional elevation through the piston of FIG.", "2(a) at right angles to the plane of that figure in the direction 2b-2b thereof, FIGS.", "2(c) and 2(d) are sectional half elevations through the outer shell of FIGS.", "2(a) and 2(b) respectively, FIG.", "3(a) is a sectional elevation through a third embodiment of an engine piston in accordance with the present invention, taken in the direction 3a-3a of FIG.", "3(b), comprising an assembly of outer shell part and mounting member bonded together, FIG.", "3(b) is a sectional elevation through the piston of FIG.", "3(a) at right angles to the plane of that figure in the direction 3b-3b thereof, FIGS.", "3(c) and 3(d) are sectional half elevations through the outer shell of FIGS.", "3(a) and 3(b) respectively, FIG.", "4 is a schematic sectional elevation through apparatus for forming the outer shell body by back extrusion from a slug of metal, FIG.", "5 is a schematic sectional elevation through apparatus for forming the outer shell body by flow forming a disc of metal centred on the crown and about the longitudinal piston axis, and FIG.", "6 is a sectional elevation through a part of a third embodiment of piston according to the invention in which the tubular side wall is of substantially uniform thickness and the ring grooves and shoulder for the mounting are defined by folding the metal of the wall radially as a function of distance along the piston axis.", "Referring to FIGS.", "1(a) to 1(d) a piston 10 for an internal combustion engine is formed from high carbon steel.", "It comprises an outer shell 12 and a mounting member 14 bonded to it metallurgically by brazing.", "The outer shell 12 comprises a crown 16 centred on a longitudinal piston axis 18, and a tubular side wall 20 extending axially with respect to peripheral region 22 of the crown to an open end 24.Centrally of the crown, and surrounded by the peripheral region 22, is a central region 26 (denoted by boundary lines 28) in the form of a combustion bowl 30 having a bowl floor 32 displaced axially with respect to the crown peripheral region, and towards the open end of the tubular side wall, by a bowl wall 34.The bowl wall conveniently has a radially reentrant form as indicated at 36.The crown 16, including both the central region 26 and peripheral region 22, is of integral formation with the tubular side wall by back extrusion onto a mandrel, as described below, to define a unitary outer shell body.", "The tubular side wall 20 includes adjacent the crown a region 40 of axially spaced, circumferentially extending ring grooves 42 machined into the wall and between the ring groove region and the open end 24, there is provided shoulder means 44 facing towards the open end effected by changes in internal diameter of the wall.", "As best seen in FIGS.", "1(c) and 1(d), the shoulder means 44 comprises a first, smaller reduction in thickness of the ring groove region at 46, defining first shoulder 48, and as second, larger reduction in thickness at 50 between the ring groove region and open end 24, defining a second shoulder 52.The tubular side wall is, apart from the ring grooves, of substantially uniform thickness in the groove region between the crown and shoulder means, and also a reduced, but substantially uniform thickness between the shoulder means and the open end; the reduction in thickness is principally defined by the shoulder means but there is also a slight tapering of wall thickness from crown to open end to minimise overall weight by having less wall thickness where less strength is required.", "The region 46 may, and not disadvantageously, lie at the same axial position as one or more of the ring grooves.", "The mounting member 14 is cast by investment casting or the like and comprises an axially thin plate 54 dimensioned to fit within the open end of the tubular side wall such that at least some points at its periphery 56, and preferably all of its periphery, interface with the wall shoulder region 46 at wall interface 57 and byway of which interface it is bonded to the outer shell.", "In this embodiment it is bonded to the tubular wall about substantially the whole of its periphery and to this end, the plate has increased axial thickness at its periphery, defining a flange 58 extending axially to one side of the plate towards the crown.", "The mounting member plate 54 also carries gudgeon boss means 60 formed integrally therewith at the side facing towards the open end 24 and axially between the flange 58 and the open end.", "The gudgeon pin boss means includes a bore 62 for the passage of a conventional gudgeon pin (not shown) transversely to the longitudinal axis 18.The mounting member plate 54 further includes a connecting rod space, in the form of aperture 64 extending through the mounting member along the longitudinal piston axis, said aperture defining from the gudgeon pin boss means two gudgeon pin bosses 66 and 68 spaced apart along the bore and, with the plate in position exposing the central region of the crown to the open end of the tubular side wall.", "The central region of the crown, in particular the junction between the combustion bowl floor 32 and wall 34, has at least one axial extension to the bowl wall, conveniently as a circumferentially complete flange 70 which provides a uniform surface extending transversely to the piston axis and against which the mounting member plate 54 can bear at crown interface 71 to define its axial position within the tubular wall.", "In this embodiment, the axial position of the flange 70 is such that the peripheral flange 58 of the plate is clear of the first shoulder 48, that is, the mounting member is positioned to one axial datum only.", "The mounting member is bonded metallurgically to the side wall at interface 57 and to the crown at interface 71 by brazing, by applying a brazing material to the interfaces between the mounting member and outer shell as they are assembled together, heating them to a temperature sufficient to melt the brazing material, followed by any heat treatment, cooling and/or quenching regime desirable to impart desired physical properties to the brazed components.", "Insofar as the mounting member and outer shell are fully heated, the individual components may be subjected to stress relieving prior to assembly and heating together.", "The flange 70 which surrounds the combustion bowl floor also surrounds the connecting rod aperture 64 such that the crown, the tubular side wall and the bonded mounting member define therebetween an annular cooling chamber 80 which is substantially closed in the axial direction by the crown and by the mounting member.", "Channel means, indicated generally at 82, permits passage of cooling fluid to and from the annular chamber.", "The channel means comprises a fluid admission aperture 84 extending through the mounting member in a substantially axial direction and disposed such that for at least part of the piston stroke a jet of fluid is directed through the aperture and into the chamber.", "A fluid drainage aperture 86 extends through the mounting member displaced about the longitudinal axis from the admission aperture.", "In keeping with producing a light weight piston, the tubular side wall is, at the open end 24 cut away about the longitudinal piston axis in line with the ends of the gudgeon pin bore to an axial level between the ends of the gudgeon pin bore and the ring groove region.", "It will be appreciated that the side wall may be cut more severely than illustrated, to the level of the shoulder means or other demarcation of the end of the ring groove region, such that there exists, to each side of the pin boss means two circumferentially discrete skirt portions essentially isolated from each other.", "It will be appreciated that having formed the shell 12 and mounting member 14, there is essentially only a single assembly operation in respect of positioning the mounting member within the outer shell and bonding it thereto, more particularly to the side wall and crown combustion bowl, but that even with the use of relatively thin-sectioned outer shell and mounting member components, the resulting structure has considerable strength and resistance to deformation of the side wall in the ring groove region.", "In the same manner that a so-called monocoque structure gives strength and stiffness to a vehicle body, the structure here is analogous and may be considered as a monocoque type of structure.", "The strength is attributable to the structural shape as well as the materials and the piston can thus be formed by relatively low cost high or medium carbon steel, that is, a low alloy steel.", "Indeed the construction is suitable for non-ferrous alloys provided they are capable of being shaped into such outer shell and mounting member and bonded together.", "Furthermore, it will be seen that the outer shell and mounting member may be made of different metals provided they can be successfully bonded or are compatible in terms of strength and thermal expansion and ability to form a metallurgical bond between them.", "The aforementioned brazing may be employed with similar or dissimilar metals.", "It may be possible to effect bonding between the mounting member and outer shell by a non-metallurgical bond.", "It may also be possible to provide one or both of the outer shell and mounting member components of non-metallic material subject to the above criteria for bonding.", "Where the outer shell is constructed with a discrete combustion bowl or other central region, that part may also be non-metallic.", "As mentioned above the outer shell 12 is formed as an integral body by back extrusion.", "Referring to FIG.", "4, this shows schematically a back extrusion apparatus 400 including a mandrel 402 having an outer surface 404 conforming to the internal shape and dimensions of the tubular side wall 20, including a step 406 corresponding to shoulder means 404 and a shallow lengthwise taper that effects minimal wall thickness according to strength requirements, as well as recess 408 corresponding to combustion bowl 30.A cylindrical sleeve 410 surrounds the mandrel, separated by gap 418 and relative movement between them exerts pressure on a metal slug 420 which deforms and flows into the gap and into conformity with the mandrel to define the shell body.", "The aforementioned small taper of mandrel surface 404 that creates the above discussed internal side wall taper also facilitates removal of the extruded shell.", "Such taper may be kept to a minimum, insofar as this is consistent with strength but may be eliminated altogether without affecting removal from the mandrel.", "It will be appreciated that such extrusion, in distinction from forging, casting and like operations, is a precision operation that permits the formation of relatively thin walls of relatively uniform thickness which in conjunction with the strength and stiffness afforded by the monocoque type of structure permits formation of a lightweight piston from a dense material such as steel.", "However it is quite possible to provide the outer body shell by forging or other metal deforming processes.", "As described hereinbefore, the combustion bowl wall has an axial extension in the form of flange 70.It will be appreciated that the mounting member could interface directly with the floor of the combustion bowl or such axial extension could be formed on the mounting member and extend to the bowl floor.", "It will also be understood that the combustion bowl may be omitted altogether, that is, have a substantially flat or domed crown, and such flange extend from the mounting member plate to the bottom of the crown surface, such arrangement still providing the support between crown and gudgeon pin boss means and annular cooling chamber.", "Although it is convenient for the central region 26 of the crown to be integral with the peripheral region 22 it need not be, and can be manufactured separately and welded into the peripheral region to effect the unitary outer shell.", "Such separate formation of a combustion bowl may be appropriate to avoid having to machine radial re-entrant features in situ, but it is, of course, not necessary for a combustion bowl to have such re-entrant features and it may have a side wall that is suitable for forming completely by the extrusion process that defines the shell.", "Although the piston 10 is essentially constructed from two components brought together in a single bonding operation, it is anticipated that there will still be machining operations required to the external surfaces of the side wall and crown, such as definition of combustion bowl re-entrant features, making valve pockets or recesses in the face of the crown, forming of ring grooves, cutting of the wall end and applying a final surface finish that also defines outside dimensions to within fine tolerances.", "Some of these may be performed before or after assembly and bonding of the outer shell and mounting member and some may be achieved during the extrusion that forms the outer shell, for example, valve pockets 90 in the crown face and ovality of cross section defined by the extrusion mandrel.", "The degree of ovality required of a piston is usually a function of its overall diameter; it is anticipated that the degree of ovality required on small diameter pistons may be achieved by the final machining of the outer surface, whereas for larger diameter pistons, such ovality may be better achieved by forming the outer shell with such cross section on a suitably shaped mandrel.", "Also, the formation of valve pockets or other shallow crown face features with the shell eliminates at least one relatively costly machining operation.", "Formation of the outer shell by back extrusion about a mandrel permits the tubular wall internal surface to be defined to a suitable degree of accuracy without further machining.", "Of particular importance in connection with forming a lightweight piston is that in addition to being able to cut away any non-essential parts of the tubular side wall, is to have all wall sections as thin as possible for the functions required thereof.", "To this end it is possible to extrude the side wall with only slight variation in thickness from end to end (other than at the shoulder means) and with the ring groove region also less thick than might be thought acceptable, because of the support from the transverse mounting member.", "It is a feature of the embodiment described that upon assembly prior to bonding, the interface 57 between the mounting member and the side wall extends axially, and thus positions the mounting member radially, whereas the crown interface 71 extends radially and positions the mounting member axially.", "The bond at interface 71, which is enclosed between the mounting member and crown, must be effected by the aforementioned brazing or some other technique which does not rely upon access to it.", "One alternative is friction welding, but that may be considered unsuitable for the axially extending wall interface 57, and although the latter is accessible from the open end, and susceptible to bonding by a different technique, it may be preferred not to have different bonding systems in use together.", "This and other alternative structural possibilities are addressed in a second exemplary embodiment of piston in accordance with the invention is shown at 110 in FIGS.", "2(a) to 2(d).", "Many of the parts are similar to those of piston 10 and the description will concentrate on the differences, For ease of reference, corresponding parts have reference numbers increased by 100.The piston 110 is a bonded assembly of outer shell 112 and mounting member 114, piston crown including a combustion bowl 130 therein.", "The outer shell 112 is generally similar to shell 12 insofar as it has tubular side wall 120 that includes a ring groove region 140 and shoulder means 144 and, integral therewith, a crown peripheral region 122.The peripheral region includes, displaced from the side wall, an axially extending combustion bowl wall 134.A central region 126 of the crown, defined by boundary lines 128 about longitudinal axis 18, comprises the floor 132 of the combustion bowl which is of discrete formation from the peripheral region and bonded thereto at interface 133 which extends around the periphery of this central region and substantially parallel to the longitudinal axis 18.The combustion bowl floor 132 is carried by the mounting member 114, being formed integrally therewith and overlying a connecting rod space 164, corresponding to the connecting rod aperture 64 of member 14 of piston 10, the interface 133 that the bowl floor makes with the bowl wall comprises a crown interface.", "The mounting member 114 comprises a plate 154 carrying on the surface opposite to the combustion bowl gudgeon pin boss means 160, including transverse bore 162 and bosses 166 and 168 spaced apart by connecting rod space 164.The outer periphery of the member at 156 is defined to be a tight fit within the tubular side wall, in particular interfacing with the wall region 146 at wall interface 157 which extends in a substantially longitudinal direction.", "The base of the combustion bowl wall, adjacent the floor, has axial extension 170 which abuts the upper surface of the mounting member plate in order to locate it axially with respect to the crown periphery.", "Radial location is effected by the crown interface 133 and wall interface 157, although the wall interface 157, insofar as it is defined at shoulder means, may provide the axial location.", "It will be appreciated that the mounting member is bonded to the tubular side wall at interface 157 and to the crown at interface 133, but significantly, in addition to the wall interface 157 being in line with the open end of the side wall the crown interface 133 is in line with the open end of the combustion bowl.", "As each interface is accessible in the axial direction, it is possible to weld each by laser beam, particle beam, plasma jet or the like by rotating the piston assembly or the welding apparatus about the longitudinal piston axis.", "To facilitate such a welded bond, the surfaces bounding each interface are formed to provide a small divergence in the direction from which such welding is effected.", "It will be understood that insofar as each interface is welded by a remote energy source in line therewith, the line of one or both interfaces may be inclined with respect to the longitudinal axis such that one or both of the interfaces are not only visible from without the piston but may have a taper that effects both radial and axial location between the mounting member and the outer shell.", "It is, of course, possible to effect bonding between the outer shell and mounting member at the interfaces by brazing as described above.", "A cooling chamber 180 is defined between the outer shell and mounting member plate 154 and the plate 154 has fluid admission channel 184 and drainage channel 186 therethrough.", "This arrangement is shown to differ from that of piston 10 in that the chamber 180 is of greater axial extent in line with the gudgeon pin bore 162, that is, overlying the bosses 166 and 168 as shown at 167 and 169.Insofar as the admission and drainage channels are at an operationally higher level, these extended regions form reservoirs for cooling fluid.", "In a modification to the above, the fluid drainage channel may comprise one or more channels 188 extending substantially radially from the cooling chamber to the connecting rod space 164.Such arrangement of cooling chamber reservoirs and drainage channels may be applied to the piston 10.It will be appreciated that the central region 126 may be defined as being other than what is substantially the whole of the combustion bowl floor.", "It may, for example be a smaller region of the floor or it may be larger and incorporate the bowl wall 134, the boundary between central and peripheral regions being at the upper crown surface, as shown by boundary lines 128′, and the mounting member/crown interface 133 coincident therewith, said central and peripheral regions defining together an essentially flat topped or domed crown.", "In a further modification, not specifically shown, the central region of the crown may be formed with the peripheral region of the crown as part of the outer shell in the manner of piston 10 and the mounting member may have an upstanding closure to a connecting rod space in the manner of piston 110, whereby the closure provides not only upstanding flange means as described above for defining the cooling chamber but also overlies, and is capable of spreading load from, the central region of the crown.", "Sectional views of a third exemplary embodiment of piston 210 in accordance with the present invention are shown in FIGS.", "3(a) to 3(d), parts corresponding to those of FIGS.", "1(a) to 1(d) having reference numbers increased by 200.The piston 210 comprises outer shell 212 and mounting member 214 bonded to each other.", "The outer shell 212 comprises a unitary body consisting of crown peripheral region 222, crown central region 226 in the form of a combustion bowl 230 and tubular side wall 220.The tubular side wall consists of a thicker ring groove region 240 adjacent the crown and a thinner skirt region, open ended at 224, separated from the ring groove region by simple shoulder means 244.The combustion bowl 230 comprises a bowl floor 232 displaced axially from the peripheral region by bowl wall 234 and the wall, at the junction with the floor, has a number of axial extensions 2701, 2702 with gaps between them and possibly of slightly different axial lengths.", "Mounting member 214 comprises a relatively thin mounting plate 254, the periphery of which is dimensioned to fit within the thinner part of the side wall adjacent the shoulder means 244; the periphery 256 of the plate defines an interface 257 with the tubular wall and has no axial flange or like projection to increase the axial length of the interface.", "The lower face of the plate, facing the open end 224, caries integral gudgeon pin boss means 260 having transverse gudgeon pin bore 262 therethrough and through the plate, along piston axis 18, is a connecting rod aperture 264 which also effects formation of separated gudgeon pin bosses 266 and 268.The upper face of the plate, indicated at 255 and facing the crown, is substantially flat and abuts the bowl wall extensions 270, and 2702 defining thereat interface 271 extending transversely with respect to the piston axis 18.Insofar as the interfaces 257 and 271 correspond in position and orientation to the interfaces 57 and 71 of piston 10, the outer shell and mounting member in the disposition shown are bonded to each other by brazing as described above, defining the strong monocoque type of structure including a closed annular cooling chamber 280 between the crown, ring groove region of the side wall and the upper surface 255 of the mounting member plate 254.Fluid channel means 284 permits admission of cooling fluid into the chamber and channels 288 permit drainage by way of the gaps between the bowl wall axial extensions 270, and 2702 to the connecting rod aperture.", "As an alternative to the equally-applicable cutting away of the open end of the side wall to the extent shown for pistons 10 and 110, the gudgeon pin bosses 266 and 268 may extend into abutment with the side wall and the latter include through apertures 290 and 292 in alignment with the gudgeon pin bore 262.Furthermore, insofar as each boss defines an interface 294, 296 respectively with the side wall, it may be bonded thereto adjacent the apertures.", "This arrangement of longer, apertured side wall and, optionally, gudgeon pin bosses extending thereto may be applied to the piston 10, and to piston 110 provided that the mounting member is bonded by brazing or the like that does not require direct access.", "The mounting means 214 is of substantially uniform cross section in the direction of the gudgeon pin bore 262, that is, as viewed in FIG.", "3(a), except of course where the connecting rod aperture 264 is cut.", "Instead of the mounting member being cast, it is formed by cutting from an extruded stock and then shaped to fit within the tubular side wall and the axially extending apertures 264 and 284 cut therein.", "It will be appreciated that a cast mounting member, having a more complex surface as seen in the above described embodiments may be employed in piston 210 or such an extruded mounting member may be employed with the pistons 10 and 110.Another difference illustrated in this embodiment is a tubular side wall which is, to each side of the shoulder means 244, of uniform thickness, that is, without the normally slightly tapering characteristic of extrusion or forging.", "The outer shell 212, although it may be formed by extrusion or forging, is produced by so-called flow forming in which, as FIG.", "5 illustrates a disc-like slug of metal 502 is caused to rotate with a profiled mandrel 504 and during rotation the peripheral regions of the disc are displaced axially to lie along and conform with the mandrel that defines the respective wall thicknesses and shoulder means.", "As with the back extrusion described above, the tubular side wall may be formed thereon and removed without an internal wall taper, where this can save weight.", "Such flow forming of the outer shell may be used in respect of piston 10 and 110.Although it may be preferred to define the tubular side wall with a thicker region into which ring grooves are subsequently machined, it will be appreciated that such flow forming permits, with the use of a radially contractible mandrel, formation of a tubular side wall 220′ of substantially uniform thickness from end to end but varying in radius as a function of axial position to define the ring grooves 242′ and shoulder means 244′, as illustrated in FIG.", "6.It will also be appreciated that whereas the above embodiments have described the mounting member as bonded about the whole of its periphery to the side wall at the shoulder means, it may be bonded only at a plurality of discrete points and the periphery of the mounting member may be other than conforming in shape to the tubular side wall, extending to contact the wall only at points of bonding.", "Although it is convenient for assembly to define a bonding interface between the periphery of the mounting member to the side wall at shoulder means which demarcates between the ring groove region and the more lightly loaded skirt, it is not essential and it may be bonded to the side wall other than at such shoulder if disposing the interface elsewhere improves stress patterns.", "It is re-iterated that the various embodiments of piston according to the invention are not limited in size and the structures and methods of manufacture are capable of being scaled to a wide variety of dimensions." ] ]

[ [ "Glazing for vehicles", "The invention relates to laminated glazing for vehicles having a limited energy transmittance.", "According to the invention, the glazing has a light transmittance of at least 70%, an energy transmittance TE that is at most equal to 51% and, for thicknesses requested by manufacturers, thicknesses for which Texe, wherein e is the total thickness of the sheets of glass in millimetres, is at most equal to 200.", "The inventive glazing provides a balanced solution that satisfies demands in terms of cost and sun-protection properties for vehicles, particularly motor vehicles." ], [ "1.Laminated glazing for vehicles comprising two sheets of coloured glass, of which the light transmission (TL) for the thicknesses required by manufacturers is at least equal to 70%, the energy transmittance (TE) is at most equal to 51% and the term TExe, wherein TE is expressed as a percentage and e is the total thickness of the two glass sheets expressed in millimetres, is at most equal to 200.2.Laminated glazing according to claim 1, wherein the energy transmittance is at most equal to 48%.", "3.", "(Canceled) 4.", "(Canceled) 5.6.", "(Canceled) 7.", "(Canceled) 8.", "(Canceled) 9.", "(Canceled) 10.", "(Canceled) 11.", "(Canceled) 12.Laminated glazing according to claim 1, and further including at least one of the following (A) through (D): A. wherein the total thickness of the two glass sheets is in the range of between 3.5 and 5.5 mm; B. wherein the two glass sheets have the same glass composition; C. wherein the glass sheets have the same thickness; D. wherein the glass sheets the following soda-lime composition: SiO2 60-75% Na2O 10-20% CaO 0-16% K2O 0-10% MgO 0-10% Al2O3 0-5% BaO 0-2% with K2O + Na2O 10-20% CaO + MgO + BaO 10-20% and comprising the following main colouring agents: Fe2O3 (total iron expressed as) 0.5-1% FeO 0.14-0.25% Co 0-0.0040% Cr2O3 0-0.0500% Vr2O5 0-0.0200% Se 0-0.0050%.", "13.Laminated glazing according to claim 12, and including at least two of the features (A) through (D).", "14.Laminated glazing according to claim 12, and including all of the features (A) through (D).", "15.Laminated glazing according to claim 1, wherein the total thickness of the two glass sheets is in the range of between 3.8 and 5.2 mm.", "16.Laminated glazing according to claim 1, for which the term TExe is at most equal to 195.17.Laminated glazing according to claim 1, wherein the soda-lime composition of the glass sheets comprises the following colouring agents: Fe2O3 (total iron expressed as) 0.5-0.6% FeO 0.16-0.20% Co 0-0.0020% Cr2O3 0.0020-0.0045%.", "18.Laminated glazing according to claim 1, wherein the soda-lime composition of the glass sheets comprises the following colouring agents: Fe2O3 (total iron expressed as) 0.7-1% FeO 0.18-0.24% V2O5 0-0.0200%." ], [ "The present invention relates to laminated glazing for vehicles.", "In particular, the invention relates to laminated glazing having a limited energy transmittance.", "There is an increasing demand from automobile manufacturers for glazing which serves several functions.", "For example, in the case of side windows of an automobile, these should contribute towards the comfort and security of the vehicle.", "With respect to comfort, this means in particular contributing to improving the temperature inside the passenger compartment when the vehicle is exposed to the sun.", "It also means improving, where necessary, the sound level of the passenger compartment.", "With respect to security, the requirement is primarily to reinforce mechanical resistance to forced entry.", "While solutions to these requirements are conceivable in principle, their industrial application raises numerous problems.", "The greatest difficulty is to meet envisaged cost levels that manufacturers are willing to accept.", "Working from the idea that an improvement in security is obtained by using laminated glazing, it must be established under what conditions such glazing enables the other desired characteristics to be achieved, in particular the cost conditions.", "It is self-evident that whatever the qualities of such glazing, it is important to remain within the most limited cost conditions possible.", "Hence, various solutions are known for providing glazing with sun-protection properties.", "In the case of monolithic glazing, the most usual solution is to use coloured glasses.", "In the case of laminated glazing, in addition to using coloured glasses, it is possible to include elements in the intermediate layer such as thin films coated with thin reflective layers, or also to deposit these same layers directly onto the surfaces of the sheets not exposed to external or internal stresses.", "The use of layers deposited on the glass or on a film incorporated as interlayer has the advantage of providing a good sun-protection effect by means of energy reflection.", "In addition, the low energy transmittance resulting from this is obtained without the light transmission (TL) necessarily being reduced too significantly.", "It must in fact be remembered that side windows of an automobile are subject to strict standards, e.g.", "a light transmission of at least 70% for the front of the vehicle.", "In spite of the advantages outlined above, the cost of solutions involving sun-protection layers is a factor which restricts the development of glazing of this type.", "The inventors have therefore proposed to develop laminated glazing for automobiles by endeavouring to meet the requirements outlined above.", "Moreover, the requirement relating to thickness must also be included with these requirements.", "Automobile manufacturers want to be able to offer their customers the choice between “traditional” monolithic glazing units “as standard” or laminated glazing units with the functions in question “as optional extra” to take into account differences in cost corresponding to these two types of installations.", "At the same time, the manufacturers require that the two types of glazing are usable without any modification of the elements on which these glazing units are assembled.", "The frames, slides etc.", "must be usable equally with monolithic glazing and laminated glazing.", "This requires that these two types of glazing are of absolutely equal thickness, or if not at least differ very little in thickness.", "The inventors have managed to combine these two different requirements and propose laminated glazing for automobiles comprising two sheets of coloured glass, of which the light transmission (TL) for the thicknesses required by manufacturers is at least equal to 70%, the energy transmittance (TE) is at most 51%, and this for a glass thickness such that the product TExe, wherein TE is expressed as a percentage and e is the total thickness of the two glass sheets expressed in millimetres, remains at most equal to 200.This expression conveys the constraints in which the glazings according to the invention are subject to.", "The rigorous conditions the glazing in question must meet are achieved partly as a result of the colourations required.", "It is relatively more difficult to achieve these conditions with glasses with a blue colouration.", "With these glasses the difficulty lies in lowering the energy transmittance to sufficiently low levels without the light transmission becoming too low at the same time.", "For this reason, the term TExe only varies within a limited range.", "Nevertheless, the blue glazing types according to the invention are preferably such that TExe is less than 195.TExe can be more easily limited with green glazing.", "This value can be brought to values equal to 180 at most without too much difficulty.", "The thickness of the glazing is preferably in the range of between 3.5 and 5.5 mm.", "Most usually, the glazing according to the invention has a total thickness in the range of between 3.8 and 5 mm.", "Moreover, the use of two distinct glasses has the disadvantage for the manufacturer of having to hold increased stocks.", "Therefore, it is preferable to assemble glass sheets of the same composition.", "For the same reasons, it is preferable to combine two glasses with identical thicknesses rather than glasses with different thicknesses.", "In the following description as well as in the claims, the TL used is that determined using the standard illuminant A as defined by the Commission Internationale de l'Éclairage.", "Illuminant A represents the radiation of a Planck radiator at a temperature of about 2856 K. This illuminant constitutes the light emitted by vehicle headlights and is essentially intended for evaluation of the optical properties of glazing intended for motor vehicles.", "TL and TE are: the total light transmission with illuminant A (TLA): this total transmission is the result of integration between the wavelengths of 380 and 780 nm of the term: ΣTλ.Eλ.Sλ/ΣEλ.Sλ, in which Tλ is the transmission at wavelength λ, Eλ is the spectral distribution of illuminant A and Sλ is the sensitivity of the normal human eye as a function of wavelength λ; the total energy transmittance (TE): this total transmittance is the result of integration between the wavelengths of 300 and 2500 nm of the term: ΣTλ.Eλ/ΣEλ, wherein Eλ is the spectral energy distribution of the sun at 30° above the horizon.", "Colours likewise come into the choice of glasses.", "They are important in the production of the glasses in question.", "They are equally important in the definition of the properties of transmission in visible, infrared or ultraviolet light, these properties by definition determining the use according to the invention.", "Moreover, the choice of colours must meet the aesthetic appeal sought by manufacturers.", "In practice, the glazing must have a predominantly green or blue colouration.", "The glasses used according to the invention match the traditional basic soda-lime compositions, in which the main components have the following proportions by weight: SiO2 60-75% Na2O 10-20% CaO 0-16% K2O 0-10% MgO 0-10% Al2O3 0-5% BaO 0-2% with K2O + Na2O 10-20% CaO + MgO + BaO 10-20%.", "In addition to the soda-lime base, the glasses used according to the invention comprise the following colouring agents, and primarily iron oxides.", "These colouring agents are contained in the following general proportions: Fe2O3 (total iron expressed as) 0.5-1% FeO 0.14-0.25% Co 0-0.0040% Cr2O3 0-0.0500% Vr2O5 0-0.0200% Se 0-0.0050%.", "The compositions may also contain other colouring agents, in particular those resulting from the raw materials used, in proportions by weight not exceeding the following: TiO2<0.1% MnO2<0.13% CeO2<0.5%.", "Preferred compositions correspond to a combination of colouring agents such as the following: Fe2O3 (total iron expressed as) 0.5-0.7% FeO 0.16-0.22% Co 0-0.0020% Cr2O3 0.0020-0.0045%.", "Even more precisely, glasses with a predominantly blue colouration preferably correspond to the following compositions of colouring agents with the glazing according to the invention: Fe2O3 (total iron expressed as) 0.5-0.6% FeO 0.16-0.20% Co 0-0.0020% Cr2O3 0.0020-0.0045%.", "Other compositions of colouring agents in the case of the glazing according to the invention with a predominantly green colouration are advantageously as follows: Fe2O3 (total iron expressed as) 0.7-1% FeO 0.18-0.25% Co 0-0.0040% Cr2O3 0-0.0250% V2O5 0-0.0200%.", "In a preferred manner, predominantly green glazing types comprise glasses with the following colouring agents: Fe2O3 (total iron expressed as) 0.7-1% FeO 0.18-0.24% V2O5 0-0.0200%.", "Glazing types exhibiting the characteristics of the invention have been formed by way of an example.", "In these examples all the glazing types are laminated with an interlayer of clear polyvinyl butyral with a thickness of 0.76 mm.", "The glasses of the formed assemblies have a soda-lime base with the following proportions by weight: SiO2 71.5-71.9% Na2O 14.1% CaO 8.8% K2O 0.1% MgO 4.2% Al2O3 0-8%.", "In these glasses the colouring agents are respectively present in the following proportions by weight: I II III IV V VI Fe2O3 0.08 0.84 0.95 0.63 0.38 0.57 FeO 0.01 0.21 0.24 0.15 0.12 0.18 Co 0.0012 0.0014 Cr2O3 0.0041 V2O5 0.0150 The glazing units are formed in different thicknesses.", "The two sheets have the same thickness in all the examples.", "Two series are formed for glazing units with a green hue, their total thickness being close to 4 mm and 5 mm respectively.", "The first series is that with a thickness of about 5 mm.", "The characteristics of the glazing units are indicated in the following table: Glass 1 Glass 2 Total thickness TL % TE % TExe 1 IV IV 4.96 76.1 50.4 212 2 III I 4.96 75.3 52.6 221 3 II IV 4.96 72.8 45.5 191 4 II II 4.96 69.8 42.1 177 5 III IV 4.96 69.7 42.8 180 In the tests reported above, it may be seen that with thicknesses close to 5 mm, the conditions sought according to the invention are only strictly met in example 3.Examples 1 and 2 have a TE which is too high and also a TExe value that is too high.", "Conversely, examples 4 and 5 have a TL that is slightly less than standard.", "In the case of the combinations of types 1 and 2, a slight increase in thickness of the glass sheets allow the TE to be brought into the ranges of the invention.", "However, at the same time the thicknesses, and therefore the term TExe, increase further.", "This shows how difficult it is to attain all the fixed conditions.", "Conversely, in the case of combinations 4 and 5, the TE is satisfactory and the light transmission is very slightly less than the standard for these glazing types.", "A slight decrease in thickness allows the TL to be brought to above 70%.", "Hence, in example 4 a variation in thickness of each sheet from 2.1 to is 2.0 mm results in values for the TL of 71.6% and for the TE 43.8%, which are completely adequate.", "The value of the TExe also remains in the limits fixed according to the invention.", "With respect to example 3 this structure has the additional advantage of combining two glass sheets that are identical and are therefore more readily matched after bending.", "The second series of glazing units has a smaller thickness of little more than 4 mm.", "In this series the thickness of each sheet is 1.7 mm.", "As above, the interlayer has a thickness of 0.76 mm.", "The resulting measurements are given in the following table: Glass 1 Glass 2 Total thickness TL % TE % TExe 6 II II 4.16 73.4 46.5 158 7 III IV 4.16 73.3 48.1 164 8 III II 4.16 70.8 44.3 151 9 III III 4.16 68.6 40.7 139 In these different examples only 9 does not meet the condition with respect to the TL.", "As previously, a slight decrease in thickness allows the TL to be brought to above the imposed limit.", "Of the other examples, 6 is of particular interest, since it allows two identical sheets to be combined.", "Similar tests to the above have been conducted with glasses with a blue hue, also with total thicknesses close to 5 and 4.5 mm.", "These assemblies were formed with two sheets of the same thickness of 2.1 mm, 1.9 mm, 1.85 mm or 1.7 mm.", "The characteristics of the glazing types produced are shown in the following table: Glass 1 Glass 2 Total thickness TL % TE % TExe 10 VI V 4.96 73.0 49.7 209 11 VI VI 4.56 72.0 48.1 183 12 VI VI 4.46 72.4 48.7 180 13 VI VI 4.16 73.4 50.8 173 Examples 11, 12 and 13 meet the various conditions demanded of glazing according to the invention.", "Example 10 has too high a TExe value, even though the TE is acceptable.", "This type of assembly shows the difficulty of attaining all the required conditions.", "In fact, a reduction in thickness of each of the sheets would result in an increase in the TE and therefore lead to an unsatisfactory glazing." ] ]

[ [ "Naadp analogues for modulating t-cell activity", "A method for modulating T cell activity by modulating the intracellular concentration and/or activity of NAADP+, compounds capable of modulating the effect of NAADP+ on T cell Ca2+ levels, and methods for identifying the same, are described." ], [ "1.A method for modulating T cell activity, which comprises the step of modulating the intracellular concentration of NAADP+ or a bioisostere thereof.", "2.A method according to claim 1, which comprises the step of stimulating a rise in intracellular Ca2+ levels by raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an activating concentration.", "3.A method according to claim 1, which comprises the step of inhibiting TCR/CD3-associated Ca2+ signalling by raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an inactivating concentration.", "4.A compound capable of antagonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, said rise being in response to stimulation of the T cell receptor/CD3 complex, for use in modulating T cell activity.", "5.A compound capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, for use in modulating T cell activity 6.A compound according to claim 5, which is capable of raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an inactivating concentration 7.A compound according to any of claims 4 to 6, for use in inducing T cell anergy.", "8.A compound according to any of claims 4 to 6, for use in blocking T cell proliferation.", "9.A compound capable of agonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, said rise being in response to stimulation of the T cell receptor/CD3 complex, for use in modulating T cell activity.", "10.A compound capable of preventing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, for use in modulating T cell activity 11.A compound according to claim 9 or 10, for stimulating T cell proliferation and/or differentiation.", "12.A compound according to any one of claims 4-11 wherein said compound is a NAADP analogue.", "13.A compound according to claim 12 wherein the NAADP analogue has the formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "14.A compound according to claim 12 or claim 13 wherein said NAADP analogue is 8-bromo-nicotinic acid adenine dinucleotide phosphate.", "15.Use of a compound as defined in any one of claims 4 to 14 in the manufacture of a medicament for use in modulating the immune response of a mammal.", "16.Use of a compound as defined in any one of claims 4 to 8 in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "17.Use according to claim 16 wherein the autoimmune disease is selected from thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rhematoid arthritis and lupus erythematosus.", "18.Use of a compound as defined in any one of claims 4 to 17 in the manufacture of a medicament for use in treating or preventing an immune disorder in a human or animal.", "19.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a compound as defined in any one of claims 4 to 14.20.A method for identifying a substance capable of antagonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell, which has been stimulated via its T cell receptor, with a candidate substance under conditions that would permit a rise in intracellular Ca2+ levels in the absence of the substance; and (ii) determining whether the substance inhibits a rise in intracellular Ca2+ levels.", "21.A method for identifying a substance capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, which method comprises: (i) contacting a T cell with a candidate substance; (ii) stimulating the T cells via TCR/CD3; and (ii) determining whether the substance inhibits TCR/CD3-associated Ca2+ signalling.", "22.A method for identifying a substance capable of agonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell with a candidate substance; and (ii) determining whether the substance elicits or enhances a rise in intracellular NAADP+ and/or Ca2+ levels.", "23.A substance identified by the method of claim 20, 21 or 22.24.A process comprising the steps of: (a) performing the method according to claim 20, 21 or 22; (b) preparing a quantity of one or more substances identified by the method.", "25.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound is a NAADP analogue.", "26.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound has the formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "27.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound is 8-bromo-nicotinic acid adenine dinucleotide phosphate.", "28.Use of a NAADP analogue in the manufacture of a medicament for use in modulating the immune response of a mammal.", "29.Use of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I) in the manufacture of a medicament for use in modulating the immune response of a mammal.", "30.Use of 8-bromo-nicotinic acid adenine dinucleotide phosphate in the manufacture of a medicament for use in modulating the immune response of a mammal.", "31.Use of a NAADP analogue in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "32.Use of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I) in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "33.Use of 8-bromo-nicotinic acid adenine dinucleotide phosphate in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "34.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a NAADP analogue.", "35.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "36.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of 8-bromo-nicotinic acid adenine dinucleotide phosphate." ], [ "<SOH> BACKGROUND TO THE INVENTION <EOH>Adaptive or specific immune responses are normally stimulated when an individual is exposed to a foreign antigen.", "Specific immunity is mediated by lymphocytes, e.g.", "B and T lymphocytes.", "During an immune response, recognition of an antigen leads to activation of lymphocytes that specifically recognise that particular antigen.", "The lymphocytes proliferate and differentiate into specialised effector cells.", "The immune response culminates in the development of mechanisms that ultimately eliminate the antigen.", "Adaptive immune responses are critical components of host defence during protection against foreign antigens, such as infectious organisms or toxins.", "However, specific immune responses are also sometimes elicited by antigens not associated with infectious agents, and this may cause serious disease.", "For example, one of the most remarkable properties of specific immunity is the ability to distinguish between self antigens and foreign antigens.", "Thus, the lymphocytes in each individual are able to recognise and respond to numerous foreign antigens but are normally unresponsive to potentially antigenic substances present in the individual itself Unresponsiveness to self antigens is an acquired process that has to be learned by the individual's lymphocytes and has to be maintained throughout life.", "Abnormalities in the induction or maintenance of self-tolerance lead to immune responses against self antigens, and debilitating diseases that are commonly called autoimmune diseases.", "The spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus.", "Another example in which specific immunity against antigens that are not associated with infections causes severe medical problems are rejections of transplanted allografts.", "In fact, adaptive immune responses to grafted tissues are the major impediment to successful transplantation in most cases.", "It is not known what causes the breakdown of tolerance and the initiation of an autoimmune response.", "However, the mechanisms of tissue destruction in autoimmune diseases and in allograft rejection are essentially the same as those operating in protective immunity.", "It is generally believed that both autoimmune reactions and allograft rejections are initiated and perpetuated by a response involving T cells.", "Thus, in the absence of a specific therapy for any of the autoimmune diseases or for allograft rejection, many therapeutic strategies currently employed aim at down modulating the activity of the immune system, in particular by reducing or preventing the activation of T cells.", "Recently, monoclonal antibodies to T cell surface antigens, that inhibit T cell activation, or substances that interfere with intracellular T cell activation pathways, such as Cyclosporin A or FK506, have been introduced for the treatment of both allograft rejection and several autoimmune diseases.", "However, current approaches for the treatment of undesirable T cell activation have been associated with a number of side effects related to general immunosuppression and therefore cannot be considered to be optimal therapy.", "Stimulation of T-lymphocytes via the T cell receptor/CD3 complex (TCR/CD3) is a critical step in T cell activation and subsequent clonal expansion.", "Previous studies have shown that activation of the TCR/CD3-complex involves the elevation of the free cytosolic Ca 2+ concentration ([Ca 2+ ] i ) by at least two mechanisms, a rapid elevation caused by Ca 2+ release from intracellular stores mediated by inositol (1,4,5) trisphosphate (Ins(1,4,5)P 3 ), and a prolonged elevation that is completely dependent on the influx of extracellular calcium (reviewed in Guse, 1998).", "Ca 2+ -release is activated by the calcium mobilizing second messengers Ins(1,4,5)P 3 (Jayaraman et al, 1995) and cADPR (Guse et al., 1999).", "Recent work indicates that Ins(1,4,5)P 3 primarily acts during the initial phase of Ca 2+ -signaling in T cells, whereas cADPR is essentially involved in the sustained phase of Ca 2+ -signaling.", "The exact mechanism of Ca 2+ signalling in T cells is still unclear, but it is of fundamental importance for proliferation and clonal expansion, and thus for a functional immune response.", "An improved understanding of the signalling pathways involved in T cell activation may be of assistance in developing strategies to stimulate a desirable adaptive immune response or to suppress inappropriate T cell activity." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present inventors have shown that NAADP+ specifically and dose-dependently stimulates Ca 2+ signalling in human T cells.", "At an activating concentration, NAADP+ either evokes repetitive and longlasting Ca 2+ oscillations or a single Ca 2+ -spike of high amplitude.", "The present inventors have also shown that NAADP+ can be self-inactivating.", "An inactivating concentration of NAADP+ inhibits subsequent stimulation of Ca 2+ signaling via the T cell receptor/CD3.For example, inactivation of the NAADP + /Ca 2+ -release system almost completely abolishes subsequent Ins(1,4,5)P 3 - or cADPR-mediated Ca 2+ -signaling.", "This shows that a functional NAADP+/Ca 2+ release system is essential for T-lymphocyte Ca 2+ signaling.", "These findings have important implications for the design of compounds capable of modulating T cell activity, since regulation of this NAADP+/Ca 2+ signalling pathway may provide an important means of stimulating T cells (and adaptive immune responses) and controlling T cell responses in a variety of T cell mediated immune disorders.", "Accordingly the present invention provides a method for modulating T cell activity, which comprises the step of modulating the intracellular concentration of NAADP+ or a bioisostere thereof.", "In one embodiment, the method involves stimulating a rise in intracellular Ca 2+ levels by raising the intracellular concentration of NAADP+ to an activating concentration.", "The present inventors have found that an intracellular concentration of 10 nM NAADP+ evokes repetitive and longlasting Ca 2+ oscillations of low amplitude, while 50 and 100 nM produces a rapid and high initial Ca 2+ peak followed by trains of smaller Ca 2+ oscillations.", "Higher concentrations of NAADP+(1 and 10 μM) gradually reduce the initial Ca 2+ peak.", "Thus an “activating concentration” of NAADP+ may be between 5 nM and 1 μM, preferably between 5 and 100 nM.", "In another embodiment, the method involves inhibiting TCR/CD3-associated Ca 2+ signaling by raising the intracellular concentration of NAADP+ to an inactivating concentration.", "The present inventors have shown that an intracellular concentration of 100 μM NAADP+ causes complete self-inactivation of Ca 2+ -signals.", "Thus an “inactivating concentration” of NAADP+ may be greater than 1 μM, preferably greater than 10 M, most preferably 100 μM or greater.", "The elucidation of a novel NAADP+-mediated T cell activation pathway also enables the identification of substances that modulate T cell activation via this pathway.", "The present invention thus also provides a compound capable of (a) antagonising the NAADP+-mediated rise in intracellular Ca 2+ levels caused by TCR/CD3 stimulation; (b) agonising the NAADP+-mediated rise in intracellular Ca 2+ levels caused by TCR/CD3 stimulation; (c) inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca 2+ signaling; or (d) preventing the NAADP+-mediated inhibition of TCR/CD3-associated Ca 2+ signaling Compounds of the invention which inhibit T cell proliferation and/or differentiation, or induce T cell anergy may be used in treating diseases characterised by an excessive or inappropriate T cell response, such as autoimmune diseases, allergies and allograft rejection.", "Candidate autoimmune diseases include thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis and lupus erythematosus.", "Compounds of the invention which induce or enhance T cell proliferation and/or differentiation or prevent the induction of T cell anergy may be used generally to boost or induce T cell immune responses.", "Virtually all adaptive immune responses require the activation of T cells and their differentiation into cytokine-producing cells.", "Thus these compounds may be used generally to prevent and treat conditions such as infectious diseases (such as viral or bacterial infections), cancers and, in particular, immunodeficiencies characterised by impaired T cell function (such as AIDS).", "The present invention further provides a method for identifying a substance capable of antagonising the NAADP+-mediated rise in intracellular Ca 2+ levels in a T cell, which method comprises: (i) contacting a T cell, which has been stimulated via its T cell receptor, with a candidate substance under conditions that would permit a sustained rise in intracellular Ca 2+ levels in the absence of the substance; and (ii) determining whether the substance inhibits a sustained rise in intracellular Ca 2+ levels.", "In one embodiment, the substance inhibits NAADP+ synthesis, for example reduces or abolishes NAADP+ synthesis.", "In another embodiment, the substance modulates, for example inhibits, binding of endogenous NAADP+ to its receptor binding site.", "The present invention further provides a method for identifying a substance capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca 2+ signalling, which method comprises: (i) contacting a T cell with a candidate substance; (ii) stimulating the T cells via TCR/CD3; and (ii) determining whether the substance inhibits TCR/CD3-associated Ca 2+ signalling.", "In one embodiment, the substance causes the intracellular concentration of NAADP+ (or a bioisostere thereof) to rise to an inactivating concentration.", "The present invention further provides a method for identifying a substance capable of agonising the NAADP+-mediated rise in intracellular Ca 2+ levels in a T cell, which method comprises: (i) contacting a T cell with a candidate substance; and (ii) determining whether the substance elicits or enhances a rise in intracellular NAADP+ and/or Ca 2+ levels.", "In one embodiment, the substance induces or enhances NAADP+ synthesis.", "In another embodiment, the substance modulates, for example enhances, binding of endogenous NAADP+ to its receptor binding site.", "A compound identified by the methods of the invention may be used in modulating the immune response of a mammal.", "Thus, for example, in another aspect of the present invention, a compound identified by a method of the invention is provided for use in treating (i) an autoimmune disease, such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rhematoid arthritis and lupus erythematosus or (ii) allograft rejection.", "The present invention also provides a pharmaceutical composition (which term also includes a veterinary formulation) comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent, excipient or carrier.", "The invention further provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a human or animal medicament." ], [ "FIELD OF THE INVENTION The present invention relates to therapeutics.", "In particular, the present invention relates to the modulation of T cell activity via a nicotinic acid adenine dinucleotide phosphate (NAADP+) mediated pathway.", "The invention also relates to compounds capable of modulating the activity of T cells via such a pathway.", "The invention also relates to treating diseases using such compounds and methods for identifying such compounds.", "BACKGROUND TO THE INVENTION Adaptive or specific immune responses are normally stimulated when an individual is exposed to a foreign antigen.", "Specific immunity is mediated by lymphocytes, e.g.", "B and T lymphocytes.", "During an immune response, recognition of an antigen leads to activation of lymphocytes that specifically recognise that particular antigen.", "The lymphocytes proliferate and differentiate into specialised effector cells.", "The immune response culminates in the development of mechanisms that ultimately eliminate the antigen.", "Adaptive immune responses are critical components of host defence during protection against foreign antigens, such as infectious organisms or toxins.", "However, specific immune responses are also sometimes elicited by antigens not associated with infectious agents, and this may cause serious disease.", "For example, one of the most remarkable properties of specific immunity is the ability to distinguish between self antigens and foreign antigens.", "Thus, the lymphocytes in each individual are able to recognise and respond to numerous foreign antigens but are normally unresponsive to potentially antigenic substances present in the individual itself Unresponsiveness to self antigens is an acquired process that has to be learned by the individual's lymphocytes and has to be maintained throughout life.", "Abnormalities in the induction or maintenance of self-tolerance lead to immune responses against self antigens, and debilitating diseases that are commonly called autoimmune diseases.", "The spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus.", "Another example in which specific immunity against antigens that are not associated with infections causes severe medical problems are rejections of transplanted allografts.", "In fact, adaptive immune responses to grafted tissues are the major impediment to successful transplantation in most cases.", "It is not known what causes the breakdown of tolerance and the initiation of an autoimmune response.", "However, the mechanisms of tissue destruction in autoimmune diseases and in allograft rejection are essentially the same as those operating in protective immunity.", "It is generally believed that both autoimmune reactions and allograft rejections are initiated and perpetuated by a response involving T cells.", "Thus, in the absence of a specific therapy for any of the autoimmune diseases or for allograft rejection, many therapeutic strategies currently employed aim at down modulating the activity of the immune system, in particular by reducing or preventing the activation of T cells.", "Recently, monoclonal antibodies to T cell surface antigens, that inhibit T cell activation, or substances that interfere with intracellular T cell activation pathways, such as Cyclosporin A or FK506, have been introduced for the treatment of both allograft rejection and several autoimmune diseases.", "However, current approaches for the treatment of undesirable T cell activation have been associated with a number of side effects related to general immunosuppression and therefore cannot be considered to be optimal therapy.", "Stimulation of T-lymphocytes via the T cell receptor/CD3 complex (TCR/CD3) is a critical step in T cell activation and subsequent clonal expansion.", "Previous studies have shown that activation of the TCR/CD3-complex involves the elevation of the free cytosolic Ca2+ concentration ([Ca2+]i) by at least two mechanisms, a rapid elevation caused by Ca2+ release from intracellular stores mediated by inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), and a prolonged elevation that is completely dependent on the influx of extracellular calcium (reviewed in Guse, 1998).", "Ca2+-release is activated by the calcium mobilizing second messengers Ins(1,4,5)P3 (Jayaraman et al, 1995) and cADPR (Guse et al., 1999).", "Recent work indicates that Ins(1,4,5)P3 primarily acts during the initial phase of Ca2+-signaling in T cells, whereas cADPR is essentially involved in the sustained phase of Ca2+-signaling.", "The exact mechanism of Ca2+ signalling in T cells is still unclear, but it is of fundamental importance for proliferation and clonal expansion, and thus for a functional immune response.", "An improved understanding of the signalling pathways involved in T cell activation may be of assistance in developing strategies to stimulate a desirable adaptive immune response or to suppress inappropriate T cell activity.", "SUMMARY OF THE INVENTION The present inventors have shown that NAADP+ specifically and dose-dependently stimulates Ca2+ signalling in human T cells.", "At an activating concentration, NAADP+ either evokes repetitive and longlasting Ca2+ oscillations or a single Ca2+-spike of high amplitude.", "The present inventors have also shown that NAADP+ can be self-inactivating.", "An inactivating concentration of NAADP+ inhibits subsequent stimulation of Ca2+ signaling via the T cell receptor/CD3.For example, inactivation of the NAADP+/Ca2+-release system almost completely abolishes subsequent Ins(1,4,5)P3- or cADPR-mediated Ca2+-signaling.", "This shows that a functional NAADP+/Ca2+ release system is essential for T-lymphocyte Ca2+ signaling.", "These findings have important implications for the design of compounds capable of modulating T cell activity, since regulation of this NAADP+/Ca2+ signalling pathway may provide an important means of stimulating T cells (and adaptive immune responses) and controlling T cell responses in a variety of T cell mediated immune disorders.", "Accordingly the present invention provides a method for modulating T cell activity, which comprises the step of modulating the intracellular concentration of NAADP+ or a bioisostere thereof.", "In one embodiment, the method involves stimulating a rise in intracellular Ca2+ levels by raising the intracellular concentration of NAADP+ to an activating concentration.", "The present inventors have found that an intracellular concentration of 10 nM NAADP+ evokes repetitive and longlasting Ca2+ oscillations of low amplitude, while 50 and 100 nM produces a rapid and high initial Ca2+ peak followed by trains of smaller Ca2+ oscillations.", "Higher concentrations of NAADP+(1 and 10 μM) gradually reduce the initial Ca2+ peak.", "Thus an “activating concentration” of NAADP+ may be between 5 nM and 1 μM, preferably between 5 and 100 nM.", "In another embodiment, the method involves inhibiting TCR/CD3-associated Ca2+ signaling by raising the intracellular concentration of NAADP+ to an inactivating concentration.", "The present inventors have shown that an intracellular concentration of 100 μM NAADP+ causes complete self-inactivation of Ca2+-signals.", "Thus an “inactivating concentration” of NAADP+ may be greater than 1 μM, preferably greater than 10 M, most preferably 100 μM or greater.", "The elucidation of a novel NAADP+-mediated T cell activation pathway also enables the identification of substances that modulate T cell activation via this pathway.", "The present invention thus also provides a compound capable of (a) antagonising the NAADP+-mediated rise in intracellular Ca2+ levels caused by TCR/CD3 stimulation; (b) agonising the NAADP+-mediated rise in intracellular Ca2+ levels caused by TCR/CD3 stimulation; (c) inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signaling; or (d) preventing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signaling Compounds of the invention which inhibit T cell proliferation and/or differentiation, or induce T cell anergy may be used in treating diseases characterised by an excessive or inappropriate T cell response, such as autoimmune diseases, allergies and allograft rejection.", "Candidate autoimmune diseases include thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rheumatoid arthritis and lupus erythematosus.", "Compounds of the invention which induce or enhance T cell proliferation and/or differentiation or prevent the induction of T cell anergy may be used generally to boost or induce T cell immune responses.", "Virtually all adaptive immune responses require the activation of T cells and their differentiation into cytokine-producing cells.", "Thus these compounds may be used generally to prevent and treat conditions such as infectious diseases (such as viral or bacterial infections), cancers and, in particular, immunodeficiencies characterised by impaired T cell function (such as AIDS).", "The present invention further provides a method for identifying a substance capable of antagonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell, which has been stimulated via its T cell receptor, with a candidate substance under conditions that would permit a sustained rise in intracellular Ca2+ levels in the absence of the substance; and (ii) determining whether the substance inhibits a sustained rise in intracellular Ca2+ levels.", "In one embodiment, the substance inhibits NAADP+ synthesis, for example reduces or abolishes NAADP+ synthesis.", "In another embodiment, the substance modulates, for example inhibits, binding of endogenous NAADP+ to its receptor binding site.", "The present invention further provides a method for identifying a substance capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, which method comprises: (i) contacting a T cell with a candidate substance; (ii) stimulating the T cells via TCR/CD3; and (ii) determining whether the substance inhibits TCR/CD3-associated Ca2+ signalling.", "In one embodiment, the substance causes the intracellular concentration of NAADP+ (or a bioisostere thereof) to rise to an inactivating concentration.", "The present invention further provides a method for identifying a substance capable of agonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell with a candidate substance; and (ii) determining whether the substance elicits or enhances a rise in intracellular NAADP+ and/or Ca2+ levels.", "In one embodiment, the substance induces or enhances NAADP+ synthesis.", "In another embodiment, the substance modulates, for example enhances, binding of endogenous NAADP+ to its receptor binding site.", "A compound identified by the methods of the invention may be used in modulating the immune response of a mammal.", "Thus, for example, in another aspect of the present invention, a compound identified by a method of the invention is provided for use in treating (i) an autoimmune disease, such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rhematoid arthritis and lupus erythematosus or (ii) allograft rejection.", "The present invention also provides a pharmaceutical composition (which term also includes a veterinary formulation) comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, together with a pharmaceutically acceptable diluent, excipient or carrier.", "The invention further provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of either entity, or a pharmaceutical composition containing any of the foregoing, for use as a human or animal medicament.", "DETAILED DESCRIPTION OF THE INVENTION A. Modulation of the Intracellular Concentration of NAADP+ or a Bioisostere Thereof NAADP+ was originally discovered as a contaminant of commercial NADP+ preparations; such preparations could also be enriched in NAADP+ content by alkaline treatment (Clapper et al., 1987).", "NAADP+ has the following formula: NAADP+ has been shown to be involved in Ca2+-mobilisation in some invertebrate cell types, such as sea urchin eggs (Lee & Aarhus 1995), ascidian oocytes (Albrieux et al., 1998), and more recently in mouse pancreatic acinar cells (Cancela et al., 1999).", "To date, NAADP+ has not been shown to have an effect in a human cell system.", "The actual mechanism of formation of NAADP+ in vivo is unknown, but at least three enzyme activities have been implicated: firstly, a deamidase enzyme which cleaves the amide group of (nicotinamide-adenine dinucleotide phosphate) NADP; and secondly a kinase enzyme that phosphorylates the 2′-position of nicotinic acid-adenine dinucleotide (NAAD).", "It is also thought that ADP-ribosyl cyclase may play a role, since it is known to be able to make NAADP+ in vitro from NADP+ by base exchange.", "The pathway by which NAADP+ may be eliminated from the cell is unclear.", "It may involve 2′-dephosphorylation, cleavage at the pyrophosphate group and/or loss of the nicotinic acid group to give ADPRP.", "The present inventors have shown that NAADP+ is capable of (a) stimulating Ca2+ signalling in human T cells, when present at an activating concentration; and (b) self-inactivating the NAADP+/Ca2+-release system when present at an inactivating concentration.", "As used herein, the term “bioisostere” is used to indicate a compound which is structurally distinct from NAADP+, but which shares some functional similarity with NAADP+.", "The bioisostere may be capable of performing one or more of the biological functions of NAADP+.", "For example, the bioisostere may be capable of stimulating Ca2+ signalling in human T cells via the NAADP+/Ca2+-release system.", "Alternatively, or in addition, the bioisostere may be capable of inhibiting the NAADP+/Ca2+-release system, for example, by mimicking the effect of NAADP+ when present at an inactivating concentration.", "A bioisostere may be endogenous to the T cell, or may be artificially introduced.", "A bioisostere may be any compound capable of exhibiting the required functional characteristics.", "For example, the bioisostere may be a small organic molecule, possibly sharing some structural features with NAADP+.", "Preferable the bioisostere shows significant structural similarity to NAADP+.", "There are a number of methods by which the intracellular concentration of NAADP+ (or a bioisostere thereof) may be modulated.", "The concentration may be increased directly by administering NAADP+ or a bioisostere thereof or a precursor thereof to the cell.", "For example, the compound may be administered by any of the known delivery routes, such as microinjection (as described in the Examples), delivery via a carrier protein, or liposome-mediated delivery.", "Alternatively, the intracellular concentration may be increased by activating one or more steps in the intracellular NAADP+ production pathway, or by inhibiting NAADP+ breakdown and/or escape from the cell.", "For example, NAADP+ levels may be raised by activating one or more of the enzymes involved in NAADP+ synthesis (such as the above-mentioned deamidase enzyme, kinase enzyme and/or ADP-ribosyl cyclase).", "Conversely, the concentration of NAADP+ may be decreased by blocking one or more steps in the intracellular NAADP+ production pathway, or by activating NAADP+ breakdown and/or escape from the cell.", "For example, NAADP+ production may be blocked by inhibiting one or more of the enzymes involved in NAADP+ synthesis (such as the deamidase enzyme, kinase enzyme and/or ADP-ribosyl cyclase).", "Also, the effective concentration of NAADP+ may be decreased by blocking the interaction between NAADP+ and its receptor.", "For example, the presence of a substance which binds to NAADP+, inhibiting or preventing its interaction with its receptor will decrease the effective concentration of NAADP+ in a cell.", "In one embodiment, the method of the present invention involves stimulating a rise in intracellular Ca2+ levels by raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an activating concentration.", "An “activating concentration” of NAADP+ is an intracellular concentration of between 5 nM and 1 μM, preferably between 5 and 100 nM, more preferably about 10 nM.", "An “activating concentration” of a bioisostere is that concentration which causes a comparable rise in intracellular Ca2+ levels to that caused by an activating concentration of NAADP+.", "The actual concentration will depend on the chemical nature of the bioisostere.", "It may be comparable to the “activating concentration” of NAADP+, especially if the bioisostere is of a similar chemical structure.", "In another embodiment, the method involves inhibiting TCR/CD3-associated Ca2+ signaling by raising the intracellular concentration of NAADP+ to an inactivating concentration.", "An “inactivating concentration” of NAADP+ is an intracellular concentration of greater than 1 μM, preferably greater than 10 μM, most preferably 100 μM or greater.", "An “inactivating concentration” of a bioisostere is that concentration which inhibits TCR/CD3-associated Ca2+ signaling by blocking the NAADP+/Ca2+ release system to a degree which is comparable to that caused by an inactivating concentration of NAADP+.", "The actual concentration will depend on the chemical nature of the bioisostere.", "It may be comparable to the “inactivating concentration” of NAADP+, especially if the bioisostere is of a similar chemical structure.", "B.", "Compounds Capable of Modulating NAADP+ Mediated Ca2+ Signalling in T Cells.", "Compounds suitable for use in the present invention are capable of modulating NAADP+-mediated Ca2+ signalling in T cells.", "They may act as antagonists, by inhibiting or blocking the NAADP+-mediated rise in intracellular Ca2+ levels which occurs in response to TCR/CD3 stimulation on the T cell surface.", "An antagonist may cause the intracellular concentration of NAADP+ to fall, or prevent the intracellular concentration to rise to an activating concentration.", "Alternatively, the antagonist may act on other components of the NAADP+ mediated Ca2+ release pathway.", "Preferably, an antagonist for use in the present invention should not substantially inhibit other pathways involved in the release of Ca2+ from intracellular stores (for example Ins(1,4,5)P3 mediated release) or involved in the influx of Ca2+ (for example cADPR-mediated influx).", "For example, a preferred antagonist will inhibit the NAADP+ pathway at least two-fold, preferably at least 5 or 10-fold more than other pathways involved in Ca2+ signalling resulting from TCR/CD3 stimulation.", "A compound for use in the present invention may act at a number of places in the NAADP+ Ca2+ signalling pathway.", "It may affect signalling between the activated TCR/CD3 complex and an NAADP+-producing enzyme (for example the above-mentioned deamidase enzyme, kinase enzyme or ADP-ribosyl cyclase).", "It may affect activation of the NAADP+-producing enzyme (such as covalent modification by an upstream effector protein, for example a kinase), or enzymatic activity of the NAADP+-metabolising enzyme (such as a competitive inhibitor having a high binding affinity for the active site of the enzyme or a non-competitive inhibitor which binds a distal site resulting in a conformational change).", "Alternative, it may affect downstream effects of NAADP+.", "In particular preferred compounds such as NAADP+ analogues may act to inhibit binding of NAADP+ to its binding site on its receptor.", "Suitable assays for identifying compounds for use in the present invention are described below in section D. One particularly preferred class of compounds for use in the present invention are NAADP+ analogues, which may bind to the receptor and compete with NAADP+.", "Compounds suitable for use in the present invention may alternatively act as agonists, by stimulating or enhancing the NAADP+-mediated rise in intracellular Ca2+ levels which occurs in response to TCR/CD3 stimulation on the T cell surface.", "An agonist may act by raising intracellular concentrations of NAADP+ to an activating concentration or by affecting other components in the NAADP+ Ca2+ signaling pathway (as explained above with regard to antagonists).", "A compound may also be capable of inducing or blocking the NAADP+-mediated inhibition of TCR/CD3-associated signalling.", "For example, the compound may be capable of raising, or blocking the elevation of, intracellular NAADP+ concentrations to an inactivating concentration.", "Alternatively, such a compound may act at another step in the auto-inactivation pathway.", "Compounds suitable for use in the present invention include NAADP analogues having the following formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "In a preferred aspect of the present invention the compound may 8-bromo-nicotinic acid adenine dinucleotide phosphate (8-Br-NAADP).", "8-Br-NAADP modulates the intracellular concentration and/or the binding affinity of NAADP+.", "The term “compound” is intended to encompass isomeric forms (such as stereoisomers and/or geometric and/or optical isomers, and mixtures thereof), chemical derivatives, mimetics, solvates and salts of the compounds.", "As used herein, the term “hydrocarbyl” refers to a group comprising at least C and H that may optionally comprise one or more other suitable substituents.", "Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, or a cyclic group.", "In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group.", "If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other.", "For example, at least two of the carbons may be linked via a suitable element or group.", "Thus, the hydrocarbyl group may contain heteroatoms.", "Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, silicon and phosphorus.", "For some embodiments, preferably the hydrocarbyl group is alkyl, alkoxy, alkenyl, alkylene, acyl and alkenylene groups—which may be unbranched- or branched-chain.", "For some embodiments, preferably the hydrocarbyl group is C1-12 alkyl, C1-12 alkoxy, C1-12 alkenyl, C1-12 alkylene, C1-12 acyl, and C1-12 alkenylene groups—which may be unbranched- or branched-chain.", "For some embodiments, preferably the hydrocarbyl group is C1-6 alkyl, C1-6 alkoxy, C1-6 alkenyl, C1-6 alkylene, C1-6 acyl, and C1-6 alkenylene groups—which may be unbranched- or branched-chain.", "It is to be appreciated that all references herein to treatment include one or more of curative, palliative and prophylactic treatment.", "Preferably, the term treatment includes at least curative treatment and/or palliative treatment.", "C. Chemical Synthesis Methods.", "The compounds of the present invention may be available commercially.", "Alternatively, the compound of the invention may be prepared by chemical synthesis techniques.", "It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention.", "This may be achieved by conventional techniques, for example as described in “Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P. J. Kocienski, in “Protecting Groups”, Georg Thieme Verlag (1994).", "It is possible during some of the reactions that any stereocentres present could, under certain conditions, be epimerised, for example if a base is used in a reaction with a substrate having an having an optical centre comprising a base-sensitive group.", "It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc.", "as is well-known in the art.", "The compounds and salts of the invention may be separated and purified by conventional methods.", "D. Synthesis of 8-bromo-nicotinic Acid Adenine Dinucleotide Phosphate (8-Br-NAADP).", "Synthesis of 8-Br-NADP To a solution of nicotinamide adenine dinucleotide phosphate sodium salt 1 (50 mg, 0.065 mmol) in 1M aqueous sodium acetate pH 4 buffer (2 ml) bromine (50 μl, 0.975 mmol) was added via syringe (Holmes et al.", "J.", "Am.", "Chem.", "Soc., 86, 1242, (1964)).", "The reaction mixture was stirred in the dark at ambient temperature for two hours, after which time high performance liquid chromatography (HPLC) analysis (Rainin Dynamix SD-200 HPLC system using a Supelcosil™ Saxi ion exchange column (25 cm×4.6 mm, 5 μm), and eluting with 50 mM aqueous potassium hydrogen phosphate pH 3 buffer with 5% methanol) indicated that all the nicotinamide adenine dinucleotide phosphate sodium salt 1 (RT=11 mins) had been consumed.", "The excess bromine was extracted into chloroform (3×5 ml), and the aqueous phase frozen and lyophilised to produce the crude 8-Br-nicotinamide adenine dinucleotide phosphate sodium salt 2 (RT=12 mins) as a pale yellow powder in 98% yield by HPLC analysis (see above).", "The crude product was purified by semi-preparative HPLC (Rainin Dynamix SD-200 HPLC system using a Supelcosil™ LC-Saxi ion exchange column (25 cm×10 mm, 5 μm), and eluting with 50 mM aqueous potassium hydrogen phosphate pH 3 buffer with 5% methanol).", "Inorganic phosphate was removed from the product by LiChroprep® reverse phase (25-40 μm) column chromatography (25 mM aqueous triethylammonium formate solution), and monitored by the use of an Aquamerck® (14661) blue colour test (Hoffmann et al.", "Bioorg.", "& Med.", "Chem.", "Lett., 6, 2571, (1996)).", "The desired product (namely 8-Br-NADP 2) eluted with Milli-Q water and was isolated as its triethylammonium salt; δH 400 MHz (D2O) 4.05 (2H, m, 2HA5′), 4.14 (3H, m, 2HN5′, HA4′), 4.26 (4H, m, HN4′, HA3′, HN3′, HN2′), 5.25 (1H, d, J 5.0, HA2′), 5.74 (1H, d, J 5.0, HA1′), 5.89 (1H, d, J 5.0, HN1′), 7.94 (1H, s, HA2), 7.99 (1H, m, HN5), 8.60 (1H, d, J 8.0, HN4), 8.88 (1H, d, J6.0, HN6), 9.12 (1H, s, HN2).", "Synthesis of 8-Br-NAADP 3 (Route 1) The title compound 3 was synthesised using a modification of the Bernoksky procedure for nicotinic acid dinucleotide phosphate synthesis (Bemofsky et al.", "Analytical Biochemistry, 67, 611, (1975) and Bemofsky Method.", "Enzymol., 66, 105, (1980)).", "NAD-ase (150 mg) was suspended in 50 mM aqueous triethanolamine acetate pH 7.6 buffer (2 ml) and sonicated for 10 mins to induce homogenisation.", "This solution was then added to 8-bromo-nicotinamide adenine dinucleotide phosphate triethylammonium salt 2 (50 mg, 0.059 mmol) and nicotinic acid (250 mg, 2.03 mmol).", "The reaction was stirred at 37° C. for 14 hrs.", "HPLC analysis (see above) showed consumption of 8-Br-nicotinamide adenine dinucleotide phosphate triethylammonium salt 2 (RT=12 mins) and formation of 8-bromo-nicotinic acid adenine dinucleotide phosphate 3 (RT=25 mins).", "The crude reaction mixture was filtered through celite and purified by semi-preparative HPLC (see above).", "Inorganic phosphate was removed from the product by LiChroprep® reverse phase (25-40 μm) column chromatography (25 mM aqueous triethylammonium formate solution), and monitored by the use of an Aquamerck® (14661) blue colour test (see above).", "The desired product (namely 8-Br-NAADP 3) eluted with Milli-Q water and was isolated as its triethylammonium salt; δH 400 MHz (D2O) 4.10 (3H, m, 2HN5′, H5A′), 4.25 (1H, m, H5A′), 4.32 (1H, m, HA4′), 4.39 (1H, m, HN4′), 4.45 (1H, m, HN3′), 4.51 (1H, m, HN2′), 4.60 (1H, m, HA3′), 5.01 (1H, m, HA2′), 5.97 (1H, br s, HN′), 6.06 (1H, d, J6.0, HA1′), 8.12 (1H, m, HN5), 8.26 (1H, s, HA2), 8.85 (1H, d, J7.5, HN4), 9.09 (1H, d, J5.0, HN6), 9.28 (1H, s, HN2).", "Synthesis of 8-Br-NAADP 3 via Direct Bromination (Route 2) i.", "Preparation of NAADP 4 Nicotinic acid adenine dinucleotide phosphate 4 was synthesised using a modification of the Bernoksky procedure (see above).", "NAD-ase (90 mg) was suspended in 50 mM aqueous triethanolamine acetate pH 7.6 buffer (2 ml) and sonicated for 10 mins to induce homogenisation.", "This solution was then added to nicotinamide adenine dinucleotide phosphate sodium salt 1 (30 mg, 0.039 mmol) and nicotinic acid (240 mg, 1.96 mmol).", "The reaction was stirred at 37° C. for 14 hrs.", "HPLC analysis (see above) showed consumption of nicotinamide adenine dinucleotide phosphate sodium salt 1 (RT=12 mins) and formation of nicotinic acid adenine dinucleotide phosphate 4 (RT=17 mins).", "The crude reaction mixture was filtered through celite and purified by AG-MP1 ion-exchange resin (150 mM aqueous TFA solution gradient).", "The desired product (namely NAADP 4) eluted with 100% 150 mM TFA solution; δH 400 MHz (D2O) 4.13 (3H, m, 2HN5′, H5A′), 4.26 (1H, m, H5A′), 4.30 (1H, m, HA4′), 4.34 (1H, m, HN4′), 4.44 (1H, m, HN3′), 4.47 (1H, m, HN2′), 4.54 (1H, m, HA3′), 5.01 (1H, m, HA2′), 6.06 (1H, d, J 5.5, HN1′), 6.17 (1H, d, J5.5, HA1′), 8.18 (1H, m, HN5), 8.31 (1H, s, HA2), 8.48 (1H, s, HA8), 8.91 (1H, d, J 8.0, HN4), 9.21 (1H, d, J6.0, HN6), 9.35 (1H, s, HN2).", "ii.", "Bromination of NAADP 4 To a solution of nicotinic acid adenine dinucleotide phosphate sodium salt 4 (20 mg, 0.026 mmol) in 1M aqueous sodium acetate pH 4 buffer (2 ml) was added bromine (20 μl, 0.388 mmol) via syringe (see Holmes detailed above).", "The reaction mixture was stirred in the dark at ambient temperature for two hours, after which time HPLC analysis (see above) indicated that all the nicotinic acid adenine dinucleotide phosphate sodium salt 4 (RT=17 mins) had been consumed.", "The excess bromine was extracted into chloroform (3×5 ml), and the aqueous phase frozen and lyophilised to produce the crude 8-Br-nicotinic acid adenine dinucleotide phosphate sodium salt 3 RT=25 mins) as a pale yellow powder.", "The crude product was purified by semi-preparative HPLC (see above).", "Inorganic phosphate was removed from the product by LiChroprep® reverse phase (25-40 μm) column chromatography (25 mM aqueous triethylammonium formate solution), and monitored by the use of an Aquamerck® (14661) blue colour test (see above).", "The desired product (namely 8-Br-NAADP 3) eluted with Milli-Q water and was isolated as its triethylammonium salt; δH 400 MHz (D2O) 4.10 (3H, m, 2HN5′, H5A′), 4.25 (1H, m, H5A′), 4.32 (1H, m, HA4′), 4.39 (1H, m, HN4′), 4.45 (1H, m, HN3′), 4.51 (1H, m, HN2′), 4.60 (1H, m, HA3′), 5.01 (1H, m, HA2′), 5.97 (1H, br s, HN1′), 6.06 (1H, d, J 6.0, HA1′), 8.12 (1H, m, HN5), 8.26 (1H, s, HA2), 8.85 (1H, d, J 7.5, HN4), 9.09 (1H, d, J 5.0, HN6), 9.28 (1H, s, HN2).", "E. Stereo and Geometric Isomers.", "Some of the compounds of the present invention may exist as stereoisomers and/or geometric isomers—e.g.", "they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.", "The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof.", "The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).", "F. Assays for Identifying Compounds Capable of Modulating NAADP+-Mediated Ca2+ Signalling in T Cells.", "A substance which affects the NAADP+ pathway may do so in several ways as discussed above.", "An antagonist may disrupt an interaction between two components of the pathway, such as disrupting the binding of NAADP+ to its binding site on the receptor.", "It may directly disrupt the binding of the two components by, for example, binding to one component and masking or altering the site of interaction with the other component.", "Candidate substances of this type may conveniently be screened by in vitro binding assays.", "Examples of candidate, substances include non-functional homologues of either of the two components as well as antibodies which recognize either of the two components.", "An agonist may enhance the interaction between two components of the pathway.", "For example, the agonist may bind to one or other component, inducing a conformational change which enhances the interaction between the two components.", "A substance which can bind directly to either of the two components may also inhibit or enhance an interaction between the two components by altering their subcellular localization thus preventing or enabling the two components from coming into contact within the cell.", "This can be tested in vivo using, for example the in vivo assays described below.", "The term ‘in vivo’ is intended to encompass experiments with cells in culture as well as experiments with intact multicellular organisms.", "Alternatively, instead of preventing or enhancing the association of the components directly, the substance may suppress or enhance the biologically available amount of one or both of the components.", "This may be by inhibiting or enhancing expression of the component, for example at the level of transcription, transcript stability, translation or post-translational stability.", "An example of such a substance would be antisense RNA which suppresses the amount of mRNA translated into protein of an enzyme involved in NAADP+ synthesis or of the NAADP+ receptor.", "Suitable candidate substances also include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for either of the two components.", "Furthermore, combinatorial libraries, peptide and peptide mimetics, defined chemical entities, oligonucleotides, and natural product libraries may be screened for activity as inhibitors or enhancers of an interaction between the two components.", "Other candidate substances include analogues of substrates or products of the NAADP+ pathway, such as NAADP+ or NAAD+ analogues.", "The candidate substances may be used in an initial screen in batches of, for example 10 substances per reaction, and the substances of those batches which show inhibition tested individually.", "Candidate substances which show activity in in vitro screens such as those described below may then be tested in in vivo systems.", "In vitro assays typically test for substances capable of affecting the interaction between particular components of the signalling pathway (see above) or the activity of a particular component, such as an enzyme.", "For example, one assay may involve testing candidate substances for the ability to inhibit synthesis of NAADP+.", "This may be performed by (i) contacting an NAADP+ producing-enzyme with a candidate substance under conditions that would allow the synthesis of NAADP+ in the absence of the candidate substance and (ii) determining if the candidate substance inhibits NAADP+ synthesis.", "Other in vitro assays may include assays for identifying substances capable of disrupting NAADP+ binding to its receptor.", "For example, membrane preparations may be obtained from T cells by methods known in the art and the binding of NAADP+ to the membrane preparation assessed in the absence or presence of a candidate substance.", "In vitro assays will generally be used as a preliminary step prior to in vivo testing since it is only in the context of an intact TCR/CD3 Ca2+ signalling pathway that the modulatory effects of a candidate substance may be completely assessed.", "In vivo assays will typically use either T cell lines, preferably human T cell lines, or T cells obtained from animal tissues, especially human tissues.", "Generally, T cells will be stimulated via the TCR/CD3 complex using standard methods such as receptor cross-linking using antibodies that recognise the receptor complex.", "For example, the TCR/CD3 complex may be stimulated by the anti-CD3 monoclonal antibody OKT3, as described in Example 4.The TCR/CD3 complex may also be stimulated using superantigens or antigen presenting cells, such as dendritic cells.", "A suitable assay method comprises stimulating a T cell via its TCR/CD3 receptor in the presence or absence of a candidate substance.", "The Ca2+ response is measured (for example using ratiometric Ca2+ imaging as described in the Examples) and compared.", "The intracellular concentration of NAADP+ may be measured by labeling of NAADP+ by a fluorescent dye (pre- or post-column derivatization) combined with a very sensitive fluorescence detector, e.g.", "laser-induced fluorescence detection (Rahavendran & Karnes, 1993).", "Alternatively, a competitive protein binding assay based on a high affinity binding protein for NAADP+ may be used.", "T cells may be contacted with a candidate substance before, concomitant with, or after stimulation of the TCR/CD3 complex.", "The concentration of candidate substance administered to the cell will vary but is typically from 0.1 to 100 μM, more preferably from 1 to 100 μM or 10 to 100 μM.", "G. Assays for Testing the Physiological Affects of Substances Capable of Modulating NAADP+/Ca2+-Release System.", "Since the intention of identifying substances that affect the NAADP+ pathway is to use them to modulate T cell activity, further assays may comprise administering a substance capable of modulating the activity of the pathway to a T cell, or a candidate substance, and determining the effect on the cell.", "For example, compounds for use in the present invention may be capable of inhibiting or enhancing the NAADP+-mediated rise in Ca2+ levels following stimulation of the cell via the TCR/CD3 complex.", "Such an affect may include inhibition or stimulation of cell proliferation in response to, for example, stimulation by an antigen presenting cell such as a dendritic cell.", "Thus one suitable assay comprises incubating a T cell with an antigen presenting cell in the presence or absence of a candidate substance and determining whether T cell proliferation is reduced or enhanced in the presence of the substance compared in the absence of the substance.", "Another suitable assay involves activating a T cell with a mitogen in the presence and absence of a candidate substance and determining whether T cell proliferation is reduced or enhanced in the presence of the substance compared in the absence of the substance.", "Examples of suitable mitogens include monoclonal antibodies to CD3 or the TCR, phorbol 12-myrstate 13-acetate, ionomycin, concanavalin A, phytohemagglutinin, superantigens and antibodies to CD2, CD3 or the T cell receptor.", "T cell activation/proliferation may be measured using a variety of techniques, for example by measuring cell number, [3H]thymidine incorporation levels of secreted cytokines such as IL-2 in the culture medium or by flow cytometric analysis of T cell surface markers indicative for activation (such as CD69, CD30, CD25 and HLA-DR).", "A preferred antagonist is capable of reducing T cell proliferation by at least 50%, more preferably at least 60, 70, 80 or 90% (for example with respect to numbers of cells expressing a cell surface marker, cytokine levels in the medium and/or numbers of cells present).", "Compounds for use in the present invention may be capable of inducing or preventing the NAADP+-inhibition of TCR/CD3 associated signalling.", "Such an effect may lead to the induction or prevention of anergy in the T cell.", "Determination of whether a T cell is in the anergic state can be performed using methods known in the art.", "For example, the T cell can be subsequently challenged with an antigen-presenting cell, and T cell activation/proliferation measured by the methods given above.", "H. Therapeutic Uses The compounds of the present invention may be used in therapy.", "In particular such compounds may be used to modulate T cell responses in vivo.", "Alternatively, T cells may be removed from a patient, treated (either with a compound of the present invention or with NAADP+ or a bioisostere thereof, or a precursor thereof, directly) and then returned to the patient (ex vivo therapy).", "Compounds capable of agonising a NAADP+-mediated rise in Ca2+ levels, or preventing NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling in T cells may be used in methods where stimulation of T cell responses, proliferation and/or differentiation is required.", "The first group of compounds may be used against any disorder which is susceptible to prevention or treatment by the induction of an adaptive immune response.", "In particular, these compounds may be used to treat immunodeficiency disorders mechanistically related to a defect in T cell activation.", "Compounds capable of antagonising a NAADP+-mediated rise in Ca2+ levels, or inducing NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling in T cells may be used to treat or prevent conditions associated with an inappropriate T cell response, for example in treating autoimmune diseases, graft rejection or allergies.", "Examples of disorders that may be treated by the second group of compounds include a group commonly called autoimmune diseases.", "The spectrum of autoimmune disorders ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic illnesses such as rheumatoid arthritis or lupus erythematosus.", "Other disorders include immune hyperreactivity, such as allergic reactions.", "In more detail: Organ-specific autoimmune diseases include multiple sclerosis, insulin dependent diabetes mellitus, several forms of anemia (aplastic, hemolytic), autoimmune hepatitis, thyroiditis, insitis, iridocyclitis, skleritis, uveitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel diseases (Crohn's disease, ulcerative colitis).", "Systemic autoimmune diseases include: rheumatoid arthritis, juvenile arthritis, scleroderma and systemic sclerosis, sjogren's syndrom, undifferentiated connective tissue syndrom, antiphospholipid syndrom, different forms of vasculitis (polyarteritis nodosa, allergic granulomatosis and angiitis, Wegner's granulomatosis, Kawasaki disease, hypersensitivity vasculitis, Henoch-Schoenlein purpura, Behoet's Syndrome, Takayasu arteritis, Giant cell arteritis, Thrombangiitis obliterans), lupus erythematosus, polymyalgia rheumatica, essentiell (mixed) cryoglobulinemia, Psoriasis vulgaris and psoriatic arthritis, diffus fasciitis with or without eosinophilia, polymyositis and other idiopathic inflammatory myopathies, relapsing panniculitis, relapsing polychondritis, lymphomatoid granulomatosis, erythema nodosum, ankylosing spondylitis, Reiter's syndrom, different forms of inflammatory dermatitis.", "A more extensive list of disorders is given in WO-A-98/09985.For ease of reference, part of that list is now provided: unwanted immune reactions and inflammation including arthritis, including rheumatoid arhritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g.", "retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g.", "following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery or organ, inflammatory and/or immune complications and side effects of gene therapy, e.g.", "due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g.", "leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.", "I.", "Administration Compounds capable of affecting a NAADP+-mediated rise in Ca2+ levels in T cells for use in immunotherapy are typically formulated for administration to patients with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition.", "The formulation will depend upon the nature of the compound identified and the route of administration but typically they can be formulated for topical, parenteral, intramuscular, intravenous, intra-peritoneal, intranasal inhalation, lung inhalation, intradermal or intra-articular administration The compound may be used in an injectable form.", "It may therefore be mixed with any vehicle which is pharmaceutically acceptable for an injectable formulation, preferably for a direct injection at the site to be treated, although it may be administered systemically.", "The pharmaceutically acceptable carrier or diluent may be, for example, sterile isotonic saline solutions, or other isotonic solutions such as phosphate-buffered saline.", "The compounds of the present invention may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).", "It is also preferred to formulate the compound in an orally active form.", "In general, a therapeutically effective daily oral or intravenous dose of the compounds of the invention, including compounds of formula (1) and their salts, is likely to range from 0.01 to 50 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg.", "The compounds of the formula (I) and their salts may also be administered by intravenous infusion, at a dose which is likely to range from 0.001-10 mg/kg/hr.", "Tablets or capsules of the compounds may be administered singly or two or more at a time, as appropriate.", "It is also possible to administer the compounds in sustained release formulations.", "Typically, the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.", "The above dosages are exemplary of the average case.", "There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.", "Alternatively, the compounds of the invention can be administered by inhalation or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.", "An alternative means of transdermal administration is by use of a skin patch.", "For example, they can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.", "They can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.", "For some applications, preferably the compositions are administered orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents.", "The compositions (as well as the compounds alone) can also be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously.", "In this case, the compositions will comprise a suitable carrier or diluent.", "For parenteral administration, the compositions are best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.", "For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.", "For oral, parenteral, buccal and sublingual administration to subjects (such as patients), the daily dosage level of the compounds of the present invention and their pharmaceutically acceptable salts and solvates may typically be from 10 to 500 mg (in single or divided doses).", "Thus, and by way of example, tablets or capsules may contain from 5 to 100 mg of active compound for administration singly, or two or more at a time, as appropriate.", "As indicated above, the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient.", "It is to be noted that whilst the above-mentioned dosages are exemplary of the average case there can, of course, be individual instances where higher or lower dosage ranges are merited and such dose ranges are within the scope of this invention.", "T cells treated ex vivo are typically administered to the patient by intramuscular, intraperitoneal or intravenous injection, or by direct injection into the lymph nodes of the patient, preferably by direct injection into the lymph nodes.", "Typically from 104 to 108 treated cells, preferably from 105 to 107 cells, more preferably about 106 cells are administered to the patient.", "The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient.", "J. Solvates.", "The present invention also includes the use of solvate forms of the compound of the present invention.", "The terms used in the claims encompass these forms.", "K Pro-Drug.", "As indicated, the present invention also includes the use of pro-drug forms of the compounds of the present invention.", "The terms used in the claims encompass these forms.", "Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form the compounds of the present invention which are pharmacologically active.", "It will be further appreciated that certain moieties known as “pro-moieties”, for example as described in “Design of Prodrugs” by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the compounds.", "Such prodrugs are also included within the scope of the invention.", "L. Mimetic.", "In one embodiment of the present invention, the compound may be a mimetic.", "As used herein, the term “mimetic” relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a reference agent.", "M. Chemical Derivative.", "In one embodiment of the present invention, the compound may be a derivative.", "The term “derivative” as used herein includes chemical modification of a compound.", "Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.", "N. Pharmaceutical Salts.", "The compounds of the present invention may be administered as pharmaceutically acceptable salts.", "Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate.", "The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.", "Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm.", "Sci.", "66, 1-19 (1977).", "Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.", "When one or more acidic moieties are present, suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically-active amines such as diethanolamine, salts.", "The compounds of the present invention may exist in polymorphic form.", "In addition, the compounds of the present invention may contain one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms.", "Where a compound contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur.", "The present invention includes the individual stereoisomers of the compound and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.", "Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g.", "by fractional crystallisation, chromatography or H.P.L.C.", "of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof.", "An individual enantiomer of the compound may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C.", "of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.", "The present invention also includes all suitable isotopic variations of the compound or a pharmaceutically acceptable salt thereof.", "An isotopic variation of a compound of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.", "Examples of isotopes that can be incorporated into the compound and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36Cl, respectively.", "Certain isotopic variations of the compound and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies.", "Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability.", "Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.", "Isotopic variations of the compound of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.", "The present invention also includes (wherever appropriate) the use of zwitterionic forms of the compounds of the present invention.", "The terms used in the claims encompass one or more of the forms just mentioned.", "O. Formulation.", "The component(s) of the present invention may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.", "P. Pharmaceutical Compositions.", "The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of the present invention and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).", "The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.", "Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit.", "1985).", "The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.", "The pharmaceutical compositions may comprise as—or in addition to—the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).", "Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.", "Examples of suitable diluents include ethanol, glycerol and water.", "Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.", "Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.", "Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.", "Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.", "Antioxidants and suspending agents may be also used.", "There may be different composition/formulation requirements dependent on the different delivery systems.", "By way of example, the pharmaceutical composition of the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.", "Alternatively, the formulation may be designed to be administered by a number of routes.", "Where the composition is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.", "Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.", "For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.", "For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.", "For some embodiments, one or more compounds may also be used in combination with a cyclodextrin.", "Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules.", "Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule.", "Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes.", "As an alternative to direct complexation with the drug the cyclodextrin may be used as an auxiliary additive, e.g.", "as a carrier, diluent or solubiliser.", "Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A-91/11172, WO-A-94/02518 and WO-A-98/55148.The pharmaceutical composition may comprise one or more additional pharmaceutically active compounds.", "The present invention will now be described by way of examples which are intended to be illustrative only and non-limiting.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1(A-P)—Concentration-response curves of Ca2+ signals in T cells microinjected with NAADP+ FIG.", "2(A,B)—Dose response curve for NAADP+ in Jurkat T cells FIG.", "3(A,B)—Concentration-response curves of Ca2+ signals in T cells microinjected with NAADP+ or NADP+ FIG.", "4(A-J)—Concentration-response curves of Ca2+ signals in T cells microinjected with NAADP+ and 8-OCH3-cADPR or cADPR FIG.", "5(A-J)—Concentration-response curves of Ca2+ signals in T cells microinjected with NAADP+ and Ins(1,4,5)P3 or Ins(1,4,6)PS3 FIG.", "6(A-C)—Concentration-response curves of Ca2+ signals in T cells microinjected with NAADP+ and then challenged with OKT3 DETAILED DESCRIPTION OF THE FIGURES FIG.", "1.Concentration-Response Curves of Ca2+ Signals in Single Intact T-Lymphocytes Microinjected with NAADP+.", "Jurkat T-lymphocytes were loaded with Fura2/AM and Ca2+ was measured as detailed under ‘Experimental Procedures’.", "Cells were injected as described in Experimental Procedures’ in the presence of 1 mM extacellular Ca2+.", "Data are presented as overlays of single tracings of individual cells (left panel).", "The right panel shows the corresponding averages from these measurements (number of cells displayed, n=5 to 17).", "As a control vehicle buffer without NAADP+ was injected (A,B).", "The time point of microinjection is indicated by arrows.", "FIG.", "2.Dose-Response Curve for NAADP+ in Jurkat T Cells.", "Data from FIG.", "1 are shown as mean values (n=5 to 17) from time point 80 sec (Ca2+ peak; A) or 400 sec (Ca2+ plateau; B).", "FIG.", "3.NADP+ Does Not Mediate Ca2+-Signaling Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under ‘Experimental Procedures’.", "Cells were injected with 50 nM NAADP+ (A) or NADP+ (B).", "Shown are the averages from 13 (A) and 5 (B) cells.", "The time point of microinjection is indicated by arrows.", "FIG.", "4 Influence of cADPR and Its Antagonist 8-OCH3-cADPR on NAADP+-Mediated Ca2+-Signaling.", "Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under ‘Experimental Procedures’.", "Left panels show the overlays of single tracings of individual cells after injection (A-E), the right panels demonstrate the corresponding averages from these overlays (F-J).", "Shown are (n=number of experiments): A/F, coinjection of NAADP (50 nM) and 8-OCH3-cADPR (100 μM) (n=7); B/G, injection of NAADP+ (50 nM, n=10); C/H, coinjection of NAADP+ (10 μM) and cADPR (10 μM) (n=7); D/I, coinjection of NAADP+ (50 nM) and cADPR (10 μM) (n=5); E/J, injection of cADPR (10 μM, n=5).", "FIG.", "5.Influence of Ins(1,4,5)P3 and Its Antagonist Ins(1,4,6)PS3 on NAADP+-Mediated Ca2+-Signaling.", "Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under ‘Experimental Procedures’.", "Left panels show the overlays of single tracings of individual cells after injection (A-E), the right panels demonstrate the corresponding averages from these overlays (F-J).", "Shown are: A/F, coinjection of NAADP+ (50 nM) and Ins(1,4,6)PS3 (40 μM) (n=7); B/G, injection of NAADP+ (50 nM) (n=10); C/H, coinjection of NAADP+ (10 μM) and Ins(1,4,5)P3 (4 μM) (n=9); D/I, coinjection of NAADP+ (50 nM) and Ins(1,4,5)P3 (4 μM) (n=3); E/J injection of Ins(1,4,5)P3 (4 μM; n=8) FIG.", "6.Effect of NAADP+ on OKT3-Induced Ca2+ Signaling in Single Jurkat T-Lymphocytes.", "Jurkat T-lymphocytes were loaded with Fura2/AM and ratiometric Ca2+ imaging and parallel microinjection in the presence of 1 mM extracellular Ca2+ was carried out as detailed under ‘Experimental Procedures’.", "The cells were injected with different concentrations of NAADP+ and then OKT3(10 μg/ml) was added.", "Injection of intracellular buffer (A), NAADP+ (50 nM [B] and 10 μM [C]), and addition of OKT3 is indicated by arrows.", "Data are presented as a typical tracing from one individual cell, for each condition at least 3 experiments were carried out.", "EXAMPLES Materials and Methods Reagents cADPR, 8-OCH3-cADPR, and Ins(1,4,6)PS3 were synthesized exactly as described (Ashamu et al., 1995; Murphy et al., 2000), purified by anion-exchange chromatography on Q-Sepharose, and used as their triethylammonium salts.", "Purity of ligands was assessed by 1H and 31P NMR spectroscopy, mass spectroscopy and, when appropriate, high performance liquid chromatography.", "NAADP+ and NADP+ were purchased from Sigma, Deisenhofen, Germany.", "The purity of NAADP+ was described by the manufacturer to be about 95%; this was confirmed by reverse phase HPLC using the method of da Silva et al.", "(1998).", "Fura 2/AM was obtained from Calbiochem, Bad Soden, Germany.", "Anti-CD3 monoclonal antibody OKT3 was purified from hybridoma supernatant on ProteinG-Sepharose.", "Cell Culture Jurkat T lymphocytes (subclone JMP) were cultured in RPMI 1640 medium containing the following additions: Glutamax I, HEPES (20 mM, pH 7.4), NCS (7.5%), penicillin (100 U/ml) and streptomycin (50 μg/ml; all obtained from Life Technologies, Eggenstein, Germany).", "The cells were cultured at 37° C. in a humidified atmosphere in the presence of 5% CO2.Ratiometric Ca2+-Imaging Batches of 107 Jurkat T cells were loaded with Fura2/AM as described (Guse et al., 1993).", "Fura2-loaded cells (107 cells/5 ml) were kept at room temperature until use.", "Glass coverslips were coated first with bovine serum albumin (5 mg/ml), and then with poly-L-lysine (0.1 mg/ml).", "Small chambers consisting of a rubber O-ring were sealed on the coverslips by silicon grease.", "Then 90 μl of extracellular buffer (140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes, 1 mM NaH2PO4, 5.5 mM glucose, pH 7.4) was added, followed by addition of 10 μl cell suspension.", "The coverslip was mounted on the stage of an inverted microscope (Axiovert 100, Zeiss, Oberkochingen, Germany).", "Ratiometric Ca2+-imaging was performed using a PhotoMed/Photon Technology (Wedel, Germany) digital imaging system built around the Axiovert 100 microscope.", "Illumination at 340 and 380 nm was carried out using a chopper/optical filter system.", "Images were captured by an intensified CCD camera (type C2400-77; spatial resolution: 525×487 pixel; Hamamatsu, Garching, Germany) and stored as individual 340 and 380 images on harddisk.", "Sampling rate was usually 5 sec for a pair of images (340 and 380 nm) using 100-fold magnification.", "Data analysis was performed off-line using PhotoMed/Photon Technology (Wedel, Germany) “Image master” analysis software.", "Ratio images (340/380) were constructed pixel by pixel, and changes in the ratio over time were measured by applying “regions-of-interest” on individual cells.", "Finally, ratio values were converted to Ca2+-concentrations by external calibration.", "Microinjection Experiments Parallel Ca2+ imaging and microin ection experiments require a firm attachment of the Jurkat T cells without preactivation of intracellular Ca2+-signaling.", "This was achieved by the above mentioned coating procedure of the glass coverslips, as detailed earlier (Guse et al., 1997).", "The cells were kept in a small chamber (100 μl volume) in extracellular buffer (140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Hepes, 1 mM NaH2PO4, 5.5 mM glucose, pH 7.4).", "Compounds to be microinjected were cleared from particles by either filtration through 0.45 μm filters, by centrifugation in an Eppendorf centrifuge at maximal speed for 10 min, or by both.", "Femtotips II (Eppendorf-Netheler-Hintz, Hamburg, Germany) were filled with 5 μl of reagent solution and inserted into the semi-automatic Eppendorf microinjection system (Transjector 5246, Micromanipulator 5171, Eppendorf-Netheler-Hintz, Hamburg, Germany).", "Injection parameters were: injection pressure: 80 hPa, compensatory pressure: 40 hPa, duration of injection: 0.5 sec, velocity of pipette: 700 μm/sec, pipette angle: 45°.", "Injections were performed into the upper part of the cell.", "Example 1 NAADP+ Activates Ca2+ Signaling at Low Concentrations in a Dose-Dependent Manner, but High Concentrations Causes Self-Inactivation of the Ca2+-Release System Microinjection of NAADP+ at a pipette concentration as low as 10 nM stimulated repetitive, longlasting Ca2+ spiking of low amplitude in intact Jurkat T cells whereas injection of intracellular buffer alone had no effect (FIG.", "1A,B,E,F).", "Microinjection of 0.1 or 1 nM NAADP+ was without effect in most of the cells (FIG.", "1C,D, and data not shown).", "At a pipette concentration of 50 nM NAADP+ an initial, rapidly occurring Ca2+-peak with a high amplitude was observed which turned into gradually lowering oscillations during the first 350 to 400 s. After this time period the calcium response changed into a low, but sustained plateau phase with very small oscillations (FIG.", "1G,H).", "At pipette concentrations of 100 nM and 1 μM similar responses were observed (FIG.", "1 I to L).", "However, the peak amplitude of the initial Ca2+-spike declined with increasing NAADP+ concentrations (FIG.", "1 J,L), and the decay of the Ca2+-signal was accelerated (FIG.", "1 H,J,L).", "At 10 μM NAADP+ the Ca2+ response appeared similar to the one at 1 nM (FIG.", "1 M,N), whereas at 100 μM NAADP+ no signal was detectable (FIG.", "1 O,P).", "The dose response relationship shows a bell shaped curve for the initial Ca2+-peak with an optimal NAADP+ concentration at 100 nM (FIG.", "2A).", "However, only minor changes of the longlasting Ca2+-signal as measured at 400 s were observed in response to 100 nM NAADP+ (FIG.", "2B).", "These data indicate that, similar to the few other cellular systems investigated so far (Lee & Aarhus, 1995; Chini et al., 1995; Albrieux et al., 1998; Cancela et al., 1999), NAADP+ at low nanomolar concentrations activates Ca2+-signaling in T cells, whereas micromolar concentrations of NAADP+ rapidly cause self inactivation of the Ca2+-release system.", "The high initial Ca2+-spike observed after microinjection of 50 nM NAADP+ was massively reduced when the extracellular Ca2+-concentration was decreased to a nominal Ca2+-free buffer indicating that Ca2+-entry is involved in the NAADP+-mediated Ca2+-response (data not shown).", "Example 2 The effect of NAADP+ on Intracellular Ca2+-Signaling in T Cells is Specific To prove the specificity of the effect of NAADP+on intracellular Ca2+-signaling in T cells, NADP+ was used in parallel microinjection experiments.", "NADP+ is a structurally very similar molecule bearing a nicotinamide group instead of the nicotinic acid group.", "In contrast to NAADP+, microinjection of NADP+ (50 nM) was completely without effect on Ca2+-signaling (FIG.", "3A,B).", "Example 3 Investigating the Relationship between the NAADP+ System and the Ins(1,4,5)P3 and cADPR Systems The Ca2+-release system which is targeted by NAADP+ has not yet been identified, but work in other cell systems indicates that neither the InsP3-R nor the RyR are involved (Lee & Aarhus, 1995; Chini et al., 1995).", "However, both these classical intracellular Ca2+-release systems have been demonstrated to be essential parts of the Ca2+-signaling machinery of T cells (Jayaraman et al., 1995; Guse et al., 1999).", "Thus, the next series of experiments were designed to investigate potential interrelations between the NAADP+ system on the one hand and both the Ins(1,4,5)P3 and cADPR systems on the other hand.", "The specific cADPR antagonist 8-OCH3-cADPR (Guse et al., 1999), when co-injected with an optimal NAADP+ concentration, did not significantly affect NAADP+-mediated Ca2+-signaling (FIG.", "4 A,F vs. B,G).", "However, when a self-desensitizing concentration of NAADP+ (10 μM) was co-injected with a stimulating concentration of CADPR (10 μM), a massive decrease of the cADPR-mediated Ca2+-signal was observed (FIG.", "4 C,H vs. E,J).", "On the other hand, an optimal stimulating concentration of NAADP+ (50 nM) microinjected together with cADPR (10 μM) did not significantly change the Ca2+ signals (FIG.", "4 D,I vs. E,J).", "These data indicate that a functional, non-desensitized NAADP+/Ca2+-release system is necessary for cADPR-mediated Ca2+-release.", "The specific Ins(1,4,5)P3 antagonist Ins(1,4,6)PS3 (Guse et al., 1997; Murphy et al., 2000) was also co-injected with an optimal NAADP+ concentration.", "Surprisingly, there was a partial reduction of the initial Ca2+ peak, but also a faster decay of this peak as compared to injection of NAADP+ alone (FIG.", "5A,F vs. B,G).", "Similar to the cADPR system, there was an almost complete inhibition of Ins(1,4,5)P3-mediated Ca2+-release when a desensitizing concentration of NAADP+ was co-injected (FIG.", "5 C,H).", "Co-injection of an optimal stimulating concentration of NAADP+ together with Ins(1,4,5)P3 resulted in a high initial Ca2+ peak (FIG.", "5 D,I), which was comparable to the peak observed in response to injection of NAADP+ alone (FIG.", "5B,C), whereas much less oscillatory activity of the cells after the initial peak was observed (FIG.", "5D,I) as compared to Ins(1,4,5)P3 alone (FIG.", "5 E,J).", "These data indicate that also the Ins(1,4,5)P3/Ca2+-release system requires a functional non-desensitized NAADP+/Ca2+-release system.", "Moreover, a part of the Ca2+-signal observed in response to microinjection of NAADP+ alone appears to be mediated by Ins(1,4,5)P3 (FIG.", "5 A,F,B,G).", "This may be explained by the co-agonistic effect of Ca2+ released by NAADP+ which then acts together with basal Ins(1,4,5)P3 at the InsP3-R; this co-agonistic effect of Ca2+ at the InsP3R has been demonstrated previously (Bezprozvanny et al., 1991).", "Example 4 Investigating the Effect of NAADP+ on Ca2+-Signaling Mediated by an Anti-CD3 mAb Both the Ins(1,4,5)P3/and the cADPR/Ca2+-release systems have been published to be essential parts of the Ca2+-signaling machinery of T cells upon stimulation of the TCR/CD3 complex (Jayaraman et al., 1995; Guse et al., 1999).", "Since the data described above indicate that a functional NAADP+/Ca2+-release system is essential for both Ins(1,4,5)P3- and cADPR mediated Ca2+-release, the present inventors investigated the effect of NAADP+ on Ca2+-signaling mediated by anti-CD3 mAb OKT3 (FIG.", "6).", "Microinjection of 50 nM NAADP+ prior to stimulation of the cells by extracellular addition of OKT3 did not significantly change the OKT3-mediated Ca2+-signal (FIG.", "6A,B).", "However, there was a dramatic inhibition of OKT3-mediated Ca2+-signaling when a desensitizing concentration of NAADP+ was microinjected before stimulation by OKT3 (FIG.", "6C).", "The main findings of this examples are (i) a dose-dependent and specific effect of NAADP+ in T cell Ca2+-signaling, (ii) the strict dependence of both Ins(1,4,5)P3- and cADPR-mediated Ca2+-release upon a functional NAADP+/Ca2+-release system, and (iii) inhibition of Ca2+-signaling mediated by ligation of the TCR/CD3 complex by prior self inactivation of the NAADP+/Ca2+-release system.", "Assuming that the NAADP+/Ca2+-release system acts as the Ca2+-providing trigger in T cells, the complex behavior of activation and inactivation opens a multitude of regulatory possibilities: simply by changing their endogenous NAADP+ concentration cells might regulate the status of the NAADP+/Ca2+-release system, e.g.", "by increasing NAADP+ the NAADP+/Ca2+-release system will become inactivated, and Ca2+-signaling will in turn be completely unresponsive.", "For T-lymphocytes, such behavior of unresponsiveness to antigenic or mitogenic stimulation is well known as anergy (Jenkins et al., 1987); however, to date it has been less clear which intracellular mechanism is responsibly involved.", "The present data indicate that the NAADP+/Ca2+-release system with its complex inactivation/activation properties may well be a mechanism underlying anergy in T cells.", "SUMMARY The present invention will now be summarised by way of numbered paragraphs: 1.A method for modulating T cell activity, which comprises the step of modulating the intracellular concentration of NAADP+ or a bioisostere thereof.", "2.A method according to paragraph 1, which comprises the step of stimulating a rise in intracellular Ca2+ levels by raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an activating concentration.", "3.A method according to paragraph 1, which comprises the step of inhibiting TCR/CD3-associated Ca2+ signalling by raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an inactivating concentration.", "4.A compound capable of antagonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, said rise being in response to stimulation of the T cell receptor/CD3 complex, for use in modulating T cell activity.", "5.A compound capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, for use in modulating T cell activity.", "6.A compound according to paragraph 5, which is capable of raising the intracellular concentration of NAADP+, or a bioisostere thereof, to an inactivating concentration.", "7.A compound according to any of paragraphs 4 to 6, for use in inducing T cell anergy.", "8.A compound according to any of paragraphs 4 to 6, for use in blocking T cell proliferation.", "9.A compound capable of agonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, said rise being in response to stimulation of the T cell receptor/CD3 complex, for use in modulating T cell activity.", "10.A compound capable of preventing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, for use in modulating T cell activity.", "11.A compound according to paragraphs 9 or 10, for stimulating T cell proliferation and/or differentiation.", "12.A compound according to any one of paragraphs 4-11 wherein said compound is a NAADP analogue.", "13.A compound according to paragraph 12 wherein the NAADP analogue has the formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "14.A compound according to paragraph 12 or paragraph 13 wherein said NAADP analogue is 8-bromo-nicotinic acid adenine dinucleotide phosphate.", "15.Use of a compound as defined in any one of paragraphs 4 to 14 in the manufacture of a medicament for use in modulating the immune response of a mammal.", "16.Use of a compound as defined in any one of paragraphs 4 to 8 in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "17.Use according to paragraph 16 wherein the autoimmune disease is selected from thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis, rhematoid arthritis and lupus erythematosus.", "18.Use of a compound as defined in any one of paragraphs 4 to 17 in the manufacture of a medicament for use in treating or preventing an immune disorder in a human or animal.", "19.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a compound as defined in any one of paragraphs 4 to 14.20.A method for identifying a substance capable of antagonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell, which has been stimulated via its T cell receptor, with a candidate substance under conditions that would permit a rise in intracellular Ca2+ levels in the absence of the substance; and (ii) determining whether the substance inhibits a rise in intracellular Ca2+ levels.", "21.A method for identifying a substance capable of inducing the NAADP+-mediated inhibition of TCR/CD3-associated Ca2+ signalling, which method comprises: (i) contacting a T cell with a candidate substance; (ii) stimulating the T cells via TCR/CD3; and (ii) determining whether the substance inhibits TCR/CD3-associated Ca2+ signalling.", "22.A method for identifying a substance capable of agonising the NAADP+-mediated rise in intracellular Ca2+ levels in a T cell, which method comprises: (i) contacting a T cell with a candidate substance; and (ii) determining whether the substance elicits or enhances a rise in intracellular NAADP+ and/or Ca2+ levels.", "23.A substance identified by the method of paragraph 20, 21 or 22.24.A process comprising the steps of: (a) performing the method according to paragraph 20, 21 or 22; (b) preparing a quantity of one or more substances identified by the method.", "25.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound is a NAADP analogue.", "26.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound has the formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "27.A compound capable of modulating the intracellular concentration and/or the binding affinity of NAADP+ wherein the compound is 8-bromo-nicotinic acid adenine dinucleotide phosphate.", "28.Use of a NAADP analogue in the manufacture of a medicament for use in modulating the immune response of a mammal.", "29.Use of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alkyl, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I) in the manufacture of a medicament for use in modulating the immune response of a mammal.", "30.Use of 8-bromo-nicotinic acid adenine dinucleotide phosphate in the manufacture of a medicament for use in modulating the immune response of a mammal.", "31.Use of a NAADP analogue in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "32.Use of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alky, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I) in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "33.Use of 8-bromo-nicotinic acid adenine dinucleotide phosphate in the manufacture of a medicament for use in treating an autoimmune disease or graft rejection.", "34.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a NAADP analogue.", "35.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of a compound having formula (I): wherein P is a substituent group independently selected from NH2, OH, SH; each of W1, W2 or W3 is independently selected from either a CH or a heteroatom, such as N, P, S or O, preferably N; X is a substituent group independently selected from OH, SH, NH2, or a halo group (preferably Br); each of R1, R2 or R3 is independently selected from H or each of Y1, Y2 or Y3 is independently selected from OH, H, NH2, halo (preferably F), SH, OR4, or wherein R4 is a hydrocarbyl; each of Z1 or Z2 is independently selected from O, S, CH2 or a halo derivative thereof, preferably CF2; and L is a linker group, suitably the linker group may have the formula (II): or may be selected from one ore more the group comprising: a phosphate, a polyphosphate, a phosphorothioate, a polyethylene glycol, an alky, an alkylaryl, a peptide and a polyamine; or isomeric forms of the compound of Formula (I).", "36.A method of treating or preventing a disease in a human or animal patient which method comprises administering to the patient an effective amount of 8-bromo-nicotinic acid adenine dinucleotide phosphate.", "All publications mentioned in the above specification are herein incorporated by reference.", "Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.", "Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.", "Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.", "REFERENCES Albrieux, M., H. C. Lee, and M. Villaz.", "1998.J.", "Biol.", "Chem.", "273:14566-14574.Bezprozvanny, I., J. Watras, and B. E. Ehrlich.", "1991 Nature 351:751-754.Cancela, J. M., G. C. Churchill, and A. Galione.", "1999.Nature (Lond) 398:74-76.Chini, E. N., K. W. Beers, and T. P. Dousa 1995.J.", "Biol.", "Chem.", "270:3216-3223.Clapper, D. L., T. F. Walseth, P. J. Dargie, and H. C. Lee.", "1987.J.", "Biol.", "Chem.", "262:9561-9568.Guse, A. H., Berg, I., da Silva, C. P., Potter, B. V. L., & Mayr, G. W., J. Biol.", "Chem.", "272, 8546-8550 (1997).", "Guse, A. H., Ca2+ signalling in T-lymphocytes, Crit.", "Rev.", "Immunol.", "18, 419-448 (1998).", "Guse, A. H., et al.", "Nature (Lond.)", "398:70-73 (1999).", "Jayaraman, T., Ondriasove, E., Ondrias, K., Harnick, D. J., & Marks, A. R, Proc.", "Natl.", "Acad.", "Sci.", "USA 92, 6007-6011(1995).", "Jenkins, M., D. M. Pardoll, J. Mizuguchi, T. M. Chused, and R. H. Schwartz.", "1987.Proc.", "Natl.", "Acad.", "Sci.", "USA 84:5409-5413.Lee, H. C., and R. Aarhus.", "1995.J.", "Biol.", "Chem.", "270:2152-2157.Murphy, C. T., A. M. Riley.", "S. J.", "Mills, C. J. Lindley, B. V. L. Potter, and J. Westwick.", "2000.Mol.", "Pharmacol.", "57:595-601 Rahavendran, S. V., and H. T. Karnes.", "1993.Pharm.", "Res.", "10:328-34." ] ]

[ [ "Method for producing concrete or mortar using a vegetal aggregate", "A method for producing concrete or mortar based on an exclusively vegetal aggregate, a mineral binding agent, and mixing water is described.", "Instead of the vegetal aggregate being premineralized in a separate step of the operation, 4 to 14 kg of a non-hydratizable, finely ground mineralizator is added for each cubic meter of vegetable aggregate when the concrete or mortar is being mixed.", "Raw calcium carbonate is a preferred mineralizator." ], [ "1.Method for producing concrete or mortar based on an exclusively vegetal aggregate, a mineral binding agent, and mixing water, the vegetal aggregate having a specific weight of 80 to 160 kg/m3, as measured at a residual moisture of approximately 15%, characterized in that 4 to 14 kg of a non-hydratizable, finely ground mineralizator is added for each cubic meter of the vegetal aggregate when the concrete or mortar is being mixed.", "2.Method as defined in claim 1, characterized in that the mineralizator is a finely ground stone dust.", "3.Method as defined in claim 1 or claim 2, characterized in that at least 80%-mass of the mineralizator that is added has a grain size of less than 0.09 mm.", "4.Method as defined in one of the claims 1 to 3, characterized in that the mineralizator essentially includes raw calcium carbonate.", "5.Method as defined in one of the claims 1 to 4, characterized in that the mineral binding agent includes Portland cement, in particular PZ 42.5 Grade Portland cement.", "6.Method as defined in one of the claims 1 to 5, characterized in that the mineral binding agent is a mixture of Portland cement and white lime.", "7.Method as defined in one of the claims 1 to 6, characterized in that between 180 and 400 kg of mineral binding agent is added for each cubic meter of the vegetal aggregate.", "8.Method as defined in one of the claims 1 to 7, characterized in that the mixing ratio of mineralizator to mixing water amounts to 25 kg to 50 kg of mineralizator to 1,000 kg of water.", "9.Method as defined in one of the claims 1 to 8, characterized in that the vegetal aggregate is composed for the most part of fibrous particles with diameters ranging from 0 mm to 5.0 mm.", "10.Method as defined in claim 9, characterized in that a light concrete or a light wash floor is produced, the vegetal aggregate being composed for the most part of fibrous particles with the lengths ranging from 5 mm to 40 mm.", "11.Method as defined in claim 9, characterized in that a light plaster or thermal insulation plaster is produced, the vegetal aggregate being composed for the most part of fibrous particles with lengths of less than 5 mm.", "12.Method as defined in claim 1 to claim 11, characterized in that coniferous wood, hemp and/or reeds are used during the production of the vegetal aggregate.", "13.Method as defined in one of the claims 1 to 12, characterized in that plants of the Miscanthus family are used for the production of the vegetal aggregate.", "14.Method as defined in claim 14, characterized in that the plant Miscanthus Gigantheus is used for production of the vegetal aggregate.", "15.Method as defined in one of the claims 1 to 14, characterized in that the method is used for producing light concrete, light mortar, light wash floor, as well as light plaster and thermal insulation plaster." ], [ "The present invention relates to a method for producing a concrete or mortar based on renewable raw materials.", "PRIOR ART The production of concrete and mortar based on an exclusively vegetal aggregate such as, for example, wood, hemp or reed fibers, is already known.", "In a first operational step during the production of such mortar, the shredded vegetal raw material is premineralized.", "During this premineralization, the vegetal particles are placed in a mineralization bath, or are wetted or sprayed with a mineralization liquid, when aluminum sulfate or cement is usually used as the mineralizator.", "The vegetal particles are then dewatered and dried.", "At the work site, the dried, premineralized particles can then be mixed with the mixing water and cement as aggregate to form a mortar.", "Premineralization of the vegetal aggregate ensures that a solid bond is formed between the vegetal aggregate and the hardened cement paste, so that the concrete or mortar possesses the desired bending tensile strength and resistance to pressure.", "In this respect, it should be noted that the known premineralization of the vegetal aggregate is costly and is, in addition, potentially harmful to the environment.", "Older methods for producing concrete or mortar are also known; in these, vegetal aggregates that have not been premineralized are mixed with mineral aggregates, mixing water, and cement.", "These methods have not been successful in practice.", "FR 1018109 (1952) describes a material for sound-absorbing floors and walls; it is preferred that this be composed as follows: 10% man-made cement, 10% ground chalk, 25% sand, 40% sawdust and 10% powdered cork.", "This particular patent specification does not explain what is meant by man-made cement.", "In addition, it should be noted that the material contains a total of 35% mineral aggregate and a very small percentage of binder.", "The patent specification in question also mentions very briefly that the mortar can be produced exclusively from cement, chalk, sand, and cork or from cement, chalk, sawdust, and cork.", "The patent specification FR 1018109 provides no additional details with respect to the mixing ratios of the last two mortars.", "DE 847 725 (1952) describes a material for producing nail-holding stones, light structural panels, and floor coverings that contain no water-soluble magnesium compounds and is relatively cheap to produce.", "The material proposed for this is a mixture of wood dust or wood chips, limestone or grey marble (grain size of 0 to 1 mm), and cement.", "It is proposed that a mixture of two parts by weight of sawdust or wood chips, four parts by weight of ground grey marble, and three parts by weight of cement be used to produce nail-holding stones.", "It is proposed that a mixture of three parts by weight of sawdust mixed with wood shavings, or wood shavings mixed with wood chips, 10 parts by weight of ground grey marble, and six to seven parts by weight of cement be used to produce light structural panels.", "A mixture of four parts by weight of wood dust, 12 parts by weight of ground grey marble, and 20 parts by weight of cement is proposed for the production of floor coverings.", "When such a material hardens, the grey marble and the cement form a relatively heavy mineral matrix in which the wood fibers are bound.", "It can be assumed that this material with its relatively heavy mineral matrix does not provide special properties with respect to thermal insulation and acoustic insulation.", "GB 638,501 (1950) describes an allegedly weatherproof material for producing panels, roofing panels, roofing tiles, pipes, and eave troughs.", "A preferred mixture for this material includes 30 parts by weight of paper or cellulose, 40 parts by weight of cement, 10 parts by weight of whiting, 10 parts by weight of hydrated lime, and 10 parts by weight of river mud.", "It can be assumed that this material does not provide good properties with respect to thermal insulation and acoustic insulation.", "OBJECTIVE OF THE PRESENT INVENTION It is the objective of the present invention to produce concrete and mortar based on a vegetal aggregate in a simple and economical manner; the hardened concrete/mortar is intended to have a relatively low specific weight, as well as good properties with respect to thermal insulation, acoustic insulation, bending tensile strength, and resistance to pressure.", "GENERAL DESCRIPTION OF THE INVENTION According to the present invention, this objective has been achieved by a method for producing concrete or mortar based on an exclusively vegetal aggregate, a mineral binding agent, and mixing water, the vegetal aggregate having a specific weight of 80-160 kg m3 as measured at a residual moisture content of approximately 15%.", "There is no premineralization of the vegetal aggregate.", "In place of this, 4 to 14 kg (in the normal case, 6 to 12 kg) of a non-hydratizable finely ground mineralizator is added for each cubic meter of vegetal aggregate when the concrete or mortar is being mixed.", "A “non-hydratizable mineralizator” is understood to be a finely ground mineral substance which, in contrast to the binding agent, forms no hydrates with the mixing water as reaction products.", "It was established that the added mineralizator is deposited on the surface of the vegetal aggregate particles when the concrete/mortar is being mixed, and that during hardening it ensures that a solid bond is established between the particles of the vegetal aggregate and the mineral binding-agent matrix, so that the concrete or mortar possesses the required bending tensile strength and resistance to pressure.", "In other words, there is, as it were, a premineralization of the surface of the vegetal aggregate, beginning with the mixing process for the mortar, up until hydratation of the binding agent.", "The finer the mineralizator is ground, the faster and better it is deposited on the surface of the vegetal aggregate.", "It should also be noted that the mineralizator is deposited only on the surface of the vegetal aggregate particles and thus does not affect the cellular structure of the particles of the vegetal aggregate.", "In addition, the quantity of mineralizator that is added is so matched to the vegetal aggregate that it is sufficient for the complete mineralization of the surface of the vegetal aggregate particles, or is just enough that only a slight surplus remains.", "This avoids a larger quantity of the mineralizator being bound into the binding agent matrix between the vegetal aggregate particles when the concrete or mortar hardens.", "In conclusion, it must be said that the method according to the present invention permits a drastic reduction of the production costs associated with the vegetal aggregate, because it renders superfluous the costly premineralization that is carried out in a separate step of the operation.", "In addition, dispensing with the separate premineralization in a separate operational step also eliminates environmental concerns with respect to the production method for concrete and mortar based on a vegetal aggregate.", "The non-vegetal constituents of the concrete/mortar according to the present invention are restricted to the mineral binding agent and the small addition of mineralizator.", "Most surprisingly, despite this, the hardened concrete/mortar displays outstanding bending tensile strength and resistance to pressure.", "Because of its large percentage of vegetal aggregate with a low specific weight and a mineral binding agent matrix without other mineral fillers, the hardened concrete/mortar is also of a relatively low specific weight and has good properties with respect to thermal insulation and acoustic insulation.", "It is an advantage that the mineralizator is a finely ground stone dust, at least 80%-mass of the stone dust having a grain size of less than 0.09 mm.", "Essentially, a preferred mineralizator includes commercially available, raw calcium carbonate.", "Advantages of the raw calcium carbonate include the facts that it is extremely inexpensive, can be very finely ground, forms a good suspension in water and, in addition, has a low specific weight of only 1.18 t/m3.The mixing ratio of mineralizator to mixing water amounts to 25 to 50 kg mineralizator in 1,000 kg water.", "An advantageous binding agent is, for example, Portland cement, in particular PZ 42.5 Grade Portland cement.", "In the event that plastering mortar is to be produced, a hydratizable lime such as white lime is added to the Portland cement.", "Depending on the type of mineral binding agent and the purpose for which the concrete/mortar is to be used, between 180 and 400 kg of mineral binding agent is added to each cubic meter of the vegetal aggregate.", "It is preferred that the vegetal aggregate be composed of fibrous particles with diameters ranging from 0 to 5.0 mm.", "If a light concrete or light wash floor is to be produced, it is advantageous if the vegetal aggregate be made up for the most part of fibrous particles 5 mm to 40 mm long.", "If light plaster or thermal insulation plaster is to be produced, it is an advantage if the vegetal aggregate be made up for the most part of fibrous particles that are shorter than 5 mm.", "It is preferred that the vegetal aggregate be produced from fibrous, rapidly growing plants, for example, by shredding.", "The following, amongst others, can be used: the wood of coniferous trees, hemp, and reeds.", "The wood from deciduous trees is not suitable because of its high sugar content.", "Above all, it is all the plants of the C4 group, which are characterized by a high level of photosynthesis performance, that are preferred.", "The rapidly growing plants of the Miscanthus family are particularly useful.", "A preferred type of Miscanthus is Miscanthus Gigantheus, which has a very high silicon content.", "The mortars and light concretes that are produced using the method according to the present invention yield products that posses a high level of dimensional stability once they have hardened.", "However, mixtures of different plants can be used as raw materials for the aggregate.", "Description of a Test A mortar made up as follows was mixed in a pan mixer: Aggregate: 1 m3 consisting of ⅓ coniferous wood+⅓ hemp+⅓ Miscanthus, shredded, particle diameter from 0 to 5 mm; length of the particles from 5-40 mm; residual moisture less than 18%.", "Binding Agent: 280 kg Portland cement (PZ 42.5 Grade) and 100 kg white lime Mineralizator: 9 kg commercial calcium carbonate consisting (according to the manufacturer) of 95% CaCO3 (raw), and 5% other material, 85%-mass being of a grain size ranging from 0 mm to 0.09 mm, maximum supergrain size 2 mm.", "Mixing Water: 250 liters, temperature approximately 18° C., to achieve a K1 consistency (slightly moist).", "During the mixing process, the aggregate materials, the mixing water, the binding agent, and the mineralizator are mixed together for two minutes in a positive mixer (e.g., a pan mixer).", "The sequence in which the additives are introduced is unimportant.", "In order to prevent the mixture sticking together and forming lumps in the pan mixer, it is, however advantageous to add the vegetal aggregate and the mixing water to the plate mixer first of all, before the mineralizator and the binding agent are added.", "A mortar, mixed as described above, was examined by the Department of Mechanical Engineering and Building Construction of the Rheinisch-Wesffalische Technische Hochschule (RWTH) [North Rhine-Westphalia College of Technology], Aachen, Germany (hereinafter: examining institute) and poured into steel molds measuring 40 mm×40 mm×100 mm, lightly tamped, and stripped.", "These molded test bodies were stored for 28 days at a temperature of 18-20° C. in the examining institute's environmental chamber so as to set up.", "The examining institute then tested 11 of these tests bodies in accordance with DIN EN 196 04 with respect to their bending tensile strength and resistance to pressure.", "The arithmetic average values for the 11 test bodies were as follows: 3.64 N/mm2, bending tensile strength; 9.43 N/mm2 resistance to pressure.", "The DIN Standard requires only 1 N/mm2 bending tensile strength and 5 N/mm2 resistance to pressure for finishing mortar.", "Use of the Concrete or Mortar The concrete or mortar produced by the method according to the present invention is excellent for the production of ecological and heat absorbing, light concrete, mortar for interior and external finishing, wash or cast plaster floors, as well as for ecological and thermal insulation and finished wall elements, building blocks, and insulating panels.", "These products are used both in new ecological structures, e.g., single and multifamily housing, as well as when renovating existing buildings, e.g., when retrofitting a wooden decks with impact-sound insulation, when the relatively low dead weight of the finished product (depending on composition, 350/550 kg/m3) is used to its best advantage." ] ]

[ [ "Peptidomimetic protease inhibitors", "The present invention relates to peptidomimetic compounds useful as protease inhibitors, particularly as serine protease inhibitors and more particularly as hepatitis C NS3 protease inhibitors; intermediates thereto; their preparation including novel steroselective processes to intermediates.", "The invention is also directed to pharmaceutical compositions and to methods for using the compounds for inhibiting HCV protease or treating a patient suffering from an HCV infection or physiological condition related to the infection.", "Also provided are pharmaceutical combinations comprising, in addition to one or more HCV serine protease inhibitors, one or more interferons exhibiting anti-HCV activity and/or one or more compounds having anti HCV activity and a pharmaceutically acceptable carrier, and methods for treating or preventing a HCV infection in a patient using the compositions.", "The present invention is also directed to a kit or pharmaceutical pack for treating or preventing HCV infection in a patient." ], [ "1.A compound of formula I wherein: R0 is a bond or difluoromethylene; R1 is hydrogen, optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R2 and R9 are each independently optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R3, R5 and R7 are each independently; optionally substituted (1,1- or 1,2-)cycloalkylene; or optionally substituted (1,1- or 1,2-)heterocyclylene; or methylene or ethylene, substituted with one substituent selected from the group consisting of an optionally substituted aliphatic group, an optionally substituted cyclic group or an optionally substituted aromatic group, and wherein the methylene or ethylene is further optionally substituted with an aliphatic group substituent; or R4, R6, R8 and R10 are each independently is hydrogen or optionally substituted aliphatic group; is substituted monocyclic azaheterocyclyl or optionally substituted multicyclic azaheterocyclyl, or optionally substituted multicyclic azaheterocyclenyl wherein the unsaturatation is in the ring distal to the ring bearing the R9-L-(N(R8)—R7—C(O)—)nN(R6)—R5—C(O)—N moiety and to which the —C(O)—N(R4)—R3—C(O)C(O)NR2R1 moiety is attached; L is —C(O)—, —OC(O)—, —NR10C(O)—, —S(O)2—, or —NR10S(O)2—; and n is 0 or 1, or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug, provided when is substituted then L is —OC(O)— and R9is optionally substituted aliphatic; or at least one of R3, R5 and R7 is ethylene, substituted with one substituent selected from the group consisting of an optionally substituted aliphatic group, an optionally substituted cyclic group or an optionally substituted aromatic group and wherein the ethylene is further optionally substituted with an aliphatic group substituent; or R4 is optionally substituted aliphatic.", "2.A compound of claim 1 wherein R0 is a bond.", "3.A compound of claim 2 wherein: optionally substituted aliphatic groups are alkyl, alkenyl or alkynyl, optionally substituted with one or more aliphatic group substituent; optionally substituted cyclic groups are cycloalkyl, cycloalkenly, heterocyclyl or heterocyclenyl groups optionally substituted with one or more ring group substituents; optionally substituted aromatic groups are aryl or heteroaryl gropus optionally substituted with one or more ring group substituents; optionally substituted (1,1- or 1,2) cycloalkylene groups are (1,1- or 1,2) cycloalkylene groups optionally substituted with one or more ring group substituents; optionally substituted (1,1- or 1,2) heterocyclylene groups are (1,1- or 1,2) heterocyclylene groups optionally substituted with one or more ring group substituents; as substituted monocyclic azaheterocyclyl is a monocyclic azaheterocyclyl group substituted directly or through a linker group by at least one substituent selected from aryl, heteroaryl, aryloxy, heteroaryloxy, aroyl or its thio analogue, heteroaryl or its thioxo analogue, aroyloxy, heteroaroyloxy, aryloxycarbonyl, heteroaryloxycarbonyl, arylsulfonyl, heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl, arylthio, heteroarylthio, aryldiazo, heteroaryldiazo, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2— wherein at least one of Y1 and Y2 is aryl or heteroaryl, wherein said linker group is selected from the group consisting of —C(O)—, —OC(O)—, lower alkyl, lower alkoxy, lower alkenyl, —O—, —S—, —C(O)C(O)—, —S(O)—, —S(O)2—, —NR80—, where R80 is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl or heteroaryl; optionally substituted multicyclic azaheterocyclyl is a multicyclic azaheterocyclyl group optionally substituted by one or more ring group substituents; optionally substituted multicyclic azaheterocyenyll is a multicyclic azaheterocyclenyll group optionally substituted by one or more ring group substituents; wherein: aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl; ring group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), acid biostere, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl, or when a ring system is saturated or partially saturated, the “ring group substituents” further include, methylene (H2C═), oxo (O═) and thioxo (S═); aryl means an aromatic monocyclic or multicyclic ring system of 6 to 14 carbon atoms; cycloalkyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms; cycloalkenyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms which contain at least one carbon-carbon double bond; cyclyl means cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl; heterocyclyl means a non-aromatic saturated monocyclic or multicyclic ring system of about 3 to about 10 carbon atomsin which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon; heterocyclenyl means a non-aromatic monocyclic or multicyclic hydrocarbon ring system of about 3 to about 10 carbon atoms in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon and which contains at least one carbon-carbon double bond or carbon-nitrogen double bond; and heteroaryl means an aromatic monocyclic or multicyclic ring system of about 5 to about 14 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon.", "4.A compound of claim 1 wherein R0 is difluoromethylene.", "5.A compound of claim 3 wherein R1 is hydrogen or optionally substituted lower aliphatic group.", "6.A compound of claims 5 wherein R1 is hydrogen or lower alkyl.", "7.A compound of claim 6 wherein R1 is hydrogen.", "8.A compound of claim 3 wherein R2 is optionally substituted lower aliphatic group or optionally substituted monocyclic group.", "9.A compound of claim 8 wherein R2 is optionally substituted lower alkyl, optionally substituted lower alkenyl, or optionally substituted monocyclic cycloalkyl.", "10.A compound of claim 9 wherein R2 is carboxymethyl, 1-carboxy-2-phenylethyl, cyclopropyl, cyclobutyl, 1-cyclohexylethyl, 1-phenylethyl, but-2-yl, 1-pyrid4-ylethyl, propen-3-yl or 3-methylbut-2-yl.", "11.A compound of claim 3 wherein R3 is optionally substituted lower aliphatic group methylene.", "12.A compound of claim 11 wherein R3is optionally halo substituted lower (alkyl or alkenyl)methylene.", "13.A compound of claim 12 wherein R3 is propylmethylene, 2,2-difluoroethylmethylene, 2,2,2-trifluoromethylene or propen-3-ylmethylene; 14.A compound of claim 13 wherein R3 is propylmethylene or 2,2-difluoroethylmethylene.", "15.A compound of claim 14 wherein R3 is propylmethylene.", "16.A compound of claim 3 wherein R4 is hydrogen or optionally substituted lower aliphatic group.", "17.A compound of claim 16 wherein R4 is hydrogen.", "18.A compound of claim 3 wherein R5 is optionally substituted lower aliphatic group methylene.", "19.A compound of claim 18 wherein R5 is optionally (phenyl, carboxy, carboxamido or alkoxycarbonyl) substituted lower (alkyl or alkenyl) methylene.", "20.A compound of claim 19 wherein R5 is methylmethylene, isopropylmethylene, t-butylmethylene, but-2-ylmethylene, butylmethylene, benzylmethylene, 3-methylbutylmethylene, 2-methylpropylmethylene, carboxymethylmethylene, carboxamidomethylmethylene, benzyloxycarbonylmethylmethylene, benzyloxycarbonylpropylmethylene or phenylpropen-3-ylmethylene.", "21.A compound of claim 20 wherein R5is isopropylmethylene or t-butylmethylene.", "22.A compound of claim 3 wherein R6 is hydrogen or optionally substituted lower aliphatic group.", "23.A compound of claim 22 wherein R6 is hydrogen or lower alkyl.", "24.A compound of claim 23 wherein R6 is hydrogen.", "25.A compound of claim 3 wherein R7 is optionally substituted lower aliphatic group methylene, optionally substituted lower cyclic group methylene or optionally substituted monocyclic (aryl or heteroaryl) methylene.", "26.A compound of claim 25 wherein R7is optionally substituted lower alkylmethylene, optionally substituted lower cycloalkylmethylene or optional substituted phenylmethylene.", "27.A compound of claim 26 wherein R7 is methylmethylene, isopropylmethylene, n-propylmethylene, phenylmethylene, cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene, s-butylmethylene, cyclohexylmethylmethylene, or phenylmethylmethylene.", "28.A compound of claom 27 wherein R7is isopropylmethylene, cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene or s-butylmethylene.", "29.A compound of claim 3 wherein each of R3, R5, and R7is mono substituted methylene.", "30.A compound of claim 29 wherein R3is mono substituted methylene and has an (S) configuration on the carbon attached to the —C(O)—R0—C(O)—NR1R2 moiety.", "31.A compound of claim 30 wherein R8 is hydrogen or optionally substituted lower aliphatic group.", "32.A compound of claim 31 wherein R8is hydrogen or lower alkyl.", "33.A compound of claim 32 wherein R8 is hydrogen.", "34.A compound of claim 3 wherein R9 is optionally substituted lower aliphatic group or optionally substituted monocyclic aromatic group.", "35.A compound of claim 34 wherein R9is optionally substituted lower alkyl or optionally substituted monocyclic heteroaryl.", "36.A compound of claim 34 wherein R9is optionally (carboxy, (loweralkyl)SO2NH—, (lower alkyl)HNCO—, hydroxy, phenyl, heteroaryl, or (lower alkyl)OC(O)NH—)-substituted lower alkyl, or optionally substituted monocyclic heteroaryl.", "37.A compound of claim 3 wherein R9 is lower alkyl substituted by (mono- or di-)MeOC(O)NH—.", "38.A compound of claim 3 wherein R9 is (carboxy, (lower alkyl)HNCO— or tetrazolyl)substituted lower alkyl.", "39.A compound of claim 3 wherein R9 is 3-carboxypropyl, 2-tetrazol-5ylpropyl, 3-(N-methylcarboxamido)propyl or 3-carboxy-2,2-dimethylpropyl.", "40.A compound of claim 3 wherein R9 is 3-carboxypropyl, 2-tetrazol-5ylpropyl or 3-(N-methylcarboxamido)propyl.", "41.A compound of claim 3 wherein R9 is optionally subtituted lower alkyl.", "42.A compound of claim 3 wherein R9 is 1-hydroxy-2-phenylethyl, isopropyl or 1-butyl.", "43.A compound of claim 3 wherein R9 is isopropyl or t-butyl.", "44.A compound of claim 3 wherein R9 is selected from the group consisting of 45.A compound of claim 3 wherein R9 is pyrazinyl.", "46.A compound of claim 3 wherein R10 is hydrogen or optionally substituted lower aliphatic group.", "47.A compound of claim 46 wherein R10 is hydrogen or lower alkyl.", "48.A compound of claim 47 wherein R10 is hydrogen.", "49.A compound of claim 3 wherein as a substituted monocyclic azaheterocyclyl is substituted pyrrolidinyl.", "50.A compound of claim 3 wherein as a substituted monocyclic azaheterocyclyl is optionally substituted or optionally substituted wherein Ar is R2 that comprises an aromatic moiety.", "51.A compound of claim 3 wherein as a substituted monocyclic azaheterocyclyl is optionally substituted 52.A compound of claim 3 wherein 53.A compound of claim 3 wherein as an optionally substituted multicyclic azaheterocyclyl is optionally substituted 54.A compound of claim 3 wherein as an optionally substituted multicyclic azaheterocyclyl is optionally substituted 55.A compound of claim 3 wherein as an optionally substituted multicyclic azaheterocyclenyl is optionally substituted 56.A compound of claim 3 wherein as an optionally substituted multicyclic azaheterocyclenyl is optionally substituted 57.A compound of claim 3 wherein as an optionally substituted multicyclic azaheterocyclenyl is optionally substituted 58.A compound of claim 3 wherein the —C(O)—N(R4)—R3—C(O)R0 C(O)NR2R1 moiety attached to is attached a carbon α to the nitrogen atom.", "59.A compound of claim 58 wherein L is —C(O)— or —OC(O)—.", "60.A compound of claim 59 wherein n is 0.61.A compound of claim 59 wherein n is 1.62.A compound as claimed in claim 1 selected from the group consisting of: or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.", "63.A pharmaceutical compositions comprising a pharmaceutically acceptable amount of the compound of claim 3 and a pharmaceutically acceptable carrier.", "64.A method for inhibiting HCV protease comprising contacting the protease with a compound claim 3.65.A method for treating a patient suffering from an HCV infection or physiological conditions related to the infection comprising administering to the patient a pharmaceutically effective amount of a compound of claim 3.66.A method for treating a patient suffering from an HCV infection or physiological conditions related to the infection comprising administering to the patient a pharmaceutically effective amount of a compound of claim 3 in combination with a pharmaceutically effective amount of another anti-HCV therapeutic.", "67.The method of claim 66 wherein the anti-HCV therapeutic is interferon or derivatized interferon.", "68.A pharmaceutical composition, comprising a hepatitis C virus serine protease inhibitor, an interferon having anti-hepatitis C virus activity, and a pharmaceutically acceptable carrier.", "69.The pharmaceutical composition of claim 68, further comprising a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "70.A pharmaceutical composition, comprising a hepatitis C virus serine protease inhibitor, a compound having anti-hepatitis C virus activity, and a pharmaceutically acceptable carrier, wherein said compound is other than an interferon.", "71.The pharmaceutical composition of claim 69, wherein said hepatitis C virus serine protease inhibitor, said interferon, and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount, and a combination thereof.", "72.A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a hepatitis C virus serine protease inhibitor compound of claim 3; an interferon selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastoid interferon, and interferon tau; and a compound having anti-hepatitis C virus activity selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, double stranded RNA, double stranded RNA complexed with tobramycin, Imiquimod, ribavirin, an inosine 5′-monophosphate dehydrogenase inhibitor, amantadine, and rimantadine and wherein said serine protease inhibitor, said interferon and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount and a combination thereof.", "73.A method of treating or preventing a hepatitis C virus infection in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a combination of a hepatitis C virus serine protease inhibitor and an interferon having anti-hepatitis C virus activity.", "74.The method of claim 73, wherein said pharmaceutically effective amount of said combination further comprises a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "75.A method of treating or preventing a hepatitis C virus infection in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a combination of a hepatitis C virus serine protease inhibitor and a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "76.The method of claim 74, wherein said hepatitis C virus serine protease inhibitor, said interferon, and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount, and a combination thereof.", "77.A method of treating or preventing a hepatitis C infection in a patient in need thereof comprising a hepatitis C virus serine protease inhibitor compound claim 3; an interferon selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastoid interferon, and interferon tau; and a compound having anti-hepatitis C virus activity selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, double stranded RNA, double stranded RNA complexed with tobramycin, Imiquimod, ribavirin, an inosine 5′-monophosphate dehydrogenase inhibitor, amantadine, and rimantadine and wherein said serine protease inhibitor, said interferon and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount and a combination thereof.", "83.A kit or pharmaceutical pack, comprising a plurality of separate containers, wherein at least one of said containers contains a hepatitis C virus serine protease inhibitor and at least another of said containers contains an interferon having anti-hepatitis C virus activity.", "84.A kit or pharmaceutical pack, comprising a plurality of separate containers, wherein at least one of said containers contains a hepatitis C virus serine protease inhibitor and at least another of said containers contains a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "85.A kit or pharmaceutical pack, comprising a plurality of separate containers, wherein at least one of said containers contains a hepatitis C virus serine protease inhibitor, at least another of said containers contains an interferon having anti-hepatitis C virus activity, and at least another of said containers contains a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "86.The kit or pharmaceutical pack of claim 85, wherein said hepatitis C virus serine protease inhibitor, said interferon, and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount, and a combination thereof.", "87.A kit or pharmaceutical pack comprising a plurality of separate containers wherein at least one of said containers contains a hepatitis C virus serine protease inhibitor compound of claims 3; at least another of said containers contains an interferon selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastoid interferon, and interferon tau; and at least another of said containers contains a compound having anti-hepatitis C virus activity selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, double stranded RNA, double stranded RNA complexed with tobramycin, Imiquimod, ribavirin, an inosine 5′-monophosphate dehydrogenase inhibitor, amantadine, and rimantadine and wherein said serine protease inhibitor, said interferon and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount and a combination thereof.", "88.A method of inhibiting hepatitis C virus replication in a cell, comprising contacting said cell, a hepatitis C virus serine protease inhibitor, and an interferon having anti-hepatitis C virus activity.", "89.The method of claim 88, further comprising contacting said cell and a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "90.A method of inhibiting hepatitis C virus replication in a cell, comprising contacting said cell, a hepatitis C virus serine protease inhibitor, and a compound having anti-hepatitis C virus activity, wherein said compound is other than an interferon.", "91.The method of claim 88, wherein said hepatitis C virus serine protease inhibitor, said interferon, and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount, and a combination thereof.", "92.A method of inhibiting heptatitis C virus replication in a cell comprising contacting said cell with a hepatitis C virus serine protease inhibitor compound of claim 3; an interferon selected from the group consisting of interferon alpha 2B, pegylated interferon alpha, consensus interferon, interferon alpha 2A, lymphoblastoid interferon, and interferon tau; and a compound having anti-hepatitis C virus activity selected from the group consisting of interleukin 2, interleukin 6, interleukin 12, a compound that enhances the development of a type 1 helper T cell response, double stranded RNA, double stranded RNA complexed with tobramycin, Imiquimod, ribavirin, an inosine 5′-monophosphate dehydrogenase inhibitor, amantadine, and rimantadine and wherein said serine protease inhibitor, said interferon and said compound having anti-hepatitis C virus activity are each present in an amount selected from the group consisting of a pharmaceutically effective amount, a subclinical pharmaceutically effective amount and a combination thereof.", "93.A compound of formula 24 wherein: is optionally substituted cycloalkyl or optionally substituted fused arylcycloalkyl; R11 is —CO2R13; R12 is an iminic glycinimide derivative adduct; and R13 is acid protecting group or optionally substituted aliphatic group.", "94.A compound of claim 93 wherein: optionally substituted cycloalkyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms optionally substituted with one or more ring group substituents; optionally substituted fused arylcycloalkyl means a fused arylcycloalkyl optionally substituted with one or more ring group substituents; optionally substituted aliphatic group are alkyl, alkenyl, or alkynyl optionally substituted with an aliphatic group substituent; an iminic glycinimide derivative adduct is a compound selected from the group consisting of wherein: R16 is an acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; R17 is optionally substituted aryl, optionally substituted aliphatic group, R18 is hydrogen, alkyl, or alkylthio; or optionally substituted aryl; wherein; ring group substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclycarbonyl or its thioxoinalue alroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl or when the ring system is saturated or partially saturated, the ring group substituents further include, methylene (H2C═), oxo (O═) and thioxo (S═); and aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl; and aryl means an aromatic monocyclic or multicyclic ring system of 6 to 14 carbon atoms.", "95.A compound according to claim 94 where R11 is —CO2R13.96.A compound according to claim 95 where R13 is an optionally substituted aliphatic group.", "97.A compound according to claim 96 where R13 is an alkyl group.", "98.A compound according to claim 97 where R13 is lower alkyl.", "99.A compound according to claim 98 where R13 is methyl.", "100.A compound according to claim 99 where R12 is wherein: R15 is optionally substituted aliphatic group; R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; R17 is optionally substituted aryl, optionally substituted aliphatic group, R18 is hydrogen, alkyl, or alkylthio; or optionally substituted aryl; R17 and R18 taken together with the carbon to which R17 and R18 are attached {circle over (S)} is a solid phase.", "101.A compound according to claim 100 where R14 is —CO2R16.102.A compound according to claim 101 where R16 is optionally substituted aliphatic.", "103.A compound according to claim 102 where R16 is alkyl.", "104.A compound according to claim 103 where R16 is lower alkyl.", "105.A compound according to claims 104 where R16 is t-Bu.", "106.A compound according to claims 105 where R17 is optionally substituted aryl.", "107.A compound according to claim 106 where R17 is phenyl.", "108.A compound according to claim 107 where R18 is optionally substituted aryl.", "109.A compound according to claim 108 where R18 is phenyl.", "110.A compound of formula 25 wherein: R15 is optionally substituted aliphatic group; and R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group.", "111.A compound according to claim 110 wherein: optionally substituted aliphatic groups are alkyl, alkenyl, or alkynyl optionally substituted with one or more aliphatic group substituents; optionally substituted aryl means an aromatic monocyclic or mylticyclic ring systems of 6 to 14 carbon atoms optionally substituted with one or more ring group substituents; wherein; ring group substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue; aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl or when the ring system is saturated or partially saturated, the “ring group substituents” further include, methylene (H2C═), oxo (O═) and thioxo (S═); and aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "112.A compound according to claim 111 where R14 is —CO2R16.113.A compound according to claim 112 where R16 is optionally substituted aliphatic.", "114.A compound according to claim 113 where R16 is alkyl.", "115.A compound according to claim 114 where R16 is lower alkyl.", "116.A compound according to claim 115 where R16 is t-Bu.", "117.A compound of formula 26 wherein: po is amide protecting group; R15 is optionally substituted aliphatic group; and R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group.", "118.A compound according to claim 117 wherein: optionally substituted aliphatic groups are alkyl, alkenyl, or alkynyl optionally substituted with one or more aliphatic group substituents; optionally substituted aryl means an aromatic monocyclic or mylticyclic ring systems of 6 to 14 carbon atoms optionally substituted with one or more ring group substituents; wherein; ring group substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl or when the ring system is saturated or partially saturated, the ring group substituents further include, methylene (H2C═), oxo (O═) and thioxo (S═); and aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "119.A compound according to claim 118 where R14 is —CO2R16.120.A compound according to claim 119 where R16 is optionally substituted aliphatic.", "121.A compound according to claim 120 where R16 is alkyl.", "122.A compound according to claim 121 where R16 is loweralkyl.", "123.A compound according to claim 122 where R16 is t-Bu.", "124.A compound according to claim 123 where p0 is selected from the group consisting of BOC, CBz, and —CO2alkyl.", "125.A compound according to claim 124 where p0 is BOC.", "126.A compound of formula 27 wherein: po is amide protecting group; R15 is optionally substituted aliphatic group; and R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group.", "127.A compound according to claim 126 wherein: optionally substituted aliphatic groups are alkyl, alkenyl, or alkynyl optionally substituted with one or more aliphatic group substituents; optionally substituted aryl means an aromatic monocyclic or mylticyclic ring systems of 6 to 14 carbon atoms optionally substituted with one or more ring group substituents; wherein; ring group substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue ,heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl or when the ring system is saturated or partially saturated, the ring group substituents further include, methylene (H2C═), oxo (O═) and thioxo (S═); and aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroaryl sulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "128.A compound according to claim 127 where R14 is —CO2R16.129.A compound according to claim 128 where R16 is optionally substituted aliphatic.", "130.A compound according to claim 129 where R16 is alkyl.", "131.A compound according to claim 130 where R16 is lower alkyl.", "132.A compound according to claim 131 where R16 is t-Bu.", "133.A compound according to claim 132 where p0 is selected from the group consisting of BOC, CBz, and —CO2alkyl.", "134.A compound according to claim 133 where p0 is BOC.", "135.A process for preparing a chiral bicycloprolinate compound of formula 28 comprising the steps of: (a) cleaving and cyclizing a compound of formula 24 wherein: is optionally substituted cycloalkyl or optionally substituted fused arylcycloalkyl; R11 is —CO2R3; R12 is an iminic glycinimide derivative adduct; R13 is acid protecting group or optionally substituted aliphatic group; under cleaving and cyclizing conditions to form a compound of formula 25 wherein: R15 is optionally substituted aliphatic group; R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; and (b) protecting the nitrogen of the lactam moiety in the compound of formula 25 with an amide protecting group to form a compound of formula 26 wherein: p0 is amide protecting group; R14 is as described herein; and (c) reducing the compound of formula 26 under reducing conditions to form a compound of formula 27 wherein: po and R14 are as described herein; and (d) deprotecting the compound of formula 27 under deprotecting conditions to form a compound of formula 28 wherein: R14 is as described herein.", "136.A compound according to claim 135 wherein: optionally substituted aliphatic groups are alkyl, alkenyl, or alkynyl optionally substituted with one or more aliphatic group substituents; optionally substituted aryl means an aromatic monocyclic or mylticyclic ring systems of 6 to 14 carbon atoms optionally substituted with one or more ring group substituents; optionally substituted cycloalkyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms optionally substituted with one or more ring group substituents; optionally substituted fused arylcycloalkyl means a fused arylcycloalkyl optionally substituted with one or more ring group substituents; an iminic glycinimide derivative adduct is a compound selected from the group consisting of wherein: R16 is an acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; R17 is optionally substituted aryl, optionally substituted aliphatic group, R18 is hydrogen, alkyl, or alkylthio; or optionally substituted aryl; wherein; ring group substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2 NC(O)O, Y1Y2NC(O)NY1— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl or when the ring system is saturated or partially saturated, the ring group substituents further include, methylene (H2C═), oxo (O═) and thioxo (S═); and aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "137.The process of claim 136 further comprising the step wherein the compound of formula 24 is prepared by effecting a Michael addition with an iminic glycinimide compound on a compound of formula 29 wherein: is optionally substituted cycloalkenyl or optionally substituted fused arylcycloalkenyl; wherein: the compound of formula 29 may be prepared by esterifying a compound of formula 29a wherein: R11a is —CHO, —COR15, —C≡N, or —CONR15R15.138.The process of claim 137 wherein the process is carried out at a temperature between 0° C. and −78° C. 139.The process of claim 138 wherein the process is carried out at −60°.", "140.The process of claim 139 wherein the process is catalyzed by a chiral phase transfer catalyst.", "141.The process of claim 139 wherein the process is catalyzed by a nonchiral phase transfer catalyst.", "142.The process of claim 141 wherein the protecting group is BOC.", "143.The process of claim 142 wherein the iminic glycinimide is (N-diphenylmethylene)-glycine tert-butyl ester.", "162.A compound of claim 1 wherein: R0 is a bond; R1 is hydrogen; R2 is lower alkyl optionally substituted with 1 to 3 aliphatic group substituents; or lower cycloalky optionally substituted with 1 to 3 cyclic group substituents; R3 and R5 are each independently methylene optionally substituted with 1 to 3 aliphatic group substitutents; R4, R6, R8 and R10 are hydrogen; R7 is methylene substituted with cycloalkyl, lower alkyl or aryl; or or (1,1- or 1,2-)cycloalkenyl optionally substituted with cycloalkyl, lower alkyl or aryl; R9 is lower alkyl optionally substituted with 1 to 3 aliphatic group substituents; or heteroaryl optionally substituted with 1 to 3 cyclic group substituents; or heterocyclic optionally substituted with 1 to 3 cyclic group substituents; is monocyclic azaheterocyclyl, multicyclic azaheterocyclyl, or multicyclic azaheterocyclenyl optionally substituted with from 1 to 3 cyclic group substituents; and L is —C(O)—, —OC(O)—.", "163.A compound of claim 162 wherein: aliphatic group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, aryl sulfonyl, heteroaryl sulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl; ring group substituents means aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), acid biostere, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2 N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl,or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl, or when a ring system is saturated or partially saturated, the “ring group substituents” further include, methylene (H2C═), oxo (O═) and thioxo (S═); aryl means an aromatic monocyclic or multicyclic ring system of 6 to 14 carbon atoms; cycloalkyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms; cycloalkenyl means a non-aromatic mono- or multicyclic ring system of 3 to 10 carbon atoms which contain at least one carbon-carbon double bond; cyclyl means cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl; heterocyclyl means a non-aromatic saturated monocyclic or multicyclic ring system of about 3 to about 10 carbon atomsin which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon; and heteroaryl means an aromatic monocyclic or multicyclic ring system of about 5 to about 14 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon.", "166.A compound of claim 163 wherein the optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group of R9 is substituted with at least one heteroaryl substituent.", "167.A compound of claim 163 wherein the optionally substituted aromatic group of R9 is optionally substituted heteroaryl.", "168.A compound of claim 166 wherein the optionally substituted aliphatic group of R9 is optionally substituted alkylheteroaryl.", "169.The process of claim 142 wherein the compound of formula 29 is 1-carboxy-1-cyclopentene methyl ester.", "170.A compound of the formula or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Infection by the HCV is a compelling human medical problem and is now recognized as the causative agent for most cases of non-A, non-B hepatitis.", "The HCV is thought to infect chronically 3% of the world's population [A. Alberti et al., “Natural History of Hepatitis C,” J. Hepatology, 31, (Suppl.", "1), 17-24 (1999)].", "In the United States alone the infection rate is 1.8% or 3.9 million people [M. J.", "Alter, “Hepatitis C Virus Infection in the United States,” J. Hepatology, 31, (Suppl.", "1), 88-91 (1999)].", "Of all patients infected over 70% develop a chronic infection that is believed to be a major cause of cirrhosis and hepatocellular carcinoma.", "[D. Lavanchy, “Global Surveillance and Control of Hepatitis C,” J.", "Viral Hepatitis, 6, 35-47 (1999)] The replication of the HCV encompasses genomic encoding a polyprotein of 3010-3033 amino acids [Q.-L. Choo, et al., “Genetic Organization and Diversity of the Hepatitis C Virus”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 88, 2451-2455 (1991); N. Kato et al., “Molecular Cloning of the Human Hepatitis C Virus Genome Prom Japanese Patients with Non-A, Non-B Hepatitis”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 87, 9524-9528 (1990); A. Takamizawa et al., “Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers”, J.", "Virol., 65, 1105-1113 (1991)].", "The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication.", "The NS proteins are derived by proteolytic cleavage of the polyprotein [R. Bartenschlager et al., “Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions”, J.", "Virol., 67, 3835-3844 (1993); A. Grakoui et al.", "“Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites”, J.", "Virol., 67, 2832-2843 (1993); A. Grakoui et al., Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products”, J.", "Virol., 67, 1385-1395 (1993); L. Tomei et al., “NS3 is a serine protease required for processing of hepatitis C virus polyprotein”, J.", "Virol., 67, 4017-4026 (1993)].", "In fact, it is the first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein [C. Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”, J.", "Virol., 68, 8147-8157 (1994)].", "The HCV NS protein 3 (NS3) contains a serine protease activity that helps in the processing of the majority of the viral enzymes, and thus is considered essential for viral replication and infectivity.", "The essentiality of the NS3 protease was inferred from the fact that mutations in the yellow fever virus NS3 protease decreases viral infectivity [T. J.", "Chambers et al., “Evidence that the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 87, 8898-8902 (1990)].", "More recently, it was demonstrated that mutations at the active site of the HCV NS3 protease could completely abolish the HCV infection in a chimpanzee model [C. M. Rice et al.", "“Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′-nontranslated region are essential for virus replication in vivo.” J.", "Virol., 74(4) 2046-51 (2000)].", "The HCV NS3 serine protease is also considered essential for viral replication as it and its associated cofactor, NS4A, help in the processing of all of the viral enzymes.", "This processing appears to be analogous to that carried out by the human immunodeficiency virus (“HIV”) aspartyl protease.", "In addition, the demonstrated use of HIV protease inhibitors as potent antiviral agents in man demonstrates that interrupting a protease protein processing stage in the viral life cycle does result in therapeutically active agents.", "Consequently, the protease enzyme is an attractive target for drug discovery.", "Several potential HCV protease inhibitors have been described.", "PCT Publications Numbers WO 00/09558, WO 00/09543, WO 99/64442, WO 99/07733, WO 99107734, WO 99/50230, WO98/46630, WO 98/17679 and WO 97/43310, U.S. Pat.", "No.", "5,990,276, M. Llinás-Brunet et al., Bioorg.", "Med.", "Chem.", "Lett., 8, 1713-1718 (1998), W. Han et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 711-713 (2000), R. Dunsdon et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 1571-1579 (2000), M. Llinás-Brunet et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 2267-2270 (2000), and S. LaPlante et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 2271-2274 (2000) each describe potential HCV NS3 protease inhibitors.", "Unfortunately, there are no serine protease inhibitors available currently as anti-HCV agents.", "In fact, there are no anti-HCV therapies except interferon-α, interferon-α/ribavirin combination and more recently pegylated inteferon-α.", "The sustained response rates for the interferon-α therapies and interferon-α/ribavirin however tend to be low (<50%) and the side effects exhibited by the therapies tend to be significant and severe [M. A. Walker, “Hepatitis C Virus: an Overview of Current Approaches and Progress,” DDT, 4, 518-529 (1999); D. Moradpour et al., “Current and Evolving Therapies for Hepatitis C,” Eur.", "J. Gastroenterol.", "Hepatol., 11, 1199-1202 (1999); H. L. A. Janssen et al., “Suicide Associated with Alfa-Interferon Therapy for Chronic Viral Hepatitis,” J.", "Hepatol., 21, 241-243 (1994); and P. F. Renault et al., “Side effects of alpha interferon”, Seminars in Liver Disease 9, 273-277, (1989)].", "Furthermore, the interferon therapies only induce long term remission in only a fraction (˜25%) of cases [O. Weiland, “Interferon Therapy in Chronic Hepatitis C Virus Infection”, FEMS Microbiol.", "Rev., 14, 279-288(1994)].", "The aforesaid problems with the interferon-a therapies has even led to the development and clinical study of pegylated derivatized interferon-α compounds as improved anti-HCV therapeutics.", "In view of the current situation regarding anti-HCV therapeutics, it is clear that there is a need for more effective and better tolerated therapies.", "Furthermore, synthesis of complex peptidomimetic compounds has long been hampered by the nonstereoselective nature of most synthetic organic processes.", "It is well known that the therapeutic activity of enantiomers of peptidomimetic compounds varies widely.", "It is therefore of great benefit to provide such stereospecific synthetic processes.", "Previous attempts to synthesize chirally specific bicycloprolinate intermediates, useful in the synthesis of the present therapeutic peptidomimetic protease inhibitors have suffered from being non enatioselective, or diasteroselective, or long encompassing synthetic pathways, or being unsuitable for preparing large quantities of product.", "Thus, there is also a need for a means of preparing large quantities of bicycloprolinates in a diastereoselective manner and enantiomerically enriched form." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a peptidomimetic compound of formula 1 wherein: R 0 is a bond or difluoromethylene; R 1 is hydrogen, optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R 2 and R 9 are each independently optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R 3 , R 5 and R 7 are each independently (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted methylene or optionally substituted ethylene), optionally substituted (1,1- or 1,2-)cycloalkylene or optionally substituted (1,1- or 1,2-)heterocyclylene; R 4 , R 6 , R 8 and R 10 are each independently hydrogen or optionally substituted aliphatic group; is substituted monocyclic azaheterocyclyl or optionally substituted multicyclic azaheterocyclyl, or optionally substituted multicyclic azaheterocyclenyl wherein the unsaturatation is in the ring distal to the ring bearing the R 9 -L-N(R 8 )—R 7 —C(O)—) n N(R 6 )—R 5 —C(O)—N moiety and to which the —C(O)—N(R 4 )—R 3 —C(O)C(O)NR 2 R 1 moiety is attached; L is —C(O)—, —OC(O)—, —NR 10 C(O)—, —S(O) 2 —, or —NR 10 S(O) 2 —; and n is 0 or 1, or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug, provided when is substituted then L is —OC(O)— and R 9 is optionally substituted aliphatic, or at least one of R 3 , R 5 and R 7 is (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted ethanediyl), or R 4 is optionally substituted aliphatic.", "This inventions also provides a compound having the structural formula: wherein: R 1 is hydrogen, optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R 2 and R 9 are each independently optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R 3 , R 5 and R 7 are each independently (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted methanediyl or optionally substituted ethanediyl); R 4 , R 6 , R 8 and R 10 are each independently is hydrogen or optionally substituted aliphatic group; is substituted monocyclic azaheterocyclyl or optionally substituted multicyclic azaheterocyclyl, or optionally substituted multicyclic azaheterocyclenyl wherein the unsaturatation is in the ring distal to the ring bearing the R 9 -L-(N(R 8 )—R 7 —C(O)—) n (R 6 )—R 5 —C(O)—N moiety and to which the —C(O)—N(R 4 )—R 3 —C(O)C(O)NR 2 R 1 moiety is attached; L is —C(O)—, —OC(O)—, —NR 10 C(O)—, —S(O) 2 —, or —NR 10 S(O) 2 —; and n is 0 or 1, or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug, provided when is substituted then L is —C(O)— and R 9 is optionally substituted aliphatic, or at least one of R 3 , R 5 and R 7 is (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted ethanediyl), or R 4 is optionally substituted aliphatic.", "The invention is also directed to a pharmaceutical composition comprising a compound of formula 1, and method for using the compound of formula 1 for inhibiting HCV protease, or treating or preventing an HCV infection in patients or physiological condition related to the infection.", "The invention is also directed to a stereoselective process for preparing a chiral bicycloprolinate compound that is an intermediate useful in preparing a compound of formula 1.The synthetic process comprises the steps of: (a) cleaving and cyclizing a compound of formula 24 wherein: is optionally substituted cycloalkyl or optionally substituted fused arylcycloalkyl; R 11 is —CO 2 R 13 ; R 12 is an iminic glycinimide adduct; R 13 is acid protecting group or optionally substituted aliphatic group; under cleaving and cyclizing conditions to form a compound of formula 25 wherein: R 15 is optionally substituted aliphatic group; R 16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; and (b) protecting the nitrogen of the lactam moiety in the compound of formula 25 with an amide protecting group to form a compound of formula 26 wherein: p o is amide protecting group; R 14 is as described herein; and (c) reducing the compound of formula 26 under reducing conditions to form a compound of formula 27 wherein: p o and R 14 are as described herein; and (d) deprotecting the compound of formula 27 under deprotecting conditions to form a compound of formula 28 wherein: R 14 is as described herein.", "The invention is also directed to the above synthetic process further comprising the step wherein the compound of formula 24 is prepared by effecting a Michael addition with an iminic glycinimide compound on a compound of formula 29 wherein: is optionally substituted cycloalkenyl or optionally substituted fused arylcycloalkenyl; R 11 is —CO 2 R 13 ; wherein: the compound of formula 29 may be prepared by esterifying a compound of formula 29a wherein: is optionally substituted cycloalkenyl or optionally substituted fused arylcycloalkenyl; R 11a is —CHO, —COR 15 , —C≡N , or —CONR 15 R 15 ; and R 15 is as described herein.", "Notably, one skilled in the art would know that conversion of ketones to esters may be accomplished, for example, by a Bayer-Villiger reaction.", "Conversion of nitrites and amides to esters may be accomplished, for example, by aqueous hydrolysis followed by further esterification.", "Conversion of aldehydes to esters may be accomplished, for example, by oxidation of the aldehyde followed by esterification.", "Another aspect of the invention is a compound of formula 1 wherein the substituents are selected from a combination of preferred or particular embodiments as defined herein.", "Another aspect of the invention is a compound of formulae 24-29 wherein the substituents are selected from a combination of preferred or particular embodiments as defined herein.", "Another aspect of the invention are pharmaceutical compositions comprising, in addition to one or more HCV serine protease inhibitors, one or more interferons or compounds that induce the production of interferons that exhibit anti-HCV activity and/or one or more compounds having anti HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, and a pharmaceutically acceptable carrier.", "Another aspect of the invention are methods of treating or preventing a HCV infection in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a combination of one or more HCV serine protease inhibitors; one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity; and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "The invention is also directed to the use of one or more HCV serine protease inhibitors in combination with one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, to prepare a medicament for treating or preventing a HCV infection in a patient in need thereof.", "The present invention is also directed to a kit or pharmaceutical pack for treating or preventing HCV infection in a patient, wherein the kit or pharmaceutical pack comprises a plurality of separate containers, wherein at least one of said containers contains one or more HCV serine protease inhibitors (alone or in combination with a pharmaceutically acceptable carrier or diluent), at least another of said containers contains one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity, (alone or in combination with a pharmaceutically acceptable carrier or diluent) and, optionally, at least another of said containers contains one or more compounds having anti-HCV activity (alone or in combination with a pharmaceutically acceptable carrier or diluent), including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "The amount of the HCV serine protease inhibitor(s), interferon(s), or anti-HCV compound(s) in any of the foregoing applications can be a pharmaceutically effective amount, a subclinical anti-HCV effective amount, or combinations thereof, so long as the final combination of HCV serine protease inhibitor(s), interferon(s), or compounds that induce the production of an interferon that exhibit anti-HCV activity, and/or anti-HCV compound(s) comprises a pharmaceutically effective amount of compounds that is effective in treating or preventing HCV infection in a patient." ], [ "This invention is directed to peptidomimetic compounds and intermediates thereto, their preparation including stereoselective synthetic processes to intermediates, pharmaceutical compositions containing the peptidomimetic compounds, and the use of the peptidomimetic compounds or compositions thereof as protease inhibitors, particularly as serine protease inhibitors, and more particularly as hepatitis C virus (“HCV”) NS3 protease inhibitors.", "The peptidomimetic compounds, as HCV NS3 protease inhibitors, are particularly useful in interfering with the life cycle of the hepatitis C virus and in treating or preventing an HCV infection or physiological conditions associated therewith.", "The present invention is also directed to methods of combination therapy for inhibiting HCV replication in cells, or for treating or preventing an HCV infection in patients using the peptidomimetic compounds or pharmaceutical compositions, or kits and pharmaceutical packs therefor.", "According to the present invention included as pharmaceutical compositions are those comprising an inhibitor of HCV serine protease in combination with an interferon having anti-HCV activity; an inhibitor of HCV serine protease in combination with a compound, other than an interferon, having anti-HCV activity; or an inhibitor of HCV serine protease in combination with both an interferon having anti-HCV activity and a compound, other than an interferon, having anti-HCV activity.", "Further the present invention is directed to stereoselective methods for preparing chiral bicycloprolinate intermediates useful in the synthesis of the peptidomimetic compounds.", "BACKGROUND OF THE INVENTION Infection by the HCV is a compelling human medical problem and is now recognized as the causative agent for most cases of non-A, non-B hepatitis.", "The HCV is thought to infect chronically 3% of the world's population [A. Alberti et al., “Natural History of Hepatitis C,” J. Hepatology, 31, (Suppl.", "1), 17-24 (1999)].", "In the United States alone the infection rate is 1.8% or 3.9 million people [M. J.", "Alter, “Hepatitis C Virus Infection in the United States,” J. Hepatology, 31, (Suppl.", "1), 88-91 (1999)].", "Of all patients infected over 70% develop a chronic infection that is believed to be a major cause of cirrhosis and hepatocellular carcinoma.", "[D. Lavanchy, “Global Surveillance and Control of Hepatitis C,” J.", "Viral Hepatitis, 6, 35-47 (1999)] The replication of the HCV encompasses genomic encoding a polyprotein of 3010-3033 amino acids [Q.-L. Choo, et al., “Genetic Organization and Diversity of the Hepatitis C Virus”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 88, 2451-2455 (1991); N. Kato et al., “Molecular Cloning of the Human Hepatitis C Virus Genome Prom Japanese Patients with Non-A, Non-B Hepatitis”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 87, 9524-9528 (1990); A. Takamizawa et al., “Structure and Organization of the Hepatitis C Virus Genome Isolated From Human Carriers”, J.", "Virol., 65, 1105-1113 (1991)].", "The HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication.", "The NS proteins are derived by proteolytic cleavage of the polyprotein [R. Bartenschlager et al., “Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions”, J.", "Virol., 67, 3835-3844 (1993); A. Grakoui et al.", "“Characterization of the Hepatitis C Virus-Encoded Serine Proteinase: Determination of Proteinase-Dependent Polyprotein Cleavage Sites”, J.", "Virol., 67, 2832-2843 (1993); A. Grakoui et al., Expression and Identification of Hepatitis C Virus Polyprotein Cleavage Products”, J.", "Virol., 67, 1385-1395 (1993); L. Tomei et al., “NS3 is a serine protease required for processing of hepatitis C virus polyprotein”, J.", "Virol., 67, 4017-4026 (1993)].", "In fact, it is the first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein [C. Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics”, J.", "Virol., 68, 8147-8157 (1994)].", "The HCV NS protein 3 (NS3) contains a serine protease activity that helps in the processing of the majority of the viral enzymes, and thus is considered essential for viral replication and infectivity.", "The essentiality of the NS3 protease was inferred from the fact that mutations in the yellow fever virus NS3 protease decreases viral infectivity [T. J.", "Chambers et al., “Evidence that the N-terminal Domain of Nonstructural Protein NS3 From Yellow Fever Virus is a Serine Protease Responsible for Site-Specific Cleavages in the Viral Polyprotein”, Proc.", "Natl.", "Acad.", "Sci.", "USA, 87, 8898-8902 (1990)].", "More recently, it was demonstrated that mutations at the active site of the HCV NS3 protease could completely abolish the HCV infection in a chimpanzee model [C. M. Rice et al.", "“Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′-nontranslated region are essential for virus replication in vivo.” J.", "Virol., 74(4) 2046-51 (2000)].", "The HCV NS3 serine protease is also considered essential for viral replication as it and its associated cofactor, NS4A, help in the processing of all of the viral enzymes.", "This processing appears to be analogous to that carried out by the human immunodeficiency virus (“HIV”) aspartyl protease.", "In addition, the demonstrated use of HIV protease inhibitors as potent antiviral agents in man demonstrates that interrupting a protease protein processing stage in the viral life cycle does result in therapeutically active agents.", "Consequently, the protease enzyme is an attractive target for drug discovery.", "Several potential HCV protease inhibitors have been described.", "PCT Publications Numbers WO 00/09558, WO 00/09543, WO 99/64442, WO 99/07733, WO 99107734, WO 99/50230, WO98/46630, WO 98/17679 and WO 97/43310, U.S. Pat.", "No.", "5,990,276, M. Llinás-Brunet et al., Bioorg.", "Med.", "Chem.", "Lett., 8, 1713-1718 (1998), W. Han et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 711-713 (2000), R. Dunsdon et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 1571-1579 (2000), M. Llinás-Brunet et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 2267-2270 (2000), and S. LaPlante et al., Bioorg.", "Med.", "Chem.", "Lett., 10, 2271-2274 (2000) each describe potential HCV NS3 protease inhibitors.", "Unfortunately, there are no serine protease inhibitors available currently as anti-HCV agents.", "In fact, there are no anti-HCV therapies except interferon-α, interferon-α/ribavirin combination and more recently pegylated inteferon-α.", "The sustained response rates for the interferon-α therapies and interferon-α/ribavirin however tend to be low (<50%) and the side effects exhibited by the therapies tend to be significant and severe [M. A. Walker, “Hepatitis C Virus: an Overview of Current Approaches and Progress,” DDT, 4, 518-529 (1999); D. Moradpour et al., “Current and Evolving Therapies for Hepatitis C,” Eur.", "J. Gastroenterol.", "Hepatol., 11, 1199-1202 (1999); H. L. A. Janssen et al., “Suicide Associated with Alfa-Interferon Therapy for Chronic Viral Hepatitis,” J.", "Hepatol., 21, 241-243 (1994); and P. F. Renault et al., “Side effects of alpha interferon”, Seminars in Liver Disease 9, 273-277, (1989)].", "Furthermore, the interferon therapies only induce long term remission in only a fraction (˜25%) of cases [O. Weiland, “Interferon Therapy in Chronic Hepatitis C Virus Infection”, FEMS Microbiol.", "Rev., 14, 279-288(1994)].", "The aforesaid problems with the interferon-a therapies has even led to the development and clinical study of pegylated derivatized interferon-α compounds as improved anti-HCV therapeutics.", "In view of the current situation regarding anti-HCV therapeutics, it is clear that there is a need for more effective and better tolerated therapies.", "Furthermore, synthesis of complex peptidomimetic compounds has long been hampered by the nonstereoselective nature of most synthetic organic processes.", "It is well known that the therapeutic activity of enantiomers of peptidomimetic compounds varies widely.", "It is therefore of great benefit to provide such stereospecific synthetic processes.", "Previous attempts to synthesize chirally specific bicycloprolinate intermediates, useful in the synthesis of the present therapeutic peptidomimetic protease inhibitors have suffered from being non enatioselective, or diasteroselective, or long encompassing synthetic pathways, or being unsuitable for preparing large quantities of product.", "Thus, there is also a need for a means of preparing large quantities of bicycloprolinates in a diastereoselective manner and enantiomerically enriched form.", "SUMMARY OF THE INVENTION The present invention relates to a peptidomimetic compound of formula 1 wherein: R0 is a bond or difluoromethylene; R1 is hydrogen, optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R2 and R9 are each independently optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R3, R5 and R7 are each independently (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted methylene or optionally substituted ethylene), optionally substituted (1,1- or 1,2-)cycloalkylene or optionally substituted (1,1- or 1,2-)heterocyclylene; R4, R6, R8 and R10 are each independently hydrogen or optionally substituted aliphatic group; is substituted monocyclic azaheterocyclyl or optionally substituted multicyclic azaheterocyclyl, or optionally substituted multicyclic azaheterocyclenyl wherein the unsaturatation is in the ring distal to the ring bearing the R9-L-N(R8)—R7—C(O)—)nN(R6)—R5—C(O)—N moiety and to which the —C(O)—N(R4)—R3—C(O)C(O)NR2R1 moiety is attached; L is —C(O)—, —OC(O)—, —NR10C(O)—, —S(O)2—, or —NR10S(O)2—; and n is 0 or 1, or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug, provided when is substituted then L is —OC(O)— and R9 is optionally substituted aliphatic, or at least one of R3, R5 and R7 is (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted ethanediyl), or R4is optionally substituted aliphatic.", "This inventions also provides a compound having the structural formula: wherein: R1 is hydrogen, optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R2 and R9 are each independently optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group; R3, R5 and R7 are each independently (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted methanediyl or optionally substituted ethanediyl); R4, R6, R8 and R10 are each independently is hydrogen or optionally substituted aliphatic group; is substituted monocyclic azaheterocyclyl or optionally substituted multicyclic azaheterocyclyl, or optionally substituted multicyclic azaheterocyclenyl wherein the unsaturatation is in the ring distal to the ring bearing the R9-L-(N(R8)—R7—C(O)—)n(R6)—R5—C(O)—N moiety and to which the —C(O)—N(R4)—R3—C(O)C(O)NR2R1 moiety is attached; L is —C(O)—, —OC(O)—, —NR10C(O)—, —S(O)2—, or —NR10S(O)2—; and n is 0 or 1, or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug, provided when is substituted then L is —C(O)— and R9is optionally substituted aliphatic, or at least one of R3, R5 and R7 is (optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group)(optionally substituted ethanediyl), or R4 is optionally substituted aliphatic.", "The invention is also directed to a pharmaceutical composition comprising a compound of formula 1, and method for using the compound of formula 1 for inhibiting HCV protease, or treating or preventing an HCV infection in patients or physiological condition related to the infection.", "The invention is also directed to a stereoselective process for preparing a chiral bicycloprolinate compound that is an intermediate useful in preparing a compound of formula 1.The synthetic process comprises the steps of: (a) cleaving and cyclizing a compound of formula 24 wherein: is optionally substituted cycloalkyl or optionally substituted fused arylcycloalkyl; R11 is —CO2R13; R12 is an iminic glycinimide adduct; R13 is acid protecting group or optionally substituted aliphatic group; under cleaving and cyclizing conditions to form a compound of formula 25 wherein: R 15 is optionally substituted aliphatic group; R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; and (b) protecting the nitrogen of the lactam moiety in the compound of formula 25 with an amide protecting group to form a compound of formula 26 wherein: po is amide protecting group; R14 is as described herein; and (c) reducing the compound of formula 26 under reducing conditions to form a compound of formula 27 wherein: po and R14 are as described herein; and (d) deprotecting the compound of formula 27 under deprotecting conditions to form a compound of formula 28 wherein: R14 is as described herein.", "The invention is also directed to the above synthetic process further comprising the step wherein the compound of formula 24 is prepared by effecting a Michael addition with an iminic glycinimide compound on a compound of formula 29 wherein: is optionally substituted cycloalkenyl or optionally substituted fused arylcycloalkenyl; R11 is —CO2R13; wherein: the compound of formula 29 may be prepared by esterifying a compound of formula 29a wherein: is optionally substituted cycloalkenyl or optionally substituted fused arylcycloalkenyl; R11a is —CHO, —COR15, —C≡N, or —CONR15R15; and R15 is as described herein.", "Notably, one skilled in the art would know that conversion of ketones to esters may be accomplished, for example, by a Bayer-Villiger reaction.", "Conversion of nitrites and amides to esters may be accomplished, for example, by aqueous hydrolysis followed by further esterification.", "Conversion of aldehydes to esters may be accomplished, for example, by oxidation of the aldehyde followed by esterification.", "Another aspect of the invention is a compound of formula 1 wherein the substituents are selected from a combination of preferred or particular embodiments as defined herein.", "Another aspect of the invention is a compound of formulae 24-29 wherein the substituents are selected from a combination of preferred or particular embodiments as defined herein.", "Another aspect of the invention are pharmaceutical compositions comprising, in addition to one or more HCV serine protease inhibitors, one or more interferons or compounds that induce the production of interferons that exhibit anti-HCV activity and/or one or more compounds having anti HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, and a pharmaceutically acceptable carrier.", "Another aspect of the invention are methods of treating or preventing a HCV infection in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a combination of one or more HCV serine protease inhibitors; one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity; and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "The invention is also directed to the use of one or more HCV serine protease inhibitors in combination with one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, to prepare a medicament for treating or preventing a HCV infection in a patient in need thereof.", "The present invention is also directed to a kit or pharmaceutical pack for treating or preventing HCV infection in a patient, wherein the kit or pharmaceutical pack comprises a plurality of separate containers, wherein at least one of said containers contains one or more HCV serine protease inhibitors (alone or in combination with a pharmaceutically acceptable carrier or diluent), at least another of said containers contains one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity, (alone or in combination with a pharmaceutically acceptable carrier or diluent) and, optionally, at least another of said containers contains one or more compounds having anti-HCV activity (alone or in combination with a pharmaceutically acceptable carrier or diluent), including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "The amount of the HCV serine protease inhibitor(s), interferon(s), or anti-HCV compound(s) in any of the foregoing applications can be a pharmaceutically effective amount, a subclinical anti-HCV effective amount, or combinations thereof, so long as the final combination of HCV serine protease inhibitor(s), interferon(s), or compounds that induce the production of an interferon that exhibit anti-HCV activity, and/or anti-HCV compound(s) comprises a pharmaceutically effective amount of compounds that is effective in treating or preventing HCV infection in a patient.", "BRIEF DESCRIPTION OF THE DRAWINGS The above and other aspects, features, and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings, all of which are given by way of illustration only, and are not limitative of the present invention, in which: FIG.", "1 shows the inhibition of HCV replicon RNA accumulation after 48 hour treatment of replicon-containing cells with Compound CU and interferon-alpha 2B, individually or in combination.", "FIG.", "2 graphically shows the isobol concavity exhibited by compounds used in combination that are antagonistic, additive, and synergistic according to the synergy calculation methods of Greco, Park and Rustom ((1990) Application of a New Approach for the Quantitation of Drug Synergism to the Combination of cis-Diamminedichloroplatinum and 1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327).", "FIG.", "3 shows the geometric relationship between a and the amount of curvature in the isobol.", "A hypothetical isobol at the E=50% effect level is displayed with a straight line isobol that would be expected under additivity.", "M is the point of intersection of the line y=x and the hypothetical isobol.", "N is the point of intersection of the line y=x and the straight line isobol.", "O is the origin (0,0).", "S gives a measure of the amount of curvature in the isobol, where S=ON/OM.", "ON is the distance from O to N and OM is the distance from O to M. The parameter α is related to S by the equation α=4(S2—S).", "FIG.", "4 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and interferon alpha-2B (Schering-Plough) using 6 dilutions of each compound in Experiment 1 FIG.", "5 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and interferon alpha-2A using 6 dilutions of each compound in Experiment 2.FIG.", "6 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and interferon alpha-2B (Schering-Plough) using 8 dilutions of each compound in Experiment 3.FIG.", "7 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and interferon alpha-2A using 8 dilutions of each compound in Experiment 4.FIG.", "8 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and ovine interferon tau using 8 dilutions of each compound in Experiment 5.FIG.", "9 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound EC and interferon alpha-2B (Schering-Plough) using 8 dilutions of each compound in Experiment 6.FIG.", "10 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound EC and interferon alpha-2A using 8 dilutions of each compound in Experiment 7.FIG.", "11 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound CU and interferon beta using 8 dilutions of each compound in Experiment 8.FIG.", "12 shows the isobol calculations using the method of Greco et al., supra, for the combination of compound EP and interferon alpha-2B (Schering-Plough) using 8 dilutions of each compound in Experiment 9.FIG.", "13 shows the isobol calculations using the method of Greco et al., supra, for the combination of Ribavirin and interferon alpha-2B (Schering-Plough) using 8 dilutions of each compound in Experiment 10.FIG.", "14 shows inhibition of HCV replicon RNA accumulation caused by treatment of replicon cells with either (A) Ribavirin alone or (B) interferon alpha-2B alone.", "In both panels, the measured inhibition as well as the inhibition corrected for cytotoxicity of the compounds is shown.", "DETAILED DESCRIPTION OF THE INVENTION The contents of each of the patent documents and other references cited herein are herein incorporated by reference in their entirety.", "As used above, and throughout the description of the invention, the following abbreviations, unless otherwise indicated, shall be understood to have the following meanings:— Designation Reagent or Fragment ACN acetonitrile AIBN 2,2′-azobisisobutyronitrile BOC or Boc tert-butyl carbamate BOP benzotriazol-1-yl-oxytris (dimethylamino)phosphonium hexafluorophosphate n-Bu3SnH tri-n-butyltin hydride t-Bu tert-butyl Cbz benzyl carbamate chiral PTC chiral phase transfer catalyst DAST (diethylamino)sulfur trifluoride (Et2NSF3) DCC dicyclocarbodiimide DCM dichloromethane (CH2Cl2) DIBAL-H Diisobutylaluminum hydride DIC 1,3-diisopropylcarbodiimide DIPEA diisopropylethylamine DMAP 4-(N,N-dimethylamino)pyridine DMP reagent Dess-Martin Periodinane reagent DMF dimethylformamide DMSO dimethylsulfoxide EA elemental analysis EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl eq equivalent(s) Et ethyl Et2O diethyl ether EtOH ethanol EtOAc ethyl acetate Et3Si triethylsilane FMOC 9-fluorenylmethoxycarbonyl H-Chg-OH HOAt 1-hydroxy-7-azabensotriazole HOBT 1-hydroxybenztriazole HOSu N-hydroxysuccinamide HPLC high performance liquid chromatography LAH lithium aluminum anhydride Me methyl MeI methyliodide MeOH methanol MeOC(O)Cl methyl chloroformate MOMCl methoxymethylchloride MOM methoxymethyl MS mass spectroscopy NaBH4 sodium borohydride Na2C4H4O6 sodium tartrate NMP N-methyl pyrrolidinone NMR nuclear magnetic resonance P- Polymer bond PyBOP benzotriazole-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphate TBD 1,5,7-triazabicyclo[4.4.0]-dec-5-ene RP-HPLC reverse phase - high pressure liquid chromatography TBSCl tert-butyldimethylsilyl chloride TCA trichloroacetic acid TFA trifluoroacetic acid Tf2O triflate anhydride THF tetrahydrofuran THP tetrahydropyran TLC thin layer chromatography As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:— “Acid bioisostere” means a group which has chemical and physical similarities producing broadly similar biological properties to a carboxy group (see Lipinski, Annual Reports in Medicinal Chemistry, “Bioisosterism In Drug Design∞ 21, 283 (1986); Yun, Hwahak Sekye, “Application Of Bioisosterism To New Drug Design” 33, 576-579, (1993); Zhao, Huaxue Tongbao, “Bioisosteric Replacement And Development Of Lead Compounds In Drug Design” 34-38, (1995); Graham, Theochem, “Theoretical Studies Applied To Drug Design:ab initio Electronic Distributions In Bioisosteres” 343, 105-109, (1995)).", "Exemplary acid bioisosteres include —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl and the like.", "“Acidic functional group” means a moiety bearing an acidic hydrogen.", "Exemplary acid functional groups include carboxyl (—C(O)OH), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, imidazolyl, mercapto, and the like, and an appropriate hydroxy such as an aromatic hydroxy, e.g., hydroxyphenyl.", "“Acid protecting group” means an easily removable group that is known in the art to protect an acidic hydrogen of a carboxyl group against undesirable reaction during synthetic procedures, e.g., to block or protect the acid functionality while the reactions involving other functional sites of the compound are carried out, and to be selectively removable.", "Such acid protecting groups are well known to those skilled in the art, having been extensively used in the protection of carboxyl groups, as described in U.S. Pat.", "Nos.", "3,840,556 and 3,719,667, the disclosures of which are hereby incorporated herein by reference.", "For suitable acid protecting groups, see T. W. Green and P. G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley and Sons, 1991.Acid protecting group also includes hydrogenation labile acid protecting group as defined herein.", "Exemplary acid protecting groups include esters such as substituted and unsubstituted C1-8 lower alkyl, e.g., methyl, ethyl, t-butyl, methoxymethyl, methylthiomethyl, 2,2,2-trichloroethyl and the like, tetrahydropyranyl, substituted and unsubstituted phenylalkyl such as benzyl and substituted derivatives thereof such as alkoxybenzyl or nitrobenzyl groups and the like, cinnamyl, dialkylaminoalkyl, e.g., dimethylaminoethyl and the like, trimethylsilyl, substituted and unsubstituted amides and hydrazides, e.g., amides and hydrazides of N,N-dimethylamine, 7-nitroindole, hydrazine, N-phenylhydrazine and the like, acyloxyalkyl groups such as pivaloyloxymethyl or propionyloxymethyl and the like, aroyloxyalkyl such as benzoyloxyethyl and the like, alkoxycarbonylalkyl such as methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl and the like, alkoxycarbonyloxyalkyl such as t-butyloxycarbonyloxymethyl and the like, alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and the like, alkylaminocarbonylaminoalkyl, such as methylaminocarbonylaminomethyl and the like, acylaminoalkyl such as acetylaminomethyl and the like, heterocyclylcarbonyloxyalkyl such as 4-methylpiperazinyl-carbonyloxymethyl and the like, dialkylaminocarbonylalkyl such as dimethylaminocarbonyl-methyl and the like, (5-(lower alkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like, and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-phenyl-2-oxo-1,3-dioxolen-4-yl) methyl and the like.", "“Acid labile amine protecting group” means an amine protecting group as defined herein which is readily removed by treatment with acid while remaining relatively stable to other reagents.", "A preferred acid labile amine protecting group is BOC.", "“Aliphatic” means alkyl, alkenyl or alkynyl as defined herein.", "“Aliphatic group substituent(s)” mean substituents attached to an aliphatic group as defined herein inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, methylene (H2C═), oxo (O═), thioxo (S═), Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3—, Y1Y2NSO2—, or Y3SO2NY1— wherein R2 is as defined herein, Y1 and Y2 are independently hydrogen, alkyl, aryl or heteroaryl, and Y3 is alkyl, cycloalkyl aryl or heteroaryl, or for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "Acidic/amide aliphatic group substituents are carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazolyl and Y12Y2NCO—.", "Non-acidic polar aliphatic group substituents are hydroxy, oxo (O═), thioxo (S═), acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—.", "Exemplary aliphatic groups bearing an aliphatic group substituent include methoxymethoxy, methoxyethoxy, ethoxyethoxy, (methoxy-, benzyloxy-, phenoxy-, or ethoxy-)carbonyl(methyl or ethyl), benzyloxycarbonyl, pyridylmethyloxy-carbonylmethyl, methoxyethyl, ethoxymethyl, n-butoxymethyl, cyclopentylmethyloxyethyl, phenoxypropyl, phenoxyallyl, trifluoromethyl, cyclopropyl-methyl, cyclopentylmethyl, carboxy(methyl or ethyl), 2-phenethenyl, benzyloxy, 1- or 2-naphthyl-methoxy, 4-pyridyl-methyloxy, benzyloxyethyl, 3-benzyloxyallyl, 4-pyridylmethyl-oxyethyl, 4-pyridylmethyl-oxyallyl, benzyl, 2-phenethyl, naphthylmethyl, styryl, 4-phenyl-1,3-pentadienyl, phenyl-propynyl, 3-phenylbut-2-ynyl, pyrid-3-ylacetylenyl and quinolin-3-ylacetylenyl, 4-pyridyl-ethynyl, 4-pyridylvinyl, thienylethenyl, pyridylethenyl, imidazolyl-ethenyl, pyrazinylethenyl, pyridylpentenyl, pyridylhexenyl and pyridylheptenyl, thienyl-methyl, pyridylmethyl, imidazolylmethyl, pyrazinylmethyl, tetrahydropyranylmethyl, tetrahydropyranyl-methyloxymethyl, and the like.", "“Acyl” means an H—CO— or (aliphatic or cyclyl)-CO— group wherein the aliphatic group is as herein described.", "Preferred acyls contain a lower alkyl.", "Exemplary acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyl, palmitoyl, acryloyl, propynoyl, cyclohexylcarbonyl, and the like.", "“Alkenoyl” means an alkenyl-CO— group wherein alkenyl is as defined herein.", "“Alkenyl” means an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain.", "Preferred alkenyl groups have 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain.", "Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkenyl chain.", "“Lower alkenyl” means about 2 to about 4 carbon atoms in the chain that may be straight or branched.", "Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexylbutenyl, decenyl, and the like.", "“Substituted alkenyl” means an alkenyl group as defined above which is substituted with one or more “aliphatic group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "Exemplary alkenyl alphatic group substituents include halo or cycloalkyl groups “Alkenyloxy” means an alkenyl-O— group wherein the alkenyl group is as herein described.", "Exemplary alkenyloxy groups include allyloxy, 3-butenyloxy, and the like.", "“Alkoxy” means an alkyl-O— group wherein the alkyl group is as herein described.", "Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.", "“Alkoxycarbonyl” means an alkyl-O—CO— group, wherein the alkyl group is as herein defined.", "Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, t-butyloxycarbonyl, and the like.", "“Alkyl” means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain.", "Preferred alkyl groups have 1 to about 12 carbon atoms in the chain, more preferred is lower alkyl as defined herein.", "Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.", "“Lower alkyl” means about 1 to about 4 carbon atoms in the chain that may be straight or branched.", "“Substituted alkyl” means an alkyl group as defined above which is substituted with one or more “aliphatic group substituents” (preferably 1 to 3) which may be the same or different, and are as defined herein.", "“Alkylsulfinyl” means an alkyl-SO— group wherein the alkyl group is as defined above.", "Preferred groups are those wherein the alkyl group is lower alkyl.", "“Alkylsulfonyl” means an alkyl-SO2-group wherein the Alkyl group is as defined above.", "Preferred groups are those wherein the Alkyl group is lower alkyl.", "“Alkylsulphonylcarbamoyl” means an alkyl-SO2—NH—C(═O)— group wherein the alkyl group is as herein described.", "Preferred alkylsulphonylcarbamoyl groups are those wherein the alkyl group is lower alkyl.", "“Alkylthio” means an alkyl-S— group wherein the alkyl group is as herein described.", "Exemplary alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio.", "“Alkynyl” means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain.", "Preferred alkynyl groups have 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain.", "Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkynyl chain.", "“Lower alkynyl” means about 2 to about 4 carbon atoms in the chain that may be straight or branched.", "The alkynyl group may be substituted by one or more halo.", "Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, octynyl, decynyl, and the like.", "“Substituted alkynyl” means alkynyl as defined above which is substituted with one or more “aliphatic group substituents” (preferably 1 to 3) which may be the same or different, and are as defined herein.", "“Amine protecting group” means an easily removable group that is known in the art to protect a nitrogen moietyof an amino or amide group against undesirable reaction during synthetic procedures and to be selectively removable.", "The use of amine/amide protecting groups is well known in the art for protecting groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley & Sons, New York (1991), incorporated herein by reference.", "Amine/amide protecting group also includes “acid labile amine/amide protecting group” and “hydrogenation labile amine/amide protecting group”.", "Exemplary amine/amide protecting groups are acyl, including formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxy-acetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl and the like, and acyloxy including methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC), 1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (CBZ), p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, and the like.", "“Amide protecting group” means an easily removable group that is known in the art to protect a nitrogen moiety of an amide group against undesirable reaction during synthetic procedures and to be selectively removable after its conversion to the amine.", "The use of amide protecting groups is well known in the art for protecting groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley & Sons, New York (1991), incorporated herein by reference.", "Amide protecting group also includes “acid labile amide protecting group” and “hydrogenation labile amide protecting group”.", "Exemplary amide protecting groups are o-nitrocinnamoyl, picolinoyl, aminocaproyl, benzoyl and the like, and acyloxy including methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, vinyloxycarbonyl, allyloxycarbonyl, t-butyloxycarbonyl (BOC), 1,1-dimethyl-propynyloxycarbonyl, benzyloxycarbonyl (CBZ), p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, and the like.", "“Amino acid” means an amino acid selected from the group consisting of natural and unnatural amino acids as defined herein.", "Amino acid is also meant to include -amino acids having L or D stereochemistry at the α-carbon.", "Preferred amino acids are those possessing an α-amino group.", "The amino acids may be neutral, positive or negative depending on the substituents in the side chain.", "“Neutral amino acid” means an amino acid containing uncharged side chain substituents.", "Exemplary neutral amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine and cysteine.", "“Positive amino acid” means an amino acid in which the side chain substituents are positively charged at physiological pH.", "Exemplary positive amino acids include lysine, arginine and histidine.", "“Negative amino acid” means an amino acid in which the side chain substituents bear a net negative charge at physiological pH.", "Exemplary negative amino acids include aspartic acid and glutamic acid.", "Preferred amino acids are α-amino acids.", "Exemplary natural amino acids are isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid and glutamic acid.", "Unnatural amino acid” means an amino acid for which there is no nucleic acid codon.", "Exemplary unnatural amino acids include, for example, the D-isomers of the natural α-amino acids as indicated above; Aib (aminobutyric acid), βAib (3-amino-isobutyric acid), Nva (norvaline), β-Ala, Aad (2-aminoadipic acid), βAad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba (γ-aminobutyric acid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutryic acid), α-aminopimelic acid, TMSA (trimethylsilyl-Ala), aIle (allo-isoleucine), Nle (norleucine), tert-Leu, Cit (citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr (2,3-diaminopropionic acid), α- or β- Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), and the like; cyclic amino acids; Na-alkylated amino acids such as MeGly (Na-methylglycine), EtGly (Na-ethylglycine) and EtAsn (Na-ethylasparagine); and amino acids in which the α-carbon bears two side-chain substituents.", "The names of natural and unnatural amino acids and residues thereof used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a-Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975).", "To the extent that the names and abbreviations of amino acids and residues thereof employed in this specification and appended claims differ from those noted, differing names and abbreviations will be made clear.", "“Amino acid protecting group” mean a group that protects an acid or amine moiety of the amino acid or other reactive moiety on the side chain of an amino acid, e.g., hydroxy or thiol.", "For examples of “corresponding protected derivatives” of amino acid side chains, see T. W. Green and P. G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley and Sons, 1991.Protecting groups for an acid group in an amino acid are described herein, for example in the sections “acidic functional group” and “hydrogenation labile acid protecting group”.", "Protecting groups for an amine group in an amino acid are described herein, for example in the sections “amine protecting group”, “acid labile amine protecting group” and “hydrogenation labile amine protecting group”.", "“Amino acid residue” means the individual amino acid units incorporated into the compound of the invention.", "“Amino acid side chain” means the substituent found on the carbon between the amino and carboxy groups in α-amino acids.", "Exemplary •-amino acid side chains include isopropyl, methyl, and carboxymethyl for valine, alanine, and aspartic acid, respectively.", "“Amino acid equivalent” means an amino acid that may be substituted for another amino acid in the peptides according to the invention without any appreciable loss of function.", "In making such changes, substitutions of like amino acids are made on the basis of relative similarity of side chain substituents, for example regarding size, charge, hydrophilicity, hydropathicity and hydrophobicity as described herein.", "“Aromatic group” means aryl or heteroaryl as defined herein.", "Exemplary aromatic groups include phenyl, halo substituted phenyl, azaheteroaryl, and the like.", "“Aroyl” means an aryl-CO— group wherein the aryl group is as herein described.", "Exemplary aroyl groups include benzoyl, 1- and 2-naphthoyl, and the like.", "“Aryl” means an aromatic monocyclic or multicyclic ring system of about 6 to about 14 carbon atoms, preferably of about 6 to about 10 carbon atoms.", "Encompassed by aryl are fused arylcycloalkenyl, fused arylcycloalkyl, fused arylheterocyclenyl and fused arylheterocyclyl as defined herein when bonded through the aryl moiety thereof.", "The aryl is optionally substituted with one or more “ring group substituents” which may be the same or different, and are as defined herein.", "Exemplary aryl groups include phenyl or naphthyl, or phenyl substituted or naphthyl substituted.", "“Substituted aryl” means an aryl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "“Aryldiazo” means an aryl-diazo- group wherein the aryl and diazo groups are as defined herein.", "“Arylene” means an optionally substituted 1,2-, 1,3-, 1,4-, bivalent aryl group, wherein the aryl group is as defined herein.", "Exemplary arylene groups include optionally substituted phenylene, naphthylene and indanylene.", "A particular arylene is optionally substituted phenylene.", "“Substituted arylene” means an arylene group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "“Aryloxy” means an aryl-O— group wherein the aryl group is as defined herein.", "Exemplary aryloxy groups include phenoxy and 2-naphthyloxy.", "“Aryloxycarbonyl” means an aryl-O—CO— group wherein the aryl group is as defined herein.", "Exemplary aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl.", "“Arylsulfonyl” means an aryl-SO2— group wherein the aryl group is as defined herein.", "“Arylsulphonylcarbamoyl” means an aryl-SO2—NH—C(═O)— group wherein the aryl group is as herein described.", "An exemplary arylsulphonylcarbamoyl group is phenylsulphonylcarbamoyl.", "“Arylsulfinyl” means an aryl-SO— group wherein the aryl group is as defined herein.", "“Arylthio” means an aryl-S— group wherein the aryl group is as herein described.", "Exemplary arylthio groups include phenylthio and naphthylthio.", "“Basic nitrogen atom” means a sp2 or sp3 hybridized nitrogen atom having a non-bonded pair of electrons which is capable of being protonated.", "Exemplary basic nitrogen atoms include optionally substituted imino, optionally substituted amino and optionally substituted amidino groups.", "“Carboxy” means an HO(O)C— (carboxylic acid) group.", "“Coupling agent” means a compound that reacts with the hydroxyl moiety of a carboxy moiety thereby rendering it susceptible to nucleophilic attack.", "Exemplary coupling agents include DIC, EDCI, DCC, and the like.", "“Cycloalkenyl” means a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms, and which contains at least one carbon-carbon double bond.", "Encompassed by cycloalkenyl are fused arylcycloalkenyl and fused heteroarylcycloalkenyl as defined herein when bonded through the cycloalkenyl moiety thereof.", "Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "“Substituted cycloalkenyl” means an cycloalkyenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "Exemplary monocyclic cycloalkenyl include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like.", "An exemplary multicyclic cycloalkenyl is norbornylenyl.", "“Cycloalkyl” means a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms.", "Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "Encompassed by cycloalkyl are fused arylcycloalkyl and fused heteroarylcycloalkyl as defined herein when bonded through the cycloalkyl moiety thereof.", "“Substituted cycloalkyl” means a cycloalkyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "Exemplary monocyclic cycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, and the like.", "Exemplary multicyclic cycloalkyl include 1-decalin, norbornyl, adamant-(1- or 2-)yl, and the like.", "“Cycloalkylene” means a bivalent cycloalkyl group as defined herein having about 4 to about 8 carbon atoms.", "Preferred ring sizes of the cycloalkylene include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "The points of binding on the cycloalkylene group include 1,1-, 1,2-, 1,3-, or 1,4-binding patterns, and where applicable the stereochemical relationship of the points of binding is either cis or trans.", "Exemplary cycloalkylene groups include (1,1-, 1,2- or 1,3-)cyclohexylene and (1,1- or 1,2-)cyclopentylene.", "“Substituted cycloalkylene” means an cycloalkylene group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein “Cyclic” or “Cyclyl” means cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl as defined herein.", "The term “lower” as used in connection with the term cyclic is the same as noted herein regarding the cycloalkyl, cycloalkenyl, heterocyclyl or heterocyclenyl.", "“Cyclyloxy” means a cyclyl-O— group wherein the cyclyl group is as herein described.", "Exemplary cycloalkoxy groups include cyclopentyloxy, cyclohexyloxy, quinuclidyloxy, pentamethylenesulfideoxy, tetrahydropyranyloxy, tetrahydrothiophenyloxy, pyrrolidinyloxy, tetrahydrofuranyloxy or 7-oxabicyclo[2.2.1]heptanyloxy, hydroxytetrahydropyranyloxy, hydroxy-7-oxabicyclo[2.2.1heptanyloxy, and the like.", "“Cyclylsulfinyl” means a cyclyl-S(O)— group wherein the cyclyl group is as herein described.", "“Cyclylsulfonyl” means a cyclyl-S(O)2— group wherein the cyclyl group is as herein described.", "“Cyclylthio” means a cyclyl-S— group wherein the cyclyl group is as herein described.", "“Diazo” means a bivalent —N═N— radical.", "“Displaceable moiety” means a group that where associated with L as defined herein is subject to being displaced by nucleophilic attack by a mono- or di-substituted amine moiety with or without the presence of an agent that facilitates said attack, e.g., coupling agent.", "Exemplary displaceable moieties include hydroxy, aliphatic oxy, halo, N-oxysuccinimide, acyloxy, and the like.", "“Effective amount” is means an amount of a compound/composition according to the present invention effective in producing the desired therapeutic effect.", "“Fused arylcycloalkenyl” means a fused aryl and cycloalkenyl as defined herein.", "Preferred fused arylcycloalkenyls are those wherein the aryl thereof is phenyl and the cycloalkenyl consists of about 5 to about 6 ring atoms.", "A fused arylcycloalkenyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "“Substituted fused arylcycloalkenyl” means a fused arylcycloalkenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "Exemplary fused arylcycloalkenyl include 1,2-dihydronaphthylene, indene, and the like.", "“Fused arylcycloalkyl” means a fused aryl and cycloalkyl as defined herein.", "Preferred fused arylcycloalkyls are those wherein the aryl thereof is phenyl and the cycloalkyl consists of about 5 to about 6 ring atoms.", "A fused arylcycloalkyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "“Substituted fused arylcycloalkyl” means a fused arylcycloalkyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "Exemplary fused arylcycloalkyl includes 1,2,3,4tetrahydro-naphthylene, and the like.", "“Fused arylheterocyclenyl” means a fused aryl and heterocyclenyl as defined herein.", "Preferred fused arylheterocyclenyls are those wherein the aryl thereof is phenyl and the heterocyclenyl consists of about 5 to about 6 ring atoms.", "A fused arylheterocyclenyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before heterocyclenyl portion of the fused arylheterocyclenyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted fused arylheterocyclenyl” means a fused arylheterocyclenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The nitrogen atom of a fused arylheterocyclenyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heterocyclenyl portion of the fused arylheterocyclenyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary fused arylheterocyclenyl include 3H-indolinyl, 1H-2-oxoquinolyl, 2H-1-oxoisoquinolyl, 1,2-di-hydroquinolinyl, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl, 3,4-dihydroisoquinolinyl, and the like.", "“Fused arylheterocyclyl” means a fused aryl and heterocyclyl as defined herein.", "Preferred fused arylheterocyclyls are those wherein the aryl thereof is phenyl and the heterocyclyl consists of about 5 to about 6 ring atoms.", "A fused arylheterocyclyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before heterocyclyl portion of the fused arylheterocyclyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted fused arylheterocyclyl” means a fused arylheterocyclyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The nitrogen atom of a fused arylheterocyclyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heterocyclyl portion of the fused arylheterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary fused arylheterocyclyl ring systems include indolinyl, 1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline, 1H-2,3-dihydroisoindol-2-yl, 2,3-dihydrobenz[f]isoindol-2-yl, 1,2,3,4-tetrahydrobenz[g]-isoquinolin-2-yl, and the like.", "“Fused heteroarylcycloalkenyl” means a fused heteroaryl and cycloalkenyl as defined herein.", "Preferred fused heteroarylcycloalkenyls are those wherein the heteroaryl thereof is phenyl and the cycloalkenyl consists of about 5 to about 6 ring atoms.", "A fused heteroaryl-cycloalkenyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before heteroaryl portion of the fused heteroarylcycloalkenyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom “Substituted fused heteroarylcycloalkenyl” means a fused heteroarylcycloalkyenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 30 which may be the same or different and are as defined herein.", "The nitrogen atom of a fused heteroarylcycloalkenyl may be a basic nitrogen atom.", "The nitrogen atom of the heteroaryl portion of the fused heteroarylcycloalkenyl may also be optionally oxidized to the corresponding N-oxide.", "Exemplary fused heteroarylcyclo-alkenyl include 5,6-dihydroquinolyl, 5,6-dihydroisoquinolyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-di-hydrobenzoxazolyl, and the like.", "“Fused heteroarylcycloalkyl” means a fused heteroaryl and cycloalkyl as defined herein.", "Preferred fused heteroarylcycloalkyls are those wherein the heteroaryl thereof consists of about 5 to about 6 ring atoms and the cycloalkyl consists of about 5 to about 6 ring atoms.", "A fused heteroarylcycloalkyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before heteroaryl portion of the fused heteroarylcycloalkyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted fused heteroarylcycloalkyl” means a fused heteroarylcycloalkyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The nitrogen atom of a fused heteroarylcycloalkyl may be a basic nitrogen atom.", "The nitrogen atom of the heteroaryl portion of the fused heteroarylcycloalkyl may also be optionally oxidized to the corresponding N-oxide.", "Exemplary fused heteroarylcycloalkyl include 5,6,7,8-tetrahydroquinolinyl, 5,6,7,8-tetra-hydroisoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl, 4,5,6,7-tetrahydrobenzoxazolyl, 1H-4-oxa-1,5-diazanaphthalen-2-onyl, 1,3-dihydroimidizole-[4,5]-pyridin-2-onyl, and the like.", "“Fused heteroarylheterocyclenyl” means a fused heteroaryl and heterocyclenyl as defined herein.", "Preferred fused heteroarylheterocyclenyls are those wherein the heteroaryl thereof consists of about 5 to about 6 ring atoms and the heterocyclenyl consists of about 5 to about 6 ring atoms.", "A fused heteroarylheterocyclenyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before the heteroaryl or heterocyclenyl portion of the fused heteroarylhetero-cyclenyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted fused heteroarylheterocyclenyl” means a fused heteroarylheterocyclenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The nitrogen atom of a fused heteroarylazaheterocyclenyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heteroaryl portion of the fused heteroarylheterocyclyl may also be optionally oxidized to the corresponding N-oxide.", "The nitrogen or sulfur atom of the heteroaryl or heterocyclyl portion of the fused heteroarylheterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary fused heteroarylheterocyclenyl include 7,8-dihydro[1,7]naphthyridinyl, 1,2-dihydro[2,7]-naphthyridinyl, 6,7-dihydro-3H-imidazo[4,5-c]pyridyl, 1,2-dihydro-1,5-naphthyridinyl, 1,2-dihydro-1,6-naphthyridinyl, 1,2-dihydro-1,7-naphthyridinyl, 1,2-dihydro-1,8-naphthyrdinyl, 1,2-dihydro-2,6-naphthyridinyl, and the like.", "“Fused heteroarylheterocyclyl” means a fused heteroaryl and heterocyclyl as defined herein.", "Preferred fused heteroarylheterocyclyls are those wherein the heteroaryl thereof consists of about 5 to about 6 ring atoms and the heterocyclyl consists of about 5 to about 6 ring atoms.", "A fused heteroarylheterocyclyl as a variable may be bonded through any atom of the ring system thereof capable of such.", "The designation of the aza, oxa or thia as a prefix before the heteroaryl or heterocyclyl portion of the fused heteroarylheterocyclyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted fused heteroarylheterocyclyl” means a fused heteroarylheterocyclyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein The nitrogen atom of a fused heteroarylheterocyclyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heteroaryl portion of the fused heteroarylheterocyclyl may also be optionally oxidized to the corresponding N-oxide.", "The nitrogen or sulfur atom of the heteroaryl or heterocyclyl portion of the fused heteroarylheterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary fused heteroarylheterocyclyl include 2,3-dihydro-1H pyrrol[3,4-b]quinolin-2-yl, 1,2,3,4-tetrahydrobenz [b][1,7]naphthyridin-2-yl, 1,2,3,4-tetrahydrobenz [b][1,6]naphthyridin-2-yl, 1,2,3,4-tetra-hydro-9H-pyrido[3,4-b]indol-2yl, 1,2,3,4-tetrahydro-9H-pyrido[4,3-b]indol-2yl, 2,3-dihydro-1H-pyrrolo[3,4-b]indol-2-yl, 1H-2,3,4,5-tetrahydroazepino[3,4-b]indol-2-yl, 1H-2,3,4,5-tetra-hydroazepino[4,3-b]indol-3-yl, 1H-2,3,4,5-tetrahydroazepino[4,5-b]indol-2 yl, 5,6,7,8-tetra-hydro[1,7]napthyridyl, 1,2,3,4-tetrhydro[2,7]naphthyridyl, 2,3-dihydro[1,4]dioxino[2,3-b]pyridyl, 2,3-dihydro-[1,4]dioxino[2,3-b]pyridyl, 3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl, 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridyl, 6,7-dihydro[5,8]diazanaphthalenyl, 1,2,3,4-tetrahydro[1,5]-napthyridinyl, 1,2,3,4-tetrahydro[1,6]napthyridinyl, 1,2,3,4-tetrahydro[1,7]napthyridinyl, 1,2,3,4-tetrahydro[1,8]napthyridinyl, 1,2,3,4-tetra-hydro[2,6]napthyridinyl, and the like.", "“Halo” means fluoro, chloro, bromo, or iodo.", "Preferred are fluoro, chloro or bromo, and more preferred are fluoro or chloro.", "“Heteroaroyl” means an heteroaryl-CO— group wherein the heteroaryl group is as herein described.", "Exemplary heteroaroyl groups include thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, 1- and 2-naphthoyl, pyridinoyl, and the like.” “Heteroaryl” means an aromatic monocyclic or multicyclic ring system of about 5 to about 14 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur.", "Preferably the ring system includes 1 to 3 heteroatoms.", "Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms.", "Encompassed by heteroaryl are fused heteroarylcycloalkenyl, fused heteroarylcycloalkyl, fused heteroarylheterocyclenyl and fused heteroarylheterocyclyl as defined herein when bonded through the heteroaryl moiety thereof.", "“Substituted heteroaryl” means a heteroaryl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The designation of the aza, oxa or thia as a prefix before heteroaryl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "A nitrogen atom of an heteroaryl may be a basic nitrogen atom and may also be optionally oxidized to the corresponding N-oxide.", "Exemplary heteroaryl and substituted heteroaryl groups include pyrazinyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl, furazanyl, pyrrolyl, 1,2,4-thiadiazolyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl, benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyridyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, benzoazaindolyl, 1,2,4-triazinyl, benzthiazolyl, furanyl, imidazolyl, indolyl, indolizinyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl, pyrazinyl, pyridazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, 1,3,4-thiadiazolyl, thiazolyl, thienyl, triazolyl, and the like.", "A preferred heteroaryl group is pyrazinyl.", "“Heteroaryldiazo” means an heteroaryl-azo-group wherein the heteroaryl and azo groups are as defined herein.", "“Heteroarylidyl” means a bivalent radical derived from a heteroaryl, wherein the heteroaryl is as described herein.", "An exemplary heteroaryldiyl radical is optionally substituted pyridinediyl.", "“Heteroarylsulphonylcarbamoyl” means a heteroaryl-SO2—NH—C(═O)— group wherein the heteroaryl group is as herein described.", "“Heterocyclenyl” means a non-aromatic monocyclic or multicyclic hydrocarbon ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur atoms, and which contains at least one carbon-carbon double bond or carbon-nitrogen double bond.", "Preferably, the ring includes 1 to 3 heteroatoms.", "Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "Encompassed by heterocyclenyl are fused arylheterocyclenyl and fused heteroarylheterocyclenyl as defined herein when bonded through the heterocyclenyl moiety thereof.", "The designation of the aza, oxa or thia as a prefix before heterocyclenyl define that at least a nitrogen, oxygen or sulfur atom is present, respectively, as a ring atom.", "“Substituted heterocyclenyl” means a heterocyclenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "The nitrogen atom of an heterocyclenyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heterocyclenyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary monocyclic azaheterocyclenyl groups include 1,2,3,4-tetrahydrohydropyridine, 1,2-dihydropyridyl, 1,4-dihydropyridyl, 1,2,3,6-tetra-hydropyridine, 1,4,5,6-tetrahydro-pyrimidine, 2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, and the like.", "Exemplary oxaheterocyclenyl groups include 3,4-dihydro-2H-pyran, dihydrofuranyl, and fluorodihydro-furanyl.", "An exemplary multicyclic oxaheterocyclenyl group is 7-oxabicyclo[2.2.1]heptenyl.", "Exemplary monocyclic thiaheterocyclenyl rings include dihydrothiophenyl and dihydrothiopyranyl.", "“Heterocyclyl” means a non-aromatic saturated monocyclic or multicyclic ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur.", "Preferably, the ring system contains from 1 to 3 heteroatoms.", "Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "Encompassed by heterocyclyl are fused arylheterocyclyl and fused heteroarylheterocyclyl as defined herein when bonded through the heterocyclyl moiety thereof.", "The designation of the aza, oxa or thia as a prefix before heterocyclyl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom.", "“Substituted heterocyclyl” means a heterocyclyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein The nitrogen atom of an heterocyclyl may be a basic nitrogen atom.", "The nitrogen or sulfur atom of the heterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide.", "Exemplary monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.", "as substituted monocyclic azaheterocyclyl is substituted directly or through a linker by at least one substituent that is, or encompasses, or is substituted by an aromatic group as defined herein; for example aryl, heteroaryl, aryloxy, heteroaryloxy, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, aroyloxy, heteroaroyloxy, aryloxycarbonyl, heteroaryloxycarbonyl, arylsulfonyl, heteroarylsulfonyl, arylsulfinyl, heteroarylsulfinyl, arylthio, heteroarylthio, aryldiazo, heteroaryldiazo, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2— wherein at least one of Y1 and Y2 is, encompasses or is substituted by an aryl or heteroaryl moiety.", "Preferred linkers include —C(O)—, —OC(O)—, lower alkyl, lower alkoxy, lower alkenyl, —O—, —S—, —C(O)C(O)—, —S(O)—, —S(O)2—, —NR80—, where R80 is hydrogen, alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl or heteroaryl.", "Particularly preferred linkers are —C(O)— and —OC(O)—.", "“Substituted multicyclic azaheterocyclyl” means a multicyclic azaheterocyclyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "“Substituted multicyclic azaheterocyclenyl” means a multicyclic azaheterocyclenyl group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "“Heterocyclylene” means a bivalent heterocyclyl group as defined herein having about 4 to about 8 carbon atoms.", "Preferred ring sizes of the heterocyclylene include about 5 to about 6 ring atoms; and such preferred ring sizes are also referred to as “lower”.", "The points of binding on the cycloalkylene group include 1,1-, 1,2-, 1,3-, or 1,4-binding patterns, and where applicable the stereochemical relationship of the points of binding is either cis or trans.", "Exemplary heterocyclylene groups include (1,1-, 1,2- or 1,3-)piperidinylene and (1,1- or 1,2-)tetrahydrofuranylene.", "“Substituted heterocyclylene” means a heterocyclylene group as defined above which is substituted with one or more “ring group substituents” (preferably 1 to 3) which may be the same or different and are as defined herein.", "“Hydrate” means a solvate wherein the solvent molecule(s) is/are H2O.", "“Hydrogenation labile amine protecting group” means an amine protecting group as defined herein which is readily removed by hydrogenation while remaining relatively stable to other reagents.", "A preferred hydrogenation labile amine protecting group is Cbz.", "“Hydrogenation labile acid protecting group” means an acid protecting group as defined herein which is readily removed by hydrogenation while remaining relatively stable to other reagents.", "A preferred hydrogenation labile acid protecting group is benzyl.", "“Hygroscopicity” means sorption, implying an acquired amount or state of water sufficient to affect the physical or chemical properties of the substance (Eds.", "J. Swarbrick and J. C. Boylan, Encyclopedia of Pharmaceutical Technology, 10, 33).", "“Iminic glycinimide derivative” means an iminic Schiff base of a glycine that is useful in the synthesis of of α-amino acids, both natural and unnatural.", "The iminic ester functionality may contain one or more assymetric centers that may aid in stereoinduction during the bond formating process.", "In addition, these iminic glycinimide derivatives may be incorporated onto polymeric supports to facilitate combinatorial synthesis.", "Iminic glycinimide derivatives may be prepared by condensing a glycine ester with the appropropriate ketone in the presence of an acid catalyst.", "The reaction is facilitated by the removal of water.", "Iminic glycinimide derivatives are well known in the art for use in Michael Addition synthetic procedures, for example as disclosed by Guillena, G., et al., J. Org.", "Chem.", "2000, 65, 7310-7322, herein incorporated by reference.", "Particular examples of iminic glycinimide derivatives according to the invention include one selected from the group of formulae wherein: M* is a transition metal, preferably CU, more preferably CUII.", "R15 is optionally substituted aliphatic group; R16 is acid protecting group, optionally substituted aryl, or optionally substituted aliphatic group; R17 is optionally substituted aryl, optionally substituted aliphatic group, R18 is hydrogen, alkyl, or alkylthio; or optionally substituted aryl; R17 and R18 taken together with the carbon to which R17 and R18 are attached {circle over (s)} is a solid phase.", "“Iminic glycinimide derivative adduct” means the resulting compound where an α-hydrogen to the nitrogen and carbonyl moiety of the Schiff base portion is removed and used to form an attachment for the bond formation thereto.", "Particular examples of iminic glycinimide derivative adducts according to the invention include one selected from the group of formulae wherein: R14, R17, and R18 are defined as described in the definition of iminic glycinimide derivative herein.", "“N-oxysuccinimide” means a moiety of the following structure “N-oxide” means a moiety of the following structure “Patient” includes both human and other mammals.", "“Peptidomimetic” mean a polymer encompassing amino acid residues joined together through amide bonds.", "“Pharmaceutically acceptable ester” refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.", "Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.", "Exemplary esters include formates, acetates, propionates, butyrates, acrylates, ethylsuccinates, and the like.", "“Pharmaceutically acceptable prodrugs” as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.", "The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood.", "Functional groups that may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this invention.", "They include, but are not limited to such groups as alkanoyl (such as acetyl, propanoyl, butanoyl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (such as trimethyl- and triethysilyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like.", "Because of the ease with which the metabolically cleavable groups of the compounds of this invention are cleaved in vivo, the compounds bearing such groups act as pro-drugs.", "The compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group.", "A thorough discussion is provided in Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods in Enzymology; K. Widder et al, Ed., Academic Press, 42, 309-396 (1985); A Textbook of Drug Design and Developement, Krogsgaard-Larsen and H. Bandaged, ed., Chapter 5; “Design and Applications of Prodrugs” 113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, 1-38, (1992); J. Pharm.", "Sci., 77, 285 (1988); Chem.", "Pharm Bull., N. Nakeya et al, 32, 692 (1984); Pro-drugs as Novel Delivery Systems, T. Higuchi and V. Stella, 14 A.C.S.", "Symposium Series, and Bioreversible Carriers in Drug Design, E. B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference.", "“Pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention.", "These salts can be prepared in situ during the final isolation and purification of the compounds.", "In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.", "Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, salicylates, propionates, methylene-bis-β-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like.", "See, for example S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm.", "Sci., 66, 1-19 (1977) which is incorporated herein by reference.", "Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed.", "Base addition salts include pharmaceutically acceptable metal and amine salts.", "Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts.", "The sodium and potassium salts are preferred.", "Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like.", "Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use.", "ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.", "“Ring group substituents” mean substituents attached to aromatic or non-aromatic ring systems inclusive of aryl, heteroaryl, hydroxy, alkoxy, cyclyloxy, aryloxy, heteroaryloxy, acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, halo, nitro, cyano, carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, cyclylthio, arylthio, heteroarylthio, cyclyl, aryldiazo, heteroaryldiazo, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, wherein Y1, Y2 and Y3 are independently hydrogen, alkyl, aryl or heteroaryl, of for where the substituent is Y1Y2N—, then one of Y1 and Y2 may be acyl, cyclylcarbonyl, aroyl, heteroaroyl, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl or heteroaryloxycarbonyl, as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2—, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered azaheterocyclyl or azaheterocyclenyl.", "When a ring system is saturated or partially saturated, the “ring group substituents” further include, methylene (H2C═), oxo (O═) and thioxo (S═).", "Acidic/amide ring group substituents are carboxy (acid), —C(O)—NHOH, —C(O)—CH2OH, —C(O)—CH2SH, —C(O)—NH—CN, sulpho, phosphono, alkylsulphonylcarbamoyl, tetrazolyl, arylsulphonylcarbamoyl, N-methoxycarbamoyl, heteroarylsulphonylcarbamoyl, 3-hydroxy-3-cyclobutene-1,2-dione, 3,5-dioxo-1,2,4-oxadiazolidinyl or hydroxyheteroaryl such as 3-hydroxyisoxazolyl, 3-hydoxy-1-methylpyrazoly and Y1Y2NCO—.", "Non-acidic polar ring group substituents are hydroxy, oxo (O═), thioxo (S═), acyl or its thioxo analogue, cyclylcarbonyl or its thioxo analogue, aroyl or its thioxo analogue, heteroaroyl or its thioxo analogue, alkoxycarbonyl, cyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, acyloxy, cyclylcarbonyloxy, aroyloxy, heteroaroyloxy, alkylsulfonyl, cyclylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, cyclylsulfinyl, arylsulfinyl, heteroarylsulfinyl, thiol, Y1Y2N—, Y1Y2NC(O)—, Y1Y2NC(O)O—, Y1Y2NC(O)NY3— or Y1Y2NSO2.“Solvate” means a physical association of a compound of this invention with one or more solvent molecules.", "This physical association includes hydrogen bonding.", "In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.", "“Solvate” encompasses both solution-phase and isolable solvates.", "Exemplary solvates include hydrates, ethanolates, methanolates, and the like.", "EMBODIMENTS With reference to inventions described herein, below are particular and preferred embodiments are related thereto.", "A particular embodiment according to the invention is where R0 is a bond.", "Another particular embodiment according to the invention is where R0 is difluoromethylene.", "A particular embodiment according to the invention is where R1 is hydrogen or optionally substituted lower aliphatic group.", "Another particular embodiment according to the invention is where R1 is hydrogen or lower alkyl.", "A preferred embodiment according to the invention is where R1 is hydrogen.", "A particular embodiment according to the invention is where R2 is optionally substituted lower aliphatic group or optionally substituted monocyclic group.", "Another particular embodiment according to the invention is where R2 is optionally substituted lower alkyl, optionally substituted lower alkenyl or optionally substituted monocyclic cycloalkyl.", "Further particular embodiment according to the invention is where R2 is carboxymethyl, 1-carboxy-2-phenylethyl, cyclopropyl, cyclobutyl, 1-cyclohexylethyl, 1-phenylethyl, but-2-yl, 1-pyrid4-ylethyl, propen-3-yl or 3-methylbut-2-yl; more preferred cyclopropyl.", "A particular embodiment according to the invention is where R3 is optionally substituted lower aliphatic group methylene.", "Another particular embodiment according to the invention is where R3 is optionally halo substituted lower (alkyl or alkenyl)methylene.", "A preferred embodiment according to the invention is where R3 is propylmethylene, 2,2-difluoroethylmethylene, 2,2,2-trifluoromethylene or propen-3-ylmethylene; more preferred R3 is propylmethylene or 2,2-difluoroethylmethylene; further preferred R3 is propylmethylene.", "A particular embodiment according to the invention is where R4 is hydrogen or optionally substituted lower aliphatic group.", "Another particular embodiment according to the invention is where R4 is hydrogen or lower alkyl.", "A preferred embodiment according to the invention is where R4 is hydrogen.", "A particular embodiment according to the invention is where R5 is optionally substituted lower aliphatic group methylene.", "Another particular embodiment according to the invention is where R5 is optionally (phenyl, carboxy, carboxamido or alkoxycarbonyl) substituted lower (alkyl or alkenyl) methylene.", "A preferred embodiment according to the invention is where R5 is methylmethylene, isopropylmethylene, t-butylmethylene, but-2-ylmethylene, butylmethylene, benzylmethylene, 3-methylbutylmethylene, 2-methylpropyl-methylene, carboxymethylmethylene, carboxamidomethylmethylene, benzyloxycarbonylmethylmethylene, benzyloxycarbonylpropylmethylene, or phenylpropen-3-ylmethylene; more preferred R5 is isopropylmethylene or t-butyl-methylene.", "A particular embodiment according to the invention is where R6 is hydrogen or optionally substituted lower aliphatic group.", "Another particular embodiment according to the invention is where R6 is hydrogen or lower alkyl.", "A preferred embodiment according to the invention is where R6 is hydrogen.", "A particular embodiment according to the invention is where R7 is optionally substituted lower aliphatic group methylene, optionally substituted lower cyclic group methylene or optionally substituted monocyclic (aryl or heteroaryl) methylene.", "Another particular embodiment according to the invention is where R7 is optionally substituted lower alkylmethylene, optionally substituted lower cycloalkylmethylene or optional substituted phenylmethylene.", "A preferred embodiment according to the invention is where R7 is methylmethylene, isopropylmethylene, n-propylmethylene, phenylmethylene, cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene, s-butylmethylene, cyclohexylmethylmethylene, or phenylmethylmethylene; more preferred is isopropylmethylene, cyclohexylmethylene, cyclopentylmethylene, t-butylmethylene or s-butylmethylene.", "A preferred embodiment according to the invention is also wherein each of R3, R5, and R7 is mono substituted methylene.", "A preferred embodiment according to the invention is also wherein R3 is mono substituted methylene and has an (S) configuration on the carbon attached to the —C(O)—R0—C(O)—NR1R2 moiety.", "A particular embodiment according to the invention is where R8 is hydrogen or optionally substituted lower aliphatic group.", "Another particular embodiment according to the invention is where R8 is hydrogen or lower alkyl.", "A preferred embodiment according to the invention is where R8 is hydrogen.", "A particular embodiment according to the invention is where R9 is optionally substituted lower aliphatic group or optionally substituted monocyclic aromatic group.", "Another particular embodiment according to the invention is where R9 is optionally substituted lower alkyl or optionally substituted monocyclic heteroaryl.", "Another particular embodiment according to the invention is where R9 is optionally (carboxy, (loweralkyl)SO2NH—, (lower alkyl)HNCO—, hydroxy, phenyl, heteroaryl, or (lower alkyl)OC(O)NH—)-substituted lower alkyl, or optionally substituted monocyclic heteroaryl.", "A further preferred embodiment according to the invention is where R9 is lower alkyl substituted by (mono- or di-)MeOC(O)NH—; more preferred is 1,2-di(MeOC(O)NH)ethyl or 1-(MeOC(O)NH)ethyl.", "A preferred embodiment according to the invention is where R9 is (carboxy, (lower allyl)HNCO— or tetrazolyl)substituted lower alkyl; more preferred 3-carboxypropyl, 2-tetrazol-5ylpropyl, 3-(N-methylcarboxamido)propyl or 3-carboxy-2,2-dimethylpropyl; further preferred is 3-carboxypropyl, 2-tetrazol-5ylpropyl or 3-(N-methylcarboxamido)propyl.", "Another preferred embodiment according to the invention is where R9 is optionally substituted lower alkyl; more preferred is 1-hydroxy-2-phenylethyl, methyl, isopropyl or t-butyl; further preferred is methyl, isopropyl or t-butyl.", "Another preferred embodiment according to the invention is where R9 is selected from the group consisting of Yet another preferred embodiment according to the invention is where R9 is pyrazinyl.", "A particular embodiment according to the invention is where R10 is hydrogen or optionally substituted lower aliphatic group.", "Another particular embodiment according to the invention is where R10 is hydrogen or lower alkyl.", "A preferred embodiment according to the invention is where R10 is hydrogen.", "A preferred embodiment according to the invention is where as a substituted monocyclic azaheterocyclyl is substituted pyrrolidinyl.", "A preferred embodiment according to the invention is where as a substituted monocyclic azaheterocyclyl is optionally substituted or optionally substituted wherein Ar is R2 that comprises an aromatic moiety; more preferred is optionally substituted further preferred is optionally substituted Further preferred optionally substituted yet further preferred Another preferred embodiment according to the invention is where as an optionally substituted multicyclic azaheterocyclyl is optionally substituted more preferred is optionally substituted Particular substituents for are hydroxy, fluoro or oxo.", "Another preferred embodiment according to the invention is where as an optionally substituted multicyclic azaheterocyclenyl is optionally substituted more preferred is further preferred is Another preferred embodiment according to the invention is where as an optionally substituted multicyclic azaheterocyclenyl is optionally substituted A preferred embodiment according to the invention is where the —C(O)—N(R4)—R3—C(O)R0C(O)NR2R1 moiety attached to is attached a carbon a to the nitrogen atom.", "A preferred embodiment according to the invention is where L is —C(O)— or —OC(O)—.", "A preferred embodiment according to the invention is where n is 0.Another preferred embodiment according to the invention is where n is 1.A preferred embodiment according to the invention is where R11 is —CO2R13.A preferred embodiment according to the invention is where A particular embodiment according to the invention is where R13 is an optionally substituted aliphatic group.", "Another particular embodiment according to the invention is where R13 is an alkyl group.", "A preferred embodiment according to the invention is where R13 is lower alkyl.", "Another preferred embodiment according to the invention is where R13 is methyl.", "A preferred embodiment according to the invention is where R14 is —CO2R16.A particular embodiment according to the invention is where R15 is alkyl.", "A preferred embodiment according to the invention is where R15 is lower alkyl.", "A preferred embodiment according to the invention is where R15 is methyl.", "A particular embodiment according to the invention is where R16 is optionally substituted aliphatic.", "Another particular embodiment according to the invention is where R16 is alkyl.", "A preferred embodiment according to the invention is where R16 is lower alkyl.", "A preferred embodiment according to the invention is where R16 is t-Bu.", "A particular embodiment according to the invention is where R17 is optionally substituted aryl.", "A preferred embodiment according to the invention is where R17 is phenyl.", "A particular embodiment according to the invention is where R18 is optionally substituted aryl.", "A preferred embodiment according to the invention is where R18 is phenyl.", "A particular embodiment according to the invention is where p0 is selected from the group consisting of BOC, CBz, and —CO2alkyl.", "A preferred embodiment according to the invention is where p0 is BOC.", "It is to be understood that this invention covers all appropriate combinations of the particular and preferred groupings referred to herein.", "Particular compounds according to the invention are selected from the group of compounds A-FH consecutively consisting of or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.", "A preferred compound is one selected from the group consisting of S, U, BW, BX, BY, BZ, CE, CU, CW, CY, DZ, EA, EC, EJ, FH, EW, EO, EZ, FG and EN a pharmaceutically acceptable salt or prodrug thereof, or solvate of such compound, its salt or its prodrug.", "Another preferred embodiment of the invention are selected from the following group of compounds: or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.", "Another preferred embodiment of the invention is a compound of the formula or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.", "Another preferred embodiment of the invention is compound of formula 1 wherein: R0 is a bond; R1 is hydrogen; R2 is lower alkyl optionally substituted with 1 to 3 aliphatic group substituents; or lower cycloalky optionally substituted with 1 to 3 cyclic group substituents; R3 and R5 are each independently methylene optionally substituted with 1 to 3 aliphatic group substitutents; R4, R6, R8 and R10 are hydrogen; R7 is methylene substituted with cycloalkyl, lower alkyl or aryl;or or (1,1- or 1,2-)cycloalkenyl optionally substituted with cycloalkyl, lower alkyl or aryl; R9is lower alkyl optionally substituted with 1 to 3 aliphatic group substituents; or heteroaryl optionally substituted with 1 to 3 cyclic group substituents; or heterocyclic optionally substituted with 1 to 3 cyclic group substituents; is monocyclic azaheterocyclyl, multicyclic azaheterocyclyl, or multicyclic azaheterocyclenyl optionally substituted with from 1 to 3 cyclic group substituents; and L is —C(O)—, —OC(O)—.", "Another preferred embodiment of the invention is a compound selected from the group consisting of: or a pharmaceutically acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.", "Another preferred embodiment of the invention is a compound of formula 1 wherein the optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group of R9 is substituted with at least one heteroaryl substituent.", "Another preferred embodiment of the invention is a compound of formula 1 wherein the optionally substituted aromatic group of R9 is optionally substituted heteroaryl.", "Another preferred embodiment of the invention is a compound of formula 1 wherein the optionally substituted aliphatic group of R9 is optionally substituted alkylheteroaryl.", "Another preferred embodiment of the invention is a compound wherein the optionally substituted heteroary group of R9 is pyrazinyl, tetrazolyl, quinolinyl, imidazolyl, isoxazolyl and pyradonyl, optionally substituted with a ring group substituent.", "The compounds of the invention optionally are supplied as salts.", "Those salts that are pharmaceutically acceptable are of particular interest since they are useful in administering the foregoing compounds for medical purposes.", "Salts that are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some instances, for use in separating stereoisomeric forms of the compounds of this invention.", "The latter is particularly true of amine salts prepared from optically active amines.", "Where the compound of the invention contains a carboxy group, or a sufficiently acidic bioisostere, base addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free acid form.", "Also, where the compound of the invention contains a basic group, or a sufficiently basic bioisostere, acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form.", "A preferred embodiment of a method according to the present invention is for treating a patient suffering from an HCV infection or physiological conditions related to the infection comprising administering to the patient a pharmaceutically effective amount of a compound of formula 1.Another preferred embodiment of a therapeutic method according to the present invention is for treating a patient suffering from an HCV infection or physiological conditions related to the infection comprising administering a pharmaceutically effective amount of a compound of formula 1 in combination with a pharmaceutically effective amount of another anti-HCV therapeutic to the patient.", "Another object of the present invention is to provide pharmaceutical compositions comprising, in addition to one or more HCV serine protease inhibitors, one or more interferons exhibiting anti-HCV activity and/or one or more compounds having anti HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, and a pharmaceutically acceptable carrier or diluent.", "It is another object of the invention to provide a pharmaceutical composition which is effective, in and of itself, for utilization in a beneficial combination therapy because it includes a plurality of active ingredients which may be utilized in accordance with the invention.", "The invention also provides kits or single packages combining two or more active ingredients useful in treating or preventing an HCV infection in a patient.", "A kit may provide (alone or in combination with a pharmaceutically acceptable diluent or carrier), the compound of formula 1 and the additional active ingredient (alone or in combination with diluent or carrier) other anti-HCV therapeutic.", "Compounds of formula 1 may be prepared by the application or adaptation of known methods as used heretofore or described in the literature, or by methods according to this invention herein.", "Another object of the present invention is to provide methods of treating or preventing a HCV infection in a patient in need thereof, comprising administering to said patient a pharmaceutically effective amount of a combination of one or more HCV serine protease inhibitors; one or more interferons exhibiting anti-HCV activity; and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "Another object of the present invention is the use of one or more HCV serine protease inhibitors in combination with one or more interferons exhibiting anti-HCV activity and/or one or more compounds having anti-HCV activity, including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity, to prepare a medicament for treating or preventing a HCV infection in a patient in need thereof.", "A further object of the present invention is a kit or pharmaceutical pack for treating or preventing HCV infection in a patient, wherein the kit or pharmaceutical pack comprises a plurality of separate containers, wherein at least one of said containers contains one or more HCV serine protease inhibitors, at least another of said containers contains one or more interferons or compounds that induce the production of an inteferon that exhibit anti-HCV activity (alone or in combination with a pharmaceutically acceptable carrier or diluent), and, optionally, at least another of said containers contains one or more compounds having anti-HCV activity (alone or in combination with a pharmaceutically acceptable carrier or diluent), including immunomodulatory compounds such as immunostimulatory cytokines exhibiting HCV antiviral activity.", "Yet another object of the present invention is to provide a method of inhibiting hepatitis C virus replication in a cell, comprising contacting said cell, a hepatitis C virus serine protease inhibitor, and optionally an interferon or compounds that induce the production of an interferon that have anti-hepatitis C virus activity.", "The amount of the HCV serine protease inhibitor(s), interferon(s), or anti-HCV compound(s) in any of the foregoing applications can be a pharmaceutically effective amount, a suboptimal anti-HCV effective amount, or combinations thereof, so long as the final combination of HCV protease inhibitor(s), interferon(s), and/or anti-HCV compound(s) comprises a pharmaceutically effective amount of compounds that is effective in treating or preventing HCV infection in a patient.", "It is a further object of the invention to provide a method for preparing a chiral bicycloprolinate compound that is useful in preparing the compound of formula 1.Preparation of Compounds of the Invention The starting materials and intermediates of compounds of the invention may be prepared by the application or adaptation of known methods, for example methods as described in the Reference Examples or their obvious chemical equivalents.", "Compounds of the invention may be prepared by the application or adaptation of known methods, by which is meant methods used heretofore or described in the literature, for example those described by R. C. Larock in Comprehensive Organic Transformations, VCH publishers (1989).", "A compound of formula 1, wherein the variables and moiety thereof are as described herein, may be prepared by treating a compound of formula 2, wherein the variables and moiety thereof are as described herein, with an appropriate oxidizing agent and under appropriate conditions.", "A particular oxidizing agent is DMP reagent.", "Particular conditions include carrying out the oxidation in an appropriate organic solvent such as dichloromethane at about room temperature.", "A compound of formula 2, wherein the variables and moiety thereof are as described herein, may be prepared by coupling a compound of formula 3, wherein the variables and moiety thereof are as described herein, and a compound of formula 4, wherein the variables thereof are as described herein, with an appropriate coupling agent and under appropriate conditions.", "Particular coupling agent and conditions include using DIC and HOAt in an appropriate organic solvent such as DMF at about 0° C. or using PyBop and DIPEA in an appropriate organic solvent such as dichloromethane at about room temperature.", "A compound of formula 3, wherein the variables and moiety thereof are as described herein, may be prepared by coupling a compound of formula 5, wherein the variables thereof are as described herein, and a compound of formula 6, wherein p2 is an acid protecting group and the moiety thereof is as described herein, with an appropriate coupling agent and under appropriate coupling conditions, followed by an appropriate deprotecting agent and under appropriate deprotecting conditions.", "Particular coupling agent and conditions include using DIC or DCC and HOAt in an appropriate organic solvent such as DMF or dichloromethane at about 0° C. to about room temperature.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the protecting agent, i.e., whether it is removable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular acid protecting agent is C1 to C8 lower alkyl; more particular methyl.", "A particular deprotecting agent is an inorganic base such as an alkali hydroxide; more particular NaOH.", "Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol at about room temperature.", "A compound of formula 5, wherein n is 0 and the other variables are as described herein, i.e., compound 5a, may be prepared by deprotecting a compound of formula 7, wherein P2 is an acid protecting group and the other variables thereof are as described herein, with an appropriate deprotecting agent and under appropriate conditions.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the protecting agent, i.e., whether it is removeable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular acid protecting agent is C1 to C8 lower alkyl; more particular methyl.", "A particular deprotecting agent is an inorganic base such as an alkali hydroxide; more particular NaOH.", "Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol at about room temperature.", "A compound of formula 7, wherein the variables thereof are as described herein, may be prepared by acylating a compound of formula 8, wherein the variables thereof are as described herein, with a compound of formula 9, wherein M is a displaceable moiety and the other variables thereof are as described herein, under appropriate conditions.", "Particular coupling conditions use DIC or DCC and HOAt in an appropriate organic solvent such as DMF or dichloromethane at about 0° C. to about room temperature, or PyBop and DIPEA in an appropriate organic solvent such as DMF or dichloromethane at about room temperature; and preferably the latter conditions.", "A particular L is carbonyl.", "A particular M is hydroxy or N-oxysuccinimide.", "A compound of formula 5, wherein n is 1 and the other variables are as described herein, i.e., compound 5b, may be prepared by deprotecting a compound of formula 10, wherein P2 is an acid protecting group and the other variables thereof are as described herein, with an appropriate deprotecting agent and under appropriate conditions.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the acid protecting agent, i.e., whether it is removeable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular acid protecting agent is C1 to C8 lower alkyl; more particular methyl.", "A particular deprotecting agent is an inorganic base such as an alkali hydroxide; more particular NaOH.", "Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol at about room temperature.", "A compound of formula 10, wherein the variables thereof are as described herein, may be prepared by acylating a compound of formula 11, wherein the variables thereof are as described herein, with a compound of formula 9, wherein the variables thereof are as described herein, under appropriate conditions.", "Particular coupling conditions use DIC or DCC and HOAt in an appropriate organic solvent such as DMF or dichloromethane at about 0° C. to about room temperature, or PyBop and DIPEA in an appropriate organic solvent such as DMF or dichloromethane at about room temperature; and preferably the latter conditions.", "A particular L is carbonyl.", "A particular M is hydroxy or N-oxysuccinimide.", "A compound of formula 11, wherein the variables are as described herein, may be prepared by deprotecting a compound of formula 12, wherein P1 is an amine protecting group and the other variables thereof are as described herein, with an appropriate deprotecting agent and under appropriate conditions.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the amine protecting agent, i.e., whether it is removeable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular amine protecting agent is Cbz or BOC; more particular Cbz.", "A particular deprotecting agent is acid such as HCl or H2/Pd(OH)2; more particular H2/Pd(OH)2.Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol or an alkyl alkanoate solvent such as ethyl acetate at about room temperature.", "A compound of formula 12, wherein the variables thereof are as described herein, may be prepared by coupling a compound of formula 13, wherein the variables thereof are as described herein, with a compound of formula 14, wherein the variables thereof are as described herein, under appropriate conditions.", "Particular coupling conditions use HOAt/DIC and DIPEA in an appropriate organic solvent such as THF at about room temperature.", "A compound of formula 4, wherein the variables are as described herein, may be prepared by deprotecting a compound of formula 15, wherein the variables thereof are as described herein, with an appropriate deprotecting agent and under appropriate conditions.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the amine protecting agent, i.e., whether it is removeable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular amine protecting agent is Cbz or BOC; more particular Cbz.", "A particular deprotecting agent is an acid such as HCl or H2/Pd(OH)2; more particular H2/Pd(OH)2.Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol or an alkyl alkanoate solvent such as ethyl acetate at about room temperature.", "A compound of formula 15, wherein the variables thereof are as described herein, may be prepared by coupling a compound of formula 16, wherein the variables thereof are as described herein, with a compound of formula 17, wherein the variables thereof are as described herein, under appropriate conditions.", "A particular amine protecting agent is Cbz or BOC; more particular Cbz.", "Particular coupling conditions use HOBT, PyBop and DIPEA in an appropriate organic solvent such as dichloromethane at about 0° C. to about room temperature.", "A compound of formula 16, wherein the variables are as described herein may be prepared by deprotecting a compound of formula 18, wherein the other variables thereof are as described herein, with an appropriate deprotecting agent and under appropriate conditions.", "The deprotecting is carried out using an appropriate deprotecting agent that depends on the nature of the acid protecting agent, i.e., whether it is removeable (labile) under acid, base, or hydrogenation conditions, and other reactive moieties in the compound undergoing deprotection, i.e., a deprotecting agent is chosen to carry out the deprotection without effecting the other reactive moieties unless a concomitant reaction is desired.", "A particular amine protecting agent is Cbz.", "A particular acid protecting agent is C1 to C8 lower alkyl; more particular methyl.", "A particular deprotecting agent is an inorganic base such as an alkali hydroxide; more particular NaOH.", "Particular deprotection conditions encompass carrying out the deprotection in an alcoholic solvent such as methanol or ethanol at about room temperature.", "A compound of formula 18, wherein R0 is a bond and the other variables thereof are as described herein, may be prepared by protecting a compound of formula 20, wherein the variables thereof are as described herein, with a compound of formula 19, wherein the variables thereof are as described herein, under appropriate conditions.", "A particular amine protecting agent is Cbz or BOC.", "Particular coupling conditions use an appropriate organic solvent such as dichloromethane at about 0° C. to about room temperature.", "A compound of formula 20, wherein R4 is hydrogen and the other variables are as described herein, may be prepared by hydrogenating a compound of formula 21, wherein the variables thereof are as described herein, with an appropriate hydrogenating agent and under appropriate conditions.", "A particular hydrogenating agent is H2/Pd(OH)2.Particular hydrogenating conditions encompass carrying out the hydrogenation in an alcoholic solvent such as methanol or ethanol or an alkyl alkanoate solvent such as ethyl acetate at about room temperature.", "A compound of formula 20 wherein R4 is optionally substituted aliphatic and the other variables are as described herein may be prepared by alkylating compound 20′ wherein the variables are as described herein with compound 22 (alkylating agent) wherein R4 is optionally substituted aliphatic and Q is a displaceable group such as a halides, tosylates or sulfonates, under appropriate conditions.", "Appropriate alkylating agents include aliphatic (halides, tosylates or sulfonates).", "Appropriate alkylating conditions encompass carrying out to alkylation in an appropriate organic solvent such as an alcoholic solvent, e.g., methanol or ethanol, or etheric solvent, e.g., ether or tetrahydrofuran at about room temparature to about reflux.", "A compound of formula 21, wherein the variables are as described herein, may be prepared by alkylating a compound of formula 22, wherein the variable thereof is as described herein, with a compound of formula 23, wherein the R3′s independently are optionally substituted aliphatic group, optionally substituted cyclic group or optionally substituted aromatic group as described herein, under appropriate conditions.", "Particular alkylating conditions encompass carrying out the alkylation using a strong base such as potassium t-butoxide in an alcoholic solvent such as methanol or ethanol at about room temperature.", "A compound of formula 24 wherein the variables thereof are as described herein, may be prepared by effecting a Michael addition on a Michael acceptor of formula 29, wherein the variable thereof is as described herein, with an iminic glycinimide derivative.", "Michael additions comprise appropriate aprotic polar solvents, alkali methyl hydroxide bases, and appropriate temperatures.", "For Michael additions, see Corey, E. J.; Noe, M. C.; Xu, F. Tetrahedron Letter 1998, 39, 5347.For the synthesis of chiral phase transfer catalysts, see Corey, E. J.; Noe, M. C.; Xu, F. J.", "Am.", "Chem.", "Soc.", "1997, 119, 12414.Appropriate solvents include DCM, ACN, or THF depending on the reaction conditions.", "Appropriate bases include CsOH, NaOH, KOH, and LiOH.", "Appropriate temperatures range from about −78° C. to about 0° C., more particularly at about −60° C. Iminic glycinimides useful in the invention are described herein.", "A preferred iminic glycinimide is N-(diphenylmethylene)glycine tert-butyl ester.", "In addition, Michael addition conditions may be affected with or without a phase transfer catalyst (PTC) (chiral and nonchiral).", "A preferred PTC is O-[9]allyl-N-9-anthracenylmethylcinchonidium bromide.", "A compound of formula 25, wherein the variables thereof are as described herein, may be prepared by imine cleavage and cyclizing of the compound of formula 24.For cleavage and cyclization procedures, see Javidan, A.; Schfer, K.; Pyne, S. Synlett 1996, 100; Tatsukawa, A.; Dan, M.; Ohbatake, M.; Kawatake, K.; Fukata, T.; Wada, E.; Kanemase, S.; Kakei, S. J. Org.", "Chem.", "1993, 58, 4221.Cleavage and cyclizing conditions include the use of polar solvents, acid reagents, and temperatures of about room temperature to about 150° C. Preferred conditions include the use of EtOH, AcONa and NH2OH.HCl, and a temperature of about boiling point for the solvent used.", "A compound of formula 26, wherein the variables thereof are as described herein, may be prepared by protecting the amide of the compound of formula 25, wherein the variables thereof are as described herein, with a suitable amide protecting group such as BOC.", "Other suitable protecting groups include CBz, —CO2alkyl.", "Also see, Greene, T. W.; P. G. M. in Protective Groups in Organic Synthesis, Wiley, New York, 1991 for other amine protecting groups.", "Protecting conditions include the use of aprotic polar solvents, organic bases as catalysts, and temperatures of about 0° C.-100° C. Preferred conditions include the use of ACN, dimethyl amino pyridine, and a temperature of about room temperature.", "A compound of formula 27, wherein the variables thereof are as described herein, may be prepared by reducing the protected compound of formula 26, wherein the variables thereof are as described herein.", "Two reductions are, in fact, done.", "The first reduction is of the amide to a hemiaminal using DIBALH or superhydride [LiBEt3H].", "The second reduction is of the hemiaminal to the amine using Et3SiH and BF3.OEt2.See Collado, I; Ezquerra, J.; Mateo, A. I.; Rubio, A., J. Org.", "Chem.", "1998, 63 1995-2001 and Ezqueera, J.; Pedregal, C.; Yruretagoyena, B.; Rubio, A.; Carreno, M. C.; Escribano, A.; Garcia Ruano, J. L. J. Org.", "Chem.", "1995, 60,2925 for reducing conditions.", "Other usual conditions for converting pyroglutamates into pyrrolidines is the use of BH3.SMe2.A compound of formula 28, wherein the variables thereof are as described herein, may be prepared by deprotecting the compound of formula 27, wherein the variables thereof are as described herein.", "See Gibson, P. G.; Bermeier, S. C.; Rapoport, H., J. Org Chem.", "1994, 59, 3216-3218 for the conditions for selective removal of N—BOC protecting group in the presence of tert-butyl ester.", "One skilled in the art would know that the deprotecuing conditions will dependent upon the choice of the protecting group.", "For example, if CBz is used, hydrogenation or basic conditions may be used.", "Preferably, if BOC is used, 1 N HCl in ethyl acetate may be used.", "See, Greene, T. W.; P. G. M. in Protective Groups in Organic Synthesis, Wiley, New York, 1991.The person of ordinary skill in the art will appreciate that a compound of formula 3 may be prepared by coupling a compound of formula 5 with a compound of formula 28 under conditions described above herein.", "Methods for preparing R3, R5 or R7 as optionally substituted ethanediyl moieties include those known to those skilled in the art, e.g., those methods described in “The organic Chemistry of β-Lactams” edited by G. Georg, VCH Publishers, Inc. (1993), e.g., pages 240-241 and 303-305.Schemes 1-11 that follow exemplify assorted methods for preparing an optionally substituted multicyclic azaheterocyclyl.", "The methods in the schemes below are also applicable to other optionally substituted multicyclic azaheterocyclyls comprising compatible like substituents.", "A compound of formula 1 including a group containing one or more nitrogen ring atoms, preferably imine (═N—), may be converted to the corresponding compound wherein one or more nitrogen ring atoms of the group are oxidized to an N-oxide, preferably by reacting with a peracid, for example peracetic acid in acetic acid or m-chloroperoxybenzoic acid in an inert solvent such as dichloromethane, at a temperature from about room temperature to reflux, preferably at elevated temperature.", "In the reactions described hereinafter it may be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions.", "Conventional protecting groups may be used in accordance with standard practice, for examples see T. W. Green and P. G. M. Wuts in “Protective Groups in Organic Chemistry” John Wiley and Sons (1991); J. F. W. McOmie in “Protective Groups in Organic Chemistry” Plenum Press, 1973.A compound that is prepared as described herein may be recovered from the reaction mixture by conventional means.", "For example, the compounds may be recovered by distilling off the solvent from the reaction mixture or, if necessary after distilling off the solvent from the reaction mixture, pouring the residue into water followed by extraction with a water-immiscible organic solvent and distilling off the solvent from the extract.", "Additionally, the product can, if desired be further purified by various well techniques, such as recrystallization, reprecipitation or the various chromatography techniques, notably column chromatography or preparative thin layer chromatography.", "According to a further feature of the present invention, compounds of the invention may be prepared by interconversion of other compounds of the invention.", "As an example of the interconversion process, compounds of formula 1 containing sulphoxide linkages may be prepared by the oxidation of corresponding compounds containing —S— linkages.", "For example, the oxidation may conveniently be carried out by means of reaction with a peroxyacid, e.g., 3-chloroperbenzoic acid, preferably in an inert solvent, e.g., dichloromethane, preferably at or near room temperature, or alternatively by means of potassium hydrogen peroxomonosulphate in a medium such as aqueous methanol, buffered to about pH 5, at temperatures between about 0° C. and room temperature.", "This latter method is preferred for compounds containing an acid-labile group.", "As another example of the interconversion process, compounds of formula 1 containing sulphone linkages may be prepared by the oxidation of corresponding compounds containing —S— or sulphoxide linkages.", "For example, the oxidation may conveniently be carried out by means of reaction with a peroxyacid, e.g., 3-chloroperbenzoic acid, preferably in an inert solvent, e.g., dichloromethane, preferably at or near room temperature.", "It will be understood that designation of aromaticity with respect to aryls and heteroaryls herein includes any highly resonant unsaturated ring structure.", "Alternatively, placement of double bonds, where indicated, represents one potential structure for the depicted compound but will be understood to include other resonant states of the compound as well as protonated and charged species, only one of which may be shown.", "It will be appreciated that compounds of the present invention may contain asymmetric centers.", "These asymmetric centers may independently be in either the R or S configuration.", "It will be apparent to those skilled in the art that certain compounds of the invention may also exhibit geometrical isomerism It is to be understood that the present invention includes individual geometrical isomers and stereoisomers and mixtures thereof, including racemic mixtures, of compounds according to the invention.", "Such isomers can be separated from their mixtures, by the application or adaptation of known methods, for example chromatographic techniques and recrystallization techniques, or they are separately prepared from the appropriate isomers of their intermediates.", "For the purpose herein it is understood that tautermeric forms are included in the recitation of a given group, e.g., thioxo/mercapto or oxo/hydroxyl.", "Acid additional salts are formed with the compounds of the invention in which a basic function such as an amino, alkylamino, or dialkylamino group is present.", "The pharmaceutically acceptable, i.e., nontoxic, acid addition salts are preferred.", "The salts chosen are chosen optimally to be compatible with the customary pharmaceutical vehicles and adapted for oral or parenteral administration.", "Acid addition salts of the compounds of this invention may be prepared by reaction of the free base with the appropriate acid, by the application or adaptation of known methods.", "For example, the acid addition salts of the compounds of this invention may be prepared either by dissolving the free base in water or aqueous alcohol solution or other suitable solvents containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and acid in an organic solvent, in which case the salt separates directly or can be obtained by concentration of the solution.", "Some suitable acids for use in the preparation of such salts are hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, various organic carboxylic and sulfonic acids, such as acetic acid, citric acid, propionic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, methanesulfonic acid, toluenesulfonic acid, fatty acids, adipate, alginate, ascorbate, aspartate, benzenesulfonate,.", "benzoate, cyclopentanepropionate, digluconate, dodecylsulfate, bisulfate, butyrate, lactate, laurate, lauryl sulfate, malate, hydroiodide, 2-hydroxy-ethanesulfonate, glycerophosphate, picrate, pivalate, pamoate, pectinate, persulfate, 3-phenylpropionate, thiocyanate, 2-naphthalenesulfonate, undecanoate, nicotinate, hemisulfate, heptonate, hexanoate, camphorate, camphersulfonate, and others.", "The acid addition salts of the compounds of this invention can be regenerated from the salts by the application or adaptation of known methods.", "For example, parent compounds of the invention can be regenerated from their acid addition salts by treatment with an alkali, e.g., aqueous sodium bicarbonate solution or aqueous ammonia solution.", "Compounds of this invention can be regenerated from their base addition salts by the application or adaptation of known methods.", "For example, parent compounds of the invention can be regenerated from their base addition salts by treatment with an acid, e.g., hydrochloric acid.", "Base addition salts may be formed where the compound of the invention contains a carboxy group, or a sufficiently acidic bioisostere.", "The bases which can be used to prepare the base addition salts include preferably those which produce, when combined with the free acid, pharmaceutically acceptable salts, that is, salts whose cations are non-toxic to the patient in pharmaceutical doses of the salts, so that the beneficial inhibitory effects inherent in the free base are not vitiated by side effects ascribable to the cations.", "Pharmaceutically acceptable salts, including those derived from alkali and alkaline earth metal salts, within the scope of the invention include those derived from the following bases: sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.", "Compounds of the present invention may be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates).", "Hydrates of compounds of the present invention may be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxan, tetrahydrofuran or methanol.", "The starting materials and intermediates may be prepared by the application or adaptation of known methods, for example methods as described in the Reference Examples or their obvious chemical equivalents.", "The compounds of the invention, their methods or preparation and their biological activity will appear more clearly from the examination of the following examples which are presented as an illustration only and are not to be considered as limiting the invention in its scope.", "Samples were analyzed by TLC, NMR, RP-HPLC or EA.", "The compounds of the invention, their methods or preparation and their biological activity will appear more clearly from the examination of the following examples which are presented as an illustration only and are not to be considered as limiting the invention in its scope.", "Samples were analyzed by TLC, NMR, RP-HPLC or EA.", "EXAMPLE 1 Compounds A-E To a DCM solution (10 mL) of compound xi (310 mg, 0.39 mmol) is added TFA (4 mL).", "The reaction is stirred at about room temperature for 5 hours.", "At this point, the solvent is removed in vacuo.", "The resulting residue is purified by reverse phase HPLC to give 195 mg (68%) of compound A, Following the above method and using the appropriate starting materials, the following consecutive compounds B-E are prepared: EXAMPLE 2 Compounds F-M To a DCM solution (10 mL) of compound xii (350 mg, 0.56 mmol) is added DMP reagent reagent (307 mg, 0.73 mmol).", "The reaction is stirred at about room temperature for 2 hours and then quenched with 10% Na2SO3 for 30 minutes.", "The reaction mixture is then extracted with EtOAc (75 mL) and washed with brine.", "The organic layer is dried and concentrated in vacuo.", "The resulting residue is purified with silica gel chromatography (80-90% EtOAc/Hexanes) to give 248 mg (71%) of compound F Following the above method and using the appropriate starting materials, the following consecutive compounds G-M are prepared: EXAMPLE 3 Compounds N-R To a DCM solution (4 mL) of compound xix (˜0.22 mmol) is added DMP reagent reagent (146 mg, 0.34 mmol).", "After stirring at about room temperature for 2 hours, the reaction is quenched with 10% Na2SO3.The reaction mixture is then diluted with DCM.", "The organic layer is separated and washed with 10% Na2SO3 twice and brine.", "The resulting organic layer is dried and concentrated in vacuo to give a residue, which is purified by silica gel chromatography (5% EtOH/EtOAc) to provide 78 mg (56%) of the desired compound N Following the above method and using the appropriate starting materials, the following consecutive compounds O-R are prepared: EXAMPLE 4 Compounds S-W To a DCM solution (10 mL) of compound xxv (320 mg, 0.5 mmol) is added DMP reagent reagent (272 mg, 0.65 mmol).", "The reaction is stirred at about room temperature for 2 hours and quenched with 10% Na2SO3 for 20 minutes.", "The resulting mixture is then extracted with EtOAc.", "The organic layer is washed with brine, dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (80% EtOAc/Hexanes) to give 170 mg (53%) of compound S, Following the above method and using the appropriate starting materials, the following consecutive compounds T-W are prepared: EXAMPLE 5 Compounds X-AD To a DCM solution (20 mL) of compound xxvi (400 mg, 0.6 mmol) is added DMP reagent reagent (329 mg, 0.78 mmol).", "The reaction is stirred at about room temperature for 1.5 hours and quenched with 10% Na2SO3 for 20 minutes.", "The resulting mixture is then extracted with EtOAc.", "The organic layer is washed with brine, dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (70-100% EtOAc/Hexanes) to give 210 mg (53%) of compound X, Following the above method and using the appropriate starting materials, the following consecutive compounds Y-AD are prepared: EXAMPLE 6 Compounds AE-AI Compound xxxiii (150 mg; 0.076 mmol) is dissolved in 5 mL TFA and stirred for two days.", "The product is purified by RP-HPLC to yield 40 mg (33% yield) of compound AE, Following the above method and using the appropriate starting materials, the following consecutive compounds AF-AI are prepared: EXAMPLE 7 Compound AJ Compound xxxviii (180 mg, 0.21 mmol) is dissolved in neat TFA (5 mL) and left for 3 day at about room temperature.", "At this point, the reaction mixture is concentrated in vacuo to give a residue, which is purified by reverse phase HPLC to give 50 mg (32%) of the compound AJ, EXAMPLE 8 Compounds AK-AM Compound xxxxiii (150 mg; 0.16 mmol) is dissolved in 4.5 mL TFA and stirred for three days.", "The product is purified by RP-HPLC to yield 70 mg (54% yield) compound AK, Following the above method and using the appropriate starting materials, the following consecutive compounds AL-AM are prepared: EXAMPLE 9 Compounds AN Compound 1ii (80 mg) is dissolved in 3 mL TFA and 3 mL DCM.", "The mixture is stirred at about room temperature for 5 hours.", "The solvent is removed by evaporation.", "The resulting residue is purified by HPLC to yield 62 mg (83%) of compound AN, EXAMPLE 10 Compounds AO Compound 1iii (160 mg; 0.2 mmol) is dissolved in 5 mL DCM and DMP reagent reagent (170 mg; 0.4 mmol) is added.", "The mixture is stirred at about room temperature for three hours.", "The solvent is removed by evaporation and the residue is dissolved in 50% acetonitrile/water and purified by RP-HPLC to yield 51 mg (32%) of compound AO, EXAMPLE 11 Compounds AP Compound 1ix (162 mg; 0.22 mmol) is dissolved in 8 mL of DCM and DMP reagent reagent (189 mg; 0.44 mmol) is added.", "The mixture is stirred at about room temperature for 3 hours.", "The solvent is removed by evaporation and product is purified by RP-HPLC to yield 41 mg (25%) of compound AP, EXAMPLE 12 Compounds AQ Compound 1x (70 mg; 0.09 mmol) is dissolved in 5 mL TFA and 5 mL DCM.", "The mixture is stirred at about room temperature for 3 hours.", "The solvent is removed by vacuum and the residue is dissolved in 50% acetonitrile/water and lyophilized to yield compound AQ as a powder, EXAMPLE 13 Compounds AR-BG Compound 1xvi (223 mg, 0.326 mmol) is stirred in a solution of TFA (5 mL) and DCM (5 mL) for 4 hours.", "TLC (silica gel: 2% MeOH/EtOAc) showed complete conversion to the slower product The solvent is removed under reduced pressure and the product lyophilized to give 198 mg (97%) compound AR, Following the above method and using the appropriate starting materials, the following consecutive compounds AS-BG are prepared: EXAMPLE 14 Compounds BH-BS Compound 1xxiii (150 mg, 0.15 mmol) is taken up in DCM (3 mL).", "To this solution is added TFA (1.5 mL).", "The resulting solution is stirred overnight.", "At this point, the reaction is concentrated in vacuo to give a residue.", "The residue is purified by reverse phase HPLC and lyophilized to give 60 mg (50%) of compound BH, Following the above method and using the appropriate starting materials, the following consecutive compounds BI-BS are prepared: EXAMPLE 15 Compounds BT-BU Following the method of Example 12 and using the appropriate starting materials, the following consecutive compounds BT-BU are prepared: EXAMPLE 16 Compound BV To a dichloromethane solution (4.2 mL) of compound 1xxvii (143 mg, 0.21 mmol) is added DMP reagent reagent (165 mg, 0.39 mmol).", "The reaction is stirred at about room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The organic layer is washed with brine, dried over MgSO4 and concentrated to a yellow oil.", "Purification by silica gel chromatography (5% EtOH/EtOAc) yielded 124 mg (79%) of compound BV, EXAMPLE 17 Compounds BW-CA To a dichloromethane solution (20 mL) of compound 1xxxix (420 mg, 0.62 mmol) is added DMP reagent reagent (342 mg, 0.81 mmol).", "The reaction is stirred at about room temperature for 1 hour and quenched with 10% Na2SO3 for 20 minutes.", "The resulting mixture is then extracted with EtOAc.", "The organic layer is washed with brine, dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (80% EtOAc/Hexanes) to give 208 mg (50%) of compound BW, Following the above method but using the appropriate starting materials, the following consecutive compounds BX-CA are prepared: EXAMPLE 18 Compounds CB-CC To a dichloromethane solution (6.5 mL) of compound 1xxxvii (200 mg, 0.3 mmol) is added DMP reagent reagent (227 mg, 0.54 mmol).", "The reaction is stirred at about room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The organic layer is washed with brine, dried over MgSO4 and concentrated to a yellow oil.", "Purification by silica gel chromatography (5% EtOH/EtOAc) yields 138 mg (70%) of compound CB, Following the above method but using the appropriate starting materials, the following compound CC is prepared: EXAMPLE 19 Compound CD Compound 1xxxxviii (40 mg, 0.05 mmol) is taken up in TFA (3 mL).", "The solution stirred over two nights and is concentrated.", "The residue is purified on reverse phase HPLC to give 25 mg (74%) of compound CD, EXAMPLE 20 Compound CE Following the method of Example 17 and using the appropriate starting materials, the following compound CE is prepared: EXAMPLE 21 Compounds CF-CG Following the method of Example 14 and using the appropriate starting materials, the following consecutive compounds CF-CG are prepared: EXAMPLE 22 Compound CH Following the method of Example 16 and using the appropriate starting materials, the following compound CH is prepared: EXAMPLE 23 Compounds CI-CM Compound cxi (490 mg, 0.75 mmol) is dissolved in DCM (6 mL).", "DMP reagent reagent (380 mg, 0.9 mmol) is added to this solution and stirred 1 hour.", "The reaction mixture is quenched with a 10% Na2SO3 solution, and then the organic phase is washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 70% EtOAc/hexanes to yield compound CI (325 mg, 66.4%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds CJ-CM are prepared: EXAMPLE 24 Compounds CN To a DCM/THF solution (3 mL/3 mL) of compound cxviii (335 mg, 0.46 mmol) is added DMP reagent reagent (300 mg, 0.69 mmol).", "The reaction mixture is stirred at room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The organic phase is washed with brine, dried over MgSO4 and concentrated to yield a yellow oil.", "Purification by silica gel chromatography (80% EtOAc/hexanes) yields compound CN (220 mg, 67%).", "EXAMPLE 25 Compounds CO-CR To a DCM/THF solution (1.5 mL/1.5 mL) of compound cxix (164 mg, 0.25 mmol) is added DMP reagent reagent (159 mg, 0.38 mmol).", "The reaction mixture is stirred at room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The organic phase is washed with brine, dried over MgSO4 and concentrated to yellow oil.", "Purification by silica gel chromatography (70% EtOAc/hexanes) yields compound CO (100 mg, 61%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds CP-CR are prepared: EXAMPLE 26 Compounds CS-CT Compound cxx is dissolved in DCM (3 mL).", "DMP reagent reagent (180 mg, 0.41 mmol) is added to the solution, and then stirred for 1 hour.", "The reaction mixture is quenched with 10% Na2SO3, and then the organic phase is washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the residue is chromatographically purified by 100% EtOAc to yield compound CS (95 mg, 43.7%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compound CT is prepared: EXAMPLE 27 Compounds CU, EI, EK-EM, EO-EZ, and FA-FG Compound cxxviii (356 mg, 0.52 mmol) is dissolved in DCM (5 mL).", "DMP reagent reagent (270 mg, 0.63 mmol) is added to this solution and stirred 1 hour.", "The reaction mixture is quenched with 10% Na2SO3, and then the organic phase is separated and washed with saturated NaHCO3 and brine.", "Following the concentration of the organic solvent, the residue is chromatographically purified by 100% EtOAc to yield compound CU (200 mg, 56.3%).", "mp 225-235° C. Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds EI, EK-EM, EO-EZ and FA-FH are prepared: EXAMPLE 28 Compounds CV-DC Compound cxxx (330 mg, 0.46 mmol) is dissolved in DCM (5 mL).", "DMP reagent reagent (240 mg, 0.56 mmol) is added to this solution and stirred 1 hour.", "The reaction mixture is quenched with a 10% Na2SO3, and the organic phase washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resulting residue is chromatographically purified by b 100% EtOAc to yield compound cxxx (280 mg, 85.9%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds CW-DC are prepared: EXAMPLE 29 Compounds DD-DE To a DCM solution (6 mL) of compound cxxxviii (400 mg, 0.57 mmol) is added DMP reagent reagent (362 mg, 0.85 mmol).", "The reaction mixture is stirred at room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The extracted organic phase is washed with brine, dried over MgSO4 and concentrated to yield yellow oil.", "Purification by silica gel (70% EtOAc/hexanes) yields compound DD (201 mg, 51%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compound DE is prepared: EXAMPLE 30 Compound DF Compound cxxxxiii (165 mg, 0.24 mmol) is dissolved in DCM (5 mL).", "DMP reagent reagent (125 mg, 0.29 mmol) is added to the solution and stirred 1 hour.", "The reaction mixture is quenched with a 10% Na2SO3, and the organic phase washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is purified chromatographically by 70% EtOAc/hexanes to yield compound DF (108 mg, 65.6%).", "EXAMPLE 31 Compounds DG-DJ To a solution of compound ci1 (0.350 g, 0.516 mmol) in DCM (15 mL) cooled by an ice bath is added DMP reagent reagent (0.281 g, 0.671 mmol).", "The mixture is stirred at room temperature for 2 hours, then quenched with 10% Na2SO3 solution and stirred for 20 minutes.", "The resulting mixture is extracted with DCM (3×20 mL) and the organic extract is dried (MgSO4).", "After filtration to remove MgSO4, the filtrate is concentrated and purified by column chromatography (70% Ethyl acetate/Hexanes) to yield the final compound DG (0.265 g, 76%) as white solid.", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds DH-DJ are prepared: EXAMPLE 32 Compounds DK-DN A DCM solution of compound c1x (108 mg, 0.123 mmol) is treated with DMP reagent reagent (78 mg, 0.185 mmol).", "After stirring at room temperature for 1 hour, the reaction mixture is diluted with EtOAc (50 mL), and then quenched with 10% Na2SO3.After stirring for 30 minutes, the organic phase is separated and washed with NaHCO3 and brine.", "The organic phase is dried and concentrated in vacuo to give a residue that is purified by silica gel chromatography (80% EtOAc/hexanes) to yield compound DK (84 mg, 78%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compounds DL-DN are prepared.", "EXAMPLE 33 Compounds DO-DS An EtOH solution (10 mL) of compound c1xii (174 mg, 0.189 mmol) is hydrogenated using Pd/C (30% eq., 60 mg, 10% palladium content) for 2.5 hours.", "The catalyst is then filtered off.", "The resulting filtrate is concentrated in vacuo to yield a residue that is purified by semi-preparative reverse phase chromatography and lyophilized to afford compound DO in 70% yield.", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds DP-DS are prepared.", "EXAMPLE 34 Compound CW Compound c1xiii (175 mg, 0.24 mmol) is taken up in DCM (3 mL).", "DMP reagent (120 mg, 0.28 mmol) is added to this solution and stirred 1 hour.", "The reaction is quenched with a 10% Na2SO3 and washed with saturated NaHCO3 and brine.", "Purification by 70% EtOAc yields compound CW (134 mg, 75%).", "EXAMPLE 35 Compounds CY and DT-DX To a DCM solution (15 mL) of c1xxii (290 mg, 0.43 mmol) is added DMP reagent (239 mg, 0.56 mmol).", "The reaction is stirred at room temperature for 1 hour and quenched with 10% Na2SO3 for 20 minutes.", "The resulting mixture is then extracted with EtOAc.", "The organic layer is washed with brine, dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (8-100% EtOAc/Hexanes) to give compound CY (151 mg, 52%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following consecutive compounds DT-DX are prepared.", "EXAMPLE 36 Compounds DY Compound 1xxxv (1.17 mmol) is taken up in DCM (5 mL).", "DMP reagent (545 mg, 1.3 mmol) is added to this solution and stirred 1 hour.", "The reaction is quenched with a P—Na2SO3 (1.5 mmol/g resin) and stirred one hour.", "P-TBD scavenger resin* (2.5 mmol/g resin) is added and stirred 45 minutes.", "The resulting mixture is filtered and purified by 50% EtOAc to give compound DY (440 mg, 50.2% over two steps).", "*Reference for P-TBD scavenger resin: J. Parlow et al.", "Tetrahedron, 55, 6785-6796 (1999).", "EXAMPLE 37 Compound DZ The starting material compound c1xxxi (94 mg, 0.14 mmole) is dissolved in a mixture of THF (10 mL) and DCM (20 mL).", "The DMP reagent (118 mg, 0.28 mmol) is then added.", "After stirring at room temperature for 2 hours.", "The reaction is dumped in a separatory funnel containing Dri Solv THF (120 mL).", "The reaction is washed with 10% Na2SO3 (50 mL), and then brine (75 mL).", "The organic layer is then separated, dried over MgSO4 and the solvent removed under reduced pressure.", "After chromatography (silica gel: elution with 50% Dri Solv THF/EtOAc, and then 4% MeOH/THF).", "Fractions are checked by MS.", "Appropriate fractions are lyopholized to yield compound DZ (38.8 mg, 41%).", "EXAMPLE 38 Compounds EA-EB The starting compound c1xxxxv (185 mg, 0.26 mmol) is dissolved in THF (20 mL).", "The DMP reagent (219 mg, 0.52 mmol) is then added.", "After stirring at room temperature for 1 hour.", "TLC shows complete conversion to ketone (5% MeOH/THF).", "The reaction is dumped in a separatory funnel containing Dri Solv THF (120 mL).", "The reaction is washed with 10% Na2SO3 (50 mL), and then brine (75 mL).", "The organic layer is then separated, dried over MgSO4 and solvent removed by reduced pressure to yield a residue that is purified by chromatography (silica gel: elution with 50% Dri Solv THF/EtOAc, and then 4% MeOH/THF) and fractions are checked by UV and MS.", "The appropriate fractions are lyopholized to yield compound EA (159 mg, 88%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compound EB is prepared: EXAMPLE 39 Compounds EC-ED To a solution of compound c1xxxxviii (0.341 g, 0.503 mmol) in DCM (15 mL) cooled in an ice bath is added DMP reagent (0.277 g, 0.654 mmol).", "The mixture is stirred at room temperature for 2 hours, then quenched with 10% Na2SO3 solution and stirred for 20 minutes.", "The resulting mixture is extracted with DCM (3×20 mL) and the organic extract is dried (MgSO4).", "After filtration to remove MgSO4, the filtrate is concentrated and purified by column chromatography (70% EtOAc/Hexane) to give compound EC (0.183 g, 54%) as white solid.", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compound ED is prepared: EXAMPLE 40 Compounds EE-EG Compound ccii (290 mg, 0.37 mmol) is taken up in DCM (5 mL).", "DMP reagent (175 mg, 0.41 mmol) is added to this solution and stirred 1 hour.", "The reaction is quenched with P—Na2SO3 (1.5 mmol/g resin) and stirred 1 hour.", "Quenched DMP reagent is scavenged with P-TBD (2.5 mmol/g resin) and stirred 1 hour.", "The resulting mixture is filtered, rinsed with DCM, before being concentrated to a residue.", "The resulting residue is purified by 50% EtOAc/Hex to yield compound EE (440 mg, 28%).", "Following the above method for preparing the above compound and methods related to preparing the intermediate thereto, but using the appropriate starting materials the following compounds EF-EG are prepared: EXAMPLE 41 Compound EH To a DCM solution (3 mL) of compound cciii (140 mg, 0.2 mmol) is added DMP reagent (133 mg, 0.3 mmol).", "The reaction is stirred at room temperature for 2 hours and quenched with 10% Na2SO3 (aq.)", "for 20 minutes.", "The resulting mixture is extracted with EtOAc.", "The organic layer is washed with brine, dried over MgSO4, concentrated to a yellow oil that is purified by silica gel (70% EtOAc/hexane), and after lypholized to yield compound EH (50 mg, 38%).", "EXAMPLE 42 Compound EJ Compound ixxxiii (520 mg, 1 mmol) is taken up in DCM (5 mL).", "PyBOP (624 mg, 1.2 mmol) is added to the above solution and stirred for 5 minutes.", "Compound cdviii (300 mg, 1.2 mmol) in THF (5 mL) is added drop-wise to this solution, followed by DIPEA (0.22 ml, 1.2 mmol).", "The reaction is stirred at room temperature overnight under nitrogen.", "At this point, the reaction is diluted with EtOAc, washed with saturated NaHCO3, and brine.", "The organic phase is dried with MgSO4, filtered, and concentrated to give the crude coupled intermediate cdix.", "cdix This intermediate cdix (˜1 mmol) is taken up in DCM (10 mL).", "Dess-Martin Periodinane (466 mg, 1.1 mmol) is added to this solution.", "After stirring for 1 hour at room temperature, the reaction is quenched with a polymer bound Na2SO3 (740 mg, 1.5 mmol DMP/g resin) and stirred 45 minutes.", "Then, the reaction mixture is scavenged with polymer bound TBD resin (440 mg, 2.5 mmol DMP/g resin).", "The resulting mixture is stirred for 45 minutes and then filtered.", "Purification is achieved in 5% EtOH/EtOAc to yield compound EJ (245 mg, 32% over 2 steps).", "Literature reference for the work-up procedure can be found in Tetrahedron 55 (1999) 6785-6796.EXAMPLE 43 Compound EN Intermediate compound cdvii (415 mg, 0.59 mmol) is taken up in DCM (10 mL) and THF (10 mL).", "t-BuOH (300 uL) is added followed by Dess-Martin Periodinane (750 mg, 1.77 mmol).", "The reaction is stirred 50 minutes and then quenched with P—Na2SO3 (1.5 mmol DMP/g resin).", "After stirring for 20 minutes at room temperature, the reaction mixture is scavenged with P-TBD (2.5 mmol DMP/g resin).", "After stirring for 1 hour, the resulting mixture was filtered and concentrated.", "Product was purified by silica gel chromatography (50% to 70% EtOAc/Hexanes) to yield compound EN (220 mg, 53%).", "Mass Spectra [M] were obtained for the following compounds as shown in Table 1 below.", "TABLE 1 LY# Example Mass Found A 733.3 B 747.2 C 657.2 D 769.4 E 733.4 F 625.4 G 639.3 H 661.4 I 643.4 J 707.3 K 641.3 L 689.3 M 639.3 N 639.4 O 731.4 P 687.4 Q 653.4 R 701.4 S 639.3 T 747.1 U 655.4 V 653.4 W 703.4 X 661.3 Y 647.3 Z 663.3 AA 667.4 AB 711.4 AC 725.4 AD 647.3 AE 779.4 AF 689.3 AG 671.4 AK 806.4 AH 687.5 AI 735.4 AJ 736.5 AM 870.4 AN 813.3 AP 724.4 AQ 653.4 AR 628.2 AW 642.2 AX 614.2 AY 628.3 BD 570.3 BE 520.2 BF 534.3 BG 584.3 BU 890.3 BV 685.4 BW 679.3 BX 695.3 BY 697.3 BZ 787.4 CA 701.3 CB 669.4 CC 733.5 CD 643.3 CE 653.5 CH 749.4 CI 653.3 CJ 717.5 CK 683.4 CL 669.3 CM 675.2 CN 717.2 CO 653.3 CP 683.3 CQ 669.3 CR 675.2 CT 661.8 CS 639.3 CU 679.2 CV 709.3 CW 743.3 CX 695.3 CY 665.2 CZ 681.3 DA 695.3 DB 701.2 DC 673.3 DD 693.3 DE 757.4 DF 682.3 DG 676.3 DH 676.2 DI 692.5 DJ 605.2 DK 874.4 DL 924.5 DM 924.2 DN 952.7 DO 830 DP 842.5 DT 667.4 DU 639.2 DV 740.3 DW 684.2 DX 678.5 DY 749.3 DZ 685.3 EA 649.3 EB 700.3 EC 702.3 ED 730.3 EE 775.3 EF 749.3 EG 722.3 EH 665.2 EI 796.4 EJ 744.3 EK 730.5 EL 730.5 EM 757.3 EN 703.5 EO 715.5 EP 679.2 EQ 651.3 ER 715.3 ES 668.5 ET 732.5 EU 743.3 EV 683.3 EW 750.4 EX 786.4 EY 744.5 EZ 780.4 FB 693.4 FC 655.3 FD 655.3 FE 774.4 FF 681.5 FG 667.5 High Resolution Mass Spectra (HRMS) of the following compounds were obtained as shown in Table 2.TABLE 2 Molecular Formula Calculated MS Mass Found Example (M + 1) (M + 1) (M + 1) L C37H52N7O6 690.3979 690.3986 M C33H50N7O6 640.3822 640.3822 Z C32H48F2N7O6 664.3634 664.3627 AB C36H48F2N7O6 712.3634 712.3649 CE C34H52N7O6 654.3979 654.3967 EN C35H52N7O6F2 704.3947 704.3945 EK C37H63N6O8S 751.4428 750.4350 (M) EC C36H59N6O8 703.4395 703.4382 CA C35H50N7O6F2 702.3790 702.3801 EZ C40H55N8O6F2 781.4213 781.4196 EU C36H52N7O6F2 716.3947 716.3929 CY C35H52N7O6 666.3979 666.3966 BX C37H58N7O6 696.4448 696.4432 S C33H50N7O6 640.3823 640.3831 BW C36H54N7O6 680.4136 680.4126 CU C36H54N7O6 680.4136 680.4128 EJ C40H57N8O6 745.4401 745.4417 EM C35H54N7O6 668.4136 668.4139 None C41H58N7O6 744.4448 744.4691 INTERMEDIATE EXAMPLE 1 Compound ii To an ethanol solution (40 mL) of compound i (8.1 g, 24.4 mmol) is added NaBH4 (924 mg, 24.4 mmol) at −10° C. The reaction is stirred at that temperature for 30 minutes, and then quenched with AcOH (3 mL).", "The reaction mixture is diluted with EtOAc (250 mL), and washed with NaHCO3 and brine.", "The organic layer is dried and concentrated in vacuo to yield a residue that is purified by silica gel chromatography (50% EtOAc/Hexanes) to provide 7.85 g (97%) of compound ii, INTERMEDIATE EXAMPLE 2 Compound iii To a THF solution (70 mL) of compound ii (4.48 g, 13.4 mmol) is added at 0° C. of NaH (699 mg, 60%, 17.42 mmol).", "After stirring at that temperature for 40 minutes, neat MeI (1.25 mL, 20.1 mmol) is added.", "The reaction is stirred at about room temperature overnight.", "At this point, the reaction is quenched carefully with saturated solution of NH4Cl at 0° C. The reaction mixture is extracted with Et2O and EtOAc.", "The organic layer is washed with water, brine and dried with Na2SO4.The organic layer thus obtained is concentrated in vacuo to provide the xanthate compound iii, INTERMEDIATE EXAMPLE 3 Compound iv The xanthate compound iii (˜13.4 mmol) is dissolved in toluene (100 mL).", "To this solution is added AIBN (216 mg, 1.34 mmol).", "The resulting solution is degassed with dry nitrogen and then treated with n-Bu3SnH (5.4 mL, 20.1 mmol).", "The reaction mixture is heated at 90° C. for 3 hours.", "At this point, the reaction is cooled to room temperature and concentrated in vacuo.", "The resulting residue is purified with silica gel chromatography (15-20% EtOAc/Hexanes) to provide 2.8 g (66% overall from compound ii) of compound iv, INTERMEDIATE EXAMPLE 4 Compound v To an ethanol solution (21 mL) of compound iv (1 g, 3.15 mmol) is added Pd(OH)2/C (655 mg, 20%, 0.95 mmol) under a stream of nitrogen.", "The resulting reaction mixture is subjected to standard hydrogenation (1.5 atm).", "After 5 hours, the hydrogen source is removed and the reaction is filtered.", "The filtrates are concentrated in vacuo to provide the free amine compound v, INTERMEDIATE EXAMPLE 5 Compound vi To a DCM solution (10 mL) of compound vii (629 mg, 1.95 mmol) is added at about room temperature HOAt (265 mg, 1.95 mmol) and followed by 1 M DCC solution in DCM (1.95 mL, 1.95 mmol).", "After stirring for 30 minutes, a DCM solution (3 mL) of compound v (1.5 mmol) is added to the above HOAt-activated acid.", "The reaction is stirred at about room temperature overnight.", "At this point, the reaction is filtered through Celite.", "The filtrates are diluted with EtOAc (75 mL) and washed with water and brine.", "The organic layer is dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (70-80% EtOAc/Hexanes) to afford 620 mg (85%) of compound vi, INTERMEDIATE EXAMPLE 6 Compound viii To an ethanol solution (10 mL) of compound vi (615 mg, 1.26 mmol) is added 2 N NaOH aqueous solution (1.26 mL, 2.52 mmol).", "The reaction is stirred overnight at about room temperature and then acidified to pH 3 using Dowex acidic resins.", "The solids are filtered off and the filtrates are concentrated in vacuo to give a residue that is redissolved in 1:1 CH3CN/H2O.", "This solution is subjected to lyophilization to provide 495 mg (85%) of compound viii, INTERMEDIATE EXAMPLE 7 Compound ix To a DCM solution (10 mL) of compound viii (230 mg, 0.5 mmol) is added PyBop (417 mg, 0.8 mmol).", "The reaction is stirred at about room temperature for 30 minutes.", "To this solution is then added a THF solution (5.25 mL) of compound x (263 mg, 0.75 mmol) and followed by DIPEA (0.174 mL, 1 mmol).", "The reaction is stirred at about room temperature overnight and then quenched with water (30 mL) for 30 minutes.", "The reaction mixture is extracted with EtOAc (100 mL).", "The organic layer is washed with brine and dried and concentrated in vacuo to afford a residue that is purified via silica gel chromatography (5% EtOH/EtOAc) to give ˜400 mg (100%) of compound ix, INTERMEDIATE EXAMPLE 8 Compound xi To a DCM solution (10 mL) of compound ix (396 mg, 0.5 mmol) is added DMP reagent reagent (278 mg, 0.65 mmol).", "The reaction is stirred at about room temperature for 1 hour and then quenched with 10% Na2SO3 for 30 minutes.", "The reaction mixture is then extracted with EtOAc (75 mL) and washed with brine.", "The organic layer is dried and concentrated in vacuo.", "The resulting residue is purified with silica gel chromatography (70% EtOAc/Hexanes) to give 320 mg (81%) of compound xi, INTERMEDIATE EXAMPLE 9 Compound xii To a DCM solution (10 mL) of compound viii (230 mg, 0.5 mmol) is added PyBop (417 mg, 0.8 mmol).", "The reaction is stirred at about room temperature for 30 minutes.", "To this solution is then added a THF solution (3.5 mL) of compound xiii (140 mg, 0.75 mmol) and followed by DIPEA (0.174 mL, 1 mmol).", "The reaction is stirred at about room temperature overnight and then quenched with water (30 mL) for 30 minutes.", "The reaction mixture is extracted with EtOAc (75 mL).", "The organic layer is washed with brine and dried and concentrated in vacuo to afford a residue that is purified via silica gel chromatography (5% EtOH/EtOAc) to give in quantitative yield compound xii, INTERMEDIATE EXAMPLE 10 Compound i′ To a methanol solution (30 mL) of compound i (5 g, 15.1 mmol) is added (BOC)2O (3.3 g, 15.1 mmol) and H2/Pd(OH)2/C (1.6 g, 10% Pd content).", "The reaction is stirred at about room temperature for 2 hours and then filtered through Celite twice.", "The Celite bed is rinsed with DCM.", "The combined filtrates are concentrated in vacuo to yield an oily residue that is purified by silica gel chromatography (40% EtOAc/Hexanes) to give 3.8 g (85%) of compound i′, INTERMEDIATE EXAMPLE 11 Compound ii′ To a methanol solution (111 mL) of compound i′ (3.7 g, 12.5 mmol) is added at 0° C. NaBH4 (0.805 g, 21 mmol).", "After stirring at 0° C. for 2.5 hours, the reaction solvent is evaporated slowly in vacuo to yield a residue that is diluted with EtOAc.", "This solution is then washed with water twice.", "The aqueous layer is extracted with EtOAc.", "The combined organic layers are dried with MgSO4 and filtered and concentrated in vacuo to yield a residue that is purified with chromatography to provide 3.76 g (99%) of compound ii′, INTERMEDIATE EXAMPLE 12 Compound xiv To a DCM solution (180 mL) of compound ii′ (3.76 g, 12.3 mmol) is added at 0° C. DMAP (5 g, 40.1 mmol) and then followed by Tf2O (4 mL, 23.7 mmol).", "The reaction is stirred at 0° C. for 1 hour and at about room temperature for additional 1.5 hours.", "The reaction mixture is then washed twice with 5% NaHCO3 and dried with MgSO4.The organic layer thus obtained is concentrated in vacuo to provide the crude triflate.", "The resulting triflate (2.7 g, 6 mmol) is dissolved in DCM (120 mL).", "To this solution is added DMAP (2.5 g, 20.5 mmol).", "The resulting reaction mixture is heated to reflux overnight At this point, the reaction is cooled to room temperature and washed with 5% NaHCO3 twice.", "The reaction mixture is dried with MgSO4 filtered and concentrated in vacuo to yield a brownish oily residue that is purified (1% MeOH/DCM) to give 500 mg (30%) of compound xiv, INTERMEDIATE EXAMPLE 13 Compound xv Compound xiv (500 mg, 1.8 mmol) is dissolved in 4 N HCl in dioxane (6.75 mL).", "The reaction is stirred at about room temperature for ˜4 hours.", "At this point, the solvent is removed in vacuo.", "The resulting residue is titrated with diethylether twice to give in almost quantitative yield the HCl salt of compound xv, INTERMEDIATE EXAMPLE 14 Compound xvi To a THF solution (7 mL) of compound vii (579 mg, 1.8 mmol) is added HOAt (245 mg, 1.8 mmol) and DCC (1.8 mL, 1 M in DCM).", "A suspension is resulted.", "After stirring at about room temperature for 15 minutes, a THF solution (6 mL) of compound xv (1.8 mmol) and DIPEA (0.63 mL, 3.6 mmol) is added to the above suspension.", "Additional DIPEA (0.8 mL) is added later.", "The reaction mixture is stirred overnight at about room temperature.", "At that point, the white solids so formed are filtered off.", "The white solids are rinsed with THF.", "The combined filtrates and washings are concentrated in vacuo to give the crude product that is purified by silica gel chromatography (100% EtOAc) to provide 665 mg (76%) of compound xvi, INTERMEDIATE EXAMPLE 15 Compound xvii To an ethanol solution (8 mL) of 7 (665 mg, 1.37 mmol) is added 1 N aqueous NaOH (2.4 mmol) at 0° C. The reaction-is stirred overnight at about room temperature, and then acidified to pH 3 using Dowex acidic resins.", "The solids are filtered.", "The resulting filtrates are concentrated in vacuo to give a pale yellow residue that is redissolved in 1:1 CH3CN/H2O and lyophilized to give 467 mg (74%) of compound xvii, INTERMEDIATE EXAMPLE 16 Compound xix A DCM solution (4 mL) of compound xvii (100 mg, 0.22 mmol) is treated with PyBop (207 mg, 0.4 mmol) at about room temperature for 20 minutes.", "At this point, the above solution is treated with a THF solution (2.6 mL) of compound xviii (65 mg, 0.32 mmol), followed by DIPEA (0.076 mL).", "After stirring at about room temperature for 7 hours, the reaction is quenched with water.", "The reaction mixture is diluted with DCM (60 mL).", "The organic layer is separated and washed twice with brine and dried with MgSO4.Upon filtration, concentrated and silica gel chromatography (5% EtOH/EtOAc), 148 mg (˜100%) of compound xix is obtained.", "INTERMEDIATE EXAMPLE 17 Compound xx To a THF solution (100 mL) of N-Cbz-L-valine (14.4 g, 57.2 mmol) is added HOBT (7.72 g, 57.2 mmol) and EDCI (10.98 g, 57.2 mmol).", "After stirring at about room temperature for 20 minutes, a THF solution (50 mL) containing tert-L-Leucine methyl ester-hydrochloride (10.4 g, 57.2 mmol) and DIPEA (11.9 mL, 68.7 mmol) is added to the above solution.", "The reaction is stirring at about room temperature overnight.", "Upon standard aqueous work-up and silica gel chromatography (30% EtOAc/Hexanes) 14 g (64%) of compound xx is afforded.", "INTERMEDIATE EXAMPLE 18 Compound xxi To a methanol solution (80 mL) of xx (6.71 g, 17.7 mmol) is added (under a stream of N2) Pd/C (1.88 g, 10% Pd content).", "The reaction vessel is subjected to hydrogenation (1 atm H2) overnight at about room temperature.", "At this point, the reaction mixture is filtered through a pad of Celite and concentrated in vacuo to provide the corresponding crude free amine for next step.", "A THF solution of the amine (˜17.7 mmol) is added to a THF (46 mL) and DMF (5 mL) solution containing 2-pyrazinecarboxylic acid (2.85 g, 23 mmol), Hobbit (3.12 g, 23 mmol) and EDCI (4.41 g, 23 mmol).", "To the resulting mixture is then added DIPEA (3.08 g, 17.7 mmol).", "The reaction is stirred overnight at about room temperature and then quenched with water.", "The reaction mixture is extracted with EtOAc.", "The organic layer is washed with brine and concentrated in vacuo to provide a residue that is purified by silica gel chromatography (40-50% EtOAc/Hexanes) to provide 3.9 g (63%) of compound xxi, INTERMEDIATE EXAMPLE 19 Compound xxii To a methanol solution (40 mL) of compound xxi (4.67 g, 13.34 mmol) is added 2 N NaOH (10 mL, 20 mmol).", "The reaction is stirred at about room temperature for 2 hours.", "At this time, an additional amount of 2 N NaOH (3.3 mL, 6.67 mmol) is added to the reaction mixture.", "After stirring at about room temperature overnight, the reaction is acidified to pH 3 using acidic resin.", "The reaction is then filtered and the filtrates are concentrated in vacuo to yield a residue that is dissolved in 1:1 CH3CN/H2O for lyophilization.", "4.15 g (93%) of compound xxii is obtained.", "INTERMEDIATE EXAMPLE 20 Compound xxiii A DCM solution (10 mL) of compound xxii (917 mg, 2.73 mmol) is treated with HOAt (371 mg, 2.73 mmol) and DCC (2.73 mL, 1 M, 2.73 mmol).", "After stirring for 30 minutes, the reaction mixture is treated with a THF solution (10 mL) of compound v (500 mg, 2.73 mmol).", "After stirring at about room temperature overnight, the white solids (urea) are filtered.", "The filtrates are concentrated in vacuo to give a residue that is purified by silica gel chromatography (60-70% EtOAc/Hexanes) to provide 1.06 g (77%) of compound xxiii, INTERMEDIATE EXAMPLE 21 Compound xxiv An ethanol solution (20 mL) of compound xxiii (1.06 g, 2.11 mmol) is treated with 2 N NaOH (2.11 mL, 4.23 mmol).", "After stirring at about room temperature overnight, the reaction mixture is acidified to pH 3 with acidic resin.", "The solids are filtered off.", "The resulting filtrates are concentrated in vacuo to give a residue that is lyophilized to give ˜1 g (100%) of compound xxiv, INTERMEDIATE EXAMPLE 22 Compound xxv A DCM solution (10 mL) of compound xxiv (236.7 mg, 0.5 mmol) is treated with PyBop (417 mg, 0.8 mmol).", "After stirring at about room temperature for 20 minutes, the reaction mixture is treated with a DMF solution (5.6 mL) of compound xiii (139.5 mg, 0.75 mmol), followed by DIPEA (0.174 mL, 1 mmol).", "After stirring at about room temperature for 8 hours, the reaction is quenched with water and extracted with EtOAc.", "The resulting organic layer is washed with brine and dried and concentrated in vacuo to give a residue that is purified by silica gel chromatography (5% EtOH/EtOAc) to afford ˜320 mg (100%) of compound xxv, INTERMEDIATE EXAMPLE 23 Compound xxvi A DCM solution (15 mL) of compound xxiv (355 mg, 0.75 mmol) is treated with PyBop (622 mg, 1.2 mmol).", "After stirring at about room temperature for 20 minutes, the reaction mixture is treated with a THF solution (10 mL) of compound xxvii′ (156 mg, 0.75 mmol), followed by DIPEA (0.26 mL, 1.5 mmol).", "After stirring at about room temperature overnight, the reaction is quenched with water and extracted with EtOAc.", "The resulting organic layer is washed with brine and dried and concentrated in vacuo to give a residue that is purified by silica gel chromatography (2% EtOH/EtOAc) to afford ˜400 mg (80%) of compound xxvi, INTERMEDIATE EXAMPLE 24 Methyl 5-cyanopentanoate Potassium Cyanide (4 g, 61.44 mmol) is dissolved in 70 mL water and 200 mL methanol.", "To the solution 10 g (51.2 mmol) of methyl 5-bromopentanoate is added and the mixture is refluxed overnight.", "The reaction mixture is concentrated to dryness.", "To the residue, 100 mL of EtOAc is added to extract the product.", "The organic is washed with water three times, dried and concentrated to yield 5.37 g (74%) of methyl 5-cyanopentanoate as an oil.", "INTERMEDIATE EXAMPLE 25 Methyl 5-tetrazol-5-ylpentanoate Methyl 5-cyanopentanoate (4.8 g, 34 mmol) is dissolved in toluene, triethylammonium chloride (14 g, 102 mmol) and sodium azide (6.63, 102 mmol) is added.", "The mixture is heated to reflux for overnight.", "The reaction mixture is cooled to room temperature, water is added to extract (3×100 mL) methyl 5-tetrazol-5-ylpentanoate from the organic.", "To the aqueous phase, concentrate HCl is added to adjust pH to 2.The product is extracted from the aqueous solution with EtOAc (3×50 mL).", "The organic is combined, dried and concentrated to yield 4.25 g (68%) of methyl 5-tetrazol-5-ylpentanoate.", "INTERMEDIATE EXAMPLE 26 Methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate Methyl 5-tetrazol-5-ylpentanoate (4.23 g, 23 mmol) and trichloroacetic acid (8.69 g, 53 mmol) are dissolved in 50 mL of CHCl3.α-Methylstyrene (2.72, 23 mmol) is added to the solution dropwise, and the reaction mixture is allowed to stirred at about room temperature fore overnight.", "The reaction mixture is diluted with EtOAc to 200 mL, and organic layer is washed with 10% aqueous KOH and brine.", "The organic layer is dried, concentrated.", "The product is purified by flash column chromatography to yield 6.6 g (95%) methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate.", "INTERMEDIATE EXAMPLE 27 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid Methyl 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoate (6.6 g, 21.8 mmol) is dissolved in methanol (100 mL) and 23 mL of 1 N aqueous NaOH is added.", "The mixture is stirred overnight and is concentrated to remove methanol.", "The residue is dissolved in water (100 mL) and the solution is neutralized by adding the same equivalent of 1 N aqueous HCl.", "The product is extracted with EtOAc (3×50 mL).", "The organic is dried and concentrated to yield 4.75 g (75%) 5-[N(1,1 dimethylbenzyl)tetrazol-5-yl]pentanoic acid.", "INTERMEDIATE EXAMPLE 28 Compound xxviii 5-[N-(1,1-dimethylbenzyl)tetrazol-5-yl]pentanoic acid (4.75 g, 16.5 mmol) is dissolved in DCM (100 mL), 4.8 g (24.8 mmol) of EDCI and 6 mL of DIPEA are added.", "To the mixture, N-hydroxylsuccinimide (3.8 g, 33 mmol) is added.", "The reaction mixture is stirred for three hours at about room temperature.", "The mixture is diluted with DCM to 200 mL and the solution is washed with water three times.", "The organic is dried and concentrated to yield 4.79 g (75%) of compound xxviii, INTERMEDIATE EXAMPLE 29 Compound xxix The dipeptide H-Val-Val-OH (3.22 g, 14.9 mmol) is suspended in 50 mL of N,N-dimethylformamide (DMF) and 4.75 g (12.42 mmol) of compound xxviii is added followed the addition of 3.4 mL (18.63 mmol) of diisopropylethylamine (DIPEA).", "The mixture is warmed up to 40° C. and stirred overnight.", "The solvent is evaporated under high vacuum.", "The residue is dissolved in EtOAc and washed with 1 N HCl and brine to yield 5.52 g (91%) of compound xxix, INTERMEDIATE EXAMPLE 30 Compound xxx 1.6 g (3.29 mmol) of compound xxix is dissolved in 20 mL of DCM, 3.3 mL of 1 M solution of DCC in THF is added.", "To the mixture, 500 mg (2.73 mmol) of compound v is added.", "The mixture is stirred at about room temperature overnight.", "The mixture is diluted with EtOAc to 100 mL and washed with 1 N HCl, NaHCO3 and brine.", "Purified by column chromatography (50% EtOAc/hexane) to yield 1.02 g (58%) compound xxx, INTERMEDIATE EXAMPLE 31 Compound xxxi Compound xxx (1.02 g, 1.57 mmol) is dissolved in 10 mL MeOH and 2 mL of 1 N aqueous NaOH is added.", "The mixture is stirred overnight The methanol is removed by evaporation and the residue is dissolved in water and neutralized with 2 mL HCl.", "Following extraction with EtOAc, 1.00 g (˜100%) of compound xxxi is afforded.", "INTERMEDIATE EXAMPLE 32 Compound xxxii Compound xxxi (300 mg, 0.48 mmol) and PyBop (300 mg, 0.58 mmol) are dissolved in 10 mL DCM.", "To the solution, compound x (201 mg, 0.58 mmol) is added and then DIPEA (104 μl) is added.", "The mixture is stirred at about room temperature overnight.", "The reaction mixture is then diluted with EtOAc to 100 mL and washed twice with 1 N HCl, twice with NaHCO3 and thrice with brine.", "The organic is dried and concentrated.", "The residue is purified by column chromatography (100% EtOAc) to yield 450 mg (98%) of compound xxxii, INTERMEDIATE EXAMPLE 33 Compound xxxiii Compound xxxii 360 mg (0.38 mmol) is dissolved in 8 mL DCM and 240 mg (0.57 mmol) of DMP reagent reagent is added.", "The mixture is stirred at about room temperature for three hours.", "The mixture is diluted with EtOAc to 50 mL and washed with brine three times.", "The product is purified by column chromatography (25% ethanol/EtOAc) to yield 300 mg (83%) of compound xxxiii, INTERMEDIATE EXAMPLE 34 Compound xxxiv To a DCM solution (10 mL) of xxxv (790 mg, 2.80 mmol) is added PyBop (1.7 g, 3.36 mmol) and Hobbit (450 mg, 3.36 mmol).", "The resulting solution is cooled to 0° C. and treated with a DCM solution (3 mL) of (s)-α-(4-pyridyl)ethylamine (410 mg, 3.36 mmol).", "This is followed by the addition of DIPEA (0.5 mL, 3.36 mmol).", "The reaction is stirred overnight at about room temperature.", "At this point, the reaction mixture is diluted with EtOAc.", "The whole is washed with saturated NaHCO3 and brine.", "The organic layer thus obtained is dried and concentrated in vacuo.", "The resulting residue is purified by silica gel chromatography (5% EtOH/EtOAc) to provide 630 mg (58%) of compound xxxiv, Note: (s)-α-(4-pyridyl)ethylamine is obtained from its D-tartrate salt by base wash (1 N NaOH) and subsequent EtOAc extraction.", "The recovery rate is 89%.", "INTERMEDIATE EXAMPLE 35 Compound xxxvi To a methanol solution (15 mL) of compound xxxiv (630 mg, 1.64 mmol) is added under N2 Pd/C (150 mg, 10% palladium content).", "The reaction is stirred under H2 overnight.", "The reaction mixture is filtered through a pad of Celite® 521.The filtrates are concentrated in vacuo to provide 420 mg (˜100%) of compound xxxvi, INTERMEDIATE EXAMPLE 36 Compound xxxvii To a DCM solution (3 mL) of compound xxxi (270 mg, 0.43 mmol) is added PyBop (270 mg, 0.52 mmol).", "This is followed by addition of compound xxxvi (160 mg, 0.64 mmol) and DIPEA (0.09 mL, 0.52 mmol).", "The reaction is stirred at about room temperature overnight.", "At this point, the reaction is diluted with EtOAc and washed with 0.1N HCl, followed by saturated NaHCO3 and brine.", "The resulting organic layer is dried and concentrated to give compound xxxvii (430 mg total mass) for next step INTERMEDIATE EXAMPLE 37 Compound xxxviii To a DCM solution (3 mL) of compound xxxvii (370 mg, 0.43 mmol) is added DMP reagent reagent (280 mg, 0.65 mmol).", "The reaction is stirred at about room temperature for 2 hours and then quenched with 10% Na2SO3.After stirring for 30 minutes, the reaction is extracted with EtOAc.", "The organic layer is washed with saturated NaHCO3 and brine.", "The resulting organic layer is dried and concentrated in vacuo to give a residue that is purified by silica gel chromatography (5% EtOH/EtOAc) to provide 180 mg (49% for 2-steps) of compound xxxviii, INTERMEDIATE EXAMPLE 38 Compound xxxx Compound xxix (2.5 g, 5 mmol) is dissolved in 40 mL of DCM, 5.1 mL of 1 M solution of DCC in THF is added to the solution.", "To the mixture, 1.08 g (3.53 mmol) of compound xxxix is added.", "The mixture is stirred at about room temperature overnight.", "The mixture is diluted with EtOAc to 100 mL, washed sequentially with 1 N HCl, NaHCO3 and brine, and then purified by column chromatography (80% EtOAc/hexane) to yield 2.59 g (95%) of compound xxxx, INTERMEDIATE EXAMPLE 39 Compound xxxxi Compound xxxx (2.59 g, 3.35 mmol) is dissolved in 20 mL MeOH and 4 mL of 1 N aqueous NaOH is added.", "The mixture is stirred overnight and then rotary evaporated to leave a residue.", "The residue is dissolved in water and neutralized with 2 mL HCl.", "The neutralized solution is then extracted with EtOAc to yield 2.49 g (˜100%) of compound xxxxi, INTERMEDIATE EXAMPLE 40 Compound xxxxii Compound xxxxi (847 mg, 1.16 mmol) and 724 mg (1.39 mmol) of PyBop are dissolved in 10 mL DCM.", "To the solution, compound xiii (260 mg, 1.39 mmol) is added and then followed by the addition of DIPEA (209 μl).", "The mixture is stirred at about room temperature overnight.", "The reaction mixture is then diluted with EtOAc to 100 mL and washed twice with 1 N HCl, twice with NaHCO3 and thrice with brine.", "The organic is dried and concentrated.", "The residue is purified by column chromatography (5% ethanol/EtOAc) to yield 930 mg (86%) of compound xxxxii, INTERMEDIATE EXAMPLE 41 Compound xxxxiii Compound xxxxii (350 mg, 0.38 mmol) is dissolved in 10 mL DCM and 242 mg (0.57 mmol) of DMP reagent reagent is added.", "The mixture is stirred at about room temperature for three hours.", "The mixture is diluted with EtOAc to 50 mL and washed thrice with brine.", "The product is purified by column chromatography (100% EtOAc) to yield 180 mg (51%) of compound xxxxiii, INTERMEDIATE EXAMPLE 42 Compound xxxxv H-Val-Val-OH (5 g, 23 mmol) is suspended in 100 mL DMF, compound xxxxiv (8.3 g, 27.6 mmol) is added, and then 6.2 mL (35.5 mmol) of DIPEA is added.", "The mixture is stirred at 40° C. for two days.", "The solvent is removed under high vacuum and the residue is dissolved in 100 mL EtOAc and washed thrice with 1 N HCl and twice with brine.", "9.14 g (99%) of compound xxxxv is afforded.", "INTERMEDIATE EXAMPLE 43 Compound xxxxvi Compound xxxxv (2.8 g, 7 mmol) and 954 mg (7 mmol) of HOAt is dissolved in 100 mL DCM.", "7 mL of 1 M DCC/DCM is added.", "To the reaction mixture, compound xxxix (2.15 g) is added and the reaction mixture is stirred at about room temperature for overnight.", "The mixture is concentrated to dryness and the residue is dissolved in EtOAc and purified by column chromatography (100% EtOAc) to yield 4.57 g (95%) of compound xxxxvi, INTERMEDIATE EXAMPLE 44 Compound xxxxvii Compound xxxxvi (4.57 g, 6.65 mmol) is dissolved in 10 mL TFA and 10 mL DCM.", "The mixture is stirred at about room temperature for 4 hours.", "The solvent is removed by vacuum and the residue is dissolved in 50:50 acetonitrile/water and lyophilized to yield as a powder compound xxxxvii, INTERMEDIATE EXAMPLE 45 Compound xxxxviii Compound xxxxvii (1 g, 1.59 mmol) and 990 mg (2.28 mmol) of PyBop is dissolved in 20 mL DCM and 1.6 mL of 1 M methylamine in THF is added.", "The mixture is stirred at about room temperature for 4 hours.", "The reaction mixture is diluted to 100 mL with EtOAc and washed with 1 N HCl, NaHCO3 and brine.", "The residue is purified by flash column chromatography (10% EtOH/EtOAc) to yield 1 g (98%) of compound xxxxviii, INTERMEDIATE EXAMPLE 46 Compound xxxxix Compound xxxxviii (1 g, 1.55 mmol) is dissolved in 10 mL of MeOH and 2 mL 1 N NaOH is added.", "The mixture is stirred at about room temperature for overnight.", "The solvent is removed by evaporation.", "The residue is dissolved in water, neutralized and extracted with EtOAc to yield 960 mg (98%) of compound xxxxix, INTERMEDIATE EXAMPLE 47 Compound li Compound xxxxix (315 mg, 0.5 mmol) and 312 mg (0.6 mmol) of PyBop are dissolved in 10 mL DCM.", "Compound 1 (56 mg, 0.6 mmol) and 108 μl of DIPEA is added.", "The mixture is stirred at about room temperature overnight, and is diluted to 100 mL with EtOAc and washed with 1 N HCl, NaHCO3 and brine.", "Purified by column chromatography (15% EtOH/EtOAc) to yield 400 mg (92%) of compound li, INTERMEDIATE EXAMPLE 48 Compound lii Compound li (400 mg, 0.46 mmol) is dissolved in 10 mL of DCM and 292 mg (0.69 mmol) DMP reagent reagent is added.", "The mixture is stirred at about room temperature for 3 hours.", "The solvent is removed by evaporation and product is purified by RP-HPLC to yield 130 mg (32%) of compound lii, INTERMEDIATE EXAMPLE 49 Compound liii Compound xxxxix (210 mg, 0.33 mmol) and 208 mg (0.4 mmol) of PyBop are dissolved in 10 mL DCM.", "Compound xiii (154 mg, 0.83 mmol) is added to the solution followed by the addition of DIPEA (72 μl, 0.4 mmol).", "The mixture is stirred at about room temperature overnight.", "The reaction mixture is diluted to 100 mL with EtOAc, washed with 1 N HCl, NaHCO3 and brine, and then purified by flash column chromatography (10% EtOH/EtOAc) to yield 250 mg (95%) of compound liii, INTERMEDIATE EXAMPLE 50 Compound liv Compound xxxxv (755 mg, 1.88 mmol) and 255 mg (1.88 mmol) of HOAt are dissolved in 20 mL DCM.", "1.88 mL of 1 M DCC/DCM is added.", "To the reaction mixture, compound v (288 mg) is added and the reaction mixture is stirred at about room temperature for 2 hours.", "The mixture is concentrated to dryness and the residue is dissolved in EtOAc and purified by column chromatography (80% EtOAc/Hexanes) to yield 800 mg (90%) of compound liv, INTERMEDIATE EXAMPLE 51 Compound lv Compound liv (800 mg, 1.41 mmol) is dissolved in 10 mL MeOH and 2 mL NaOH is added.", "The mixture is stirred at about room temperature overnight.", "The solvent is removed by vacuum and the residue is dissolved in water and neutralized with 2 mL 1 N HCl.", "The product is extracted with EtOAc.", "Evaporation of the extraction solvent afforded 760 mg (˜100%) lv, INTERMEDIATE EXAMPLE 52 Compound lvii Compound lv (760 mg, 1.41 mmol) and 880 mg (1.69 mmol) of PyBop are dissolved in 5 mL DCM.", "Compound lvi (530 mg, 2.12 mmol) is added to the solution and then 0.31 of DIPEA is added.", "The mixture is stirred at about room temperature overnight.", "The reaction mixture is diluted to 100 mL with EtOAc, washed with 1 N HCl, NaHCO3 and brine, and then purified by flash column chromatography (100% EtOAc) to yield 870 mg (80%) of compound lvii, INTERMEDIATE EXAMPLE 53 Compound lviii Compound lvii (350 mg, 0.45 mmol) is dissolved in 5 mL TFA and 5 mL DCM and the mixture is stirred at about room temperature for 3 hours.", "The solvent is removed by evaporation and the product is purified by RP-HPLC to yield 220 mg (69%) of compound lviii, INTERMEDIATE EXAMPLE 54 Compound lix Compound lviii (200 mg, 0.28 mmol) and 218 mg (0.42 mmol) of PyBop are dissolved in 5 mL DCM.", "Methylamine (0.28 mL of 2 M in TBF) is added.", "The mixture is stirred at about room temperature overnight.", "The mixture is diluted to 100 mL with EtOAc, washed with 1 N HCl, NaHCO3 and brine, and then purified by column chromatography (15% EtOH/EtOAc) to yield 168 mg (79%) of lix, INTERMEDIATE EXAMPLE 55 Compound lx Compound lviii (200 mg, 0.26 mmol) is dissolved in 4 mL of DCM and 165 mg (0.39 mmol) of DMP reagent reagent is added.", "The mixture is stirred at about room temperature for 3 hours.", "The solvent is removed by evaporation.", "The residue is dissolved in 50% acetonitrile/water, and filtered purified by RP-HPLC to yield 140 mg (70%) of compound lx, INTERMEDIATE EXAMPLE 56 Compound ii A DCM (30 mL) and EtOH (30 mL) solution of compound i (4 g, 12.1 mmol), under N2, is cooled down to −10° C. NaBH4 (458 mg, 12.1 mmol) is added and the solution is stirred at −10° C. for 50 minutes.", "TLC (50% EtOAc/Hexane) showed total conversion to a slower running spot.", "The reaction is carefully quenched with ice and then with a cold saturated solution of NH4Cl (10 mL).", "The mixture is dumped in DCM (300 mL).", "The organic layer is washed once with saturated solution of NH4Cl (60 mL) and twice with brine (60 mL).", "The organic layer is then separated, dried over MgSO4 and concentrated in vacuo, to yield 3.5 g of compound ii (87%) INTERMEDIATE EXAMPLE 57 Compound lxi In a 250 mL round bottom flask equipped with a H2 balloon, an ethanolic solution (50 mL) of compound ii (3.5 g, 10.5 mmol) is subjected to standard hydrogenation conditions [20% Pd(OH)2/C (1.47 g, 2.1, mmol)] for 5 hours at about room temperature.", "The catalyst is filtered off through Celite and washed with DCM.", "The solvent is then removed under reduced pressure to yield 2 g (96%) of compound lxi, INTERMEDIATE EXAMPLE 58 Compound lxii Under inert atmosphere, a solution of compound lxi (200 mg, 1 mmol), compound lxiii, (233 mg, 1.1 mmol), HOAt (1-hydroxy-7-azabenzotriazole) (156 mg, 1.15 mmol) in anhydrous DMF (6 mL) is stirred for 20 minutes.", "The temperature is then taken down to 0° C., followed by the addition of DIC(0.18 mL, 1.15 mmol).", "The reaction is stirred overnight at about room temperature.", "The solution is diluted with EtOAc and then washed twice with 1 N HCl, twice with saturated aqueous NaHCO3, and brine.", "The organic layer is separated dried over MgSO4 and the solvent removed under reduced pressure.", "The residue is cleaned by chromatography (silica gel: 70% EtOAc/DCM) to give in 45% yield compound lxii.", "INTERMEDIATE EXAMPLE 59 Compound lxiv A solution of compound ixii (777 mg, 2 mmol) in dioxane (6 mL) and 0.5 M NaOH (6 mL) is stirred for 5 hours at about room temperature.", "Examination by TLC (100% EtOAc) shows complete conversion to a spot at the origin.", "The reaction is cooled down with an ice bath followed by the addition of 1 N HCl (4 mL).", "Solid NaCl is then added and the whole mixture is extracted twice with EtOAc (2×150 mL).", "The organic extracts are then combined, dried over MgSO4 and the solvent removed under reduced pressure to give compound lxiv in 92% yield.", "INTERMEDIATE EXAMPLE 60 Compound lxv Under an inert atmosphere, a solution of compound x (203 mg, 0.58 mmol), compound lxiv (276 mg, 0.775 mmol), HOAt (1-hydroxy-7-azabenzotriazole) (126 mg, 0.93 mmol) in anhydrous DMF (6 mL) is stirred for 20 minutes.", "The temperature is then taken down to 0° C., followed by the addition of DIC (0.14 mL, 0.93 mmol).", "The reaction is stirred overnight at about room temperature.", "The solution is diluted with EtOAc and then washed twice with 1 N HCl, twice with saturated aqueous NaHCO3, and brine.", "The organic layer is separated dried over MgSO4 and the solvent removed under reduced pressure.", "The residue is purified by chromatography (silica gel: 50% EtOAc/DCM to 80:19:1 EtOAC/DCM/MeOH) to give compound lxv in 62% yield.", "INTERMEDIATE EXAMPLE 61 Compound lxvi Under an inert atmosphere, to a solution of compound lxv (287 mg, 0.42 mmol) in anhydrous DCM (15 mL) is added the DMP reagent reagent (605 mg, 1.43 mmol) The reaction is stirred for 2 hours at about room temperature.", "(Note.—The doubling of the amount of the DMP reagent reagent and the reaction time is to assure that both alcohol groups are completely oxidized to the corresponding keto groups).", "Examination by TLC (silica gel: 2% MeOH/EtOAc) shows complete conversion to the faster product.", "The reaction is diluted with DCM (150 mL) and then washed twice with a 10% aqueous sodium sulfite solution (2×50 mL), twice with saturated aqueous NaHCO3, and with brine.", "The organic layer is separated dried over MgSO4 and the solvent removed under reduced pressure.", "The residue is purified by chromatography (silica gel: 50% EtOAC/DCM to 80:19:1 EtOAC/DCM/MeOH) to give in 77% yield compound lxvi.", "INTERMEDIATE EXAMPLE 62 Compound lxvii To a DCM solution (60 ml) of L-3 phenyl lactic acid (2 g, 12 mmol) is added PYBOP (7.5 g, 14.4 mmol).", "To this solution is added a DCM solution (20 mL) containing L-valine methyl ester HCl (2.4 g, 14.4 mmol) and DIPEA (2.6 mL, 14.4 mmol).", "The resulting reaction mixture is stirred overnight at about room temperature.", "At this point, the reaction is diluted with EtOAc (30 mL), washed with NaHCO3 (30 mL) and brine (15 mL).", "The organic layer is dried over Na2SO4, filtered and concentrated.", "Purification is achieved in 50% EtOAc/Hex on silica gel to give 2.97 g (89%) of comnpound lxvii, INTERMEDIATE EXAMPLE 63 Compound lxviii Compound lxvii (2.97 g, 10.6 mmol) is taken up in DCM (50 mL) and cooled with an ice bath.", "TBSCI (2.1 g, 13.8 mmol) is added to this solution followed by imidazole (0.94 g, 13.8 mmol).", "The resulting solution is stirred overnight.", "The reaction is then diluted with EtOAc (50 mL), washed with NaHCO3 and brine.", "The organic layer is dried over Na2SO4, filtered and concentrated.", "Purification is achieved in 20% EtOAc/Hexane on silica gel to give 3.79 g (90%) of compound lxviii, INTERMEDIATE EXAMPLE 64 Compound lxix To a methanol (50 ml) solution of compound lxviii (3.78 g, 9.6 mmol) is added 1 N aqueous NaOH (14.4 mL, 14.4 mmol).", "The resulting solution is stirred overnight.", "The solvent is partially removed in vacuo.", "The pH of the reaction mixture is then lowered to 3 using 1 N HCl aqueous solution.", "The solution is diluted with EtOAc and brine.", "The desired product is extracted with EtOAc (3×50 ml).", "The organic layers are combined, dried over Na2SO4, filtered and concentrated to give 3.5 g (96%) of compound lxix, INTERMEDIATE EXAMPLE 65 Compound lxx To a DCM (15 mL) solution containing compound lxix (1.1 g, 2.9 mmol) is added HOAt (0.44 g, 3.2 mmol) followed by a 1 M solution of DCC (3.2 mL, 3.2 mmol) in DCM.", "After sting at about room temperature for 20 minutes, a DCM (15 mL) solution of compound xxxix (970 mg, 3.2 mmol) is added.", "This reaction is stirred overnight under N2.The reaction is then diluted with EtOAc (30 mL), filtered through a pad of silica gel, washed with 0.1 N HCl, NaHCO3, and brine.", "The organic layer is dried over Na2SO4, filtered and concentrated in vacuo.", "Purification is achieved in 50% EtOAc/Hex on silica gel to give 1.5 g (77%) of compound lxx, INTERMEDIATE EXAMPLE 66 Compound lxxi To a methanol solution (30 mL) of compound xx (1.5 g, 2.4 mmol) is added 1 N aqueous NaOH (3.6 mL, 3.6 mmol).", "The resulting solution stirred overnight.", "At this point, the solvent is partially removed, and the pH of the reaction mixture is adjusted to 3 using 1 N aqueous HCl.", "The reaction is then diluted with EtOAc (50 mL) and brine (20 mL).", "The aqueous layer is extracted with EtOAc (3×50 mL).", "The organic layers are combined, dried over Na2SO4, filtered and concentrated to provide 1.3 g (92%) of compound lxxi, INTERMEDIATE EXAMPLE 67 Compound lxxii To a solution of DCM (2 mL) containing compound lxxi (180 mg, 0.28 mmol) is added PyBOP (175 mg, 0.34 mmol) and DIPEA (0.06 mL, 0.34 mmol), followed by a DCM solution (3 nL) of compound x (150 mg, 0.41 mmol).", "The resulting solution is stirred overnight under N2.The reaction is then diluted with EtOAc (30 mL), washed with NaHCO3 and brine.", "The organic layer is dried over Na2SO4, filtered and concentrated.", "Purification is achieved in 100% EtOAc on silica gel to give 270 mg (98%) of compound lxxii, INTERMEDIATE EXAMPLE 68 Compound lxxiii To a DCM (3 mL) solution of compound lxxii (270 mg, 0.27 mmol) is added DMP reagent reagent (140 mg, 0.33 mmol).", "After stirring at about room temperature for 1.5 hours, the reaction is quenched with 10% Na2SO3 (10 mL).", "The reaction is diluted with EtOAc (30 mL) and stirred for 10 minutes.", "The organic layer is washed with NaHCO3 and brine.", "The organic layer is dried over Na2SO4, filtered and concentrated.", "Purification is achieved in 60% EtOAc/Hex.", "to give 150 mg (56%) of compound lxxiii, INTERMEDIATE EXAMPLE 69 Compound lxi To an ethanol solution (50 mL) of compound ii (3.5 g, 10.5 mmol) is added under a stream of nitrogen Pd(OH)2/C (1.47 g, 20% Pd content, 2.1 mmol).", "The reaction is subjected to hydrogenation under 1 atm pressure.", "Upon completion, the catalysts are filtered through a pad of Celite and washed with dichloromethane.", "The filtrates are concentrated in vacuo to give 2 g (96%) of compound lxi.", "INTERMEDIATE EXAMPLE 70 Compound lxxiv To a DMF solution (60 mL) of compound vii (9.1 g, 28.2 mmol) is added HOAt (4 g, 29.4 mmol) and 1,3-disiopropylcarbodiiide (3.7 g, 29.4 mmol).", "After stirring at about room temperature for 30 minutes, a DMF solution (10 mL) of compound lxi (5.1 g, 25.6 mmol) is added to the above solution.", "The reaction is stirred at about room temperature overnight.", "At this point, the white solids are filtered off.", "The filtrates are concentrated in vacuo to give a residue that is purified by silica gel chromatography to give 9.5 g (67%) of compound lxxiv, INTERMEDIATE EXAMPLE 71 Compound lxxv To a solution of compound lxxiv (1.5 g, 3 mmol) in anhydrous THF (25 mL) is added EtiPr2N (0.78 mL, 4.5 mmol) at about room temperature.", "The mixture is cooled to 0° C. and MOMCl (1.5 mL, 19.7 mmol) is added in a dropwise fashion.", "The reaction is allowed to warm to room temperature and stirred overnight.", "The solution is then diluted with ether and washed with water (3 times).", "The aqueous layers are extracted further by ether and all the organic layers are dried over MgSO4 before being concentrated to afford a yellow oil.", "The desired isomer of compound lxxv is isolated by silica gel chromatography (EtOAc/Hexanes 5/2) in 40% yield with clear separation of diastereomers.", "INTERMEDIATE EXAMPLE 72 Compound lxxvi To a solution of compound lxxv (502 mg, 0.9 mmol) in EtOH (5 mL) is added 2 N aqueous NaOH (0.9 mL, 1.8 mmol) dropwise at 0° C. The reaction is allowed to warm to room temperature and stirred overnight.", "Upon completion of the saponification, the solution is acidified to pH 3 with Dowex 50W8X-200 acidic resin.", "The solids are filtered off and the resulting filtrate is concentrated in vacio to give a oily residue that is lyophilized to give 370 mg (80%) compound lxxvi, INTERMEDIATE EXAMPLE 73 Compound lxxvii A dichloromethane solution (4 mL) of compound lxxvi (110 mg, 0.21 mmol) is treated with PyBOP (200 mg, 0.38 mmol).", "After stirring at about room temperature for 30 minutes, the reaction mixture is charged with a THF solution (3.2 mL) of compound xiii (60 mg, 0.32 mmol), followed by EtiPr2N.", "After stirring overnight at about room temperature, the reaction is quenched with water and extracted with EtOAc.", "The resulting organic layer is washed with brine and dried over MgSO4, before being concentrated to a yellow oil.", "Purification by silica gel chromatography (5% EtOH/EtOAc) yieids 143 mg (100%) of compound lxxvii, INTERMEDIATE EXAMPLE 74 Compound lxxviii To a THF solution (50 mL) of H-Chg-OH 2 (5 g, 19.4 mmol) is added HOBt (2.63 g, 19.4 mmol) and EDCI (3.72 g, 19.4 mmol).", "After stiring at about room temperature for 20 minutes, a THF (19 mL) and DMF (10 mL) solution containing tert-L-Leucine methyl ester-hydrochloride (19.4 mmol) and DIPEA (6.75 mL, 38.8 mmol) is added to the above solution.", "The reaction is stirred at about room temperature overnight.", "Standard aqueous work-up and silica gel chromatography (15-20% EtOAc/Hexanes) affords 2.27 g (30%) of compound lxxviii, INTERMEDIATE EXAMPLE 75 Compound lxxix To a THF solution (12 mL) of compound lxxviii (2.27 g, 5.91 mmol) is added 4 N HCl solution in dioxane (7.38 mL, 29.5 mmol).", "The reaction is stirred at about room temperature overnight.", "At this point, the solvent is removed under reduced pressure to yield the compound lxxix that is used directly for next reaction.", "INTERMEDIATE EXAMPLE 76 Compound lxxx To a THF solution of compound lxxix (5.9 mmol) is added to a THF (20 mL) solution containing 2-pyrazinecarboxylic acid (878 mg, 7.08 mmol), HOBt (957 mg, 7.08 mmol) and EDCI (1.36 g, 7.08 mmol).", "To the resulting mixture is then added DIPEA (2.05 mL, 11.8 mmol).", "The reaction is stirred overnight at about room temperature and then quenched with water.", "The reaction mixture is extracted with EtOAc.", "The organic layer is washed with brine and concentrated in vacuo to provide a residue that is purified by silica gel chromatography (40-50% EtOAc/Hexanes) to provide 1 g (36%) of compound lxxx, INTERMEDIATE EXAMPLE 77 Compound lxxxi To a methanol solution (20 mL) of compound lxxx (1 g, 2.56 mmol) is added 2 N NaOH 3.2 mL, 6.4 mmol).", "The reaction is stirred at about room temperature overnight.", "At this point, the reaction is acidified to pH 3 using 5 N HCl.", "The reaction is diluted with EtOAc (75 mL), and washed with water and brine.", "The organic layer thus obtained is dried and concentrated in vacuo to give a residue that is dissolved in 1:1 CH3CN/H2O for lyophilization.", "A total of ˜1 g (100%) of compound lxxxi is obtained.", "INTERMEDIATE EXAMPLE 78 Compound lxxxii A dichloromethane solution (10 mL) of compound lxxxi (2.56 mmol) is treated with HOAt (348 mg, 2.56 mmol) and DCC (2.56 mL, 1M, 2.56 mmol).", "After stirring for 30 minutes, the reaction mixture is treated with a THF solution (5 mL) of compound v (2.56 mmol).", "After stirring at about room temperature overnight, the white solids (urea) are removed by filtration.", "The filtrates are concentrated in vacuo to give a residue that is purified by silica gel chromatography to provide 1.4 g (100%) of the compound lxxxii, INTERMEDIATE EXAMPLE 79 Compound lxxxiii An ethanol solution (15 mL) of compound lxxxii (1.4 g, 2.58 mmol) is treated with 2 N NaOH (2.58 mL, 5.17 mmol).", "After stirring at about room temperature overnight, the reaction mixture is acidified to pH 3 with acidic resin.", "The solids are filtered off.", "The resulting filtrates are concentrated in vacuo to give a residue that is lyophilized to give 1.32 g (˜100%) of compound lxxxiii, INTERMEDIATE EXAMPLE 80 Compound lxxxiv A dichloromethane solution (15 mL) of compound lxxxiii (360 mg, 0.7 mmol) is treated with PyBOP (582 mg, 1.12 mmol).", "After stirring at about room temperature for 20 minutes, the reaction mixture is treated with a THF solution (10 mL) of compound xiii (195.6 mg, 1.05 mmol), followed by DIPEA (0.25 mL, 1.40 mmol).", "After stirring overnight at about room temperature, the reaction is quenched with water and extracted with EtOAc.", "The resulting organic layer is washed with brine and dried and concentrated in vacuo to give a residue that is purified by silica gel chromatography (3% EtOH/EtOAc) to afford 420 mg (88%) of compound lxxxiv, INTERMEDIATE EXAMPLE 81 Compound ii′″ A mixture of anhydrous dichloromethane and ether (20 mL:20 mL) is cooled to −78° C. under N2 (g).", "To the solution is added TiCI4 (1 M in dichloromethane, 10 mL, 10 mmol) and then MeLi (1.4 M in ether, 7.1 mL, 10 mmol) is added subsequently with stirring for another 30 minutes at −78° C. A solution of compound i (2 g, 6 mmol) in 10 mL dichloromethane is added to the mixture dropwise at the same temperature over 15 minutes.", "The solution is slowly warmed up to −40° C. for 10 minutes and then stirred at 0° C. for 2 hours.", "The reaction is quenched by pouring the mixture into a water/ether mixture (1:1) and then the layers are allowed to separate.", "The aqueous layer is further extracted by ether twice.", "All organic layers is washed by water, brine and dried over MgSO4 before being concentrated to a yellow oil.", "The desired compound ii′″ is isolated by silica gel chromatography (EtOAc/Hexanes 2/1) in 83% INTERMEDIATE EXAMPLE 82 Compound lxi′ To the compound ii′″ (1.7 g, 5 mmol) is added to 10 wt % Pd on C (0.53 g, 0.5 mmol), followed by addition of MeOH (17 mL).", "Hydrogen gas is flushed through the reaction mixture and hydrogen gas is maintained for reaction at 1 atm overnight.", "The reaction mixture is then filtered and concentrated to afford 929 mg (87%) of compound lxi′ as a colorless oil.", "INTERMEDIATE EXAMPLE 83 Compound lxxxv To a THF solution (16 mL) of compound xxii (1 g, 3 mmol) is added at about room temperature HOAt (0.41 g, 3 mmol) and followed by 1 M DCC solution of dichloromethane (3 mL, 3 mmol).", "After stirring for 30 minutes at about room temperature, a dichloromethane solution (6 mL) of compound lxi′ is added to the above HOAt-activated acid.", "The reaction is stirred at about room temperature overnight.", "At this point, the reaction is filtered through Celite.", "The filtrate is diluted with EtOAc (120 mL) and washed with water and then brine.", "The organic layer is dried and concentrated to an yellow oil which is purified by silica gel chromatography (100% EtOAc) to yield 1 g (65%) of compound lxxxv, INTERMEDIATE EXAMPLE 84 Compound lxxxvi To an ethanol solution (8 mL) of compound lxxxv (920 mg. 1.7 mmol) is added 2 N NaOH aqueous solution (1.7 mL, 3.4 mmol).", "The reaction is stirred overnight at about room temperature and then acidified to pH 3 by Dowes acidic resin.", "The solids are filtered off and the filtrate is concentrated to give a colorless oil, which is redissolved in 1:1 CH3CN/H2O and lyophilized to provide 800 mg (93%) of compound lxxxvi.", "HPLC shows a single product peak.", "INTERMEDIATE EXAMPLE 85 Compound lxxxvii To a dichloromethane solution (4 mL) of compound lxxxvi (150 mg, 0.3 mmol) is added by PyBOP (250 mg, 0.47 mmol).", "The solution is stirred at about room temperature for 30 minutes.", "To this solution is then added a THF (4.5 mL) solution of compound xiii (84 mg, 0.45 mmol) followed by EtiPr2N (0.1 mL, 0.6 mmol).", "The reaction is stirred at about room temperature overnight and then quenched with water (25 mL) for 30 minutes.", "The mixture is then extracted with EtOAc.", "The resulting organic layer is washed with brine and dried over MgSO4, before being concentrated to a yellow oil.", "Purification by silica gel chromatography (5% EtOH/EtOAc) yields 200 mg (100%) of compound lxxxvii, INTERMEDIATE EXAMPLE 86 Compound lxxxix Compound lxxxviii, N-Cbz-L-Valine, (2.5 g, 9.9 mmol) is taken up in THF (30 mL).", "EDCI (2.29 g, 11.9 mmol) and HOBT (1.62 g, 11.9 mmol) are added and the mixture stirred five minutes.", "L-tert-Leucine methyl ester hydrochloride (2.17 g, 11.9 mmol) is added in THF (23.9 mL) followed by DIPEA (2.1 mL).", "The reaction mixture is stirred overnight under nitrogen.", "The reaction mixture is diluted with ethyl acetate, washed with 1 N HCl, saturated sodium bicarbonate, and brine.", "The organic phase is dried over sodium sulfate, filtered and concentrated.", "The concentrate residue is purified in 25% ethyl acetate/hexane to afford 1.1 g (29%) of compound lxxxix, INTERMEDIATE EXAMPLE 87 Compound lxxxx Compound lxxxix is hydrolyzed under standard conditions using methyl alcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 1.03 g (95%) of compound lxxxx, INTERMEDIATE EXAMPLE 88 Compound lxxxxi Compound lxxxx (385 mg, 1.06 mmol) is taken up in dichloromethane (3 mL).", "DCC (1.4 mmol) is added followed by HOAt (190 mg, 1.4mmol).", "Compound v (260 mg, 1.4 mmol) is then added in dichloromethane (3 mL).", "The resulting mixture is stirred overnight under nitrogen.", "The reaction is diluted with ethyl acetate, filtered through silica gel, and concentrated.", "The residue is purified in 50% ethyl acetate/hexane to afford 440 mg (80%) of compound lxxxxi, INTERMEDIATE EXAMPLE 89 Compound lxxxxii Compound lxxxxi is hydrolyzed under standard conditions using ethyl alcohol (0.3 M) and 1 N NaOH (1.5 eq) to afford 390 mg of compound lxxxx.ii, INTERMEDIATE EXAMPLE 90 Compound lxxxxiii Compound lxxxxii (350 mg, 0.7 mmol) is taken up in dichloromethane (3 mL).", "PyBOP (480 mg, 0.91 mmol) is added followed by compound xiii (170 mg, 0.91 mmol).", "DIPEA (0.16 mL, 0.91 mmol) is added and reaction mixture stirred overnight.", "The reaction mixture is concentrated and purified in 100% ethyl acetate to afford 420 mg (90%) of compound lxxxxiii, INTERMEDIATE EXAMPLE 91 Compound lxxxxiv Compound lxxxxiii is hydrogenated using 10% Pd/C (1% mol) in methyl alcohol under hydrogen to afford 335 mg (100%) of compound lxxxiv, INTERMEDIATE EXAMPLE 92 Compound lxxxxv Ethyl 1H-tetrazole-5-acetate (5 g, 32 mmol) is taken up in chloroform (80 mL).", "Trichlioroacetic acid (12.03 g, 73.65 mmol) is added followed by alpha methyl styrene (3.78 g, 32 mmol).", "The reaction mixture is stirred overnight.", "The next day, the solution is diluted with ethyl acetate, washed with 10% KOH and brine.", "The organic phase is dried over magnesium sulfate, filtered and concentrated to afford 8 g (96%) of the corresponding N-protected ethyl tetrazole-5-acetate.", "This material is subjected to standard hydrolysis conditions using ethyl alcohol (0.3 M) and 1 N NaOH (3 eq) to afford 7 g (99%) of compound lxxxxv, INTERMEDIATE EXAMPLE 93 Compound lxxxxvi Compound lxxxxv (3.62 g, 14.7 mmol) is taken up in dichloromethane (50 mL).", "EDCI (4.32 g, 22.1 mmol) and DIPEA (5.1 mL, 29.4 mmol) are added and stirred for five minutes.", "N-hydroxy succinimide (3.38 g, 29.4 mmol) is added and stirred three hours.", "The reaction is diluted with dichloromethane and washed with water three times.", "The organic phase is dried over sodium sulfate, filtered and concentrated to afford 3.66 g (73%) of compound lxxxxvi, INTERMEDIATE EXAMPLE 94 Compound lxxxxvii Compound lxxxxiv (335 mg, 0.62 mmol) and compound lxxxxvi (343 mg, 1 mmol) are taken up in dichloromethane (6 mL).", "DIPEA (0.17 mL, 1 mmol) is added and reaction mixture stirred overnight.", "The reaction is diluted with ethyl acetate, washed with saturated sodium bicarbonate, brine and concentrated.", "The residue is purified in 5% ethyl alcohol/ethyl acetate to give 80 mg (16%) of compound lxxxxvii, INTERMEDIATE EXAMPLE 95 Compound lxxxxviii Compound lxxxxvii (80 mg, 0.11 mmol) is taken up in dichloromethane (3 mL).", "DMP reagent reagent (55 mg, 0.13 mmol) is added and stirred for one hour.", "The reaction mixture is diluted with ethyl acetate and quenched with 10% solution of sodium sulfite.", "The organic phase is washed with saturated sodium bicarbonate and brine.", "The organic phase is concentrated and the reulting residue is purified in 100% ethyl acetate to afford 40 mg (48%) of compound lxxxxvii, INTERMEDIATE EXAMPLE 96 Compound xxxix Compound ic, N-Cbz-4-Hydroxy Pro methyl ester, (2.1 g, 7.9 mmol is prepared in quantitative yield from compound c, N-Cbz-4-hydroxy Pro), is dissolved in DCM (25 mL).", "CDI (1.54 g, 9.5 mmol) and DIPEA (1.7 mL, 9.5 mmol) are added to the solution and stirred for 10 minutes.", "1,2,3,4-Tetrahydroisoquinoline (TIQ) (1.2 mL, 9.5 mmol) is added drop-wise to the reaction mixture and stirred five hours.", "The organic phase is washed with water, 1 N HCl, and brine.", "Following the concentration of thee organic phase, the resultant residue is chromatographically purified by 40% EtOAc/Hexanes to yield compound ci, N-Cbz4-TIQcarbonyloxy-Pro methyl ester, (2.5 g, 75%).", "Compound ci (2.5 g, 5.9 mmol) is dissolved in MeOH (75 mL).", "The solution is flushed with N2 and Pd/C (10%, 300 mg) is added.", "The reaction mixture is flushed with H2 and stirred overnight.", "The reaction mixture is filtered through Celite and concentrated to yield compound compound xxxix, 4-(TIQ-carbonyloxy)-Pro, methyl ester,(1.49 g, 83%).", "INTERMEDIATE EXAMPLE 97 Compound vii Compound cii, N-pyrazin-2-ylcarbonyl-Val-Val methyl ester, (10.9 g, 32.4 mmol) is dissolved in THF (80 mL), and then aqueous NaOH (48.6 mL, 48.6 mmol) is added.", "The resulting mixture is stirred 48 hours, and then additional NaOH (16.3 mL, 16.3 mmol) is added and mixture is heated to 40° C. for three hours.", "The pH of the reaction mixture is then lowered to 3, and the acqueous phase extracted with EtOAc and then concentrated to yield crude compound vii, N-pyrazin-2-ylcarbonyl-Val-Val acid (10.6 g, 100%).", "INTERMEDIATE EXAMPLE 98 Compound ciii Compound cii (4.1 g, 12.7 mmol) is dissolved in DCM (20 mL).", "HOAt (1.73 g, 12.7 mmol) and DCC (12.7 mmol) are added to this solution, and the solution stirred for one hour.", "Compound xxxix (3.22 g, 10.6 mmol) is added to reaction mixture in DCM (10 mL).", "The resulting mixture is stirred overnight under N2.The reaction mixture is filtered through silica gel and concentrated.", "The resulting residue is purified by silica gel chromatography (50% to 80% EtOAc/Hexanes gradient) to yield compound ciii, N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro methyl ester, (5.27 g, 81.7%).", "INTERMEDIATE EXAMPLE 99 Compound civ Compound ciii (650 mg, 1.29 mmol) is dissolved in THF (5 mL).", "Aqueous NaOH (1.42 mL, 1.42 mmol) is added to the solution and then stirred overnight.", "The pH of the solution is lowered to 3, and the organic phase is isolated and concentrated to yield a residue.", "The residue is purifed using reverse phase HPLC in acetonitrile/water to yield compound civ, N-pyrazin-2-ylcarbonyl-Val-Val-4-(TIQcarbonyloxy)-Pro acid, (600 mg, 95%).", "INTERMEDIATE EXAMPLE 100 Compound cv N-Boc-L-tert-Leucine (2.3 g, 10 mmol) and L-tert-Leucine methyl ester hydrochloride (2 g, 11 mmol) are combined in DMF (30 mL).", "HOAt (1.6 g, 11.5 mmol) is then added to the solution.", "The resulting mixture is stirred for 20 minutes under N2 and then lowered to 0° C. whereupon DIC (1.8 mL, 11.5 mmol) and 2,4,6-collidine (1.45 mL, 11 mmol) are added.", "The resulting solution is stirred overnight with warming to room temperature.", "The reaction mixture is diluted with EtOAc, and the organic phase washed with 1 N HCl, saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 20% -30% EtOAc/hexanes gradient to yield compound cv (3.3 g, 92%).", "INTERMEDIATE EXAMPLE 101 Compound cvi Compound cv (3.3 g, 9.2 mmol) is hydrolyzed using dioxane (40 mL) and 0.5 N NaOH (37 mL, 18.4 mmol) to yield compound cvi (2.9 g, 92%).", "INTERMEDIATE EXAMPLE 102 Compound cvii Compound cvi (2 g, 5.8 mmol) and compound v (1 g, 5.5 mmol) are dissolved in DMF (20 mL).", "HOAt (832 mg, 6.6 mmol) and DIC (1.1 mL, 6.6 mmol) are then added to the solution.", "The resulting solution is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with 1 N HCl, saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 20%-30% EtOAc/hexanes gradient to yield compound cvii (2.4 g, 81%).", "INTERMEDIATE EXAMPLE 103 Compound cviii Compound cvii (2.4 g, 4.72 mmol) is dissolved in DCM (10 mL).", "TFA (10 mL) is added to the solution.", "The resulting solution is stirred for 4 hours.", "The reaction mixture is concentrated, dissolved in EtOAc, and then the organic phase is washed with 1 N NaOH and brine.", "The organic phase is concentrated to yield compound cviii (1.084 g, 56.1%).", "INTERMEDIATE EXAMPLE 104 Compound cix 2-Pyrazinecarboxylic acid (181 mg, 1.46 mmol) and compound cviii (541 mg, 1.325 mmol) are dissolved in DMF (15 mL).", "HOAt (207 mg, 1.52 mmol) and DIC (0.24 mL, 1.52 mmol) are added to the solution.", "The resulting solution is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with 1 N HCl, saturated.", "NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 20%-30%-35% EtOAc/hexanes gradient to yield compound cix (430 mg, 63%).", "INTERMEDIATE EXAMPLE 105 Compound cx Compound cix is hydrolyzed using EtOH (7 mL) and 1 N NaOH (4.7 mL, 4.7 mmol) to yield compound cx (700 mg, 91.6%).", "INTERMEDIATE EXAMPLE 106 Compound cxi Compound cx (690 mg, 1.42 mmol) is dissolved in DCM (9 mL).", "PYBOP (890 mg, 1.7 mmol) is then added to the solution, followed by the addition of Compound xiii′ (320 mg, 1.7 mmol).", "To the resulting mixture is added DIPEA (0.3 mL, 1.7 mmol).", "The reaction mixture is stirred overnight under N2.The reaction mixture is then diluted with EtOAc, washed with saturated NaHCO3, and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 100% EtOAc to yield compound cxi (490 mg, 52.7%).", "INTERMEDIATE EXAMPLE 107 Compound cxiv Compound cxii (1.2 g, 3.06 mmol) is dissolved in MeOH (12 mL).", "After thoroughly flushing with N2, 10 wt % Pd(OH)2 on carbon (0.6 g) is added and the mixture is hydrogenated for overnight, whereupon a complete reaction mixture is shown by TLC (30% EtOAc/hexanes).", "The solution is isolated from solid material by filtration and concentrated to the corresponding deprotected compound cxiiias a colorless oil (100%) that is used in the next step without further purification.", "2-Pyrazinecarboxylic acid (400 mg, 3.2 mmol, 1.1 eq) is dissolved in DCMP/THF (4 mL/4 mL), and then HOAt (440 mg, 3.2 mmol) and DCC (343 mL, 1 M in DCM) are added.", "After stiring at room temperature for 20 minutes, the compound cxiii (0.96 g, 3.2 mmol) obtained previously is dissolved in DCM (6.4 mL) and added to the activated mixture.", "After stirring overnight at room temperature, the reaction mixture is filtered through Celite and compound cxiv is purified by column chromatography (30% EtOAc/hexanes) to yield a white solid (0.8 g, 80%).", "INTERMEDIATE EXAMPLE 108 Compound cxv Compound cxiv (0.8 g, 2.2 mmol) is dissolved in MeOH (10 mL), and then 2 N NaOH (aq) (3.3 mL, 6.6 mmol) is added.", "The solution is stirred at room temperature overnight, whereupon the completion of the reaction mixture is indicated by TLC (50% EtOAc/hexanes).", "Acidification to pH 3 by 5 N HCl and diluted with EtOAc is followed by extraction of the organic phase.", "The extracted organic phase is washed with brine and dried over MgSO4 to yield compound cxv (0.74, 95%) upon concentration.", "INTERMEDIATE EXAMPLE 109 Compound cxvi To a DCM solution (6 mL) of compound cxv (0.74 g, 2.1 mmol) at room temperature is added HOAt (290 mg, 2.1 mmol), followed by the addition of 1 M DCC solution in DCM (2.2 mL, 2.2 mmol).", "After stirring for 30 minutes at room temperature, a THF solution (10.5 mL, 0.2 M) of compound v (2.1 mmol) is added to the above HOAt-activated acid.", "The reaction mixture is stirred at room temperature overnight.", "At this point, the reaction mixture is filtered through celite.", "The filtrate is diluted with EtOAc (120 mL) and washed with water and brine.", "The organic phase is dried and concentrated to a yellow oil that is purified by silica gel chromatography (50% EtOAc/hexanes) to yield compound cxvi (0.714 g, 66%).", "INTERMEDIATE EXAMPLE 110 Compound cxvii To an EtOH solution of compound cxvi (0.7 g, 1.4 mmol) is added 2 N NaOH aqueous solution (2 mL, 4 mmol).", "The reaction mixture is stirred overnight at room temperature, then acidified to pH 3 by 5 N HCl and diluted with EtOAc is followed by extraction of the organic phase.", "The extracted organic phase is washed with brine and dried over MgSO4 to yield compound cxvii (95%) upon concentration.", "INTERMEDIATE EXAMPLE 111 Compound cxviii To a DCM/THF solution (10 mL/2 mL) of compound cvii (300 mg, 0.6 mmol) is added PyBOP (416 mg, 0.8 mmol).", "The solution is stirred at room temperature for 30 minutes.", "To this solution is then added compound xxxvi′ (200 mg, 0.8 mmol), followed by DIPEA (0.22 mL, 1.2 mmol).", "The reaction mixture is stirred at room temperature overnight and then quenched with water (25 mL) for 30 minutes.", "The mixture is then extracted with EtOAc.", "The resulting organic phase is washed with brine and dried over MgSO4, before being concentrated to yield a yellow oil.", "Purification by silica gel chromatography (3-5% EtOH/EtOAc) yields compound cxviii (335 mg, 76%).", "INTERMEDIATE EXAMPLE 112 Compound cxix To a DCM solution (10 mL) of compound cxvid (340 mg, 0.6 mmol) is added PyBOP (470 mg, 0.9 mmol).", "The solution is stirred at room temperature for 30 minutes.", "To this solution is then added compound xiii′ (170 mg, 0.9 mmol), followed by DIPEA (0.24 mL, 1.2 mmol).", "The reaction mixture is stirred at room temperature overnight and then quenched with water (25 mL) for 30 minutes.", "The mixture is then extracted with EtOAc.", "The resulting organic phase is washed with brine and dried over MgSO4, before being concentrated to yellow oil.", "Purification by silica gel chromatography (3-5% EtOH/EtOAc) yields compound cxix (164 mg, 36%).", "INTERMEDIATE EXAMPLE 113 Compound xx N-Cbz-L-Valine (6.28 g, 25 mmol) is dissolved in DCM (30 mL).", "HOBT (3.38 g, 25 mmol) and DCC (25 mL, 1 M solution) are added to this solution and stirred five minutes.", "L-tert-Leucine methyl ester hydrochloride (25 mL, 1 M solution) is added to this mixture and stirred overnight under N2.The reaction mixture is diluted with EtOAc, washed with 1 N HCl, saturated NaHCO3, and brine.", "The organic phase is dried over Na2SO4, filtered and concentrated.", "The residue is chromatographically purified by 20%-30% EtOAc/hexanes to yield compound xx (2.96g, 31%).", "INTERMEDIATE EXAMPLE 114 Compound xxi Compound xx (2.95 g, 7.8 mmol) is hydrogenated using 10% Pd/C (800 mg) in MeOH (40 mL) under H2 to yield the below corresponding free amine (1.9 g, 100%).", "2-Pyrazine-carboxylic acid (970 mg, 7.8 mmol) is dissolved in DCM (20 mL).", "PyBOP (4.06 g, 7.8 mmol) is added to this solution.", "The free amine (1.9 g, 7.8 mmol) in DCM (15 mL) is added to the solution, and then DIPEA (1.36 mL, 7.8 mmol) is added.", "The resulting mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase is washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the residue is chromatographically purified by 30%40% EtOAc/Hexanes to yield compound xxi (2.07 g, 75.8%).", "INTERMEDIATE EXAMPLE 115 Compound xxii Compound xxi is hydrolyzed using MeOH (20 mL) and 1 N NaOH (3 eq) to yield compound xxii (1.82g, 93.9%).", "INTERMEDIATE EXAMPLE 116 Compound xxiii Compound xxii (895 mg, 2.66 mmol) is dissolved in DCM (10 mL).", "DCC (3.2 mmol) is added to the solution, and then HOAt (435 mg, 3.2 mmol) is added.", "Compound v (3.2 mmol) in THF (16 mL) is then added.", "The resulting mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, filtered through silica gel, and concentrated.", "The resulting residue is chromatographically purified by 50% EtOAc/hexanes to yield compound xxiii (730 mg, 54.8%).", "INTERMEDIATE EXAMPLE 117 Compound xxiv Compound xxiii is hydrolyzed using EtOH (5 mL) and 1 N NaOH (1.5 eq) to yield compound xxiv (690 mg, 100%).", "INTERMEDIATE EXAMPLE 118 Compound cxx Compound xxiv (245 mg, 0.52 mmol) is dissolved in DCM (3 mL).", "PyBOP (330 mg, 0.62 mmol) is added to the solution, and then compound xiii′ (120 mg, 0.62 mmol) is added.", "To the resulting mixture is added DIPEA (0.11 mL, 0.62 mmol).", "The reaction mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the residue is chromatographically purified by 5% EtOH/EtOAc to yield compound cxx (220 mg, 60%).", "INTERMEDIATE EXAMPLE 119 Compound xiii′ Boc-NVA-OH (24.96 g, 114.9 mmol) is dissolved in THF (200 mL).", "CDI (22.35, 137.8 mmol) is added portion-wise to the solution, and the solution is stirred for 30 minutes.", "N,O-Dimethylhydroxylamine hydrochloride (12.33 g, 126.4 mmol) is dissolved in DMF (50 mL) and then DIPEA (22 mL, 126.4 mmol) is added to the solution.", "The DMF solution is allowed to stir at room temperature for 20 minutes and then added to THF solution.", "The resulting mixture is stirred over a weekend under N2.The reaction mixture is concentrated in vacuo to 100 mL total volume.", "This organic phase is washed with 1 N HCl, saturated NaHCO3 and brine.", "The organic phase is concentrated to yield crude compound cxxi (25.3 g).", "LAH (107.3 mmol) is added to a dry 1-L round bottom flask under N2 in a 1 M Et2O solution.", "This solution is lowered to 0° C., and then compound cxxi (97.5 mmol) is added drop-wise in Et2O (100 mL).", "Upon completion of the addition, the resulting mixture is stirred for 30 minutes.", "The reaction mixture is quenched at 0° C. by slowly adding EtOAc (50 mL), followed by slowly adding a 5% KHSO4 (50 mL) solution.", "This mixture is stirred for 30 minutes.", "The organic phase is washed with 1 N HCl, saturated NaHCO3, and brine.", "The organic phase is concentrated to yield crude compound cxxii (22.28 g).", "Compound cxxii is dissolved in MeOH (100 mL).", "Na2S2O4 (16.82 g, 96.6 mmol) is dissolved in water (100 mL and then added to solution of compound cxxii at 0° C. This mixture is stored in the refrigerator (5° C.) overnight.", "KCN (7.53 g, 115.9 mmol) in water (100 mL) is added to reaction mixture and stirred for 1.5 hours at room temperature.", "The compound is extracted with EtOAc (3×100 mL).", "The organic phase is washed with brine (3×50 mL), dried over MgSO4, filtered and concentrated to yield crude compound cxxiii (15.86 g).", "Compound cxxiii (15.86 g) is dissolved in dioxane (100 mL).", "Concentrated HCl (37%, 100 mL) is added to this solution followed by anisole (10 mL) and reflux is established (110° C.).", "The reaction stirred for 1.5 hours.", "When the reaction mixture is cooled to room temperature, the solvent is removed in vacuo to yield a dry paste.", "The residue is dried overnight under high vacuum to yield crude compound cxxiv.", "Compound cxxiv (69.6 mmol) is dissolved in DMF (60 mL) and THF (60 mL).", "N-(Benzyl-oxycarbonyloxy)succinimide (17.33 g, 69.6 mmol) is added to the mixture, followed by the addition of DIPEA (12.1 mL, 69.6 mmol).", "The reaction mixture is stirred overnight under N2.The mixture is concentrated to a reduced volume (50 mL) and diluted with EtOAc.", "The organic phase is washed with 0.1 N HCl (2×100 mL) and brine to yield compound cxxv (17.5 g, 54.2% over five steps).", "Compound cxxv (5.66 g, 20.14 mmol) is dissolved in DCM (60 mL).", "PyBOP (12.57 g, 24.2 mmol) and HOBT (3.27 g, 24.2 mmol) are added to this solution and stirred five minutes.", "The resulting mixture is lowered to 0° C., and then cyclopropylamine (1.67 mL, 24.2 mmol) and DIPEA (4.2 mL, 24.2 mmol) are added.", "The reaction mixture is stirred overnight warning to room temperature.", "The reaction mixture is washed with 0.1 N HCl, saturated NaHCO3, and brine.", "The organic phase is then concentrated and chromatographically purified using 70% EtOAc/Hexanes to yield compound cxxvi (3.18 g, 49.3%).", "Compound cxxvi (3.18 g, 9.94 mmol) is hydrogenated using 10% Pd/C (600 mg) in MeOH (70 mL).", "The reaction mixture is stirred overnight under H2, filtered through celite and concentrated to yield crude compound xiii′ (2.1 g, 100%).", "INTERMEDIATE EXAMPLE 120 Compound cxxvii N-Cbz-L-Cyclohexylglycine (3 g, 10.3 mmol) is dissolved in DCM (36 mL).", "HOAt (1.5 g, 11.28 mmol) and DCC (11.28 mL, 11.28 mmol) are added to this solution and stirred five minutes.", "L-tert-Leucine methyl ester hydrochloride (103 mL, 1 M solution, 10.3 mmol) is added to this mixture and stirred overnight under N2.The reaction mixture is filtered through celite, rinsed with EtOAc and concentrated to a residue that is purified chromatographically using 20%-30% EtOAc/hexanes to yield compound cxxvii (2.2 g, 52%).", "INTERMEDIATE EXAMPLE 121 Compound lxxix′ Compound cxxvii (2.2 g, 5.2 mmol) is hydrogenated using 20% Pd(OH)2/C (1 g) in MeOH (15 mL) under H2 to yield compound lxxix′ (1.4 g, 98%).", "INTERMEDIATE EXAMPLE 122 Compound lxxx 2-Pyrazinecarboxylic acid (360 mg, 2.9 mmol) is dissolved in DCM (10 mL).", "PyBOP (1.81 g, 3.5 mmol) is added to the solution.", "Compound lxxix′ (825 mg, 2.9 mmol) in THF (10 nL) is then added to the solution, followed by the addition of DIPEA (0.5 mL, 2.9 mmol).", "The resulting mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with saturated NaHCO3 and brine.", "The residue resulting from the concentration of the organic phase is chromatographically purified by 30% EtOAc/Hexanes to yield Compound lxxx (780 mg, 69%).", "INTERMEDIATE EXAMPLE 123 Compound lxxxi Compound lxxx is hydrolyzed using MeOH (10 mL) and 1 N NaOH (3 eq) to yield compound lxxxi (615 mg, 81.8%).", "INTERMEDIATE EXAMPLE 124 Compound lxxxii Compound lxxxi (610 mg, 1.6 mmol) is dissolved in DCM (10 mL).", "DCC (1.94 mL, 1.94 mmol) is then added to the solution, followed by the addition of HOAt (270 mg, 1.94 mmol).", "Compound v (1.94 mmol) in THF (19.4 mL) is then added to the solution.", "The resulting mixture is stirred over two nights under N2.The reaction mixture is diluted with EtOAc, filtered through silica gel, and concentrated.", "The resulting residue is purified chromatographically by 40% EtOAc/hexanes to yield compound lxxxii (450 mg, 83.4%).", "INTERMEDIATE EXAMPLE 125 Compound lxxxiii Compound lxxxi is hydrolyzed using EtOH (10 mL) and 1 N NaOH (3 eq) to yield compound lxxxiii (650 mg, 99%).", "INTERMEDIATE EXAMPLE 126 Compound cxxviii Compound lxxxiii (400 mg, 0.78 mmol) is dissolved in DCM (5 mL).", "PyBOP (610 mg, 1.2 mmol) is added to the solution, followed by Compound xiii′ (230 mg, 1.2 mmol).", "To the resulting mixture is added DIPEA (0.2 mL, 1.2 mmol).", "The reaction mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the residue is chromatographically purified by 100% EtOAc to 5% EtOH/EtOAc gradient to yield compound cxxviii (365 mg, 68.7%).", "INTERMEDIATE EXAMPLE 127 Compound cxxx Compound lxxxiii (365 mg, 0.7 mmol) is dissolved in DCM (5 mL).", "PYBOP (440 mg, 0.84 mmol) is added to the solution, followed by the addition of compound cxxix (0.84 mmol) in THF (8.4 mL).", "To the resulting mixture is added DIPEA (0.1 mL, 0.84 mmol).", "The reaction mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase is washed with satutrated NaHCO3 and brine.", "Following the concentration of the organic phase, the resulting residue is chromatographically purified by 100% EtOAc to yield compound cxxx (350 mg, 70%).", "INTERMEDIATE EXAMPLE 128 Compound cxxxi Compound cxxv (2.54 g, 9.05 mmol) is dissolved in DCM (30 mL).", "PYBOP (5.65 g, 10.9 mmol) and HOBT (1.47 g, 10.9 mmol) are added to the solution and stirred five minutes.", "The resulting mixture is lowered to 0° C., whereupon (S)-(+)-3-Methyl-2-butylamine (1.27 mL, 10.9 mmol) and DIPEA (1.9 mL, 10.9 mmol) are added.", "The reaction mixture is stirred overnight with warming to room temperature.", "The organic phase is washed with 0.1 N HCl, saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 30% EtOAc/hexanes to yield compound cxxxi (1.44 g, 45.5%).", "INTERMEDIATE EXAMPLE 129 Compound cxxix Compound cxxxi (1.3 g, 3.7 mmol) is hydrogenated using 10% Pd/C (500 mg) in MeOH (40 mL).", "The reaction mixture is stirred overnight under H2.The reaction mixture is filtered through celite and the organic phase concentrated to yield crude compound cxxix (800 mg, 100%).", "INTERMEDIATE EXAMPLE 130 Compound cxxxiv Compound cxxxii (1.6 g, 3.7 mmol) is dissolved in MeOH (12 mL).", "After thoroughly flushing with N2, 10 wt % Pd(OH)2 on carbon (0.74 g) is added and the mixture is hydrogenated overnight, whereupon a complete reaction mixture is shown by TLC (30% EtOAc/hexanes).", "The solution is isolated from solid material by filtration and concentrated to yield compound cxxxiii as colorless oil (100%) that is used in the next step without further purification.", "2-Pyrazinecarboxylic acid (400 mg, 3.2 mmol, 1.1 eq) is dissolved in DCM/THF (4 mL/4 mL), and then HOAt (440 mg, 3.2 mmol) and DCC (3.3 mL, 1 M in DCM) is added.", "After stirring at room temperature for 20 minutes, compound cxxxiii (0.96 g, 3.2 mmol) obtained previously is dissolved in DCM (6.4 mL) and added to the activated mixture.", "After stirring over 2 days at room temperature, the reaction mixture is filtered through Celite, and concentrated to a residue that is purified by column chromatography (50% EtOAc/hexanes) to yield compound cxxxiv as a white solid (1.06 g, 83%).", "INTERMEDIATE EXAMPLE 131 Compound cxxxv Compound cxxxiv (1.06 g, 2.6 mmol) is dissolved in MeOH (10 mL), and then 2 N NaOH (aq) (4 mL, 8 mmol) is added.", "The solution is stirred at room temperature overnight, whereupon the completion of the hydrolysis is indicated by TLC (50% EtOAc/hexanes).", "The solution is acidified to pH 3 by 5 N HCl, diluted with EtOAc and then the organic phase is extracted.", "The extracted organic phase is washed with brine and dried over MgSO4 to yield compound cxxxv (100%) upon concentration.", "INTERMEDIATE EXAMPLE 132 Compound cxxxvi To a DCM solution (8 mL) of compound cxxxv (1.44 g, 3.7 mmol) at room temperature is added HOAt (500 mg, 3.7 mmol), and then 1 M DCC solution in DCM (3.7 mL, 3.7 mmol) is added.", "After stirring for 30 minutes at room temperature, a THF solution (18.5 mL, 0.2 M) of compound v (3.7 mmol) is added to the above HOAt-activated acid.", "The reaction mixture is stirred at room temperature overnight.", "The reaction mixture is filtered through Celite.", "The filtrates are diluted with EtOAc (120 mL) and washed with water and brine.", "The organic phase is dried and concentrated to yield yellow oil that is purified by silica gel chromatography (70% EtOAc/hexanes) to yield compound cxxxvi (1 g, 71%).", "INTERMEDIATE EXAMPLE 133 Compound cxxxvii To an EtOH solution (8 mL) of compound cxxxvi (1 g, 1.8 mmol) is added 2 N NaOH aqueous solution (2.7 mL, 5.4 mmol).", "The reaction mixture is stirred overnight at room temperature, then acidified to pH 3 by 5 N HCl, diluted with EtOAc, and then the organic phase is extracted.", "The extracted organic phase is washed with brine and dried over MgSO4 to yield compound cxxxvii (88%) upon concentration.", "INTERMEDIATE EXAMPLE 133 Compound cxxxviii To a DCM solution (10 mL) of compound cxxxvii (350 mg, 0.6 mmol) is added PyBOP (450 mg, 0.86 mmol).", "The solution is stirred at room temperature for 30 minutes.", "To this solution is then added compound xiii′ (160 mg, 0.86 mmol) followed by DIPEA (0.23 mL, 1.3 mmol).", "The reaction mixture is stirred at room temperature overnight and then quenched with water (25 mL) for 30 minutes.", "The mixture is then extracted with EtOAc.", "The extracted organic phase is washed with brine and dried over MgSO4, before being concentrated to yield yellow oil.", "Purification by silica gel chromatography (5% EtOH/tOAc) yields compound cxxxviii (407 mg, 88%).", "INTERMEDIATE EXAMPLE 134 Compound cxxxix 5-Methylisoxazole-3-carboxylic acid (200 mg, 2.05 mmol) is dissolved in DCM (5 mL).", "PyBOP (1.07 g, 2.05 mmol) is added to the solution.", "Compound lxxix′ (582 mg, 2.05 mmol) in DCM (5 mL) is added to the solution, followed by the addition of DIPEA (0.36 mL, 2.05 mmol).", "The resulting mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with saturated NaHCO3 and brine.", "The organic phase is concentrated, and the resultant residue is purified chromato-graphically by 30% EtOAc/hexanes to yield Compound cxxxix (495 mg, 61.4%).", "INTERMEDIATE EXAMPLE 135 Compound cxxxx Compound cxxxix is hydrolyzed using MeOH (10 mL) and 1 N NaOH (3 eq) to yield compound cxxxx (430 mg, 90%).", "INTERMEDIATE EXAMPLE 136 Compound cxxxxi Compound cxxxx (380 mg, 1 mmol) is dissolved in DCM (5 mL).", "DCC (1.2 mmol) is then added to the solution, followed by the addition of HOAt (165 mg, 1.2 mmol).", "Compound v (1.2 mmol) is then added in THF (12 mL).", "The resulting mixture is stirred overnight under N2.The reaction mixture is diluted with EtOAc, filtered through silica gel, and concentrated.", "The resultant residue is chromatographically purified by 35% EtOAc/hexanes to yield compound cxxxxi (320 mg, 58%).", "INTERMEDIATE EXAMPLE 137 Compound cxxxxii Compound cxxxxi is hydrolyzed using EtOH (10 mL) and 1 N NaOH (3 eq) to yield compound cxxxxii (730 mg, 94.3%).", "INTERMEDIATE EXAMPLE 138 Compound cxxxxiii Compound cxxxxii (240 mg, 0.46 mmol) is dissolved in DCM (5 mL).", "PyBOP (295 mg, 0.56 mmol) is then added to the solution, followed by the addition of Compound xiii′ (110 mg, 0.56 mmol).", "To the resulting mixture is added DIPEA (0.1 mL, 0.56 mmol).", "The reaction mixture is stirred over two nights under N2.The reaction mixture is diluted with EtOAc, and the organic phase washed with saturated NaHCO3 and brine.", "Following the concentration of the organic phase, the resultant residue is chromatographically purified by 90% EtOAc/hexanes to yield Compound cxxxxiii (168 mg, 53%).", "INTERMEDIATE EXAMPLE 139 Compound cxxxxiv To a solution of NaOH (2N, 42.1 mL, 84.2 mmol) at 5° C. is added L-alanine (5.00 g, 56.1 mmol).", "After stirring for 10 minutes, methyl chloroformate (6.5 mL, 84.2 mmol) and NaOH (2 N, 42.1 mL, 84.2 mmol) are added dropwise simultaneously.", "The solution is stirred in ice bath for 2 hours, then at room temperature for 1 hour.", "The mixture is washed with Et2O (2×50 mL), the aqueous layer is neutralized to pH ˜2 with 5 N HCl, and extracted with EtOAc (3×50 mL).", "The extracted organic phase is washed with brine, dried by MgSO4 and concentrated to yield compound cxxxxiv, N-carbomethoxy-L-alanine, (4.54 g, 54%) as colorless oil.", "INTERMEDIATE EXAMPLE 140 Compound cxxxxvi A solution of compound cxxxxv (3.57 g, 9.44 mmol) in THF at 5° C. is treated with HOAt (1.28 g, 9.44 mmol), and then DCC (9.50 mL, 9.50 mmol) is added.", "After stirring in ice bath for 45 minutes, a solution of compound v (104 mL, 10.4 mmol) in THF is added.", "The mixture is stirred at room temperature overnight.", "The mixture is cooled to 5° C. and quenched with saturated NaHCO3.After filtration to remove the precipitated DCU, the mixture is dissolved in EtOAc (100 mL), washed with saturated NaHCO3, brine, and then dried by MgSO4 and concentrated to a residue purified by silica column chromatography (25% EtOAc/Hexanes) to yield compound cxxxxvi (2.91 g, 57%) as gummy foam.", "INTERMEDIATE EXAMPLE 141 Compound cviii To a solution of compound cxxxxvi in MeOH (25 mL) cooled by an ice bath under a stream of N2 is added slowly Pd/C.", "The mixture is hydrogenated at 1 atm overnight.", "The catalyst is removed by filtration, the filtrate is combined with 5 mL DMF and dried under vacuum to yield compound cviii.", "INTERMEDIATE EXAMPLE 142 Compound cxxxxvii To a solution of compound cxxxxiv (0.298 g, 2.03 mmol) and HOAt (0.276 g, 2.03 mmol) in THF cooled in ice bath is treated with DCC (2.05 mL, 2.05 mmol).", "After stirring in an ice bath for 0.5 hour, a solution of compound cviii in THF is added, and then DIPEA (0.39 mL, 2.2 mmol) is added.", "The mixture is stirred at room temperature overnight, then cooled in an ice bath, and quenched with saturated NaHCO3.The precipitated DCU is filtered and the filtrate is dissolved in EtOAc (100 mL).", "The organic phase is washed with saturated NaHCO3, brine, and then dried by MgSO4.After removal of the organic solvent, the residue is purified by silica column chromatography (60% EtOAc/Hexanes) to yield compound cxxxxvii (0.47 g, 48%) as gummy foam.", "INTERMEDIATE EXAMPLE 143 Compound cxxxxviii To a solution of compound cxxxxvii (0.47 g, 0.847 mmol) in EtOH (5 mL) at 5° C. is added NaOH (2 N, 1.31 mL, 2.62 mmol).", "The mixture is stirred at room temperature for 4 hours.", "The solution is acidified to pH ˜2 with HCl (1N) and the EtOH is removed by rotary evaporation.", "The mixture is extracted with EtOAc (3×30 mL) and the combined extract is washed with brine, and then dried by MgSO4.The solvent is removed and the residue is dried under vacuum to yield compound cxxxxviii (0.366 g, 82%) as gummy foam.", "INTERMEDIATE EXAMPLE 144 Compound cil A solution of compound cxxxxviii (0.366 g, 0.718 mmol) in DCM is cooled in an ice bath and treated with PyBop (0.599 g, 1.15 mmol).", "After stirring at room temperature for 0.5 hour, the mixture is cooled by an ice bath and treated with a solution of compound xiii′ (0.200 g, 1.08 mmol) in THF and DIPEA (0.250 mL, 1.44 mmol).", "The mixture is stirred at room temperature overnight and then quenched with NH4Cl solution.", "The solvent is concentrated and the mixture is dissolved in EtOAc (100 mL).", "The organic phase is washed with saturated NaHCO3, brine, and then dried by MgSO4.After removal of the organic solvent, the residue is purified by column chromatography (5% EtOH/EtOAc) to yield compound cil (0.35 g, 72%) INTERMEDIATE EXAMPLE 145 Compound cxxi To a THF solution (85 mL) of N-Boc-Nva-OH (compound 1) (8.68 g, 40 mmol) is added CDI (7.79 g, 48 mmol).", "After stirring at room temperature for 30 minutes, the above solution is treated with a DMF solution (25 mL) containing N,O-dimethyl-hydroxylamine hydrochloride (4.25 g, 44 mmol) and DIPEA (7.66 mL, 44 mmol).", "The reaction mixture is stirred overnight at room temperature.", "The reaction mixture is then concentrated in vacuo.", "The resulting residue is diluted with EtOAc (300 mL).", "This solution is washed sequentially with 0.1 N HCl (50 mL), saturated NaHCO3 (3×50 mL) and brine.", "The organic phase is concentrated in vacuo to yield a residue that is purified with silica gel chromatography (40% EtOAc/Hexanes) to compound cxxi (9.38 g, 94%).", "INTERMEDIATE EXAMPLE 146 Compound cxxii To a diethyl Et2O solution (50 mL) of compound cxxi (9.38 g, 31.9 mmol) cooled to 0° C. is added (slowly) LAH (34.7 mL, 1 M, 34.7 mmol).", "The temperature of the reaction flask is maintained below 5° C. during LAH addition.", "Upon completion of the addition, EtOAc (20 mL) is added to the reaction to quench the excess LAH.", "Aqueous KHSO4 (5%, 20 mL) is then added in a dropwise fashion in order to keep the temperature below 5° C. The organic phase is separated and then washed sequentially with 1 N HCl (3×30 mL), saturated NaHCO3 (3×30 mL) and brine.", "The organic phase is concentrated and dried in vacuo to yield crude compound cxxii (5.18 g, 69%).", "INTERMEDIATE EXAMPLE 147 Compound cl To a THF (25 mL) suspension of Zn (2.75 g, 42 mmol) is added at reflux 0.2 mL of EtOC(O)CF2Br.", "This is followed by slowly adding a THF solution (25 mL) of compound cxxii (3.05 g, 15.0 mmol) and EtOC(O)CF2Br (4.84 mL, 37.5 mmol).", "Upon completion of the addition of both reagents, the reaction mixture is further refluxed for 30 minutes.", "The reaction mixture is cooled to room temperature and diluted with DCM (200 mL).", "The organic phase is washed with 1 N KHSO4.The organic phase is concentrated and dried in vacuo to yield a residue that is purified by silica gel chromatography (20% EtOAc/Hexane) to yield compound cl (2.78 g, 57%).", "This preparation is essentially the same as that disclosed by Thaisrivongs et al., J. Med.", "Chem., 29, 2080-2087 (1986).", "INTERMEDIATE EXAMPLE 148 Compound cli A THF solution (40 mL) of compound cl (2.78 g, 8.53 mmol) is treated with 1 N NaOH (12.8 mL, 12.8 mmol).", "After stirring at room temperature overnight, thie solvent is partially removed in vacuo.", "The remaining reaction mixture is diluted with water (50 mL) and lyophilized to yield crude compound cli (2.82 g, >100%) as its sodium salt.", "This preparation is essentially the same as that disclosed by Thaisrivongs et al., J. Med.", "Chem., 29, 2080-2087 (1986).", "INTERMEDIATE EXAMPLE 149 Compound clii A DCM solution (10 mL) of the crude compound cli (516 mg, 1.61 mmol) is treated with HOBT (436 mg, 3.23 mmol) and DIC (0.328 mL, 2.09 mmol).", "After stirring at room temperature for 30 minutes, the reaction mixture is treated with a DCM solution (5 mL) containing glycine benzylester-TsOH salt (815 mg, 2.42 mmol) and DIPEA (0.422 mL, 2.42 mmol).", "After stirring at room temperature for 12 hours, the reaction mixture is quenched with water and extracted with EtOAc.", "The organic phase is dried and concentrated in vacuo and purified by silica gel chromatography (40% EtOAc/hexanes) to yield compound ciii (495 mg, 69%).", "1H NMR of compound clii (400 MHz, CDCl3): δ7.29-7.21 (i, 5H), 5.16 (bs, 2H), 4.89 (bs, 1H), 4.20-3.90 (m, 4H), 3.80 (bs, 1H), 1.75-1.42 (m, 4H), 1.38 (s, 9H), 0.87 (m, 3H).", "Starting from crude compound cli, compounds cliii (83%) and cliv (50%) are prepared in an identical method to that described for compound clii.", "1H NMR of compound cliii (400 Mz, CDCl3): δ7.49 (bs, 1H), 7.34-7.24 (m, 5H), 5.13 (AB q, J=12.2 Hz, J′=23.9 Hz, 2H), 4.88 (bd, J=8.8 Hz, 1H), 4.53 (m, 1H), 3.98-3.91 (m, 2H), 3.82 (m, 1H), 1.65-1.20 [m, 16H, including singlet at 1.37 (9H)], 0.86 (t, J=7.3 Hz, 3H).", "1H NMR of compound cliv (400 MHz, CDCl3): δ7.60-7.0 (m, 10H), 5.30-5.00 (m, 2H), 5.00-4.75 (m, 2H), 4.15-3.70 (m, 3H), 3.30-3.00 (m, 2H), 1.75-1.20 [m, 13H, including singlet at 1.36 (9H)], 0.86 (bs, 3H).", "INTERMEDIATE EXAMPLE 150 Compound clv To a DCM (10 mL) and THF (5 mL) solution of the crude compound cli (1 g, 3.13 mmol) is added HOBT (634 mg, 4.69 mmol) and EDCI (781 mg, 4.07 mmol), and then (s)-α-methylbenzylamine (0.604 mL, 4.69 mmol).", "The reaction mixture is stirred overnight at room temperature and then quenched with water.", "The reaction mixture is extracted with EtOAc.", "The organic phase is washed with brine and dried by Na2SO4.The organic phase is concentrated in vacuo to yield a residue that is purified by silica gel chromatography (20% EtOAc/hexanes) to yield compound clv (459 mg, 37%).", "1H NMR of compound clv(400 MHz, CDCl3): δ7.32-7.21 (m, 6H), 5.00 (m, 1H), 4.75 (m, 1H), 3.94 (m, 2H), 3.70 (ma, 1H), 1.65-1.15 [m, 16H, including doublet at 1.51 (J=6.8 Hz, 3H), singlet at 1.39 (9H)], 0.82 (m, 3H).", "INTERMEDIATE EXAMPLE 151 Compound clvi Compound clv (220 mg, 0.55 mmol) is dissolved in 4 N HCl in dioxane (10 mL).", "The reaction mixture is stirred at room temperature for 2 hours and then concentrated in vacuo to give the crude compound clvi (˜100%) as its HCl salt.", "Following the procedure described for preparing compound clvi, compounds clvii, clviii, and clix are prepared in almost quantitative yield from the crude compound cli.", "INTERMEDIATE EXAMPLE 152 Compound clx A DCM solution (4 mL) of the HCl salt of compound vii (96 mg, 0.144 mmol) is treated with PyBOP (120 mg, 0.23 mmol) and DIPEA (0.1 mL, 0.576 mmol).", "After stirring at room temperature for 30 minutes, the solution is treated with a THF solution (4 mL) containing compound clv (0.288 mmol) and DIPEA (0.2 mL, 1.152 mmol).", "The reaction mixture is stirred at room temperature overnight.", "The reaction mixture is then diluted with EtOAc (50 mL), and the organic phase then washed with NaHCO3 and brine.", "The organic phase is concentrated in vacuo and the residue purified by silica gel chromatography (80% EtOAc/hexanes) to yield compound clx (113 mg, 89%).", "INTERMEDIATE EXAMPLE 153 Compound clxi A DCM solution (6 mL) of compound vii (140 mg, 0.235 mmol) is treated with PyBOP (196 mg, 0.376 mmol) for 30 minutes.", "A THF solution (6 mL) of compound clvii (˜0.47 mmol) and DIPEA (0.327 mL, 1.88 mmol) is then added to the above solution.", "The reaction mixture is stirred at room temperature overnight and quenched with water (30 minutes).", "The reaction mixture is extracted with EtOAc (50 mL).", "The organic phase is washed with NaHCO3 and brine.", "The combined aqueous layers are back extracted with EtOAc (50 mL).", "The combined organic phases are dried and concentrated in vacuo.", "The resultant residue is purified by silica gel chromatography (80-100 EtOAc/hexanes) to yield compound clxi (104 mg, 48%).", "INTERMEDIATE EXAMPLE 154 Compound clxii To a DCM solution (10 mL) of compound clxi (280 mg, 0.304 mmol) is added DMP reagent reagent (193 mg, 0.456 mmol).", "The reaction mixture is stirred at room temperature for 3 hours and quenched with 10% Na2SO3.The organic phase is washed with NaHCO3 and brine.", "The resulting organic phase is dried and concentrated in vacuo to yield a residue that is purified with silica gel chromatography (80-100% EtOAc/hexanes) to yield compound clxii (271 mg, 97%).", "INTERMEDIATE EXAMPLE 155 Compound clxiii Compound lxxxiii (220 mg, 0.43 mmol) is taken up in DCM (5 mL).", "PyBOP (270 mg, 0.51 mmol) is added to the DCM solution and stirred 5 minutes.", "Compound xxxvi′ (0.51 mmol) in THF (5.1 mL) is added drop-wise to this solution.", "DIPEA (0.09 mL, 0.51 mmol) is added to reaction mixture and stirred overnight under N2.The next day, the reaction mixture is diluted with EtOAc, washed with saturated NaHCO3, washed with brine.", "Purification by 70% to 90% EtOAc/Hexane gradient yields compound clxiii (180 mg, 56%).", "INTERMEDIATE EXAMPLE 156 Compound clxiv Compound cxxv (2.09 g, 7.4 mmol) is taken up in DCM (20 mL).", "PyBOP (4.64 g, 8.9 mmol) and HOBt (1.2 g, 8.9 mmol) are added to this solution and stirred five minutes.", "The resulting mixture is lowered to 0° C where S(−)-α-Methylbenzylamine (1.15 mL, 8.9 mmol) and DIPEA (1.55 mL, 8.9 mmol) are added.", "The reaction is stirred overnight with warming to room temperature.", "The reaction mixture is washed with 0.1 N HCl, sat NaHCO3, and brine.", "Purification by 30% EtOAc/Hexanes yields compound clxiv (1.6 g, 56.3%).", "INTERMEDIATE EXAMPLE 157 Compound xxxvi′ Compound clxiv (1.48 g, 3.8 mmol) is hydrogenated using 10% Pd/C (300 mg) in MeOH (50 mL).", "The reaction mixture stirred overnight under H2.The reaction mixture is filtered through celite and concentrated to give compound xxxvi′ (895 mg, 94.2%).", "INTERMEDIATE EXAMPLE 158 Compound clxvi To a DCM solution (15 mL) of compound clxv (2 g, 8.2 mmol) is added HOAt (1.34 g, 9.84 mmol) and DCC (9.84 mL, 1 M, 9.84 mmol).", "After stirring at room temperature for 20 minutes, a THF solution (9.84 mL) containing tert-L-Leucine methyl ester-hydrochloride (9.84 mmol) and DIPEA (1.72 mL, 9.84 mmol) is added to the above solution.", "Then DMAP (1 g, 8.2 mmol) is added at room temperature.", "The reaction is stirred at room temperature overnight.", "Following standard aqueous work-up and silica gel chromatography (20% EtOAc/Hexanes), compound clxvi (1.75 g, 58%) is obtained.", "INTERMEDIATE EXAMPLE 159 Compound clxvii To a THF solution (35 mL) of compound clxvi (1.75 g, 4.73 mmol) is added 4 N HCl solution in dioxane (11.8 mL, 47.3 mmol).", "The reaction is stirred at room temperature overnight.", "At this point, the solvent is removed under reduced pressure to yield crude clxvii (˜100%), which is redissolved in DMF and used directly in the next reaction.", "INTERMEDIATE EXAMPLE 160 Compound clxviii To a DCM solution (15 mL) containing 2-pyrazinecarboxylic acid (447 mg, 3.6 mmol), PyBOP (1.87 g, 3.6 mmol) is added a DMF solution (15 mL) of compound clxvii (811 mg, 3 mmol).", "To the resulting mixture is then added DIPEA (0.63 mL, 3.6 mmol).", "The reaction is stirred overnight at room temperature and then quenched with water.", "The reaction mixture is extracted with EtOAc.", "The organic layer is washed with brine and concentrated in vacuo to provide a residue that is purified by silica gel chromatography (40% EtOAc/Hexanes) to yield compound clxviii (0.93 g, 82%).", "INTERMEDIATE EXAMPLE 161 Compound clxix To a MeOH solution (10 mL) of compound clxviii (0.93 g, 2.47 mmol) is added 2 N NaOH (3.71 mL, 7.41 mmol).", "The reaction is stirred at room temperature overnight.", "Then the reaction is acidified to pH 3 using 1 N HCl.", "The reaction is diluted with EtOAc (75 mL), and washed with water and brine.", "The organic layer thus obtained is dried and concentrated in vacuo to give compound clxix (˜100%).", "INTERMEDIATE EXAMPLE 162 Compound clxx A DCM solution (10 mL) of compound clxix (2.47 mmol) is treated with HOAt (436 mg, 3.21 mmol) and DCC (3.2 mL, 1 M, 3.2 mmol).", "After stirring for 30 minutes, the reaction mixture is treated with a THF solution (13.6 mL) of compound v (499 mg, 2.72 mmol).", "After stirring at room temperature overnight, white solids (urea) are filtered.", "The filtrates are concentrated in vacuo to give a residue that is purified by silica gel chromatography to yield compound clxx (0.99 g, 76%).", "INTERMEDIATE EXAMPLE 163 Compound clxxi An EtOH solution (20 mL) of compound clxx (0.99 g, 1.88 mmol) is treated with 2 N NaOH (2.81 mL, 5.63 mmol).", "After stirring at room temperature overnight, the reaction mixture is acidified to pH 3 with 1 N HCl.", "The reaction mixture is extracted with EtOAc (75 mL).", "The organic layer is dried and concentrated in vacuo to give compound clxxi (772 mg, 82%).", "INTERMEDIATE EXAMPLE 164 Compound clxxi A DCM solution (10 mL) of compound clxxi (290 mg, 0.58 mmol) is treated with PyBOP (484 mg, 0.93 mmol).", "After stirring at room temperature for 20 minutes, the reaction mixture is treated with a THF solution (7.5 mL) of compound xiii′ (140 mg, 0.75 mmol), followed by DIPEA (0.13 mL, 0.75 mmol).", "After stirring overnight at room temperature, the reaction is quenched with water and extracted with EtOAc.", "The resulting organic layer is washed with brine and dried and concentrated iin vacuo.", "The resulting residue is purified by silica gel chromatography (5% EtOH/EtOAc) to yield compound clxxii 290 mg (75%).", "INTERMEDIATE EXAMPLE 165 Compound clxxiv Compound lxxxiii (600 mg, 1.17 mmol) is taken up in DCM (4 mL).", "PyBOP (670 mg, 1.3 mmol) is added, stirred five minutes, and cooled to 0° C. Compound clxxiii (333 mg, 1.3 mmol) in THF (13 mL) is added drop-wise to this solution.", "DIPEA (0.23 mL, 1.3 mmol) is added to reaction mixture and allowed to warm to ambient temperature with stirring for two nights.", "The next day, the reaction is concentrated and purified by 2% EtOH/EtOAc to give crude compound clxxiv (900 mg, excess of 100%).", "INTERMEDIATE EXAMPLE 166 Compound clxxxv Compound cxxv (3.01 g, 10.7 mmol) is taken up in DCM (30 mL) and the temperature lowered to −78° C. PyBOP(6.1 g, 11.7 mmol) and HOBT (1.58 g, 11.7 mmol) are added to this solution followed by (S)-(+)-1-cyclohexylethylamine, compound clxxv, (1.74 mL, 11.7 mmol) and DIPEA (2.1 mL, 11.7 mmol).", "The resulting mixture stirred overnight at room temperature.", "The next day, the reaction mixture is diluted with EtOAc, washed with 0.1 N HCl, saturated NaHCO3, and brine.", "The product is purified in 40% EtOAc/Hex to give 2 g (47.8%) of compound clxxvi.", "INTERMEDIATE EXAMPLE 167 Compound clxii Compound clxxvi (2 g, 5.13 mmol) is hydrogenated using 10% Pd/C (500 mg) in MeOH (40 mL).", "The reaction mixture stirred overnight under H2.The reaction mixture is filtered through celite and concentrated to give compound clxxiii (1.31 g, 99.8%).", "INTERMEDIATE EXAMPLE 168 Compound clxxix: In a round bottom flask under inert atmosphere, compound clxxvii [(S)-(−)-2-oxo 1,5 imidazoline dicarboxylic acid 1-benzyl ester] (290 mg, 1.1 mmol) is dissolved in anhydrous DMF (6 mL).", "HOAt (151 mg, 1.2 mmol) is added and the reaction is stirred at room temperature for 25 minutes.", "The reaction is then cooled down in an ice bath.", "DIC (0.2 mL, 0.16 g, 1.2 mmol) is then added followed by the addition of compound clxxviii (1 mmol, 435 mg.) in anhydrous DMF (4 mL).", "The rtertion is allowed to rise slowly to room temperature and stirred for 2 days.", "The reaction is then dumped in a separatory funnel containing 120 mL of EtOAc and washed 2× with 1 N HCl (50 mL) and 1× brine.", "The organic layer is separated, dried over MgSO4.The solvent evaporated under reduced pressure and the residue purified by chromatography on silica gel (load in DCM and elute with 30% then 50% EtOAc/DCM then 2% MeOH(EtOAc) to yield product clxxix (434 mg, 64%).", "INTERMEDIATE EXAMPLE 169 Compound clxxx The starting material clxxix (434 mg, 0.64 mmol) is dissolved in Dioxane (6 mL) and 0.5 M aqueous NaOH solution (4 mL, 3 eq.).", "The reaction is run overnight.", "TLC in 100% EtOAc (using PMA stain) shows in addition to the expected acid product at the origin, a faster running product.", "The reaction mixture is acidified to pH 2 with 1 N HCl, and then extracted 2× with EtOAc.", "Solid NaCl is added to the aqueous solution to facilitate the extraction.", "The organic extracts are then combined, dried over MgSO4 and evaporated under reduced pressure.", "MS indicates that the CBZ group is removed by the hydrolysis.", "The resulting compund clxxx (quantitative yield) is used as is in the next step.", "INTERMEDIATE EXAMPLE 170 Compound clxxxi In a round bottom flask under inert atmosphere, compound clxxx (279 mg, 0.54 mmol) is dissolved in anhydrous DMF (6 mL).", "HOAt (82 mg, 0.65 mmol) is added and the reaction is stirred at room temperature for 25 minutes.", "The reaction is then cooled down in an ice bath.", "DIC (0.11 mL, 0.65 mmol) is then added, followed by the addition of compound xiii′ (0.7 mmol) in anhydrous DMF (4 mL).", "The reaction is allowed to rise slowly to room temperature and stirred for 21 hours.", "The reaction is then dumped in a separatory funnel containing 120 mL of EtOAc and washed 2× with 1 N HCl (50 mL) and 1× brine.", "The organic layer is separated, dried over MgSO4.The solvent evaporated by reduced pressure and the product cleaned by chromatography on silica gel (load in DCM and elute with 50% EtOAc/Hexane, then 3% MeOH/EtOAc, then 20% EtOH/EtOAc).", "After removal of solvent, the residue is redissolved in Dri Solv THF and filtered to remove any silica gel.", "Removal of the solvent then yields compound clxxxi (434 mg, 64% yield).", "INTERMEDIATE EXAMPLE 171 Compound clxxxiii In a round bottom flask under inert atmosphere, 6-hydroxy picolinic (153 mg, 1.1 mmol) is dissolved in anhydrous DMF (6 mL).", "HOAt (151 mg, 1.2 mmol) is added and then thereaction is stirred at room temperature for 25 minutes.", "The reaction is then cooled down in an ice bath.", "DIC (0.2 mL, 0.16 g, 1.2 mmol) is then added followed by the addition of the compound clxxxii (1.0 mmol, 435 mg.) in anhydrous DMF (4 mL).", "The reaction is allowed to rise slowly to room temperature and stirred for 2 days.", "The reaction is then dumped in a separatory funnel containing 120 mL of EtOAc and washed 2× with 1 N. HCl (50 mL) and 1× with brine.", "The organic layer is separated, dried over MgSO4.The solvent is evaporated by reduced pressure and the product purified by chromatography on silica gel (load in DCM, elute with 30%, then 50% EtOAc/DCM, and then 2% MeOH/EtOAc) to yield compound clxxxiii collected (314 mg, 56%).", "INTERMEDIATE EXAMPLE 172 Compound clxxxiv The starting material clxxxiii (314 mg, 0.56 mmol) is dissolved in dioxane (5 mL) and 0.5 M NaOH (3.4 mL, 3 eq).", "The reaction is run overnight TLC in 100% EtOAc (using UV) shows complete conversion to the slow running acid product at the origin.", "The reaction is acidified to pH 2 with 1 N HCl, and then extracted 2× with EtOAc.", "Solid NaCl is added to the aqueous to facilitate the extraction.", "The organic extracts are then combined, dried over MgSO4, and then evaporated under reduced pressure to yield compound clxxxiv (0.5 mmol, 89%) that is used as is in the next step.", "INTERMEDIATE EXAMPLE 173 Compound clxxxv In a round bottom flask under inert atmosphere, acid compound clxxxiv (265 mg, 0.5 mmol) is dissolved in anhydrous DMF (6 mL).", "HOAT (75.6 mg, 0.6 mmol) is added and the reaction is stirred at room temperature for 25 minutes.", "The reaction is then cooled down in an ice bath.", "DIC (0.1 mL, 0.6 mmol) is then added followed by the addition of the compound xiii′ (0.65 mmol) in anhydrous DMF (4 mL).", "The reaction is allowed to rise slowly to room temperature and stirred for 21 hours.", "The reaction is then dumped in a separatory funnel containing EtOAc (120 mL) and washed 2× with 1 N HCl (50 mL) and 1× with brine.", "The organic layer is separated, dried over MgSO4.The solvent is evaporated by reduced pressure and the product purified by chromatography on silica gel (load in DCM, elute with 50% EtOAc/Hexane, then pure EtOAc, and then 4% MeOH/EtOAc) to yield product compound clxxxv (185 mg, 52%).", "INTERMEDIATE EXAMPLE 174 Compound cxxxxiv′ To a solution of D-alanine (5 g, 56.1 mmol) in 1 N NaOH (152 mL, 152 mmol) at 0° C. is added a solution of MeOC(O)Cl (6.5 mL, 84.2 mmol) in diethyl ether (30 mL).", "The mixture is stirred in ice bath for 3 hours and then adjusted to pH 9 with 1 N NaOH.", "After stirring at room temperature for 1 hour, the mixture is washed with ether (3×50 mL), acidified to pH ˜2 with 5 N HCl, extracted with EtOAc (5×50 mL).", "The organic extract is washed with water, brine, and then dried (MgSO4).", "The solvent is removed to yield compound cxxxiv, N-methoxycarbonyl-D-alanine, as colorless oil (6.48 g, 79%).", "INTERMEDIATE EXAMPLE 175 Compound clxxxvi To a solution of N-methoxycarbonyl-D-alanine (0.193 g, 1.31 mmol) and HOAt (0.177 g, 1.31 mmol) in DCM (10 mL) cooled in ice bath is treated with DCC (1.31 mL, 1.31 mmol).", "After stirring in an ice bath for 0.5 hour, a solution of prepared compound clxxxii (0.88 mmol) in THF (8.8 mL) is added.", "The mixture is warmed up to room temperature and stirred overnight, then cooled in ice bath, and quenched with saturated NaHCO3 solution.", "The precipitates are filtered and the filtrate is taken up in EtOAc (100 mL).", "The organic layer is washed with saturated NaHCO3 solution, brine, and then dried (MgSO4).", "After removal of the solvent, the residue is purified by silica column chromatography (60% EtOAc/Hexane) to yield compound clxxxvi as gummy foam (0.321 g, 68%).", "INTERMEDIATE EXAMPLE 176 Compound clxxxvii To a solution of compound clxxxvi (0.321 g, 0.597 mmol) in EtOH (5 mL) at 5° C. is added 2 N NaOH (1.05 mL, 2.1 mmol).", "The mixture is stirred at room temperature for 4 hours.", "The solution is acidified to pH ˜2 with 1 N HCl and EtOH is removed by rotary evaporation.", "The mixture is extracted with EtOAc (3×30 mL) and the combined extract is washed with brine, and then dried (MgSO4).", "Solvent is removed and the residue is dried under vacuum to give compound clxxxvii as gummy foam (0.235 g, 77%).", "INTERMEDIATE EXAMPLE 177 Compound clxxxviii A solution of compound clxxxvii (0.363 g, 0.712 mmol) in DCM (10 mL) is cooled in an ice bath and treated with PyBOP (0.594 g, 1.14 mmol).", "After stirring at room temperature for 0.5 hour, the mixture is cooled in ice bath and treated with a solution of compound xiii′ (1.1 mmol) in THF (11 mL) and DIPEA (0.249 mL, 1.42 mmol).", "The mixture is stirred at room temperature overnight and quenched with NH4Cl solution.", "The solvent is concentrated and the mixture is taken up in EtOAc (100 mL).", "The organic layer is washed with saturated NaHCO3 solution, brine, and then dried (MgSO4).", "After removal of the solvent, the residue is purified by column chromatography (5% EtOH/EtOAc) to give clxxxviii (0.341 g, 71%).", "INTERMEDIATE EXAMPLE 178 Compound clxxxix Diaminopropionic acid (3 g, 28.7 mmol) is taken up in 1 M NaOH (86.2 mL, 86.2 mmol) and cooled to 0° C., and then MeOC(O)Cl (5.54 mL, 71.75 mmol) is added in Et2O (25 mL).", "The resulting mixture stirred overnight warming to room temperature.", "The reaction mixture pH is lowered to 2 and aqueous layer is extracted 3× with EtOAc.", "Extracts are combined and dried over Na2SO4, filtered and concentrated to yield compound clxxxix (3.09 g, 48.9%).", "INTERMEDIATE EXAMPLE 179 Compound cc Compound clxxxix (340 mg, 1.55 mmol) is taken up in DCM (4 mL).", "DCC (1.7 mmol) and HOAt (235 mg, 1.7 mmol) are added followed by compound clxxi (1.7 mmol) in DCM (3.4 mL).", "The reaction mixture stirred overnight.", "The next day, the reaction mixture is filtered through a pad of silica and concentrated.", "Purification is achieved in 75% EtOAc/Hex to give compound clxxxx (715 mg, 72.4%).", "INTERMEDIATE EXAMPLE 180 Compound clxxxxi Compound clxxxx (715 mg, 1.12 mmol) is hydrolyzed under standard conditions using EtOH (4 mL) and 1 N NaOH (3 eq) to yield compound clxxxxi (600 mg, 88.0%).", "INTERMEDIATE EXAMPLE 181 Compound clxxxxii Compound clxxxxi (550 mg, 0.9 mmol) is taken up in DCM (8 mL).", "PyBOP (675 mg, 1.3 mmol) is added followed by compound xiii′ (1.3 mmol) in THF (1.3 mL).", "DIPEA (0.23 mL, 1.3 mmol) is added and the resulting solution stirred overnight.", "The next day, the reaction is diluted with EtOAc, washed with saturated NaHCO3, and then brine, before being concentrated to yield a residue.", "The resulting residue is purified by 5% EtOH/EtOAc to yield compound clxxxxii (290 mg, 41.5%).", "INTERMEDIATE EXAMPLE 182 Compound clxxxxii Cbz-cyclohexyglycine-tert-leucine methyl ester (7.36 g, 17.6 mmol) is hydrolyzed under standard conditions using MeOH (60 mL) and 1 N NaOH (52.8 mL, 3 eq) to yield intermediate clxxxxii (92%).", "INTERMEDIATE EXAMPLE 183 Compound clxxxxiv Compound clxxxxiii (3.82 g, 9.46 mmol) is taken up in DCM (30 mL).", "DCC (11.35 mmol) in DCM (11.35 mL) is added, followed by the addition of HOAt (1.54 g, 11.35 mmol).", "The resulting mixture stirred five minutes and compound v (9.46 mmol) in THE (40 mL) is added.", "The resulting mixture is stirred overnight.", "The next day, the reaction mixture is diluted with EtOAc, washed with 1 N HCl, saturated NaHCO3, and then brine, before being concentrated to yield a residue.", "The reulting reside is purified by 20% to 30% gradient on silica gel to give compound clxxxxiv (3.03 g, 56.3%).", "INTERMEDIATE EXAMPLE 183 Compound clxxxii Compound clxxxxiv (3.03 g, 5.33 mmol) is hydrogenated using 10% Pd/C (500 mg) in MeOH (30 mL) under H2 for 4 hours to yield compound clxxxii (2.3 g, 99%).", "INTERMEDIATE EXAMPLE 184 Compound clxxxxv To a solution of 1-amino-1-cyclohexanecarboxylic acid (2.86 g, 20 mmol) in MeOH (40 mL) is added dropwise SOCl2 (3 mL) at 0° C. The mixture is slowly warmed up to room temperature and then refluxed for 5 hours.", "Et2O is then added to the clear solution and the precipitate is isolated.", "The solid is further dried over vacuum to yield compound clxxxxv (95%) as white powder.", "INTERMEDIATE EXAMPLE 185 Compound clxxxxvi 2-Pyrazinecarboxylic acid (1 g, 8 mmol, 1 eq) is dissolved in DCM (15 mL) with addition of HOAt (1.1 g, 8 mmol) and DCC (8 mL, 1 M) in DCM.", "After stirring at room temperature for 20 minutes, compound clxxxxv (1.3 g, 8 mmol) is added to the activated mixture.", "DIPEA (2 mL, 12 mmol) is added subsequently, followed by DMAP (1.5 g, 12 mmol).", "After stiring over 3 days at room temperature, the reaction mixture is filtered through celite, concentrated and the desired product clxxxxvi is purified by column chromatography (50% EtOAc/hexane) as yellow oil (2.1 g, 100%).", "INTERMEDIATE EXAMPLE 186 Compound clxxxxvii Compound clxxxxvi (1.06 g, 2.6 mmol) is dissolved in MeOH (30 mL) with addition of 2 N NaOH (aq) (12 mDL, 24 mmol).", "The solution is stirred at room temperature overnight before TLC (50% EtOAc/hexane) indicates complete hydrolysis.", "The solution is then acidified to pH 3 by 5 N HCl and diluted with EtOAc and followed by extraction of the organic layer.", "The organic layer is subsequebtly washed with brine and dryed over MgSO4 to yield compound clxxxvii (84%) upon concentration.", "INTERMEDIATE EXAMPLE 187 Compound clxxxxviii Compound clxxxvii (1.6 g, 6.4 mmol) is dissolved in DCM (18 mDL) and then HOAt (0.96 g, 7 mmol) and DCC (7 mL, 1 M in DCM) are subsequently at room temperature.", "After stirring at room temperature for 20 minutes, L-tert-leucine methyl ester hydrochloride (7 mL, 1 M in THF) is added to the activated mixture.", "DIPEA (1.2 mL, 7 mmol) is added subsequently, followed by DMAP (1.2 g, 9.8 mmol).", "After stirring over 3 days at room temperature, the reaction mixture is filtered through celite, purified by column chromatography and concentrated to yield compound clxxxxviii (60% EtOAc/hexane) as white solid (1.74 g, 72%).", "INTERMEDIATE EXAMPLE 188 Compound cic Compound clxxxxviii (1.74 g, 4.6 mmol) is dissolved in MeOH (22 mL) with addition of 2 N NaOH (aq) (7 mL, 14 mmol).", "The solution is stirred at room temperature overnight before TLC (50% EtOAc/hexane) indicated complete hydrolysis.", "The solution is acidified to pH 3 by 5 N HCl and diluted with EtOAc and then the organic layer is extracted.", "The organic layer is washed with brine and dried over MgSO4 and then concentrated to yield compound cic (100%).", "INTERMEDIATE EXAMPLE 189 Compound cc To a DCM solution (15 mL) of compound cic (1.5 g, 4.1 mmol) at room temperature is added HOAt (610 mg, 4.5 mmol), followed by 1 M DCC solution in DCM (4.5 mL, 4.5 mmol).", "After stirring for 30 minutes at room temperature, then a THF solution (20 mL, 0.2 M) of compound v (4 mmol) is added.", "The reaction is stirred at room temperature overnight.", "Then, the reaction is filtered through celite.", "The filtrate is concentrated to a yellow oil which is purified by silica gel chromatography (50% EtOAc/hexane) to yield compound cci (660 mg, 32%).", "INTERMEDIATE EXAMPLE 190 Compound cci To an EtOH solution (6 mL) of compound cc (600 mg, 1.13 mmol) is added 2 N NaOH (1.7 mL, 3.4 mmol).", "The reaction is stirred for 2 hours at room temperature, then acidified to pH 3 by 5 N HCl.", "The mixture is then diluted with EtOAc, followed by extraction of the organic layer.", "Subsequently, the organic layer is washed with brine and then dried over MgSO4 to yield compound cci (92%) upon concentration.", "INTERMEDIATE EXAMPLE 191 Compound ccii To a DCM solution (8 mL) of ccii (310 mg, 0.62 mmol) is added PyBOP (420 mg, 0.8 mmol).", "The solution is stirred at room temperature for 30 minutes.", "To this solution is then added compound xiii′ (8 mL, 0.1 M) in THF, followed by the addition of DIPEA (0.23 mL, 1.3 mmol).", "The reaction is stirred at room temperature overnight and then quenched with water (25 mL) for 30 minutes.", "The mixture is then extracted with EtOAc.", "The resulting organic layer is washed with brine and then dried over MgSO4, before being concentrated to yield a yellow oil.", "Purification by silica gel chromatography (3% EtOH/EtOAc) yields compound ccii (140 mg, 33%).", "INTERMEDIATE EXAMPLE 192 Compound ccxiv To a solution of compound cciii, tert-butyl (N-diphenylmethylene)-glycine ester, (6 g, 0.0206 mmol) and chiral PTC (1.08 g, 0.00206 mmol) in dry D-wi (48 mL), under N2 atmosphere, at −60° C., is added CsOH.H2O (6.9 g, 0.0412 mmol).", "To the reaction mixture is added dropwise 1-carboxy-1-cyclopentene methyl ester (5.2 mL, 0.0412 mmol) in 10 mL of DCM.", "The mixture is stirred for 4 days at −60° C., then diluted with 200 mL of Et2O and 15 mL of saturated NH4Cl aqueous solution is added.", "Phases are separated and the organic phase is washed with 15 mL water and 15 mL brine.", "The aqueous phases are extracted with 100 mL of Et2O.", "The organics phases are joined and dried over Na2SO4.Crude product is obtained by removal of the solvent dissolved in 100 mL of EtOH and then NH2OH.HCl (1.43 g, 0.0206 mmol) and NaOAc (1.68 g, 0.0206 mmol) are added.", "The mixture is refluxed for 48 hours.", "Then the solvent is removed and the crude residue obtained is directly purified by flash chromatography eluting with 30%-50% EtOAc/hexane to yield compound cciv (65%) as a white solid.", "C12H19NO3 (MW=225.29); MS: m/z (M++1)=226.5.Enantiomeric excess: 18% ee, determined by Chiral HPLC.", "INTERMEDIATE EXAMPLE 193 Compound ccv To a solution of compound cciv (2 g, 0.0088 mmol) in 60 mL of ACN is added a catalytic amount of DMAP (0.216 g, 0.0017 mmol) and a solution of di-tert-butyl-d-carbonate (2.49 g, 0.011 mmol) in 30 mL of ACN.", "The mixture is stirred for 14 hours at room temperature, then diluted with 100 mL of DCM, and washed with saturated NaHCO3 (10 mL) and with brine (10 mL).", "The organic phase is dried over Na2SO4.Evaporation of the solvent yields a crude product that is purified on a silica gel column eluting with 15% EtOAc/hexane to give compound ccv (86%) as white solid.", "C17H27NO5 MW=325,40 MS: m/z (M++1)=326.2 INTERMEDIATE EXAMPLE 194 Compound ccvi To a solution of compound ccv (1.7 g, 0.0052 mmol) in 50 mTL of THF (0.14 M) at −78° C., is added DIBAL-H (7.8 mL, 0.0078 mmol).", "The mixture is stirred for 1 hour, then 10 mL of MeOH are added.", "The mixture is diluted with 25 mL of EtOAc and 25 mL of saturated aqueous solution of sodium tartrate, and then stirred at room temperature for an hour.", "The phases are separated and the aqueous phase is extracted once with 50 mL of EtOAc.", "The organic phases are combined and dried over Na2SO4.Evaporation of solvent gave a crude residue that is used without any purification.", "The crude is dissolved in 25 mL of DCM, Et3Si (0.84 mL, 0.0052 mmol) is added, and then the mixture is cooled to −78° C. before the dropwise addition of BF3OEt2 (0.71 mL, 0.0061 mmol).", "After 30 minutes Et3Si (0.84 mL) and BF3OEt2 (0.71 mL) are added and the mixture stirred for 2 hours to −78° C. The reaction is then quenched with saturated aqueous NaHCO3 (10 mL) and extracted with DCM (2×20 mL).", "The organic phases are combined and dried over Na2SO4.Evaporation of solvent gives a crude residue that is purified by flash chromatography eluting with 13% EtOAc/hexane to yield compound ccvi (87%).", "C17H29NO4 MW=311,42 MS: m/z (M++1)=312.6 INTERMEDIATE EXAMPLE 195 Compound ccvii Compound ccvi (0.5 g, 0.0016 mmol) is dissolved in 8 mL of 1 N HCl in EtOAc (prepared by bubbling dry HCl into dry EtOAc then diluting to 1 N with additional EtOAc).", "The mixture is stirred for 6 hours at room temperature.", "Solvent is removed in vacuo and the resulting precipitate is dissolved in Et2O.", "After stirring the mixture for 15 minutes, the solvent is removed under reduced pressure.", "The resulting white solid is washed with Et2O and the compound ccvii (0.27 g, 80% yield) is isolated by filtration.", "C12H21NO2 MW 211,15 MS: m/z (M++1)=212.6 INTERMEDIATE EXAMPLE 196 Compound v To a solution of compound ccxvi (0.230 g, 0.74 mmol) in DCM(3.7 mL) is added TFA (2.85 mL).", "The mixture is stirred overnight, then solvent is removed iit vacuo to dryness and the residue is dissolved in EtOH (7.5 mL).", "The mixture is cooled at 0° C. and SOCl2 (0.22 mL, 2.96 mmol) is added dropwise and then refluxed for 2 hours.", "EtOH is removed at reduced pressure and the residue dissolved in DCM (10 mL).", "The resulting solution is washed twice with a saturated aqueous solution of NaHCO3 (5 mL).", "Phases are separated and the organic phase is dried over Na2SO4 and solvent removed in vacuo to yield compound v (80%) as oil.", "C10H17NO2 M.W.", ":183.25 MS: m/z (M++1)=184.2 INTERMEDIATE EXAMPLE 197 Compound cd 1-Benzylimidazole (6 g, 37.9 mmol) is taken up in Et2O (180 mL).", "The resulting solution is lowered to −60° C. and treated with n-BuLi (1.6 M, 24 mL).", "The reaction is stirred for 30 minutes and then CO2 is bubbled through mixture for 15 minutes.", "The precipitate is filtered, rinsed with Et2O and then taken up in H2O.", "This aqueous solution is acidified to pH 3 with 5 N HCl.", "The desired product, cd, is isolated after lyophilization as a white solid.", "INTERMEDIATE EXAMPLE 198 Compound cdi A DCM solution (100 mL) of compound i (9.25 g, 27.9 mmol) is treated at 0° C. with DAST (9.2 mL, 69.8 mmol).", "After stirring at room temperature overnight, the reaction is quenched with ice and extracted with DCM (200 mL).", "The organic layer is washed with brine and concentrated.", "in vacuo.", "The residue is purified with silica gel chromatography (30% EtOAc/hexanes) to yield 8.5 g (86%) of the desired fluorinated intermediate.", "A portion of this intermediate (4.5 g, 14.2 mmol) is dissolved in EtOH (75 mL).", "This solution is subjected to standard hydrogenation conditions using Pd(OH)2/C (2.98 g, 20% Pd content, 4.26 mmol).", "After stiring overnight at room temperature, the reaction mixture is filtered through Celite.", "The filtrates are concentrated in vacuo to yield compound cdi (2.5 g, 96%).", "INTERMEDIATE EXAMPLE 199 Compound cdii To a solution of compound cd (890 mg, 4.4 mmol) taken up in DCM (15 mL).", "HOBT (595 mg, 4.4 mmol) and DCC (4.4 mmol, 1 M in DCM) are added and stirred for 20 minutes.", "A DCM solution (15 mL) of lxxix′ (990 mg, 3.5 mmol) is added to this mixture.", "The resulting mixture is stirred overnight under nitrogen.", "The reaction mixture is diluted with EtOAc, washed with saturated NaHCO3 and brine.", "The organic layer is concentrated in vacuo to give a residue, which is purified in 30% EtOAc/Hexanes to yield compound cdii (666 mg, 41%).", "INTERMEDIATE EXAMPLE 200 Compound cdiii Compound cdiii is prepared from compound cdii under standard hydrolysis conditions using methyl alcohol (10 mL) and 1 N NaOH (3 eq).", "565 mg of compound cdiii are recovered (88%).", "INTERMEDIATE EXAMPLE 201 Compound cdiv Compound cdiii (1.24 mmol) is taken up in DCM (5 mL).", "DCC (1.6 mmol, 1 M DCM) is added followed by HOAT (1.6 mmol).", "The resulting mixture is stirred 20 minutes and compound cdi (1.6 mmol) is added dropwise in THF (8 mL).", "The reaction is stirred overnight.", "The reaction is filtered and rinsed with EtOAc.", "The combined organic layer is washed with saturated NaHCO3, brine, dried over MgSO4, and concentrated.", "Purification is achieved in 30% EtOAc/Hexanes to yield compound cdiv (565 mg, 70%).", "cdiv INTERMEDIATE EXAMPLE 202 cdv Compound cdv (565 mg, 0.86 mmol) is prepared from compound cdiv under standard hydrolysis conditions using ethyl alcohol (10 mL) and 1 N NaOH (3 eq).", "490 mg (91%) of compound cdv is recovered.", "INTERMEDIATE EXAMPLE 203 cdvi Compound cdv (490 mg, 0.78 mmol) is taken up in DCM (10 mL).", "PyBOP (520 mg, 1 mmol) is added to DCM solution followed by a THF solution (10 mL) of xiii (186 mg, 1 mmol).", "DIEA (0.18 mL, 1 mmol) is added to the reaction mixture and stirred overnight under nitrogen.", "The next day, the reaction is diluted with EtOAc, washed with saturated NaHCO3 and brine.", "Purification is achieved in 100% EtOAc to yield compound cdvi (478 mg, 77%).", "INTERMEDIATE EXAMPLE 204 cdxi Compound cdvi (478 mg, 0.6 mmol) is hydrogenated using Pd(OH)2/C (20% dry basis, 100 mg) in MeOH (40 mL).", "The reaction mixture is stirred overnight under hydrogen.", "At this point, the reaction mixture is filtered through Celite and concentrated to yield compound cdvii (417 mg, 98%).", "INTERMEDIATE EXAMPLE 205 cdx Compound cxxv (Purchased from Albany Molecular Research Inc., 1.5 g, 5.2 mmol) is taken up in DCM (15 mL).", "PyBOP (2.7 g, 5.2 mmol) and HOBT (700 mg, 5.2 mmol) are added to this solution.", "A THF solution (15 mL) of (−)-alpha-(4-pyridyl)ethyl amine (640 mg, 5.2 mmol) is added to above solution, followed by DIEA (0.93 ml, 5.2 mmol).", "[The (−)-alpha-(4-pyridyl)ethyl amine is obtained from the tartrate salt of (−)-alpha-(4-pyridyl)ethyl amine (Aldrich) by stirring with 1 N NaOH (2 eq) for 1 hour followed by extraction with EtOAc (3×) 70% recovery].", "The reaction is stirred overnight at room temperature.", "The reaction mixture is washed with saturated NaHCO3, and brine.", "The product is purified in 5% EtOH/EtOAc to yield 2 g (99%) of intermediate compound cdx.", "INTERMEDIATE EXAMPLE 206 cdviii Compound cdx (2 g, 5.2 mmol) is hydrogenated using 10% Pd/C (500 mg) in MEOH (50 mL).", "The reaction mixture is stirred overnight under hydrogen.", "The product is filtered through celite and concentrated to give compound cdviii (1.3 ,g 98%).", "Pharmacology Compounds according to the invention as described herein as being useful for being able to inhibit HCV protease, and thus, are also useful for inhibiting HCV replication.", "Accordingly, an invention herein is directed to a method of inhibiting HCV protease comprising contacting an anti-HCV protease inhibitory amount of a compound of formula 1 with a composition comprising HCV protease.", "Yet another invention herein is directed to a method of inhibiting replication of HCV comprising contacting HCVwith an effective amount of a compound of formula 1.Furthermore, another invention herein is directed to a method of treating a patient suffering from or subject to an HCV infection comprising administering to the patient a pharmaceutically effective amount of compound of formula 1.References herein to treating an HCV infection should be understood to include prophylactic therapy to prevent or inhibit the infection as well as the treatment of an established acute or chronic HCV infection or physiological conditions associated with HCV infection to essentially cure the patient of the infection, inhibit the degree (amount) of infection or ameliorate the physiological conditions associated therewith.", "“Effective amount” is meant to describe an amount of the compound of the present invention effective within the scope of reasonable biological judgement, suitable for use in contact with the cells of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio in treating an HCV infection and thus producing the desired therapeutic effect.", "Physiological conditions discussed herein include some, but not all, of the possible clinical situations where an anti-HCV treatment is warranted.", "Those experienced in this field are well aware of the circumstances requiring either an anti-HCV treatment.", "A particular aspect of the invention provides for a compound according to the invention to be administered in the form of a pharmaceutical composition, though the compound may be administered alone.", "“Pharmaceutical composition” means a composition comprising a compound of formula 1 and at least one component selected from the group comprising pharmaceutically acceptable carriers, diluents, coatings, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, emulsion stabilizing agents, suspending agents, isotonic agents, sweetening agents, flavoring agents, perfuming agents, coloring agents, antibacterial agents, antifungal agents, other therapeutic agents, lubricating agents, adsorption delaying or promoting agents, and dispensing agents, depending on the nature of the mode of administration and dosage forms.", "The compositions may be presented in the form of tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs or syrups.", "Examplary suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances.", "Examplary antibacterial and antifungal agents for the prevention of the action of microorganisms include parabens, chlorobutanol, phenol, sorbic acid, and the like.", "Examplary isotonic agents include sugars, sodium chloride and the like.", "Examplary adsorption delaying agents to prolong absorption include aluminum monosterate and gelatin.", "Examplary adsorption promoting agents to enhance absorption include dimethyl sulphoxide and related analogs.", "Examplary carriers, diluents, solvents, vehicles, solubilizing agents, emulsifiers and emulsion stabilizers, include water, chloroform, sucrose, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, tetrahydrofurfuryl alcohol, benzyl benzoate, polyols, propylene glycol, 1,3-butylene glycol, glycerol, polyethylene glycols, dimethylformamide, Tween® 60, Span® 80, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate, fatty acid esters of sorbitan, vegetable oils (such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil) and injectable organic esters such as ethyl oleate, and the like, or suitable mixtures of these substances.", "Examplary excipients include lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate phosphate.", "Examplary disintegrating agents include starch, alginic acids and certain complex silicates.", "Examplary lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.", "Other therapeutic agents may be used in combination with a compound of the present invention, including other anti-HCV agents.", "Some Examplary known anti-HCV agents include immunomodulatory agents, such as α-, β- or γ-interferons; pegylated derivatized interferon-α compounds, other antiviral agents such as ribavirin and amantadine; other inhibitors of hepatitis C protease; inhibitors of other targets in the HCV life cycle including the helicase, polymerase, metalloprotease, internal ribosome entry, or broad-spectrum antiviral compounds such as VX-497, an inhibitor of cellular inosine monophosphate dehydrogenase, IMPDH, covered by U.S. Pat.", "No.", "5,807,876; or combinations thereof.", "Therapeutic agents used in combination with a compound of the present invention may be administered separately, simultaneously or sequentially.", "The choice of material in the pharmaceutical composition other than the compound of formula 1 is generally determined in accordance with the chemical properties of the active compound such as solubility, the particular mode of administration and the provisions to be observed in pharmaceutical practice.", "For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets.", "The pharmaceutical compositions may be presented in assorted forms such as tablets, pills, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs or syrups.", "“Liquid dosage form” means the dose of the active compound to be administered to the patient is in liquid form, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.", "In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such solvents, solubilizing agents and emulsifiers.", "Solid compositions may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.", "When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension.", "The oily phase of the emulsion pharmaceutical composition may be constituted from known ingredients in a known manner.", "While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.", "Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer.", "It is also preferred to include both an oil and a fat.", "Together, the emulsifier(s) with or without stabilizer(s) make up the emulsifying wax, and the way together with the oil and fat make up the emulsifying ointment base which forms the oily dispersed phase of the cream formulations.", "If desired, the aqueous phase of the cream base may include, for example, a least 30% w/w of a polyhydric alcohol, i.e.", "an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof.", "The topical formulations may desirably include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas.", "The choice of suitable oils or fats for a formulation is based on achieving the desired cosmetic properties.", "Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.", "Straight or branched chain, mono- or dibasic alkyl esters such as di-isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used.", "These may be used alone or in combination depending on the properties required.", "Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.", "In practice, a compound/pharmaceutical compositions of the present invention may be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, inhalational, rectal, nasal, buccal, sublingual, vaginal, colonic, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal.", "It will be appreciated that the preferred route may vary with for example the condition of the recipient.", "“Pharmaceutically acceptable dosage forms” refers to dosage forms of the compound of the invention, and includes, for example, tablets, dragees, powders, elixr, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.", "Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition.", "“Formulations suitable for oral administration” may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.", "The active ingredient may also be presented as a bolus, electuary or paste.", "A tablet may be made by compression or moulding, optionally with one or more accessory ingredients.", "Compressed tables may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.", "Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compounds moistened with an inert liquid diluent.", "The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.", "Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of the invention.", "If desired, and for more effective distribution, the compounds can be microencapsulated in, or attached to, a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices (e.g., poly(d,l-lactide co-glycolide)), liposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compound(s) for a period of 2 weeks or longer.", "The compounds may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.", "“Formulations suitable for nasal or inhalational administration” means formulations which are in a form suitable to be administered nasally or by inhalation to a patient.", "The formulation may contain a carrier, in a powder form, having a particle size for example in the range 1 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc.)", "Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents.", "Inhalational therapy is readily administered by metered dose inhalers.", "37 Formulations suitable for oral administration” means formulations which are in a form suitable to be administered orally to a patient.", "The formulations may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.", "The active ingredient may also be presented as a bolus, electuary or paste.", "“Formulations suitable for parenteral administration” means formulations that are in a form suitable to be administered parenterally to a patient.", "The formulations are sterile and include emulsions, suspensions, aqueous and non-aqueous injection solutions, which may contain suspending agents and thickening agents and anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic, and have a suitably adjusted pH, with the blood of the intended recipient.", "“Formulations suitable for rectal or vaginal administrations” means formulations that are in a form suitable to be administered rectally or vaginally to a patient.", "The formulation is preferably in the form of suppositories that can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.", "“Formulations suitable for systemic administration” means formulations that are in a form suitable to be administered systemically to a patient.", "The formulation is preferably administered by injection, including transmuscular, intravenous, intraperitoneal, and subcutaneous.", "For injection, the compounds of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.", "In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use.", "Lyophilized forms are also included.", "Systematic administration also can be by transmucosal or transdermal means, or the compounds can be administered orally.", "For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.", "Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration.", "In addition, detergents may be used to facilitate permeation.", "Transmucosal administration may be through use of nasal sprays, for example, or suppositories.", "For oral administration, the compounds are formulated into conventional oral administration forms such as capsules, tablets, and tonics.", "“Formulations suitable for topical administration” means formulations that are in a form suitable to be administered topically to a patient.", "The formulation may be presented as a topical ointment, salves, powders, sprays and inhalants, gels (water or alcohol based), creams, as is generally known in the art, or incorporated into a matrix base for application in a patch, which would allow a controlled release of compound through the transdermal barrier.", "When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.", "Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.", "Formulations suitable for topical administration in the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.", "Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.", "“Solid dosage form” means the dosage form of the compound of the invention is solid form, for example capsules, tablets, pills, powders, dragees or granules.", "In such solid dosage forms, the compound of the invention is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders as for example starches, lactose, sucrose, glucose, mannitol and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, () opacifying agents, (k) buffering agents, and agents which release the compound(s) of the invention in a certain part of the intestinal tract in a delayed manner.", "Actual dosage levels of active ingredient(s) in the compositions of the invention may be varied so as to obtain an amount of active ingredient(s) that is (are) effective to obtain a desired therapeutic response for a particular composition and method of administration for a patient.", "A selected dosage level for any particular patient therefore depends upon a variety of factors including the desired therapeutic effect, on the route of administration, on the desired duration of treatment, the etiology and severity of the disease, the patient's condition, weight, sex, diet and age, the type and potency of each active ingredient, rates of absorbtion, metabolism and/or excretion and other factors.", "Total daily dose of the compounds of this invention administered to a patient in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 mg/kg body weight daily and preferably 0.01 to 10 mg/kg/day.", "For example, in an adult, the doses are generally from about 0.01 to about 100, preferably about 0.01 to about 10, mg/kg body weight per day by inhalation, from about 0.01 to about 100, preferably 0.1 to 70, more especially 0.5 to 10, mg/kg body weight per day by oral administration, and from about 0.01 to about 50, preferably 0.01 to 10, mg/kg body weight per day by intravenous administration.", "The percentage of active ingredient in a composition may be varied, though it should constitute a proportion such that a suitable dosage shall be obtained.", "Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose.", "Obviously, several unit dosage forms may be administered at about the same time.", "A dosage may be administered as frequently as necessary in order to obtain the desired therapeutic effect.", "Some patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate.", "For other patients, it may be necessary to have long-term treatments at the rate of 1 to 4 doses per day, in accordance with the physiological requirements of each particular patient.", "It goes without saying that, for other patients, it will be necessary to prescribe not more than one or two doses per day.", "The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy.", "Such methods include the step of bringing into association the active ingredient with the carrier that constitutes one or more accessory ingredients.", "In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.", "The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials with elastomeric stoppers, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.", "Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.", "Compounds within the scope of the present invention exhibit marked pharmacological activities according to tests described in the literature and below, which tests results are believed to correlate to pharmacological activity in humans and other mammals.", "In Vitro Enzyme Assay Procedure Inhibition of HCV NS3 Serine Protease HCV NS3 protease domain was expressed and purified as described previously (Vertex, PCT publication WO98/17679; which is incorporated herein by reference).", "The chromogenic peptide substrate, EDVV AbuC-p-nitroanilide, and the NS4A cofactor fragment (-KKGSVVIVGRIVLSGK-) for NS3 protease was custom synthesized by American Peptide Corn (Ca).", "The compounds of this invention were tested for their ability to inhibit HCV NS3 protease activity using a spectrophotometric assay with EDVV AbuC-p-nitroanilide as substrate.", "The assay was run in a 96-well microtiter plate using a SpectraMax 250 reader (Molecular Devices, Sunnyvale, Calif.) with kinetic capability.", "Cleavage of EDVV AbuC-p-nitroanilide (500 μM) substrate by purified HCV NS3 protease (0.5 μM) was performed at 30° C. in the buffer containing 30 μM NS4A fragment, 46 mM Hepes, pH 8.0, 92 mM NaCl, 18% glycerol, 5 mM DTT, and 7.5% DMSO in the absence or presence of the testing compound.", "The reaction was monitored for pNA (p-nitroaniline) release at 405 nm.", "The determination of the kinetic parameters including Vmax, Km and Vmax/Km is performed under the conditions as described above.", "Ki values are calculated from rate vs. [inhibitor] plots, at fixed concentrations of enzyme and substrate, by a nonlinear least squares fit of the data to the equation of Morrison for tight binding competitive inhibition [J. F. Morrison, Biochim.", "Biophys.", "Acta., 185, 269-286 (1969)].", "The Prism program (GraphPad Software, Inc.) is used for this procedure.", "The HCV serine protease inhibitors disclosed herein can be used in combination with other molecules that directly exhibit or indirectly elicit anti-HCV activity either prophylactically in patients at risk for contracting HCV infection, or to treat patients that are already infected.", "The term “anti-HCV activity” refers to the capacity of a molecule, when present, to completely inhibit or reduce accumulation of HCV virions compared to HCV virion accumulation in the absence of such molecule, and/or the capacity of a molecule to reduce or ameliorate conditions or symptoms associated with HCV infection or pathogenesis in patients.", "Molecules having anti-HCV activity include those that disrupt one or more steps in HCV infection or replication, as well as those that evoke immunomodulating and antiproliferative actions in host cells.", "Molecules having anti-HCV activity can inhibit HCV-specific replicative events such as, but not limited to, HCV-directed nucleic acid or protein synthesis.", "Stages of HCV replication at which molecules having anti-HCV activity can act include cell entry (e.g., attachment; penetration); uncoating and release of the HCV genome; replication of the HCV genome (e.g., replication of either strand of the viral RNA genome; transcription of viral messenger RNA); translation of HCV proteins; posttranslational modification of HCV proteins (e.g., proteolytic cleavage; glycosylation); intracellular transport of viral proteins; assembly of virion components; and release of viral particles (e.g., budding).", "Classes of molecules having anti-viral activity include, but are not limited to, soluble receptor decoys and antireceptor antibodies; ion channel blockers, capsid stabilizers, and fusion protein inhibitors; inhibitors of viral polymerases, reverse transcriptase, helicase, primase, or integrase; antisense oligonucleotides and ribozymes; immunomodulating and immunostimulating agents, including cytokines such as interferons, as well as peptide agonists, steroids, and classic drugs such as levamisole; inhibitors of regulatory proteins; protease inhibitors; assembly protein inhibitors; and antiviral antibodies and cytotoxic lymphocytes.", "The term “anti-HCV effective amount” or “pharmaceutically effective amount” refers to an amount of a compound, or combination of compounds as disclosed herein, effective in reducing or ameliorating conditions or symptoms associated with HCV infection or associated pathogenesis in patients, or in reducing viral levels in vitro or in vivo.", "In vitro applications include the Replicon Assay system, described below, where such amounts are effective in reducing HCV replicon RNA accumulation and/or the accumulation of proteins encoded by genes contained therein.", "Compounds having anti-HCV activity contemplated for use in the compositions and methods of combination therapy disclosed herein include, but are not limited to, immunomodulatory molecules, including immunostimulatory cytokines, and other compounds known to have HCV antiviral activity, such as various antiviral nucleosides and nucleotides.", "Immunomodulatory molecules contemplated for use in combination with the HCV serine protease inhibitors disclosed herein include, but are not limited to, interferon-alpha 2B (Intron A, Schering Plough); Rebatron (Schering Plough, Interferon-alpha 2B+Ribavirin); pegylated interferon alpha (Reddy, K. R. et al.", "Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C. Hepatology 33, 433-438 (2001)); consensus interferon (Kao, J. H., Chen, P. J., Lai, M. Y.", "& Chen, D. S. Efficacy of consensus interferon in the treatment of chronic hepatitis C. J. Gastroenterol.", "Hepatol.", "15, 1418-1423 (2000)); interferon-alpha 2A (Roferon A; Roche); lymphoblastoid or “natural” interferon; interferon tau (Clayette, P. et al.", "IFN-tau, a new interferon type I with antiretroviral activity.", "Pathol.", "Biol.", "(Paris) 47, 553-559 (1999)); interleukin 2 (Davis, G. L., Nelson, D. R. & Reyes, G. R. Future options for the management of hepatitis C. Seminars in Liver Disease 19, 103-112 (1999)); Interleukin 6 (Davis, G. L., Nelson, D. R. & Reyes, G. R. Future options for the management of hepatitis C. Seminars in Liver Disease 19, 103-112 (1999)); Interleukin 12 (Davis, G. L., Nelson, D. R. & Reyes, G. R. Future options for the management of hepatitis C. Seminars in Liver Disease 19, 103-112 (1999)); Ribavirin; and compounds that enhance the development of a type 1 helper T cell response (Davis, G. L., Nelson, D. R. & Reyes, G. R. Future options for the management of hepatitis C. Seminars in Liver Disease 19, 103-112 (1999)).", "Interferons may ameliorate viral infections by exerting direct antiviral effects and/or by modifying the immune response to infection.", "The antiviral effects of interferons are often mediated through inhibition of viral penetration or uncoating, synthesis of viral RNA, translation of viral proteins, and/or viral assembly and release.", "Compounds that stimulate the synthesis of interferon in cells (Tazulakhova, E. B., Parshina, O. V., Gusev, T. S. & Ershov, F. I. Russian Experience in Screening, Analysis, and Clinical Application of Novel Interferon Inducers.", "J. Interferon Cytokine Res.", "21, 65-73)) include, but are not limited to, double stranded RNA, alone or in combination with tobramycin, and Imiquimod (3M Pharmaceuticals) (Sauder, D. N. Immunomodulatory and pharmacologic properties of imiquimod.", "J.", "Am.", "Acad.", "Dermatol.", "43, S6-11 (2000)).", "Other compounds known to have, or that may have, HCV antiviral activity by virtue of non-immunomodulatory mechanisms include, but are not limited to, Ribavirin (ICN Pharmaceuticals); inosine 5′-monophosphate dehydrogenase inhibitors (VX497, being developed by Vertex Pharmaceuticals); amantadine and rimantadine (Younossi, A. M. and Perillo, R. P. The roles of amantadine, rimantadine, ursodeoxycholic acid, NSAIDs, alone or in combination with alpha interferons, in the treatment of chronic hepatitis C. Seminars in Liver Disease 19, 95-102 (1999)); LY217896 (U.S. Pat.", "No.", "4,835,168) (Colacino, J. M. et al.", "Evaluation of the anti-influenza virus activities of 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and its sodium salt.", "Antimicrobial Agents & Chemotherapy 34, 2156-2163 (1990)); and 9-Hydroxyimino-6-methoxy-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydro-phenanthrene-1-carboxylic acid methyl ester; 6-Methoxy-1,4a-dimethyl-9-(4-methyl-piperazin-1-ylimino)-1,2,3,4,4a,9,10,10a-octahydro-phenanthrene-1-carboxylic acid methyl ester-hydrochloride; 1-(2-Chloro-phenyl)-3-2,2-diphenyl-ethyl)-urea (U.S. Pat.", "No.", "6,127,422).", "Formulations, doses, and routes of administration for the foregoing molecules are either taught in the references cited below, or are well-known in the art as disclosed, for example, in F. G. Hayden, in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 50, pp.", "1191-1223, and the references cited therein.", "Alternatively, once a compound that exhibits HCV antiviral activity has been identified, a pharmaceutically effective amount of that compound can be determined using techniques that are well-known to the skilled artisan.", "Note, for example, Benet et al., in Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, Hardman et al., Eds., McGraw-Hill, New York (1996), Chapter 1, pp.", "3-27, and the references cited therein.", "Thus, the appropriate forumulations, dose(s) range, and dosing regimens, of such a compound can be easily determined by routine methods.", "The drug combinations of the present invention can be provided to a cell or cells, or to a human patient, either in separate pharmaceutically acceptable formulations administered simultaneously or sequentially, formulations containing more than one therapeutic agent, or by an assortment of single agent and multiple agent formualtions.", "However administered, these drug combinations form an anti-HCV effective amount of components.", "A large number of other immunomodulators and immunostimulants that can be used in the methods of the present invention are currently available and include: AA-2G; adamantylamide dipeptide; adenosine deaminase, Enzon; adjuvant, Alliance; adjuvants, Ribi; adjuvants, Vaxcel; Adjuvax; agelasphin-11; AIDS therapy, Chiron; algal glucan, SRI; algammulin, Anutech; Anginlyc; anticellular factors, Yeda; Anticort; antigastrin-17 immunogen, Ap; antigen delivery system, Vac; antigen formulation, IDBC; antiGnRH immunogen, Aphton; Antiherpin; Arbidol; azarole; Bay-q-8939; Bay-r-1005; BCH-1393; Betafectin; Biostim; BL-001; BL-009; Broncostat; Cantastim; CDRI-84-246; cefodizime; chemokine inhibitors, ICOS; CMV peptides, City of Hope; CN-5888; cytokine-releasing agent, St; DHEAS, Paradigm; DISC TA-HSV; J07B; I01A; I01Z; ditiocarb sodium; ECA-10-142; ELS-1; endotoxin, Novartis; FCE-20696; FCE-24089; FCE-24578; FLT-3 ligand, Immunex; FR-900483; FR-900494; FR-901235; FTS-Zn; G-proteins, Cadus; gludapcin; glutaurine; glycophosphopeptical; GM-2; GM-53; GMDP; growth factor vaccine, EntreM; H-BIG, NABI; H-CIG, NABI; HAB439; Helicobacter pylori vaccine; herpes-specific immune factor; HIV therapy, United Biomed; HyperGAM+CF; ImmuMax; Immun BCG; immune therapy, Connective; immunomodulator, Evans; immunomodulators, Novacell; imreg-1; imreg-2; Indomune; inosine pranobex; interferon, Dong-A (alpha2); interferon, Genentech (gamma); interferon, Novartis (alpha); interleukin-12, Genetics Ins; interleukin-15, Immunex; interleukin-16, Research Cor; ISCAR-1; J005X; L-644257; licomarasminic acid; LipoTher; LK-409; LK-410; LP-2307; LT (R1926); LW-50020; MAF, Shionogi; MDP derivatives, Merck; met-enkephalin, TNI; methylfurylbutyrolactones; MIMP; mirimostim; mixed bacterial vaccine, Tem; MM-1; moniliastat; MPLA, Ribi; MS-705; murabutide; murabutide, Vacsyn; muramyl dipeptide derivative; muramyl peptide derivatives myelopid; -563; NACOS-6; NH-765; NISV, Proteus; NPT-16416; NT-002; PA-485; PEFA-814; peptides, Scios; peptidoglycan, Pliva; Perthon, Advanced Plant; PGM derivative, Pliva; Pharmaprojects No.", "1099; No.", "1426; No.", "1549; No.", "1585; No.", "1607; No.", "1710; No.", "1779; No.", "2002; No.", "2060; No.", "2795; No.", "3088; No.", "3111; No.", "3345; No.", "3467; No.", "3668; No.", "3998; No.", "3999; No.", "4089; No.", "4188; No.", "4451; No.", "4500; No.", "4689; No.", "4833; No.", "494; No.", "5217; No.", "530; pidotimod; pimelautide; pinafide; PMD-589; podophyllotoxin, Conpharm; POL-509; poly-ICLC; poly-ICLC, Yamasa Shoyu; PolyA-PolyU; Polysaccharide A; protein A, Berlox Bioscience; PS34WO; Pseudomonas MAbs, Teijin; Psomaglobin; PTL-78419; Pyrexol; pyriferone; Retrogen; Retropep; RG-003; Rhinostat; rifamaxil; RM-06; Rollin; romurtide; RU40555; RU41821; Rubella antibodies, ResCo; S-27609; SB-73; SDZ-280-636; SDZ-MRL-953; SK&F-107647; SL04; SL05; SM-4333; Solutein; SRI-62-834; SRL-172; ST-570; ST-789; staphage lysate; Stimulon; suppressin; T-150R1; T-LCEF; tabilautide; temurtide; Theradigm-HBV; Theradigm-HPV; Theradigm-HSV; TBF, Pharm & Upjohn; THF, Yeda; thymalfasin; thymic hormone fractions; thymocartin; thymolymphotropin; thymopentin; thymopentin analogues; thymopentin, Peptech; thymosin fraction 5, Alpha; thymostimulin; thymotrinan; TMD-232; TO-115; transfer factor, Viragen; tuftsin, Selavo; ubenimex; Ulsastat; ANGG−; CD4+; Collag+; COLSF+; COM+; DA-A+; GAST−; GF-TH+; GP-120−; IF+; IF-A+; IF-A-2+; IF-B+; IF-G+; IF-G-1B+; EL-2+; IL-12+; IL-15+; IM+; LHRH−; LIPCOR+L LYM-B+; LYM-NK+; LYM-T+; OPI+; PEP+; PHG-MA+; RNA-SYN−; SY-CW−; TH-A-1+; TH-5+; TNF+; UN.", "Representative nucleoside and nucleotide compounds useful in the present invention include, but are not limited to: (+) -cis-5-fluoro-1-[2-(hydroxy-methyl) -[1, 3-oxathiolan -5-yl]cytosine; (−)-2′-deoxy-3′-thiocytidine-5′-triphosphate (3TC); (−)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosine (FFC); (−) 2′,3′, dideoxy-3′-thiacytidine[(−) -SddCl; 1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC); 1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine triphosphate (FIACIP); 1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU); 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; 2′,3′-dideoxy-3′-fluoro-5-methyl-dexocytidine (FddMeCyt); 2′,3′-dideoxy-3′-chloro-5-methyl-dexocytidine (ClddMeCyt); 2′,3′-dideoxy-3′-amino-5-methyl-dexocytidine (AddMeCyt); 2′,3′-dideoxy-3′-fluoro-5-methyl-cytidine (FddMeCyt); 2′,3′-dideoxy-3′-chloro-5-methyl-cytidine (ClddMeCyt); 2′,3′-dideoxy-3′-amino-5-methyl-cytidine (AddMeCyt); 2′,3′-dideoxy-3′-fluorothymidine (FddThd); 2′,3′-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC); 2′,3′-dideoxy-beta-L-5-thiacytidine; 2′,3′-dideoxy-beta-L-5-cytidine (beta-L-ddC); 9-(1,3-dihydroxy-2-propoxymethyl) guanine; 2′-deoxy-3′-thia-5-fluorocytosine; 3′-amino-5-methyl-dexocytidine (AddMeCyt); 2-amino-1,9-[(2-hydroxymethyl-1-(hydroxymethyl)ethoxy]methyl]6H-purin-6-one (gancyclovir); 2-[2-(2-amino-9H-purin-9y)ethyl]-1,3-propandil diacetate (famciclovir); 2-amino-1,9 dihydro-9-[(2-hydroxy-ethoxy)methyl]6H-purin-6-one (acyclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine (penciclovir); 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxy-guanine diacetate (famciclovir); 3′-azido-3′-deoxythymidine (AZT); 3′-chloro-5-methyl-dexocytidine (ClddMeCyt); 9-(2-phosphonyl-methoxyethyl)-2′,6′-diaminopurine-2′,3′-dideoxyriboside; 9-(2-phosphonylmethoxyethyl)adenine (PMEA); acyclovir triphosphate (ACVTP); D-carbocyclic-2′-deoxyguanosine (CdG); dideoxy-cytidine; dideoxy-cytosine (ddC); dideoxy-guanine (ddG); dideoxy-inosine (ddI); E-5-(2-bromovinyl)-2′-deoxyuridine triphosphate; fluoro-arabinofuranosyl-iodouracil; 1-(2′-deoxy-2′-fluoro-1-beta-D-arabinofuranosyl)-5-iodo-uracil (FIAU); stavudine; 9-beta-D-arabinofuranosyl-9H-purine6-amine monohydrate (Ara-A); 9-beta-D-arabinofuranosyl-9H-purine-6-amine-5′-monophosphate monohydrate (Ara-AMP); 2-deoxy-3′-thia-5-fluorocytidine; 2′,3′-dideoxy-guanine; and 2′,3′-dideoxy-guanosine.", "Synthetic methods for the preparation of nucleosides and nucleotides useful in the present invention are well known in the art as disclosed in Acta Biochim Pol., 43, 25-36 (1996); Swed.", "Nucleosides Nucleotides 15, 361-378 (1996); Synthiesis 12, 1465-1479 (1995); Carbohzyd.", "Chem.", "27, 242-276 (1995); Chem.", "Nucleosides Nucleotides 3, 421-535 (1994); Ann.", "Reports in Med.", "Chem., Academic Press; and Exp.", "Opin.", "Invest.", "Drugs 4, 95-115 (1995).", "The chemical reactions described in the references cited above are generally disclosed in terms of their broadest application to the preparation of the compounds of this invention.", "Occasionally, the reactions may not be applicable as described to each compound included within the scope of compounds disclosed herein.", "The compounds for which this occurs will be readily recognized by those skilled in the art.", "In all such cases, either the reactions can be successfully performed by conventional modifications known to those skilled in the art, e.g., by appropriate protection of interfering groups, by changing to alternative conventional reagents, by routine modification of reaction conditions, and the like, or other reactions disclosed herein or otherwise conventional will be applicable to the preparation of the corresponding compounds of this invention.", "In all preparative methods, all starting materials are known or readily preparable from known starting materials.", "While nucleoside analogs are generally employed as antiviral agents as is, nucleotides (nucleoside phosphates) must sometimes have to be converted to nucleosides in order to facilitate their transport across cell membranes.", "An example of a chemically modified nucleotide capable of entering cells is S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine (HPMPC, Gilead Sciences).", "Nucleoside and nucleotide compounds of this invention that are acids can form salts.", "Examples include salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium, or magnesium, or with organic bases or basic quaternary ammonium salts.", "Immunomodulators and immunostimulants useful in the combination therapy methods of the present invention can be administered in amounts lower than those conventional in the art.", "For example, interferon alpha is typically administered to humans for the treatment of HCV infections in an amount of from about 1×106 units/person three times per week to about 10×106 units/person three times per week (Simon et al., Hepatology 25: 445-448 (1997)).", "In the methods and compositions of the present invention, this dose can be in the range of from about 0.1×106 units/person three times per week to about 7.5×106 units/person three times per week; more preferably from about 0 5×106 units/person three times per week to about 5×106 units/person three times per week; most preferably from about 1×106 units/person three times per week to about 3×106 units/person three times per week.", "Due to the enhanced hepatitis C virus antiviral effectiveness of immunomodulators and immunostimulants in the presence of the HCV serine protease inhibitors of the present invention, reduced amounts of these immunomodulators/immunostimulants can be employed in the methods and compositions disclosed herein.", "Similarly, due to the enhanced hepatitis C virus antiviral effectiveness of the present HCV serine protease inhibitors in the presence of immunomodulators and immunostimulants, reduced amounts of these HCV serine protease inhibitors can be employed in the methods and compositions disclosed herein.", "Such reduced amounts can be determined by routine monitoring of hepatitis C virus titers in infected patients undergoing therapy.", "This can be carried out by, for example, monitoring HCV RNA in patients' serum by slot-blot, dot-blot, or RT-PCR techniques, or by measurement of HCV surface or other antigens.", "Patients can be similarly monitored during combination therapy employing the HCV serine protease inhibitors disclosed herein and other compounds having anti-HCV activity, for example nucleoside and/or nucleotide antiviral agents, to determine the lowest effective doses of each when used in combination.", "In the methods of combination therapy disclosed herein, nucleoside or nucleotide antiviral compounds, or mixtures thereof, can be administered to humans in an amount in the range of from about 0.1 mg/person/day to about 500 mg/person/day; preferably from about 10 mg/person/day to about 300 mg/person/day; more preferably from about 25 mg/person/day to about 200 mg/person/day; even more preferably from about 50 mg/person/day to about 150 mg/person/day; and most preferably in the range of from about 1 mg/person/day to about 50 mg/person/day.", "Doses of compounds can be administered to a patient in a single dose or in proportionate multiple subdoses.", "In the latter case, dosage unit compositions can contain such amounts of submultiples thereof to make up the daily dose.", "Multiple doses per day can also increase the total daily dose should this be desired by the person prescribing the drug.", "The regimen for treating a patient suffering from a HCV infection with the compounds and/or compositions of the present invention is selected in accordance with a variety of factors, including the age, weight, sex, diet, and medical condition of the patient, the severity of the infection, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic, and toxicology profiles of the particular compounds employed, and whether a drug delivery system is utilized.", "Administration of the drug combinations disclosed herein should generally be continued over a period of several weeks to several months or years until virus titers reach acceptable levels, indicating that infection has been controlled or eradicated.", "Patients undergoing treatment with the drug combinations disclosed herein can be routinely monitored by measuring hepatitis viral RNA in patients' serum by slot-blot, dot-blot, or RT-PCR techniques, or by measurement of hepatitis C viral antigens, such as surface antigens, in serum to determine the effectiveness of therapy.", "Continuous analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of each component in the combination are administered, and so that the duration of treatment can be determined as well.", "Thus, the treatment regimen/dosing schedule can be rationally modified over the course of therapy so that the lowest amounts of each of the antiviral compounds used in combination which together exhibit satisfactory anti-hepatitis C virus effectiveness are administered, and so that administration of such antiviral compounds in combination is continued only so long as is necessary to successfully treat the infection.", "The present invention encompasses the use of the HCV serine protease inhibitors disclosed herein in various combinations with the foregoing and similar types of compounds having anti-HCV activity to treat or prevent HCV infections in patients.", "For example, one or more HCV serine protease inhibitors can be used in combination with: one or more interferons or interferon derivatives having anti-HCV activity; one or more non-interferon compounds having anti-HCV activity; or one or more interferons or interferon derivatives having anti-HCV activity and one or more non-interferon compounds having anti-HCV activity.", "When used in combination to treat or prevent HCV infection in a human patient, any of the presently disclosed HCV serine protease inhibitors and foregoing compounds having anti-HCV activity can be present in a pharmaceutically or anti-HCV effective amount.", "By virtue of their additive or synergistic effects, when used in the combinations described above, each can also be present in a subclinical pharmaceutically effective or anti-HCV effective amount, i.e., an amount that, if used alone, provides reduced pharmaceutical effectiveness in completely inhibiting or reducing the accumulation of HCV virions and/or reducing or ameliorating conditions or symptoms associated with HCV infection or pathogenesis in patients compared to such HCV serine protease inhibitors and compounds having anti-HCV activity when used in pharmaceutically effective amounts.", "In addition, the present invention encompasses the use of combinations of HCV serine protease inhibitors and compounds having anti-HCV activity as described above to treat or prevent HCV infections, where one or more of these inhibitors or compounds is present in a pharmaceutically effective amount, and the other(s) is(are) present in a subclinical pharmaceutically effective or anti-HCV effective amount(s) owing to their additive or synergistic effects.", "As used herein, the term “additive effect” describes the combined effect of two (or more) pharmaceutically active agents that is equal to the sum of the effect of each agent given alone.", "A syngergistic effect is one in which the combined effect of two (or more) pharmaceutically active agents is greater than the sum of the effect of each agent given alone.", "EXAMPLE 42 Current standard therapy for hepatitis C virus (HCV) infection is treatment with the immunomodulator alpha-interferon (Chronic Hepatitis C: Current Disease Management, U.S. Department of Health and Human Services, National Institutes of Health, 1999).", "This therapy is ineffective in most HCV patients, who show either no response or a relapse even after prolonged interferon therapy.", "Additionally, there are severe side effects associated with interferon therapy.", "In view of the pressing need for new, more effective antiviral drugs to treat HCV infected patients, the present inventors have developed a series of compounds that inhibit the serine protease of HCV (a complex of HCV viral proteins NS3 and NS4A).", "These compounds can be used alone, together with one another, and in combination with other classes of compunds to treat or prevent HCV infection.", "This example describes the testing of three representative HCV serine protease inhibitors, i.e., Compound CU, Compound EP, and Compound EC, alone and in combination with individual members of a set of interferons (interferon alpha-2B (Schering-Plough), interferon alpha-2A (PBL Biomedical Laboratories, New Brunswick, N.J.), interferon beta (Research Diagnostics Inc, Flanders, N.J.), and ovine-interferon tau (Research Diagnostics Inc, Flanders, N.J.)) in an HCV subgenomic RNA replicon assay (Replicon Assay) to determine if the two compounds act in concert to diminish HCV RNA accumulation.", "The Replicon Assay measures the amount of HCV subgenomic RNA (replicon RNA) remaining in replicon cells (Lohmann et al.", "Science 285:110-113 (1999)) after two days of drug treatment relative to the amount of replicon RNA in untreated cells.", "In this assay, the potency of compounds as HCV antiviral drugs is directly proportional to the level of inhibition of replicon.", "RNA accumulation.", "The two drugs are tested in combinations in the in vitro Replicon Assay system to determine whether, when used together, they exhibit additive or synergistic anti-HCV activity.", "The Replicon Assay is employed as a surrogate model for in vitro HCV infection to evaluate the combined effects of the immunomodulator, for example interferon-alpha 2B ((Intron A); Schering Plough), and the HCV serine protease inhibitor, for example Compound CU.", "As shown below, the results demonstrate that there is a clear synergistic anti-HCV effect of these two types of drugs as measured using formal mathematical determinations of synergy to analyze their capacity to reduce HCV RNA levels in the Replicon Assay.", "The Replicon Assay The Replicon Assay employing a cell line containing the self-replicating HCV subgenomic RNA (replicon) is described in Lohmann et al.", "Science 285:110-113 (1999).", "The Genbank accession number for the sequence of the replicon used in the experiments described herein is listed in this reference as AJ242654.This paper discloses methods for in vitro transcription of RNA from the replicon cDNA, transfection of the replicon RNA into Huh7 cells by electroporation, and selection of cells containing the replicon RNA using the antibiotic G418.Huh7 cells are a hepatoma cell line obtained from Dr. William Mason at Fox Chase Cancer Research Center (Philadelphia).", "These cells are publicly available from Fox Chase, and have been extensively described in the scientific literature (Nakabayashi et al.", "Cancer Res.", "42:3858-3863 (1982)).", "In the experiments described herein, all of the template DNA is removed from the in vitro transcribed replicon RNA preparation prior to electroporation of this RNA into Huh7 cells by multiple treatment with DNase (three sequential treatments).", "The Replicon Assay is performed as described in detail below.", "Briefly, Replicon cells are placed in 96 well trays at a density of 10,000 cells per well and incubated 37° C. The cells are incubated in DMEM (Dulbecco's Minimal Essential Media) supplemented with 10% fetal bovine serum, glutamine, nonessential amino acids, and the antibiotic G418 (0.25 mg/ml).", "After overnight incubation, the medium is replaced with DMEM containing 2% fetal bovine serum and various concentrations of the serine protease inhibitor, such as Compound CU, and/or an interferon such as interferon-alpha 2B (Intron A, Schering Plough).", "Each compound is tested at six to eight different concentrations.", "For one extreme of the range of concentrations, high concentrations of the compounds that will result in almost complete inhibition of replicon RNA accumulation after two days of treatment are selected.", "From these starting concentrations, serial dilutions are made so that the concentration ranges tested in the Replicon Assay include concentrations at which the compounds are highly effective, as well as concentrations at which there is no significant effect.", "Each HCV serine protease inhibitor concentration is tested without any added interferon, as well as with the addition of six to eight different interferon doses.", "Similarly, interferon is tested without any added HCV serine protease inhibitor.", "After a 48-hour incubation with the compounds, the medium is removed from the plates, and total cellular RNA is extracted from the cells using the RNeasy-96 kit manufactured by Qiagen Inc. (Valencia, Calif.).", "This RNA is then analyzed by quantitative RT-PCR, or TaqMan® (Applied Biosystems, Foster City Calif.).", "The TaqMan® RT-PCR target is the neomycin resistance gene in the replicon RNA.", "The plates are configured so that there are 5 replicates of each drug treatment sample, and 16 replicates of the untreated samples.", "This permits greater statistical confidence in the quantitative RT-PCR data.", "Analysis of the Replicon Assay data yields two values that are useful in assessing the potency of potential HCV antiviral agents.", "At each compound concentration tested, the level of inhibition in replicon RNA accumulation caused by the compound during two days of treatment relative to the amount of replicon RNA in untreated cells is determined.", "This is reported as percent inhibition.", "When a series of data points generated by treatments of cells at a range of concentrations has been obtained, IC50 values, i.e., the compound concentration at which HCV replicon RNA accumulation is diminished 50% by the compound, are generated.", "Through repeated testing of the HCV serine protease inhibitors in the Replicon Assay, it is determined that the IC50 has a percent coefficient of variation (% CV or 100% x standard deviation in the IC50/mean IC50) of about 20%.", "The IC50 is the value used to rank individual compounds tested in this assay based on their potency as HCV antiviral agents.", "Simple IC50 determinations are inadequate to assess the utility of compounds used in combination.", "The most effective analysis of the array of data generated using all the combinations of different interferons and serine protease inhibitors requires evaluation of the percent inhibitions as shown in Table 7 using mathematical methods described below that are designed to determine if combination treatments are agonistic, additive, or synergistic.", "Details of the Replicon Assay are as follows: Procedure for Quantitative Analysis of HCV Replicon RNA in the HCV Replicon Assay Using TaqMan®® RT-PCR The Replicon Assay is used to measure the capacity of potential HCV antiviral compounds to inhibit the accumulation of a HCV subgenomic RNA replicon molecule in a Huh7 cell line (Lohmann et al.", "Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line.", "Science 285, 110-113 (1999)).", "This assay comprises three operational components: (1) Replicon cell maintenance, assay plate set up, and compound application; (2) Extraction of total cellular RNA from replicon cells; and (3) Real time RT-PCR (TaqMa®) to measure the mount of replicon RNA in each sample.", "The Replicon Assay requires at least 4 days to perform; however, the process can be interrupted and samples frozen between steps.", "Each assay component is described below.", "1.Replicon Cell Maintenance, Assay Plate Setup, and Compound Application 1.1 Replicon Cell Line Maintenance The cell line used in the Replicon Assay is produced as described in Lohmann et al.", "(Replication of Subgenomic Hepatitis C Virus RNAs in a Hepatoma Cell Line.", "Science 285, 110-113 (1999)).", "After 150 cm2 cell culture flasks (Costar) containing Replicon cells are incubated at 37° C. and 5% CO2 and become confluent, the cells are diluted 1:10, v/v, into fresh 150 cm2 cell culture flasks.", "The medium is DMEM containing 10% fetal bovine serum (PBS), 1× non-essential amino acids (NEAA), 1× Glutamine (Glu), and 0.25 mg/ml G418.Three serial passages are performed, each time allowing the cells to become confluent, followed by dilution of the cells into fresh 150 cm2 cell culture flasks.", "These cells, referred to as “original cells,” are then aliquoted and stored for future use in the Replicon Assay.", "TaqMan®-based analysis is performed to determine the number of HCV replicon genomes per cell, which reveals the presence of ˜150 copies of the replicon per cell.", "This is based on the ratio of copies of replicon RNA to two times the copies of the human apoB gene (number of haploid genomes).", "1.1.1 Original cells are stored in liquid N2.For cells used in the Replicon Assay, after 20 serial passages, cells are abandoned, and a fresh lot is revived from liquid N2 storage.", "1.2 Plating of cells in 96-Well Trays for the Replicon Assay 1.2.1.For preparation of 96-well plates, a 75% confluent 75 cm2 flask of replicon-containing cells are trypsmized, and resuspended in 10 ml Medium A. Trypsinization is performed by removing the medium, adding 1 ml of trypsin-EDTA 0.25%, w/v, and then removing the trypsin-EDTA.", "After 5-10 minutes the cells release from the flask and are resuspended in medium.", "1.2.2.Cells are counted using a hemacytometer, and the cell concentration is adjusted to 105 cells/ml.", "1.2.3.Each well is seeded with a 100 μl cell suspension using an Impact2 multi-channel pipette (Matrix), never plating more than four 96-well plates from a single cell suspension.", "1.2.4.96-well plates are incubated at 37° C. overnight.", "1.3.Compound Dilution and Application to Replicon Cell Trays 1.3.1.HCV serine protease inhibitor compounds are dissolved in dimethylsulfoxide (DMSO) to a final concentration of 20 mM.", "Interferons are suspended in phosphate buffered saline solution containing 0.1% w/v bovine serum albumin.", "1.3.2.The 20 mM compound solution is diluted to 1 mM with DMSO.", "1.3.3.50 μl of compound dissolved in DMSO are added to 10 ml Medium B (the compound concentration is 5 mM, and the DMSO concentration is now 0.5%), or 20 μl of 1 mM compound and 30 μl DMSO are added to 10 ml Medium B (compound concentration 2 μM.)", "1.3.4.Compound dilution to final concentration is completed by mixing compound/Medium B solution with Medium C (contains 0.5% DMSO).", "Serial one to five dilutions of the compound are made with Medium C in a 2 ml polypropylene 96-well block to obtain the desired final concentrations of compound.", "1.3.5.The cell plate is removed from the 37° C. incubator and labeled on the top right corner of the lid and the right side of the base.", "The medium is poured off of the 96-well plates.", "1.3.6.100 μl compound/medium solutions from each well of the 96-well dilution block are added to the 96well cell plate using an Impact2 Pipette.", "1.3.7.100 μl medium C are added to all the untreated wells according to Table 3 for testing compounds at either 1, 3, or 6 different concentrations.", "“Untx” refers to mock-treated cells (DMSO added at the same concentration as in treated cells); “Con.” refers to compound concentration.", "TABLE 3 Compound 1 Compound 2 1 2 3 4 5 6 7 8 9 10 11 12 2 compounds, 6 concentrations, 5 replicates A untx untx untx untx untx untx untx untx untx untx B con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 C con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 D con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 E con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 con.", "4 F con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 con.", "5 G con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 con.", "6 H untx untx untx untx untx untx untx untx untx untx 4 compounds, 3 concentration, 5 replicates A untx untx untx untx untx untx untx untx untx untx B con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 C con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 D con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 E con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 con.", "1 F con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 con.", "2 G con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 con.", "3 H untx untx untx untx untx untx untx untx untx untx Compound 3 Compound 4 1 2 3 4 5 6 7 8 9 10 11 12 16 compounds, 1 concentration, 4 replicates.", "A untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx B untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx C untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx D untx cpd 1 cpd 2 cpd 3 cpd 4 cpd 5 cpd 6 cpd 7 cpd 8 untx E untx cpd 9 cpd 10 cpd 11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx F untx cpd 9 cpd 10 cpd 11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx G untx cpd 9 cpd 10 cpd 11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx H untx cpd 9 cpd 10 cpd 11 cpd 12 cpd 13 cpd 14 cpd 15 cpd 16 untx 12 compounds, 1 concentration, 5 replicates.", "A untx untx untx untx untx untx untx untx untx untx B cpd 1 cpd 1 cpd 1 cpd 1 cpd 1 cpd 7 cpd 7 cpd 7 cpd 7 cpd 7 C cpd 2 cpd 2 cpd 2 cpd 2 cpd 2 cpd 8 cpd 8 cpd 8 cpd 8 cpd 8 D cpd 3 cpd 3 cpd 3 cpd 3 cpd 3 cpd 9 cpd 9 cpd 9 cpd 9 cpd 9 E cpd 4 cpd 4 cpd 4 cpd 4 cpd 4 cpd 10 cpd 10 cpd 10 cpd 10 cpd 10 F cpd 5 cpd 5 cpd 5 cpd 5 cpd 5 cpd 11 cpd 11 cpd 11 cpd 11 cpd 11 G cpd 6 cpd 6 cpd 6 cpd 6 cpd 6 cpd 12 cpd 12 cpd 12 cpd 12 cpd 12 H untx untx untx untx untx untx untx untx untx untx 1.4.The Plates are Incubated for 48 Hours at 37° C., and then Subjected to RNA Extraction.", "TABLE 4 Summary of equipment and supplies used for cell culture and compound set up 8 channel Impact2 Pipette, 1250 μl cat no 2004 Matrix 2 ml polypropylene deep-well block, cat no 4222 Matrix 96-well, sterile 25 ml Reagent Reservoirs, Sterile cat no.", "8096 Matrix 1250 μl X-tra long pipet tips cat no.", "8255 Matrix 96-well plate cat no.", "3595 Costar Hemacytometer Bright line Reichert improved Neubauer 0.1 mm deep DMEM cat no.", "51444-79P JRH L-glutamine (Glu) cat no.", "12403-010 GIBCO-BRL Non-essential amino acids (NEAA) cat no.", "11140-050 GIBCO-BRL Fetal Bovine Serum (FBS) cat no.", "16250-078 GIBCO-BRL G418 cat no.", "55-0273 Invitrogen DMSO cat no.", "D-2650 Sigma Medium A DMEM, 10% FBS, 1× NEAA, 1× Glu, 0.25 mg/ml G418 Medium B DMEM, 2% FBS, 1× NEAA, 1× Glu Medium C DMEM, 2% FBS, 1× NEAA, 1× Glu, 0.5% DMSO Trypsin-EDTA 0.25% GIBCO-BRL 2.Extraction of Total Cellular RNA from Replicon Cells 2.1 Introduction The goal of the procedure is to extract RNA from in vitro tissue culture samples so that the viral or cellular RNA is quantitatively recovered and pure enough to be analyzed by quantitative HCV RT-PCR assay.", "To permit detection of variations in the efficiency of the RNA extraction, standard amounts of bovine viral diarrhea virus (BVDV), an RNA virus with some similarity to HCV, are added to each cell sample before RNA extraction.", "Thus, the level of BVDV RNA detected in the final multiplex RT-PCR reaction should be consistent among all wells within the variability limits associated with the Replicon Assay.", "This RNA extraction efficiency internal control is discussed further in the TaqMan® section, below.", "The RNA extraction approach used is the RNeasy-96 method manufactured by Qiagen Inc. (Valencia, Calif.).", "This method employs 96 silica-based mini-columns that are positioned in an array compatible with 96-well tissue culture operations.", "The RNA extraction technology is a modification of the Boom method, in which all cellular proteins and nucleic acid, including nucleases, are first denatured with a strong chaotropic salt (guanidinium thiocyanate).", "In this environment, nucleic acids have a strong affinity for silica, the material in the mini-column discs; however, proteins and other contaminants do not bind to silica, and pass through the columns.", "After washing the columns with chaotropic/ethanol solutions, the samples are partially dried, and the nucleic acid is then released from the column in a small volume of water.", "To reduce variability in recovering HCV RNA, care is taken with the column washing and partial drying conditions.", "The presence of a small amount of ethanol on a column will contaminate the final RNA and interfere with the RT-PCR detection system-L Caution is required in all phases of this procedure because the starting samples may be biohazardous, the chaotropic salt is highly caustic, and as a thiocyanate, it can generate poisonous cyanide gas if allowed to come in contact with acidic environments.", "TABLE 5 Summary of Equipment and Supplies Needed for HCV RNA Extraction Procedures RNeasy 96 Kit (24) cat no.", "74183 Qiagen QIAvac 96 manifold cat no.", "19504 Qiagen Centrifuge 4-15C, for 2×96 plates, cat no.", "81010 Qiagen 6000× g plate rotor for 2×96 plates cat no.", "81031 Qiagen 200 Proof Ethyl Alcohol 8 channel Impact2 Pipette, 250 μl cat no 2002 Matrix 8 channel Impact2 Pipette, 1250 μl cat no 2004 Matrix 2 ml polypropylene deep-well block, cat no 4222 Matrix 96-well, sterile 25 ml Reagent Reservoirs, Sterile cat no.", "8096 Matrix 1250 μl X-tra long pipet tips cat no.", "8255 Matrix 200 μl pipet tips cat no.", "7275 Matrix serum free MEM medium cat no.", "11095-80 GIBCOBRL 2.2 Procedure: 2.2.1 Cell Lysis 2.2.1.1.Prepare lysis buffer.", "For one 96-well plate, add 150 μl β-mercaptoethanol (β-ME) and 1 μl BVDV stock (vortex stock before adding) to 15 ml RLT buffer (a component of the RNeasy kit, Qiagen).", "This stock is prepared by infecting MDBK cells (bovine kidney cells , #CCL-22, available from the American Type Culture Collection, Manassas Va.) with BVDV and harvesting the culture at peak cytopathic effect (CPE).", "This stock has an infectious titer of approximately 1×107 pfu/ml.", "This gives BVDV a threshold cycle (Ct) of about 22 in the TaqMan® assay.", "The BVDV stock is stored in a −80° C. freezer.", "2.2.1.2.Cells are washed with 150 μl serum-free MEM medium (program 4 on 8 channel electronic pipette P1250: Fill 1250, Disp 150×8).", "150 μl lysis buffer are added to each well (same program).", "2.2.1.3.RNA is extracted immediately, or cells are frozen at −80° C. 2.2.2.Preparation of reagents and materials for RNA extraction.", "2.2.2.1.Note the lot # of the RPE and RNeasy 96 Kit.", "2.2.2.2.RPE: 720 ml of 100% ethanol are added.", "to one bottle of RPE (Qiagen), and mixed well; RPE bottles are always shaken well before use.", "2.2.2.3.70% Ethanol: 150 ml diethylpyrocarbonate (DEPC) water are added to 350 ml 100% ethanol and mixed well.", "2.2.3.Preparation of RNA with RNeasy 96 kit 2.2.3.1.Frozen samples are thawed at room temperature for 40 min.", "At the same time, one column of Extraction Controls is thawed for each plate (Extraction Controls: The RNeasy Extraction Controls are a set of 8 tubes all connected together.", "Inside of each tube is 170 μl of cell lysate with a certain ratio of HCV positive and negative cells.", "From the top to the bottom are two each of a low, medium, high, and zero number controls, respectively.", "(See section 2.3 of the protocol below.)", "2.2.3.2.The samples are mixed by pipetting 100 μl up and down five times.", "The entire sample is transferred into columns 1-10 of the 2 ml Matrix square-well block (program 1 on P250: Mix 100×5, Fill 170, Purge).", "2.2.3.3.150 μl of the replicon standard is transferred into column 1 l(no samples in column 12).", "2.2.3.4.150 μl of 70% ethanol (EtOH) are added to each sample (program 4 on P1250: Fill 1250, Disp 150).", "2.2.3.5.An RNeasy 96 plate labelled with the appropriate plate number is placed in the vacuum manifold.", "Mix and transfer the lysate/EtOH to the RNeasy 96 plate (program 1 on P1250: Mix 200, Times 5, Fill 330, and Purge).", "Any unused wells are sealed with transparent tape (supplied by Qiagen), usually column 12.2.2.3.6.Vacuum (approximately 800 mbar) is applied to load the sample onto the mini-columns.", "2.2.3.7.The RNeasy-96 plate is washed with 1000 μl of RW1 buffer (Qiagen)/well (program 2 on P1250: Fill 1000, Disp 1000).", "2.2.3.8.Vacuum is applied to the filter through the RW1 buffer, and the flow-through is emptied.", "2.2.3.9.The RNeasy-96 plate is washed with 1000 μl of RPE buffer/well (program 2 on P1250).", "2.2.3.10.Vacuum is applied to filter through the RPE buffer.", "2.2.3.11.Repeat Step 2.2.3.9 2.2.3.12.Vacuum is applied to the filter through the RPE buffer, keeping the vacuum applied for 3 min.", "2.2.3.13.Dry the RNeasy 96 plate: The RNeasy-96 plate is placed in a collection microtube rack (supplied by Qiagen), covered with the supplied AirPore tape, and the unit is centrifuged for 10 min at 6000× g (Qiagen Sigma centrifuge; 4-15° C.).", "2.2.3.15.Elute the RNA from the RNeasy 96-well plate: The RNeasy-96 plate is transferred onto the top of a new collection microtube rack.", "70 μl of RNase-free water are added to the middle of each well (program 3 on P1250: Fill 850, Disp 70).", "2.2.3.16.Incubate 1 min at room temperature, and then place a fresh AirPore tape over the plate.", "2.2.3.17.The unit is then centrifuged for 4 min at 6000× g in a Sigma 4-15° C. centrifuge.", "The eluted volume measures between 28 μl and 50 μl.", "2.2.3.18.The RNeasy-96 plate is discarded, and the collection tube rack is sealed with the Qiagen-provided caps (8 per strip).", "2.2.3.19.The eluted RNA is stored at −80° C. or immediately analyzed in the TaqMan® assay.", "2.3 Extraction Controls Preparation Day 1 2.3.1.1.Plate out 2.5×107 replicon-producing cells in a 150 cm2 tissue culture flask (T-150).", "2.3.1.2.Plate out 2.0×106 Huh7 cells in a 75 cm2 tissue culture flask (T-75).", "2.3.1.3 Incubate overnight at 37° C. Day 2 2.3.1.4.Lyse the cells with lysis buffer.", "2.3.1.5.Remove the supernatant from the Huh7 and replicon-producing cells, and wash the monolayer with 10 ml serum-free medium (MEM).", "2.3.1.6.Add 30 ml of lysis buffer (with 1 μl of BVDV stock/15 ml of lysis buffer) to the Huh7 cells, mix by repeated pipetting, and place the cell lysate in a 50 ml conical-bottomed tissue culture centrifuge tube.", "2.3.1.7.Add 10.5 ml of lysis buffer to the replicon-producing cells, mix by repeated pipetting, and place the cell lysate in a 15 ml conical-bottomed tissue culture centrifuge tube.", "2.3.2.For the HIGH Extraction Standard: Aliquot 170 μl of the replicon-producing cells cell lysate into rows 5 and 6 of two Matrix 0.75 ml tube racks.", "2.3.3.For the MEDIUM Extraction Standard: Add 1.0 ml of the replicon-producing cells cell lysate to 9 ml of the Huh7 lysate, and mix well.", "Aliquot 170 μl of this mixture to rows 3 and 4 of two Matrix 0.75 ml tube racks.", "2.3.4.For the LOW Extraction Standard: Add 50 μL of the replicon-producing cells cell lysate to 10 ml of the Huh7 lysate, and mix well.", "Aliquot 170 μl of this mixture to rows 1 and 2 of two Matrix 0.75 ml tube racks.", "2.3.5.ZERO Extraction Control: Aliquot 170 μL of the Huh7 cell lysate to rows 7 and 8 of two Matrix 0.75 ml tube racks.", "2.3.6.Store controls at −90° C. 3.TaqMan® RT-PCR and Data Analysis 3.1 Introduction: Real-time quantitative RT-PCR is used to measure the amount of HCV replicon RNA in each sample.", "This technology is also referred to as the PCR-based 5′ nuclease assay, and TaqMan®.", "The analytic instrument is the Applied Biosystems 7700 Prism Sequence Detection System (Applied Biosystems, Foster City, Calif.).", "This instrument is essentially a time-multiplexed laser-induced fluorescence spectrograph coupled with a thermal cycler.", "It monitors the accumulation of PCR amplicon in each well of a 96-well sample tray throughout the course of the PCR process.", "3.2.Use of BVDV Internal Control: As mentioned in the previous section, an internal positive control is incorporated into every sample.", "This serves as a measure of RNA extraction efficiency, and shows if the sample contains contaminants that inhibit TaqMan® PCR.", "BVDV is mixed with the chaotropic cell lysis buffer prior to applying the lysis buffer to the cells.", "Although the positive control is in every sample, the BVDV internal positive control assay is only performed when the HCV replicon RNA assay data fall outside of expected limits, suggesting that there could be a problem with the samples.", "The 7700 is capable of simultaneously monitoring the accumulation of two different PCR amplicons in the same tube by using detection probes labeled with two different fluorescent reporter dyes (“multiplexing”).", "Specific criteria that elicit a TaqMan® analysis for the BVDV internal positive control of a sample plate are described in the section on data analysis (3.6).", "3.3 HCV Replicon RNA TaqMan® probe and primers.", "Because of the expected genetic stability and general lack of RNA secondary structure in the neomycin resistance gene (neo) encoded in the replicon, primers and a probe that bind in that region are employed.", "This segment of the replicon RNA extends from bases 342-1193 of the 8001 base pair replicon (SEQ ID NO: 1): 301 gtgcttgcga gtgccccggg aggtctcgta gacegtgcac catgagcacg aatcctaaac 361 ctcaaagaaa aaccaaacgt aacaccaacg ggcgcgccat gattgaacaa gatggattgc 421 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga 481 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt 541 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat 601 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg 661 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg 721 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc 781 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga 841 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag 901 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc 961 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg CCGCTTTTCT GGATTCATCG forward primer 1021 aCTGTGGCCG GCTGGGTGTG Gcggaccgct atcaggacat agcgttggct acccgtgata TaqMan ® probe 1081 ttgctgaaga gcTTGGCGGC GAATGGGctg accgcttcct cgtgctttac ggtatcgccg reverse primer 1141 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagtttaaa 3.4.Procedures 3.4.1.Method for Preparing 1× Master Mixtures for NEO and BVDV RT-PCR TABLE 6 Summary of equipment and supplies for preparing RT-PCR 10-plate Master Mix Order No.", "Supplier Equipment and supplies 0.5-10 μl pipette 22 47 005-1 2000 Series Eppendorf 2-20 μl pipette 22 47 015-9 2000 Series Eppendorf 10-100 μl pipette 22 47 020-5 2000 Series Eppendorf 50-200 μl pipette 22 47 025-6 2000 Series Eppendorf 100-1000 μl pipette 22 47 030-2 2000 Series Eppendorf 1250 μl Matrix tips cat no.", "8255 Matrix 200 μl Matrix tips cat no.", "7275 Matrix 10 μl ART tips cat no.", "2140 Molecular Bioproducts 20 μl ART tips cat no.", "2149P Molecular Bioproducts 100 μl ART tips cat no.", "2065E Molecular Bioproducts 200 μl ART tips cat no.", "2069 Molecular Bioproducts 1000 μl ART tips cat no.", "2079E Molecular Bioproducts Electronic pipette, Impact2 cat no.", "2001 Matrix 1.5 ml RNase-free microfuge tubes cat no.", "12450 Ambion 14 ml Polypropylene tubes cat no.", "352059 Falcon 25 ml reagent reservoir cat no.", "8096 Matrix 96-well reaction plate cat no.", "N801-0560 Applied Biosystems optical cap strips cat no.", "N801-0935 Applied Biosystems Disposable Sterile Gowns cat no.", "9515-E Baxter Reagents Acid 0.1 N HCl Fisher RNAseZap cat no.", "9780 Ambion RNAse away cat no.", "7005 Molecular Bioproducts 10-pak, EZ RT-PCR core reagents cat no.", "403028 Applied Biosystems kit, 5× reaction buffer, 25 mM Manganese Acetate, deoxy NTPs VIC NEO probe, 2 μM (=10×), cat no.", "450003, custom, Applied Biosystems 550 μl per aliquot 5′-VIC-CTG TGG CCG GCT GGG TGT GG-TAMRA-3′ (SEQ ID NO: 2) VIC BVDV probe, 2 μM (=10×), cat no.", "450003, custom, Applied Biosystems 550 μl per aliquot (Vertex) 5′-VIC-CCC TCG TCC ACG TGG CAT CTC GA-TAMRA-3′ (SEQ ID NO: 3) NEO forward primer, 3 μM (=10×) cat no.", "4304972, custom, Applied Biosystems forward/reverse primer mix, 550 μl 5′-CCG CTT TTC TGG ATT per aliquot CAT CG-3′ (SEQ ID NO: 4) NEO reverse primer, 3 μM (=10×) cat no.", "4304972, custom, Applied Biosystems forward/reverse primer mix, 550 μl 5′-CCC ATT CGC CGC CAA-3′ per aliquot (SEQ ID NO: 5) BVDV forward primer, 3 μM (=10×) custom, 5′-CAG GGT AGT Oligos etc forward/reverse primer mix, 550 μl CGT CAG TGG TTC G-3′ per aliquot (SEQ ID NO: 6), 1.0 μM scale w/gel purification BVDV reverse primer, 3 μM (=10×) custom, 5′-GGC CTC TGC Oligos etc forward/reverse primer mix, 550 μl AGC ACC CTA TC-3′ (SEQ per aliquot ID NO: 7), 1.0 μM scale w/gel purification NEO RNA standards In vitro transcribed RNA from a plasmid containing the neo gene portion of the HCV replicon RNA using T7 RNA polymerase.", "The in vitro transcribed RNA is quantitated based on known molecular weight of the transcripts and the UV-absorbance of the purified transcript solution.", "This RNA is diluted, aliquoted, and stored at −80° C. Individual aliquots are thawed for each TaqMan ® assay.", "RNA samples to be tested isolated from HCV replicon cells (section 2 of this Protocol), 10 μl/96-well plate Nuclease-Free Water cat no.", "9930 Ambion (Not DEPC Treated) 3.4.2.Preparation of Reagents for Master mix 3.4.2.1.Clean the bench according to the two steps below, and wipe the pipettes with RNAse away.", "RNAse Zap (Ambion, Austin, Tex.)", "RNAse Away (Molecular Bioproducts, San Diego, Calif.) 3.4.2.2.Open core EZ RT-PCR reagents (Applied Biosystems) and put the 5× buffer on ice, thaw frozen reagents at room temperature for about 15 minutes, and then put them on ice.", "One EZ RT-PCR reagents kit can be used to analyze two 96-well RNA extractions.", "3.4.2.3.Take one tube of 2 μM VIC probe (NEO or BVDV, 550 μl per tube) from −20° C. and put on ice.", "3.4.2.4.Take one tube 3 μM forward/reverse primer mix (NEO or BVDV, 550 μl per tube) from −20° C. and put on ice.", "3.4.2.5.Take one tube (30 μl) of standards RNA transcript (108 copies/10 μl) from −80° C. and place on ice.", "3.4.2.6.Take one tube of room temperature Ambion water.", "3.4.3.Assembly of Master Mixture for One 96-Well Plate Reaction.", "3.4.3.1.Use a 1 ml pipette to transfer 5× buffer (Applied Biosystems) to a 14 ml tube; total volume added is 1100 μl.", "3.4.3.2.Use a 1 ml pipette to add 25 mM Mn(OAc)2 (Applied Biosystems) to a 14 ml tube; total volume added is 660 μl.", "3.4.3.3.Use a 200 μl pipette to add 165 μl of 10 mM DATP to the 14 ml tube.", "Do the same for 10 mM dCTP, 20 mM dUTP, and 10 mM dGTP.", "3.4.3.4.Use a 1 ml pipette to add 550 μl 10×3 μM forward/reverse primer mix.", "3.4.3.5.Use a 1 ml pipette to add 550 μl 10×2 μM probe.", "3.4.3.6.Use a 1 ml pipette to add 220 μl rTth DNA polymerase (Applied Biosystems).", "3.4.3.7.Use a 100 μl pipette to add 55 μl AmpErase UNG (Applied Biosystems).", "3.4.3.8.Use a 1 ml pipette to add 605 μl Ambion H2O to the 14 ml tube; the final volume is 4400 μl total.", "3.4.3.9.Transfer the 4400 μl master mix to a 25 ml reagent reservoir.", "3.4.3.10.Dispense 40 μl per well for all 96 wells using an 8-channel pipette.", "3.4.3.11.Transfer 10 μl of extracted unknown samples to wells of the reaction plate using an 8-channel pipette, column by colulmn, column 1 through column 11.Cap each column after transfer.", "3.4.3.12.Add 270 μl Ambion H2O to the 30 μl 108 copies/10 μl RNA transcript for use in the standard curve and mix.", "There are now 107 copies of the HCV replicon quantitation standard RNA/10 μl.", "3.4.4.Setting Up the ABI 7700 for Each Run 3.4.4.1 Before each run, reboot the computer for the ABI 7700 and rebuild the desktop.", "3.4.4.2 Close and remove any redundant programs from the hard drive; overuns data to trash.", "3.4.4.3 Open Sequence Detector v1.7 program (SDS software).", "3.4.4.5 Open the “Replicon Assay Runs” folder.", "3.4.4.6 Open the “Replicon Assay” template plate.", "The thermal cycler conditions programmed into the template are as follows: Stage 1: 50° C. for 2 min.", "Stage 2: 60° C. for 30 min.", "Stage 3: 95° C. for 5 min.", "Stage 4: 95° C. for 15 sec.", "Stage 5: 60° C. for 60 sec.", "Cycle repeat number of stages 4-5: 40.Template instrument:diagnosis: advanced options: Select views: display mse.", "Select views: display best fit.", "Select miscellaneous: reference dye ROX.", "3.4.4.7 “Save” (not “save as”) the file in the “Replicon Assay Runs” folder.", "3.4.4.8 Show setup: hit RUN 3.5 Preparing the ABI7700 Data After a Run Using SDS Software.", "3.5.1.The assay plates are removed from the ABI7700 and discarded without ever being opened.", "This greatly reduces laboratory problems with PCR cross contamination.", "3.5.2.The data are analyzed using the Sequence Detector System software V1.7.3.5.3.The threshold levels are initially set using default settings.", "3.5.4.Data rejection criteria: Data points or series of whole plates can be rejected.", "If there has been a significant deviation from protocol, reagent failure or mishap, or ABI 7700 run failure, data can be discarded.", "For rejection of any data points from an apparently normal run, one or more of these criteria must be met.", "3.5.4.1.Threshold cycle calculations.", "Normally use the default values for the SDS software.", "If the Ct of the most concentrated sample is less than 15, then change the threshold value stop limit as needed to a lower value so that the Ct of the highest concentration sample is greater than the stop value.", "Update calculations after making this change.", "3.5.3.2.Consider rejecting an entire abnormal TaqMan® run as indicated by a deviation from the mean values for the slope and y-axis intercept of the line generated by analysis of the neo RNA standards.", "The acceptable ranges for those values are: Slope values should be between 3.0 and 3.6 y-intercept cycles should be between 36 and 41 cycles.", "3.5.4.3.Aberrant individual TaqMan® wells as indicated by extreme Rn/ΔRn can be deleted prior to data analysis so that they do not affect the SDS software calculations.", "3.5.4.4.Examine and record the no-template control Ct values and confirm that they are >7.0 Ct (>100×) higher than the Ct for any compound treated sample.", "3.5.5.The HCV RNA standards Ct values are compared with previous results.", "3.5.6.The HCV RNA standard curve is compared with previous results.", "3.5.7.If aberrant amplification is evident in individual wells, those wells are identified and noted.", "3.5.8.The “results” file is exported and transferred from the 7700 computer to another computer for analysis using Microsoft Excel.", "3.5.9.Any of the following changes in reagent preparations or dilution used is reported.", "New probe or primer synthesis from vendor.", "New probe or primer dilution and aliquots.", "New standards RNA transcript preparation.", "New standards RNA transcript dilution and aliquots.", "New BVDV viral preparation.", "New column 11 standards preparation.", "3.6 TaqMan® Data Analysis.", "3.6.1.Copy and paste TaqMan® HCV Ct number and copy number from the TaqMan® results file into the appropriate cells of the Replicon Assay data analysis Microsoft Excel macro, and run the macro.", "3.6.2.Copy the TaqMan® results table from the macro sheet onto another sheet, input compound serial number and lot number.", "3.6.3 From this excel sheet, the mean, standard deviation, and percentage CV of Compound inhibition activity, as well as HCV copy number, HCV Ct number, and BVDV Ct number (if available), of all dilution points in 5 replicates and no-compound control, will be calculated.", "3.6.4.Criteria for data rejection and implementation of BVDV Control TaqMan®.", "Check all the calculations.", "Data points or series of whole plates can be rejected.", "If there is a significant deviation from protocol, reagent failure or mishap, or ABI 7700 run failure, data can be discarded.", "For rejection of any data points from an apparently normal run, then one or more of these criteria must be met.", "The standard deviation of percentage inhibition should be less than 30% in active compounds.", "The % CV of HCV copy number should be less than 30%.", "The standard deviation of HCV Ct of all samples should be less than 0.5; this is usually about 0.1 to 0.3 in most samples.", "If the HCV Ct standard deviation is more than 0.5, then go back to the raw data table, and check the Ct numbers of 5 replicates.", "If the Ct number of any one well is 2 Ct different from the average Ct number of 5 replicates, then this well should omitted from the analysis.", "If more than 3 wells (not on same column) have unusual Ct numbers, then the BVDV TaqMan® internal control assay should be carried out.", "If the BVDV data show irregularity, then the compound should be tested again.", "3.6.5.IC50 calculation: Copy and paste the data of average inhibition and standard deviation into a sigmoid dose response with a variable slope calculator that uses non-linear regression methods.", "Using this tool, calculate the IC5o by using both of two methods: fixing the top at 100% inhibition only, or fixing the top at 100% inhibition and the bottom at 0% inhibition.", "The method that gives the clearest fit is then reported for each compound.", "The most reliable IC50 comes from the calculation having the lowest standard error.", "If IC50s calculated from these two curve fit options show more than one fold difference, or if the IC50 SD is greater than the IC50, the compound should be tested again at adjusted concentrations.", "Calculation of the Effect of HCV Serine Protease Inhibitors in Combination with Interferons The effect of a HCV serine protease inhibitor (HSPI) and an interferon in combination can be assessed in the Replicon Assay by generating a dose response curve for the HSPI in the presence of various levels of interferon, or by determining a dose response curve for an interferon in the presence of various levels of HSPI.", "The goal is to assess whether there is more or less inhibition of viral RNA accumulation than would be expected if the two drugs produce additive effects on the RNA.", "More specifically, the additivity definition of Lowe ((1928) Die Quantitation Probleme der Pharmakologic, Ergebn.", "Physiol., 27, 47-187) is used.", "This is defined as follows.", "Let D be the concentration of interferon that results in effect E, and let DE,HSPI be the concentration of protease inhibitor that results in effect E. 1 = D 1 D E , INF + D 2 D E , HSPI ( 1 ) Then no interaction or Lowe additivity is defined by the following relationship, where the combination of concentration D1 of INF and D2 of HSPI produces the effect E. The degree of synergy or antagonism is expressed in terms of iso-effect curves or Isobols.", "The combination (D1,D2) is a point on a graph where the axes are the concentrations of interferon and HSPI (FIG.", "2).", "All such combinations that produce an effect level E form the E effect Isobol.", "It is necessarily the case that (DE,INF,0) and (0, DE,HSPI) are points on the Isobol.", "The Isobols are straight lines connecting points (DE,INF,0) and (0, DE,HSPI) when the additivity relationship (1) is satisfied.", "Concave-up Isobols indicate synergy, and concave-down Isobols indicate antagonism.", "Following the guidelines of Berenbaum, M. C. ((1985) The expected effect of a combination of agents: the general solution.", "J. Theor.", "Biol., 114, 413-431), and Greco, Park and Rustom ((1990) Application of a New Approach for the Quantitation of Drug Synergism to the Combination of cis-Diamminedichloroplatinum and 1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327), add a term to (1) to account for synergy or antagonism.", "The equation defines a response surface that can be fit to the percent control values at all treatment combinations.", "Contour plots from this fitted response surface are the Isobols.", "The response surface model assumes a sigmoidal dose response for each compound defined by (2).", "E = E max 1 + ( [ Drug ] IC50 ) m + B ( 2 ) The concentrations that give a specified level of activity E alone are given by (3) D E , INF = IC50 INF ⁡ ( E - B E max - E + B ) 1 / m INF ( 3 ) D E , HSPI = IC50 HSPI ⁡ ( E - B E max - E + B ) 1 / m 310 To satisfy the model of Greco et al.", "(1990), the combined action of the drugs must then satisfy equation (4) for every combination of drugs that produces response level E. 1 = [ INF ] IC50 INF ⁡ ( E - B E max - E + B ) 1 / m INF + ( 4 ) ⁢ [ HSPI ] IC50 HSPI ⁡ ( E - B E max - E + B ) 1 / m 310 + ⁢ α ⁡ [ INF ] ⁡ [ HSPI ] IC50 INF ⁢ IC50 HSPI ⁡ ( E - B E max - E + B ) 1 / 2 ⁢ m INF ( E - B E max - E + B ) 1 / 2 ⁢ m 310 The parameter a measures the amount of interaction.", "A zero value of alpha means no interaction or additivity since the equation reduces to (1) when α=0.Given IC50s, Hill slopes (m), maximum value (Emax), and minimum value (B), this equation can be solved to give the effect that results from any treatment combination [INF] and [HSPI].", "Therefore, this equation defines a response surface.", "Given an experiment where [INF] and [HSPI] are varied, the parameters can be chosen using nonlinear weighted least squares regression.", "The parameter a can be related to a synergy measure S (Hewlett, P. S. (1969) Measurement of potencies of drug mixtures.", "Biometrics, 25, 477-487), which is taken directly from the Isobols at a 50% effect.", "S is the ratio, of the distance from the origin to the Isobol defining additivity, to the distance from the origin to Isobol of the fitted data, along the line at 45 degrees from the axes.", "(S=ON/OM see FIG.", "3).", "The relationship is α=4(S2−S).", "The method discussed in Greco et al.", "(1990), above, for fitting the response surface and determining synergy parameter a with its significance level is followed in assessing the degree of synergy in a series of experiments testing HSPIs in combination with several different interferons.", "However, there is a need to weight observations with lower counts more than those with higher counts.", "The counts relate directly to the percent control, which is the effect E. Using methods described in Carroll, R. J. and Rupert, D. ((1988) (Transformation and Weighting in Regression, Chapman and Hall, New York), the well to well variability can be seen to increase with the square of the mean percent control value.", "Therefore, the observations are weighted by one over the fitted percent control value (E) squared.", "The variance and weighting used to analyze these experiments is consistent with variability relationships observed by researchers investigating methods for analyzing Radioligand assays (Finney, D. J., (1976), Radioligand Assay, Biometrics, 32,721-740, and Dudley, R. A. Edwards, P., Ekins, R. P., McKinzie, I. G. M., Raab, G. M., Rodbard, D. and Rodgers, R. P. C. (1985), Guidelines for Immunoassay Data Processing, Clinical Chemistry, 31/8, 1264-1271).", "Results In an initial experiment, HCV serine protease inhibitor Compound CU is tested over a concentration range from 3 μM to 0.0123 μM, i.e., a,244-fold range.", "The interferon-alpha 2B concentrations vary from 30 units per sample to 0.0096 units per sample, i.e., a 3125-fold range.", "As shown in Table 7, when used as a single drug treatment, Compound CU exhibits an IC50 of 0.48 μM, and the interferon IC50 is 2.19 U.", "Within the precision of the Replicon Assay, which is approximately 20%, addition of interferon-alpha 2B results in an increase in the inhibition of replicon RNA accumulation in a dose-dependent manner.", "For example, treatment of cells with 0.333 μM Compound CU results in 28% inhibition of replicon RNA accumulation.", "Treatment of cells with a combination of 0.333 μM Compound CU, which is 71% of the IC50 dose (0.469 μM) and 0.24 U of interferon-alpha 2B, which is 11% of the interferon-alpha 2B IC50 (2.05 μM) results in a 49% inhibition of replicon RNA accumulation.", "Thus, 71% of one IC50 dose in combination with 11% of the other results in 49% inhlbition of replicon RNA accumulation.", "Using an intuitive approach to determining if a combination treatment is synergistic or additive or antagonistic, one could predict that if effect of combination treatment were only additive, one would expect the combined fractions of the two IC50 doses needed to obtain a 49% inhibition of replicon RNA accumulation to be 98%.", "Our experimental results demonstrate that the level of inhibition of replicon RNA accumulation is achieved using 71% plus 11%, i.e.", "82% of the IC50 dose rather than 98%, as predicted for additive effects of combination treatment.", "Thus at these concentrations of compounds, the effect appears to be synergistic because smaller fractional doses of the IC50 dose of each compound are used to obtain 49% inhibition of HCV replicon RNA than would be required of either compound alone, where 98% of the IC50 doses would be needed.", "The results of this combination treatment are shown in Table 8 and graphically in FIG.", "1.TABLE 7 Inhibition of replicon RNA accumulation after 48 hour treatment with Compound CU and interferon-alpha 2B, individually or in combination Interferon-alpha 2B(units) Compound 30 6 1.2 0.24 0.048 0.0096 0 CU (conc.)", "U U U U U U U 3 μM 99% 99% 99% 99% 98% 98% 98% 1 μM 99% 98% 96% 95% 92% 93% 88% 0.333 μM 94% 87% 66% 49% 33% 27% 28% 0.1111 μM 93% 79% 46% 29% 12% 15% 11% 0.0370 μM 92% 78% 44% 21% 2% 7% 8% 0.0123 μM 92% 78% 44% 20% 19% 19% 5% 0 μM 89% 73% 38% 16% 8% 12% 0% These initial results, derived as stated earlier via use of the in vitro Replicon Assay and a simple additivity analysis of the data generated by that assay, demonstrate that combination treatment of replicon cells with an HCV serine protease inhibitor and an interferon yields at least an additive antiviral effect, and likely a synergistic antiviral effect.", "The foregoing data have been reanalyzed using the formal mathematical tools described above to determine if the relationship between HCV serine protease inhibitor CU and interferon alpha-2B is synergeistic, additive, or antagonistic.", "The reanalyzed data are shown numerically in Table 8, and graphically in FIG.", "4.Table 8 summarizes further results obtained in the Replicon Assay after treatment of replicon-containing cells for 48 hours with various HCV serine protease inhibitors of the present invention and several different interferons, individually or in combination.", "We point out that the standard deviation of values measured for inhibition of HCV replicon RNA in the Replicon Assay is ˜20%.", "Compounds are tested over a broad concentration range and at lower compound concentrations that cause no significant inhibition of HCV replicon RNA concentration.", "Because of the ˜20% standard deviation of the assay, some data points will generate negative numbers.", "Negative inhibition numbers indicate in a particular experiment there is on average more HCV replicon RNA molecules in the compound treated samples than in the mock treated samples.", "TABLE 8 INHIBITION OF REPLICON RNA ACCUMULATION AFTER 48 HOUR TREATMENT WITH HCV SERINE PROTEASE INHIBITORS AND DIFFERENT INTERFERONS, INDIVIDUALLY OR IN COMBINATION EXPERIMENT 1 Compound IFN alpha-2B (units) CU (μM) 30.00 6.00 1.20 0.24 0.048 0.0096 0.000 0.000 89% 73% 38% 16% 8% 12% 0% 0.012 92% 78% 44% 20% 19% 19% 5% 0.037 92% 78% 44% 21% 2% 7% 8% 0.111 93% 79% 46% 29% 12% 15% 11% 0.333 94% 87% 66% 49% 33% 27% 28% 1.000 99% 98% 96% 95% 92% 93% 88% 3.000 99% 99% 99% 99% 98% 98% 98% EXPERIMENT 2 Compound IFN alpha-2A (units) CU (μM) 30 6 1.2 0.24 0.048 0.0096 0 0 86% 61% 27% 4% −7% 5% 0% 0.0123 87% 66% 17% −23% 8% 8% 10% 0.37 85% 62% 13% −2% 0% −1% 1% 0.1111 87% 68% 37% 20% −6% 12% 10% 0.333 92% 77% 58% 41% 26% 25% 44% 1 98% 96% 90% 86% 84% 83% 85% 3 99% 99% 98% 98% 98% 98% 98% EXPERIMENT 3 Interferon alpha-2B Compound CU (μM) (units) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 0 98% 93% 62% 23% 12% −2% −4% −2% 0 0.049 98% 95% 70% 39% 12% 2% 6% 9% 3% 0.123 98% 95% 70% 43% 15% 7% 2% 5% 2% 0.307 98% 95% 73% 46% 16% 14% 7% 19% −3% 0.768 98% 95% 82% 56% 43% 34% 28% 32% 28% 1.920 98% 98% 87% 71% 51% 54% 49% 52% 45% 4.8 99% 98% 92% 82% 74% 71% 69% 71% 59% 12.0 99% 98% 96% 89% 87% 85% 85% 85% 80% 30.0 99% 99% 98% 95% 93% 92% 92% 93% 89% EXPERIMENT 4 Interferon alpha-2A Compound CU (μM) (units) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 0 98% 94% 74% 38% 17% 3% −1% 6% 0% 0.049 98% 93% 60% 22% 29% 21% −9% −6% 6% 0.123 98% 93% 67% 29% 21% 12% 3% 2% −8% 0.307 98% 93% 66% 29% 22% 4% −3% −4% 10% 0.768 98% 95% 67% 46% 24% 21% 20% 9% 15% 1.920 98% 96% 73% 48% 43% 44% 27% 33% 29% 4.8 98% 97% 82% 61% 61% 59% 52% 55% 43% 12.0 99% 98% 91% 75% 76% 72% 71% 74% 73% 30.0 99% 98% 96% 89% 86% 85% 84% 84% 83% EXPERIMENT 5 Ovine Interferon Compound CU (μM) tau (units) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 0.0 98% 95% 65% 24% −1% −14% −14% −12% 0 0.9375 97% 95% 72% 41% 17% 11% 12% 6% 17% 1.875 97% 95% 71% 40% 31% 18% 18% 11% 4% 3.75 98% 96% 75% 44% 38% 25% 34% 18% 17% 7.5 98% 96% 82% 61% 42% 37% 25% 26% 36% 15 98% 97% 84% 64% 59% 61% 56% 51% 53% 30 98% 98% 90% 79% 72% 68% 65% 68% 68% 60 98% 98% 93% 87% 80% 80% 74% 77% 82% 120 98% 98% 95% 92% 86% 87% 86% 86% 87% EXPERIMENT 6 Interferon- alpha 2B Compound EC (μM) (units) 3 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.0234 0 0 96% 93% 81% 56% 29% 23% 19% 1% 0 0.0492 96% 92% 80% 60% 31% 15% 19% 29% 6% 0.1229 96% 94% 78% 58% 32% 13% 20% 20% 4% 0.3072 97% 95% 82% 60% 38% 32% 34% 42% 23% 0.768 97% 95% 87% 66% 43% 41% 46% 43% 25% 1.92 98% 97% 90% 73% 62% 51% 54% 58% 47% 4.8 98% 97% 94% 87% 76% 73% 78% 76% 69% 12.0 98% 98% 96% 92% 86% 86% 86% 85% 84% 30.0 98% 98% 96% 96% 93% 92% 92% 95% 91% EXPERIMENT 7 Interferon- alpha-2A Compound EC (μM) (units) 3.0 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.02344 0 0 96% 92% 81% 47% 28% 17% −1% −8% 0 0.0492 96% 93% 78% 58% 21% 8% −12% 10% −17% 0.1229 95% 93% 79% 64% 14% 5% 14% 7% −22% 0.3072 95% 91% 80% 64% 22% 15% 5% 2% −5% 0.768 96% 95% 81% 64% 34% 21% 19% 20% 4% 1.92 96% 95% 88% 78% 44% 41% 19% 33% 21% 4.8 97% 95% 91% 85% 60% 58% 60% 53% 49% 12.0 97% 97% 95% 91% 77% 72% 76% 70% 71% 30.0 98% 98% 97% 94% 91% 86% 85% 85% 84% EXPERIMENT 8 Interferon- Compound CU (μM) beta (units) 3.0 1.5 0.75 0.375 0.1875 0.0938 0.0469 0.02344 0 0 97% 95% 77% 34% 16% 6% −7% 0% 0 0.2344 98% 97% 83% 49% 31% 19% −21% −7% 1% 0.4688 98% 96% 84% 56% 39% 27% 10% −3% 21% 0.9375 98% 97% 91% 73% 54% 42% 31% 15% 30% 1.875 98% 98% 95% 80% 65% 58% 65% 60% 60% 3.75 98% 98% 97% 92% 86% 81% 77% 73% 79% 7.5 99% 98% 98% 96% 93% 93% 93% 90% 92% 15.0 99% 99% 99% 97% 97% 96% 97% 95% 96% 30.0 99% 99% 99% 99% 98% 99% 98% 98% 97% EXPERIMENT 9 Interferon- alpha-2B Compound EP (μM) (units) 8 4 2 1 0.5 0.25 0.125 0.0625 0 0 94% 96% 96% 92% 64% 36% 23% 8% 0 0.0492 95% 96% 96% 91% 67% 25% 28% 8% 3% 0.1229 95% 97% 96% 91% 65% 44% 4% 11% 4% 0.3072 95% 97% 96% 91% 71% 46% 20% 8% 20% 0.7680 96% 97% 97% 93% 75% 49% 36% 24% 24% 1.92 96% 97% 97% 94% 82% 67% 49% 52% 54% 4.8 96% 98% 97% 96% 90% 79% 75% 75% 70% 12 97% 98% 98% 97% 94% 89% 89% 87% 83% 30 97% 98% 98% 98% 96% 94% 94% 95% 92% EXPERIMENT 10 Interferon- alpha-2B Ribavirin (μM) (units) 200 80 32 12.8 5.12 2.048 0.8192 0.3277 0 0 85% 62% 43% 3% −8% −17% −22% −6% 0 0.0492 87% 66% 48% 44% 11% −4% −10% 11% −7% 0.1229 84% 64% 53% 40% 26% −12% −5% 11% −9% 0.3072 86% 70% 62% 44% 28% 1% 6% 14% 7% 0.7680 90% 80% 72% 65% 38% 30% 28% 44% 29% 1.92 93% 85% 77% 76% 61% 57% 58% 50% 46% 4.8 96% 92% 87% 83% 82% 74% 71% 77% 72% 12 97% 95% 93% 91% 90% 89% 90% 89% 85% 30 98% 97% 96% 95% 94% 94% 93% 95% 94% As shown in FIGS.", "4-13, which graphically depict the data in Table 8 plotted using the above-described mathematical method for measuring synergy, the Isobol curves for all combinations of HCV serine proteae inhibitors and interferons tested are concave-up, indicating that the antiviral effect of the treatments in the Replicon Assay is synergistic.", "These results are tabulated in Table 9, which shows relative levels of synergy for combination treatment and IC50 values for antiviral compounds used individually.", "The key elements in Table 9 are the a values, and the p-values for the determinations.", "The α term is a measure of the maximum inflection of the Isobols for each combination treatment.", "An α value of zero indicates additivity, a negative value indicates antagonism, and as is the case in the combination treatments with the HCV serine protease inhibitors and interferons shown above, a value greater than one indicates synergy.", "The larger the a parameter, the greater the synergy.", "As shown in Table 9 for the combinations of HCV serine protease inhibitors and interferons, even ignoring significance levels in each experiment, a t test based on the 9 experiments for the average alpha value being 0 (no interaction) has a p-value of 0.00014, indicating that the results are highly significant.", "The calculation of synergy based on the method of Greco Rustom ((1990) Application of a New Approach for the Quantitation of Drug Synergism to the Combination of cis-Diamminedichloroplatinum and 1-β-D-Arabinofuranosylcytosine, Cancer Research, 50, 5318-5327) used in this analysis is an ideal tool for evaluation of the kind of experimental data that can be generated using the HCV Replicon Assay.", "There are other methods that are applied to studies of antiviral compounds such as Pritchard and Shipman (Prichard, M. N., and Shipman, C. Jr., (1990) “A three-dimensional model to analyze drug-drug interactions (review),” Antiviral Res.", "14: 181-206).", "Application of their synergy calculation method to the data shown in Table 8 also indicates combination treatment of the replicon cells with an HCV serine protease inhibitor and interferon will result in a synergistic inhibition of HCV replicon RNA accumulation (data not shown).", "TABLE 9 RELATIVE LEVELS OF SYNERGY FOR COMBINATION TREATMENT AND IC50 VALUES FOR ANTIVIRAL COMPOUNDS USED INDIVIDUALLY HCV Serine Protease IC50 INF IC50 HSPI α P-value Inhibitor (HSPI) Interferon (units) (μM) (SE)1 α > 0 Experiment 1 Compound CU IFN alpha-2B 2.05 0.469 0.477 (0.09) <0.0001 Experiment 2 Compound CU IFN alpha-2A 3.72 0.446 0.770 (0.12) <0.0001 Experiment 3 Compound CU IFN alpha-2B 2.36 0.587 0.730 (0.08) <0.0001 Experiment 4 Compound CU IFN alpha-2A 5.67 0.633 0.438 (0.08) <0.0001 Experiment 5 Compound CU IFN tau 13.22 0.605 0.328 (0.07) <0.0001 Experiment 6 Compound EC IFN alpha-2B 2.53 0.384 0.516 (0.10) <0.0001 Experiment 7 Compound EC IFN alpha-2A 5.50 0.312 1.24 (0.20) <0.0001 Experiment 8 Compound CU IFN beta 1.82 0.466 0.551 (0.09) <0.0001 Experiment 9 Compound EP IFN alpha-2B 3.06 0.426 0.490 (0.12) <0.0001 Experiment 10 Ribavirin IFN alpha-2B 1.22 145 −0.24 (0.067) 0.0004 1(SE) Standard Error Another measure for evaluating the synergistic nature of anti-HCV drug treatment using the present HCV serine protease inhibitors and interferons is to use the same methods described above to evaluate the current standard combination therapy for HCV, i.e., interferon alpha-2B in combination with Ribavirin in the Replicon Assay.", "The last line of Table 9 shows that the α parameter for a mixture of interferon alpha-2B and Ribavirin is a negative number, indicating that there is a small amount of antagonism between these two drugs.", "This further emphasizes the significance of the combination treatments disclosed herein employing the present HCV serine protease inhibitors in combination with interferons in that these treatments clearly produce synergy, while the standard combination therapy in use for HCV (interferon alpha-2B in combination with Ribavirin) is not synergistic in the Replicon Assay.", "The foregoing comparison of combination treatments employing the present HCV serine protease inhibitors plus interferons versus Ribavirin plus interferon in the Replicon Assay clearly indicates that the former are synergistic, while the latter is not.", "The experimental results obtained using the Replicon Assay indicate that a much lower dose of interferon would be efficacious if the interferon is used in combination with a HCV serine protease inhibitor than is needed when interferon alpha-2B is used in combination with Ribavirin.", "The Replicon Assay is a useful model system in which to test potential anti-HCV compounds, and is currently widely relied upon as an effective predictor of compound anti-HCV activity.", "Note, for example, Blight et al.", "(2000) Efficient Initiation of HCV RNA Replication in Cell Culture.", "Science 8; 290:1972-1974, and Chung et al.", "(2001) Hepatitis C virus replication is directly inhibited by IFN-α in a full-length binary expression system.", "Proc.", "Nat.", "Acad.", "Sci.", "U.S.A. 98(17):9847-52.Ribavirin alone is marginally effective in reducing the accumulation of HCV replicon RNA in the Replicon Assay (Table 8, Experiment 10 and last line of Table 9).", "This result is in apparent conflict with in vivo studies where, when used by itself, Ribavirin has no significant therapeutic value for the treatment of HCV.", "In contrast, in the Replicon Assay, correcting for cytotoxicity as discussed below, Ribavirin has an IC50 of 145 μM.", "This result can be explained by recognizing that the Replicon Assay permits evaluation of high Ribavirin concentrations that would not be possible in human therapy due to in vivo cytotoxicity (Chutaputti A.", "(2000) Adverse effects and other safety aspects of the hepatitis C antivirals.", "Journal of Gastroenterology and Hepatology.", "15 Suppl:E156-63).", "This evaluation necessarily requires assessment of the cytotoxicity of Ribavirin.", "Such toxicity occurs in patients and in cellular assays (Shiffman M. L., Verbeke S. B., Kimball P. M. (2000) Alpha interferon combined with ribavirin potentiates proliferative suppression but not cytokine production in mitogenically stimulated human lymphocytes.", "Antiviral Research.", "48(2):91-9).", "In the experiments disclosed herein, Ribavirin cytotoxicity in the Replicon Assay is observed and measured in two ways.", "In both the XTT metabolic assay to determine replicon cell viability (Roehm N. W., Rodgers G. H., Hatfield S. M., Glasebrook A. L. (1991) An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.", "Journal of Immunol.", "Methods.", "142(2):257-65) and in the TaqMan® quantitiative RT-PCR assay that measures glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in treated versus untreated cells in the Replicon Assay (Drink N., Szamel M., Young A. R., Wittern K. P., Bergemann J.", "(2000) Comparative quantification of IL-1beta, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human skin in vivo.", "Inflammation Research.", "49(6):290-6), significant Ribavirin-induced cytotoxicity is observed, but is corrected for as follows.", "It is assumed that the level of GAPDH mRNA, which is a constitutively expressed housekeeping gene, is the same in all viable cells.", "It is known from measurements of GAPDH mRNA levels in cells treated with the transcription inhibitor actinomycin D that the half life of GAPDH mRNA is only a few hours (data not shown).", "Thus, it is postulated, as done by others using TaqMan® technology to determine the levels of particular mRNAs in human cells, that GAPDH mRNA levels are proportional to viable cell numbers (VCN) in any given sample, with the relationship of VCN=2{circumflex over ( )}(40-CtGAPDH mRNA).", "The VCN is computed for each of the Replicon Assay sample wells, and then we divide the HCV Replicon RNA copy number for a specific well by the VCN for that well.", "Once computed, this ratio is used instead of the HCV copy number to compute inhibition (“Avg.", "Inh using ratio”; FIG.", "14.A).", "Without correcting the Replicon Assay data for this cytotoxicity, such cytotoxicity is read as false positive inhibition of HCV RNA replicon accumulation.", "In the Replicon Assay, it is assumed that the measured inhibition of HCV RNA replicon accumulation is the sum of the actual inhibition of HCV RNA replicon accumulation and the apparent inhibition of HCV RNA replicon accumulation due to cytoxicity.", "It is furthermore assumed that, based on the close correlation of the XTT and TaqMan® GAPDH mRNA measures of cytotoxicity, the inhibition of accumulation of GAPDH mRNA caused by compounds tested in the Replicon Assay is a reliable measure of apparent inhibition of HCV RNA replicon accumulation due to cytoxicity.", "Thus, the true anti-HCV activity of a compound in the Replicon Assay corrected for general cytotoxicity can be estimated by dividing the number of HCV replicon RNA molecules measured in each sample by the VCN, thus normalizing to the number of viable cells in each sample.", "Using this method, FIG.", "14.A shows an estimate of true Ribavirin anti-HCV activity in the Replicon Assay (“Avg.", "Inh using ratio”).", "The estimate of the IC50 for Ribavirin is best calculated using this method.", "In FIG.", "14.A, “Avg.", "Inh original” shows the uncorrected IC50 for Ribavirin, which is approximately 80 μM, whereas the corrected IC50 value calculated from the “Avg.", "Inh using ratio” curve is approximately 145 μM.", "Note that the difference between corrected and measured inhibition of HCV RNA replicon accumulation as a result of interferon alpha-2B treatment (FIG.", "14.B) is insignificant in view of the ˜20% % CV of the Replicon Assay.", "Like interferon alpha-2B, the HCV serine protease inhibitors tested in the present example exhibit no significant cytotoxicity at the concentrations employed.", "This is determined using XTT assays, in which the TC50 values for the various compounds are: CU=64.7 μM, EP>10 μM, and EC>50 μM.", "These TC50 values are 20-140 fold greater than the IC50 values shown in Table 9.Thus, cytotoxicity of these compounds has no significant effect on HCV RNA accumulation in the Replicon Assay within the precision of the assay because such cytotoxicity occurs only at HCV serine protease inhibitor concentrations significantly greater than those tested in the Replicon Assay.", "Conclusions Regarding the Efficacy of HCV Serine Protease Inhibitors and Interferons, Individually and in Combination The anti-HCV activities of the present HCV serine protease inhibitors and various interferons when used alone in the HCV Replicon Assay are shown in the columns and lines of the individual experiments making up Table 8 that employ only one antiviral agent.", "Table 9 lists the IC50 values measured for each antiviral compound when tested alone.", "The foregoing results, derived via use of the in vitro Replicon Assay, also demonstrate that combination treatment of replicon cells with HCV serine protease inhibitors of the present invention and various interferons yields synergistic antiviral effects.", "It is fully expected that these effects will translate into in vivo effectiveness.", "Combination therapy employing HCV serine protease inhibitors of the present invention possesses several major advantages over single drug therapy.", "First, by making treatment possible with lower doses of the individual drugs than would be possible if used alone, one would expect a reduction in toxicity and side effects associated with treatment.", "This is especially important in the case of interferon therapy, where the side effects are severe, and have been shown to be proportional to the dose administered to patients.", "The foregoing data indicate that a dose of HCV serine protease inhibitor such as CU at the IC95 level could be combined with a dose of interferon alpha, for example at the IC50 level, and the result would be much more effective therapy than could be achieved with the HCV serine protease inhibitor alone without the adverse side effects caused by high doses of interferon alpha.", "A second major benefit of combination therapy is that because the two drugs act independently, there is less chance of development of mutant HCV strains that resist treatment.", "Development of resistance is a major concern with RNA viruses like HCV.", "Because of their high rate of mutation, such viruses can rapidly adapt to environmental stresses.", "A third benefit of combination therapy may be reduced cost, due to the need for lower amounts of therapeutic agents required for effective treatment.", "Additional immunomodulators that can be employed in the methods disclosed herein include, for example, alpha interferon 2A, consensus interferon, tau interferon, interferon+Ribavirin (Rebatron), pegylated interferon, and promoters of interferon gene expression.", "It is fully anticipated that the anti-HCV activity of these compounds will be improved when used in combination with HCV serine protease inhibitors such as those disclosed herein.", "As interferons are known to be active in vivo and in the Replicon Assay, it is expected that the present HCV serine protease inhibitors will also be active in vivo, and more importantly, be capable of eliciting synergistic activity when used in combination with interferons, immune system stimulators thereof, or other compounds having HCV antiviral activity that act by a mechanism other than inhibition of the HCV serine protease.", "The best current therapy for HCV employs interferon alpha and the nucleoside analog Ribavirin.", "This treatment is only marginally effective, and results in significant side effects that diminish patient compliance (Chronic Hepatitis C: Current Disease Management, U.S. Department of Health and Human Services, National Institutes of Health, 1999).", "Additionally, in -transplant patients, it is not clear the Ribavirin-interferon combination works, and may in fact be worse than interferon alone (Chronic Hepatitis C: Current Disease Management, U.S. Department of Health and Human Services, National Institutes of Health, 1999).", "The results presented above demonstrate a synergistic combination effect when interferons are used with a new class of HCV antivirals, the serine protease inhibitors of the present invention.", "We fully expect that the in vitro results disclosed herein will lead to more effective treatment of HCV patients than is currently possible using interferon alone.", "Sub-therapeutic doses of interferon could mobilize the patient's immune system to better fight the virus, and the serine protease inhibitor could attack the virus directly, dealing the virus a two-pronged attack via different mechanisms of action.", "Treatment of HCV infection could thus be achieved at reduced cost to the patient in terms of both diminished side effects and lower payments for necessary pharmaceutical agents as less of both drugs would be needed for effective HCV antiviral therapy.", "The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof." ] ]

[ [ "Novel diamines having a casr modulating activity", "The invention concerns diamines of general formula (I), wherein: A represents a group A1 or A2 of general formula (II); B represents a group B1 or B2 of general formula (III); X represents a SO2, CH2, C═O or COO; YZ represents a group of formula CH(R25)—CH(R26) or CH(R27)═(R28), and R1 to R28, identical or different, represent independently of one another, a hydrogen or halogen atom or an alkyl, cycloalkyl, CN, NO2, hydroxy, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino diarylalkylamino, trihalogenoalkyl or trihalogenoalkoxy group, provided that in the group A1, at least one of the radicals R1, R2, R3, R4 or R5 represents the hydrogen atom when the other four do not represent the hydrogen atom and in the group B1, at least one of the radicals R13, R14, R15, R16 or R17 represents the hydrogen atom when the other four do not represent the hydrogen atom, and their salts with a pharmaceutically acceptable acid, in the form of racemic mixture or their optically pore isomers.", "The invention also concerns their preparation, pharmaceutical compositions comprising them and their use as CaSR activity modulator and as medicine particularly designed for the treatment of psychological diseases and disorders involving CaSR activity modulation." ], [ "1-20.", "(canceled) 21.A compound comprising a diamine of general formula (I): wherein: A is A1 or A2 having the following general formula: wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that when A is A1 at least one of the radicals R1, R2, R3, R4 or R5 is hydrogen when the other four radicals are not hydrogen, B is B1 or B2 having the following general formula: wherein R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that when B is B1, at least one of the radicals R13, R14, R15, R16 or R17 is hydrogen when the other four radicals are not hydrogen, X is SO2, CH2, C═O or COO, is CH(R25)—CH(R26) or CH(R27)═CH(R28), wherein R25, R26, R27 and R28 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy; and salts thereof optionally further comprising a pharmaceutically acceptable acid, wherein the diamine is a racemic mixture of isomers or a purified isomer.", "22.The compound of claim 21, wherein A is A1, B is B2 and is CH(R25)—CH(R26), wherein R25 and R26 are both hydrogen.", "23.The compound of claim 21, wherein X is SO2.24.The compound of claim 21, wherein the diamine is selected from the group consisting of the following formulae: and the salts thereof further comprising a pharmaceutically acceptable acid.", "25.A method for preparing the compound of claim 21, wherein is CH(R25)—CH(R26) and at least one of the radicals R25 and R26 is not hydrogen, wherein at least one of the radical(s) R25 and R26 is selectively introduced into the molecule to yield the following general formula II: A is A1 or A2 having the following general formula: wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that when A is A1 at least one of the radicals R1, R2, R3, R4 or R5 is hydrogen when the other four radicals are not hydrogen, B is B1 or B2 having the following general formula: wherein R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that when B is B1, at least one of the radicals R13, R14, R15, R16 or R17 is hydrogen when the other four radicals are not hydrogen, and X is SO2, CH2, C═O or COO.", "26.A method for preparing the compound of claim 21, wherein is CH2—CH2 or CH(R27)═CH(R28) and X is CH2, C═O or COO comprising the steps of: a) selectively introducing an arylmethyl, aroyl or aryloxycarbonyl group, said groups being optionally substituted, to generate the compound depicted in formula III: wherein B is B1 or B2 having the following general formula: wherein R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that when B is B1, at least one of the radicals R13, R14, R15, R16 or R17 is hydrogen when the other four radicals are not hydrogen, and R27 and R28 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy; and b) deprotecting the compound obtained in step (a).", "27.A method for preparing the compound of claim 21, wherein X is SO2 and is CH2—CH2 or CH(R27)═CH(R28), comprising the steps of: a) subjecting the compound of formula III to a deprotection reaction, and b) introducing an optionally substituted arylsulfonyl group into the NH2 functional group of the compound obtained in step (a).", "28.The method of claim 25, wherein X is CH2, C═O or COO and the compound of formula II is prepared by the method of claim 26.29.The method of claim 25 wherein X is SO2, and the compound of formula II is prepared by the method of claim 27.30.The method of claim 26, wherein the compound of formula III is prepared by the step of nucleophilic opening of an aziridine of general formula IV: wherein is CH2—CH2 or CH(R27)═CH(R28), wherein R27 and R28 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, said nucleophilic opening being performed by a compound having the following general formula V: wherein B is B1 or B2 having the following general formula: wherein R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24 independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy, provided that in the group B1, at least one of the radicals R13, R14, R15, R16 or R17 represents the hydrogen atom when the other four radicals do not represent hydrogen.", "31.The method of claim 30, wherein the compound of formula IV is prepared by reaction between an olefin of general formula VI: wherein is CH2—CH2 or CH(R27)═CH(R28) and a compound having the following formula VII: 32.A pharmaceutical composition comprising at least one compound as claimed in claim 21 and an appropriate pharmaceutical excipient.", "33.A method of using the compound of claim 21 as a CaSR activity modulator.", "34.A method of using the compound of claim 21 as a CaSR antagonist, wherein X is selected from the group consisting of SO2, C═O and COO group.", "35.A method of using the compound of claim 21 as a CaSR agonist, wherein X is CH2.36.A method of using the compound of claim 21 for the treatment of physiological diseases or disorders linked to disturbances in CaSR activity.", "37.A method of using the compound of claim 21, wherein X is SO2, C═O or COO for the treatment of demyelinating diseases associated with the expression of CaSRs in the oligodendrocytes, osteoporosis, Paget's disease, rheumatoid arthritis, tumors associated with humoral hypercalcemia, osteoarthritis, osteosarcomas, fractures, cardiovascular, gastrointestinal, endrocrin or neurodegenerative diseases or cancers where (Ca2+)e ions are abnormally high.", "38.A method of using the compound of claim 21, wherein X is CH2 for the treatment of diseases linked to hypercalcemia, primary or secondary hyperparathyroidism, osteoporosis, cardiovascular, gastrointestinal, endocrin or neurodegenerative diseases or certain cancers where the (Ca2+)e ions are abnormally high.", "39.A compound comprising an aziridine of general formula IV: wherein is CH(R27) CH(R28), and wherein R27 and R28 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl and trihaloalkoxy." ], [ "The present invention relates to compounds having extracellular calcium ((Ca2+)e) and extracellular magnesium ((Mg2+)e) ion receptor or Calcium Sensing Receptor (CaSR) modulating activity.", "It relates in particular to a novel class of amine-containing compounds, their preparation, the pharmaceutical compositions containing them and their use as modulator of the activity of CaSRs and as medicament intended in particular for the treatment of physiological diseases or disorders involving the modulation of CaSR activity.", "The CaSR modulating activity corresponds to the capacity to produce, induce or antagonize biological responses observed by the variations in the concentration of extracellular calcium ions (Ca2+)e and of extracellular magnesium ions (Mg2+)e. This activity may be of the calcimimetic type or of the calcilytic type.", "The (Ca2+) e and (Mg2+)e ions play an important role in the body because they regulate calcium homeostasis on which depend the vital functions.", "Thus, hypercalcemia, that is to say states where the (Ca2+)e ions are above the mean threshold, have a major incidence on numerous functions such as cardiac, renal or intestinal functions.", "They deeply affect the central nervous system (Chattopadhyay et al., Endocr.", "Review, 1998, vol.", "17, p. 289-307).", "The CaSRs are proteins which are sensitive to (Ca2+)e and (Mg2+)e ions, and are present in the parathyroid glands, the kidney, the intestine, the lungs, the bone cells, the brain, the spinal cord, sensitive neurons the pituitary gland, the stomach, the keratinocytes (Brown et al., Nature, 1993, vol.", "366, p. 575-580; Ruat et al., Proc.", "Natl.", "Acad.", "Sci., USA, 1995, vol.", "92, p. 3161-3165; Brown et al., Ann.", "Rev.", "Med., 1998, vol.", "49, p. 15-29).", "These proteins are encoded by a single gene isolated from various animal species.", "They belong to the family of G protein-coupled receptors with seven transmembrane domains, and exhibit structural homologies with the metabotropic glutamate receptors, GABAB receptors, hypothetical pheromone and taste receptors.", "Activating or inhibitory mutations of the gene in humans are responsible for extremely serious genetic diseases which cause hypocalcemia or hypercalcemia (Pollack et al., Cell, 1993, vol.", "75, p. 1297-1303; Pollack et al., Nature Genetics, 1994, vol.", "8, p. 303-307; Brown et al., Ann.", "Rev.", "Med., 1998, vol.", "49, p. 15-29).", "The functions linked to the expression of these proteins in the tissues are not yet all known and are the subject of a very high research activity, particularly as regards the CaSRs present in the parathyroid and thyroid glands, the kidney, the intestine, the spinal cord, the brain and the bone cells.", "In the parathyroid gland, the CaSRs modulate the secretion of the parathyroid hormone (PTH) which is the main regulator of calcium homeostasis: the increase in (Ca2+)e ions in the serum will activate the CaSRs present on the cells of the thyroid gland and reduce the secretion of the PTH hormone.", "Complementary DNA encoding rat CaSR has been isolated from a rat striatum cDNA library (Ruat et al., Proc.", "Natl.", "Acad.", "Sci., 1995, vol.", "92, p. 3161-3165).", "This receptor is identical with respect to its amino acid sequence to that expressed in the other tissues.", "Transfected Chinese hamster ovary_(CHO) cells expressing rat CaSR(CHO(CaSR)) have been characterized and the chemical signals (second messengers) induced by the activation of this receptor have been analyzed.", "Thus, a biochemical test which makes it possible to measure the accumulation of tritiated inositol phosphates [3H]IP in response to the activation of the receptor has been developed (Ruat et al., J. Biol.", "Chem., 1996, vol.", "271, p. 5972-5975; Ferry et al., Biochem.", "Biophys.", "Res.", "Commun., 1997, vol.", "238, p. 866-873).", "It has been shown that the Ca2+, Mg2+, but also Ba2+ ions in millimolar concentration ranges stimulate the CaSRs.", "The activation of the CaSRs could be induced in the brain by the β-amyloid peptides which are involved in neurodegenerative diseases such as Alzheimer's disease (Ye et al., J. Neurosci.", "Res., 1997, vol.", "47, p. 547-554).", "Disruption of the CaSR activity is associated with biological disorders such as osteoporosis, Paget's disease, rheumatoid arthritis, tumors associated with humoral hypercalcemia, osteoarthritis, osteosarcomas, fractures, primary and secondary hyperparathyroidism, osteoporosis, cardiovascular, gastrointestinal, endrocrine or neurodegenerative diseases, or certain cancers in which (Ca2+)e ions are abnormally high.", "Secondary hyperparathyroidism is observed during chronic renal insufficiency and is characterized by hyperplasia of the parathyroid glands and an increase in circulating PTH.", "Renal insufficiency is also accompanied by renal osteodystrophy which is characterized by bone disorders with a high or low renewal of the bone mass (osteitis fibrosa, osteomalacia).", "Osteoporosis is a multifactorial disease which depends in particular on the age and the gender.", "While menopausal women are very highly affected, osteoporosis is found increasingly to be a problem in old men, and there are currently no truly satisfactory treatments.", "Its social cost could increase further in the coming years, particularly in our European society where the lifespan is increasing.", "Osteoporosis is currently treated with estrogens, calcitonin or biphosphonates which prevent bone resorption without stimulating new bone growth.", "More recent data demonstrate that intermittent increases in PTH or its derivatives are effective in the treatment of osteoporosis and make it possible to remodel the bone by stimulating bone formation (Whitfield et al., R.G.", "Landes Co., Austin, USA, 1998).", "This novel therapeutic route for the treatment of osteoporosis appears very advantageous although major problems are linked to the use of the PTH hormone such as the route of injection, but also the appearance of tumors which have been recently observed during clinical trials in humans.", "The intermittent secretion of endogenous PTH may be obtained by blocking the calcium receptor with the aid of an antagonist molecule, which is beneficial in the treatment of osteoporosis.", "The secretion of PTH may be blocked by CaSR agonists.", "This blocking may be followed by a rapid increase in PTH (rebound effect), which is also beneficial in the treatment of osteoporosis.", "Taking into account the important role of calcium homeostasis, numerous CaSR modulators have already been used.", "Thus, the company NPS Pharmaceutical has developed two main types of family of organic compounds as CaSR agonists, namely polyamines such as NPS 019 (3), and the arylalkylamines, small size molecules of which the best known representative to date is NPS R-568 (2).", "The compound NPS R-568 was developed from the structure of Fendiline (1), a potent activator of CaSR of the parathyroid gland.", "The compound NPS-R-568 reduces or eliminates osteitis fibrosa in rats (Wada et al., Kidney International, 1998, vol.", "53, p. 448-453) and reduces the PTH concentrations in patients (men) suffering from chronic renal insufficiency (Antansen et al., Kidney International, 1998, vol.", "53, p. 223-227).", "This compound was successfully used orally to reduce the concentrations of PTH and of free serum Ca2+ ions in menopausal women suffering from primary hyperparathyroidism (Silverberg et al., New Engl.", "J.", "Med., 1997, vol.", "337, p. 1506-1510).", "In another study, the compound NPS-R-568 made it possible to reduce between 20-50% the cell proliferation observed in the parathyroid gland in a rat model reproducing chronic renal insufficiency (Wada et al., J. Clin.", "Invest., 1997, vol.", "100, p. 2977-2983).", "These studies demonstrate that a calcimimetic compound, which is active toward the calcium receptor present on the parathyroid gland, may be considered as an advantageous therapeutic tool for treating certain forms of primary and secondary hyperparathyroidism.", "During clinical trials (Phase I-II), the company NPS Pharmaceutical observed a low bioavailability of the compound NPS-R-568 as well as variable clinical effects according to the individuals which could result from polymorphism of the gene encoding CaSR in humans (Nemeth et al., Trends Endoc.", "Metab, 1999, vol.", "10, p. 66-71).", "Furthermore, during experimental trials in rats, the NPS R-467 compound, (an analog of NPS R-568 (2) in which the chlorine is replaced with a hydrogen) (Nemeth et al., PNAS (USA), 1998, vol.", "95, p. 4040-4045) a compound having a structure similar to NPS R-568, proved more selective toward the receptors for the parathyroid compared to those for the thyroid gland.", "This selectivity can be explained by differences linked to the tissues, which suggests that calcimimetic molecules specific for a tissue may be synthesized and may have considerable clinical importance.", "In parallel, the inventors recently reported the preparation and the calcimimetic activity of arylalkyl-1,2-diamines having the general structure described below (4), and among which the compound PHD 321 (5) constitutes one of the most active products.", "The inventors also recently reported the synthesis of compounds of the following general formula (6): in which: X represents a group —NR4, —CH═N— or —CH(R5)—N(R4)—, Y represents an oxygen or sulfur atom or a group —CR5, —CH(R5), —C(R5)═C(R6)—, —CH(R5)—CH(R6)— or NR provided that when X represents the group —CH═N— or —CH(R5)—N(R4)—, Y represents an oxygen or sulfur atom or the group NR, —C(R5) or —CH(R5), R represents a hydrogen atom, an alkyl, aryl or aralkyl group, R1, R5 and R6, which may be identical or different, each represent a hydrogen or halogen atom or an alkyl or alkoxy group, R2 represents a hydrogen atom or an alkyl group, R3 represents an aryl group and R4 represents a hydrogen atom, an alkyl, aryl, aralkyl, alkylsulfonamide, arylsulfonamide or aralkylsulfonamide group.", "As regards the CaSR antagonists, the companies SMITHKLINE BEECHAM and NPS Pharmaceutical have reported in recent patents the preparation of the compounds having the general structure 7 (WO9737967; WO9845255; WO9951241; WO9951569) and of compounds of the pyridinium type (WO9844925).", "Among these, the most active as calcilytic agents have an IC50 of less than 10−7 M. Such products open new therapeutic windows for the treatment of osteoporosis.", "However, their arylopropanolamine structure, close to those of β-adrenergics induces an undesirable residual activity at this level.", "The complete absence of CaSR modulating molecules in clinical medicine and the problems encountered in phase I-II for first-generation calcimimetics underline the need to find novel molecules which modulate CaSR activity.", "Consequently, the inventors set themselves the aim of preparing compounds which modulate CaSR activity and which do not possess the abovementioned disadvantages.", "The present invention therefore relates to diamines of the following general formula (I): in which: A represents a group A1 or A2 of the following general formula: where R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 and R12, which are identical or different, represent, independently of each other, a hydrogen or halogen atom or an alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl or trihaloalkoxy group, provided that in the group A1, at least one of the radicals R1, R2, R3, R4 or R5 represents the hydrogen atom when the other four do not represent the hydrogen atom, B represents a group B1 or B2 of the following general formula: where R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23 and R24, which are identical or different, represent, independently of each other, a hydrogen or halogen atom or an alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl or trihaloalkoxy group, provided that in the group B1, at least one of the radicals R13, R14, R15, R16 or R17 represents the hydrogen atom when the other four do not represent the hydrogen atom, X represents an SO2, CH2, C═O or COO group, represents a group of formula CH(R25)—CH(R26) or CH(R27)═CH(R28) where the groups —R25, R26, R27 and R28, which are identical or different, represent, independently of each other, a hydrogen or halogen atom or an alkyl, cycloalkyl, CN, NO2, hydroxyl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, arylalkylamino, diarylamino, diarylalkylamino, trihaloalkyl or trihaloalkoxy group, and their salts with a pharmaceutically acceptable acid, in the form of a racemic mixture or of their optically pure isomers.", "Advantageously, A represents the group A1, B represents the group B2 and represents the group CH(R25)—CH(R26) where R25 and R26 each represent a hydrogen atom.", "Still more advantageously, X represents the SO2 group.", "Particular examples of diamines according to the present invention are those chosen from the group consisting of the diamines of formulae (Ia) to (Id): and their salts with a pharmaceutically acceptable acid.", "The term “pharmaceutically acceptable acid” is understood to mean, for the purposes of the present invention; any nontoxic acid, including organic and inorganic acids.", "Such acids include acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric and para-toluenesulfonic acids.", "Hydrochloric acid is particularly preferred.", "The term “alkyl group” is understood to mean, for the purposes of the present invention, any substituted or unsubstituted, linear or branched C1-C6 alkyl group, in particular the methyl group.", "The term “cycloalkyl group” is understood to mean, for the purposes of the present invention, any substituted or unsubstituted, linear or branched C3-C6 cycloalkyl group.", "The term “alkoxy group” is understood to mean, for the purposes of the present invention, any alkoxy group of 1 to 6 carbon atoms, which are linear or branched, substituted or unsubstituted, in particular the —OMe group.", "The term “aryl group” is understood to mean, for the purposes of the present invention, one or more aromatic rings having 5 to 8 carbon atoms, which may be joined or fused, it being possible for said rings to be substituted.", "In particular, the aryl groups may be phenyl or naphthyl groups.", "The term “aralkyl group” is understood to mean, for the purposes of the present invention, any aryl group as defined above, linked via an alkyl group as defined above.", "In particular, an aralkyl group is a benzyl or naphthylmethyl group.", "The term “aryloxy group” is understood to mean, for the purposes of the present invention, any aryl group as defined above, linked to an oxygen atom.", "The term “alkylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with a single alkyl group as defined above.", "The term “dialkylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with two alkyl groups, which are identical or different, as defined above.", "The term “cycloalkylamino” is understood to mean, for the purposes of the present invention any amine group in which the two substituents are linked to each other to form a C2-C6 cyclic aliphatic chain.", "The term “arylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with a single aryl group as defined above.", "The term “arylalkylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with a single arylalkyl group as defined above.", "The term “diarylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with two aryl groups, which are identical or different, as defined above.", "The term “diarylalkylamino” is understood to mean, for the purposes of the present invention, any amine group substituted with two aralkyl groups, which are identical or different, as defined above.", "The term “trihaloalkyl” is understood to mean, for the purposes of the present invention, any alkyl group as defined above, substituted with three halogen atoms.", "The term “trihaloalkoxy” is understood to mean, for the purposes of the present invention, any alkoxy group as defined above, substituted with three halogen atoms.", "Preferred examples of a halogen atom are Cl and F. The compounds according to the invention all have an asymmetric carbon and can therefore exist in the form of optical isomers.", "The present invention comprises these isomers pure or as a mixture.", "The present invention also relates to the method of preparing these compounds.", "The preparation of the molecules may be simply carried out in 4 or 5 stages depending on of the general formula I.", "In all cases, the first two stages are the following: a) reaction, advantageously in the presence of CuI or II, between an olefin of general formula VI: in which: represents the group CH2—CH2 or the group CH(R27)═CH(R28) where R27 and R28 are as defined in formula I, and the compound of formula VII: to give the aziridine of general formula IV: in which represents the group CH2—CH2 or the group CH(R27)═CH(R28) where R27 and R28 are as defined in formula I, b) nucleophilic opening of the aziridine of general formula IV by the compound of the following general formula V: in which B is as defined in formula I, to give the diamine of general formula III in which: represents the group CH2—CH2 or the group CH(R27)═CH(R28) and B, R27 and R28 are as defined in formula I.", "These two stages are carried out by methods well known to persons skilled in the art.", "The following stages depend on the type of radical X desired.", "To obtain the diamines of general formula I in which X represents the CH2, C═O or COO group, respectively, and represents the group CH2—CH2 or the group CH(R27)═CH(R28), method a)b) comprises, in addition, the following stages: c1) selective introduction of an arylmethyl, aroyl or aryloxycarbonyl group, respectively, depending on the radical X which it is desired to introduce, said groups being optionally substituted, into the compound of formula III, and d1) deprotection of the compound obtained.", "Stages c1) and d1) are carried out by methods well known to persons skilled in the art.", "To obtain the diamines of general formula I in which X represents respectively the SO2 group and represents the group CH2—CH2 or the group CH(R27)═CH(R28), method a)b) comprises, in addition, the following stages: c2) the compound of formula III undergoes a deprotection reaction, and d2) an optionally substituted arylsulfonyl group is introduced into the NH2 functional group of the compound thus obtained.", "Stages c2) and d2) are carried out by methods well known to persons skilled in the art.", "To obtain the diamines of general formula I in which represents the group CH(R25)—CH(R26) where at least one of the radicals R25 and R26 does not represent the hydrogen atom, the method comprises, in addition, either after stage d1), or after stage d2), depending on the radical X desired, a final stage consisting in the selective introduction of the R25 and R26 radical(s) which do not represent the hydrogen atom into the molecule of the following general formula II: in which: A, B and X are as defined in formula I.", "The introduction of the R25 and/or R26 radical(s) may be carried out by methods well known to persons skilled in the art depending on the type of radical desired, in particular by epoxidation, aziridination, dihydroxylation, aminohydroxylation or Heck reaction.", "The olefins of general formula VI and the compound of general formula V are easily commercially available.", "The compound of formula VII may be easily produced according to the method described by Evans et al.", "in Journal of the American Society, 1994, vol.", "116, p. 2742.The simplicity of the method of preparation of the compounds of formula I as described above and its very good yield make it possible to introduce a large variety of substituents R1 to R28.The present invention also relates to pharmaceutical compositions comprising, as active ingredient, at least one of the diamines according to the present invention and an appropriate excipient.", "Such compositions may also comprise other active ingredients.", "These compositions may be formulated for administration to mammals, including humans.", "The dosage varies according to the treatment and according to the condition in question.", "These compositions are prepared so as to be administrable by the digestive or parenteral route.", "In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredient may be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals or to human beings.", "The appropriate unit forms for administration comprise the forms for administration by the oral route such as tablets, gelatin capsules, powders, granules and oral solutions or suspensions, the forms for sublingual and buccal administration, the forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and the forms for rectal administration.", "When a solid composition is prepared in the form of tablets, the main active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like.", "It is possible to coat the tablets with sucrose or other appropriate materials or it is possible to treat them such that they have a prolonged or delayed activity and they continuously release a predetermined quantity of active ingredient.", "A preparation in gelatin capsules is obtained by mixing the active ingredient with a diluent and by pouring the mixture obtained into soft or hard gelatin capsules.", "A preparation in syrup or elixir form may contain the active ingredient together with a sweetener, an antiseptic, as well as a taste enhancer and an appropriate coloring agent.", "The water-dispersible powders or granules may contain the active ingredient mixed with dispersing agents or wetting agents, or suspending agents, and with flavor correctors or sweeteners.", "For rectal administration, suppositories are used which are prepared with binders which melt at rectal temperature, for example cocoa butter or polyethylene glycols.", "For parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically acceptable dispersing agents and/or wetting agents are used.", "The active ingredient may also be formulated in the form of microcapsules, optionally with one or more carrier additives.", "The present invention also relates to the use of these diamines and of the pharmaceutical compositions containing them as modulator of CaSR activity.", "Advantageously, the diamines according to the invention, for which X represents the SO2, C═O or COO group, may be used as CaSR antagonists and those for which the group X represents the CH2 group as CaSR agonists.", "CaSRs can in particular be found in the parathyroid gland, the thyroid, the bone cells, sensitive neurones, the stomach, the lungs, the kidney, the pituitary gland, the brain, the hypothalamus, the olfactory surfaces or the hippocampus.", "The diamines according to the invention and the pharmaceutical compositions comprising them can be used as a medicament, in particular for the treatment of physiological diseases or disorders linked to disturbances in the CaSR activity.", "Still more particularly, in the case of the diamines for which X represents the SO2, C═O or COO group, these physiological diseases or disorders are of the type including demyelinating diseases associated with the expression of CaSRs in the oligodendrocytes, osteoporosis, Paget's disease, rheumatoid arthritis, tumors associated with humoral hypercalcemia, osteoarthritis, osteosarcomas, fractures, cardiovascular, gastrointestinal, endrocrin or neurodegenerative diseases or cancers where (Ca2+)e ions are abnormally high and in the case of the diamines for which X represents the CH2 group, these physiological diseases or disorders are of the type including diseases linked to hypercalcemia, primary or secondary hyperparathyroidism, osteoporosis, cardiovascular, gastrointestinal, endocrin or neurodegenerative diseases or certain cancers where the (Ca2+) e ions are abnormally high.", "Secondary hyperparathyroidism is more particularly observed during chronic renal insufficiency.", "The present invention also relates to the aziridines of general formula IV: in which: represents the group CHR27=CHR28 where R27 and R28 are as defined in formula I.", "The following example of synthesis of a compound according to the invention is given without limitation and illustrates the invention.", "Synthesis of N-p-(methoxybenzenesulfonyl)-7-azabicyclo-[4.1.0]heptane To a solution of copper trifluoromethanesulfonate (56 mg; 0.01 mmol) in 4 ml of acetonitrile distilled over CaCH2, in the presence of an activated molecular sieve, are successively added, at 0° C. under argon, cyclohexene (0.51 ml; 5 mmol) and, in portions over a period of 3 hours, [N-(p-methoxyphenylsulfonyl)imino]-phenyliodinane (Ph1=NSO2Ph-p-MeO) (0.390 g; 1 mmol).", "The heterogeneous mixture, which is green in color, is stirred at 0° C. for 6 hours before being filtered on silica (eluent: ethyl acetate) in order to remove the molecular sieve and the copper salts.", "After evaporation of the solvents, the yellow oily residue is purified by chromatography on silica (eluent: heptane/ethyl acetate: 4/1) to give 0.120 g (0.45 mmol; 45%) of an impure white solid.", "Mass spectroscopy (Es)=m/z=267 [M+H]+ Synthesis of N1-(4-methoxybenzenesulfonyl)-N-2-[1-(1-naphthyl)ethyl]cyclohexane-1,2-diamine: PHD 263 To a solution of aziridine prepared beforehand (80 mg; 0.30 mmol) in 1 ml of tetrahydrofuran (THF), are successively added triethylamine (0.009 ml; 0.06 mmol) and 1-(1-naphthyl)ethylamine (0.145 ml; 0.90 mmol).", "After stirring for 3 days at 60° C., the medium is concentrated before being purified on a silica column (eluent: heptane/ethyl acetate: 1/1).", "125 mg (0.285 mmol; 95%) of the compound of aziridine opening are isolated in the form of a colorless foam.", "The diamine is then converted to its hydrochloride by treatment with a solution of HCl in methanol.", "Mass spectrometry (ES): m/z: 439 [M+H]+ Melting point: 135-140° C. (decomposition) The other compounds of Table 1 are prepared in the same manner as PHD 263.TABLE 1 Structure of various compounds according to the invention Empirical formula Reference Molar mass Structure PHD 263 C25H30N2O3S.HCl 475.05 PHD 346 C25H29ClN2.2HCl 485.89 PHD 348 C25H29FN2.2HCl 449.44 PHD 349 C26H32N2O4S.HCl 505.08 PHD 350 C24H27ClN2O3S.HCl 479.47 PHD 397 C24H27N3O4S 453.57 PHD 401 C25H27F3N2O3S.HCl 529.03 PHD 403 C25H27F3N2O2S.HCl 513.03 PHD 404 C28H30N2O2S 458.63 PHD 406 C24H27ClN2O2S.HCl 479.47 PHD 408 C24H28N2O2S.HCl 445.02 PHD 511 C25H26N2O3S.HCl 473.04 AK 115 C26H32N2O4S.HCl 505.08 AK 116 C24H27N2O2SCl.HCl 479.47 AK 117 C24H25N2O2SCl3.HCl 548.35 Activity on Transfected Cells Expressing the Receptor Sensitive to Extracellular Calcium (Ca2+)e Ions 1.Procedure The calcilytic or calcimimetic activity of compounds according to the invention was estimated by measuring the inhibition of the accumulation of tritiated inositol phosphates induced by 9 mM extracellular calcium ions in the presence of 10 μM of each of the compounds in CHO(CaS) cells.", "The technique for measuring the accumulation of tritiated inositol phosphates [3H]IP which is used is that described in Ruat et al., J. Biol.", "Chem., 1996, vol.", "271, p. 5972-5975.Only the mode of incubating the compounds is modified.", "After a preincubation of 15 min of the compounds according to the invention in the presence of a basal concentration of (Ca2+)e equal to 2 mM, the compounds are incubated for 30 min in the presence of a (Ca2+)e concentration of 4 or 9 mM.", "The results, mean of 2 to 4 independent experiments each carried out in triplicate, are grouped together in Table 2.2.Result The activity of the compounds is expressed as a percentage of the control activity measured in the presence of (Ca2+)e alone of concentration 4 mM or 9 mM.", "The compound PHD 350 leads to a 25% inhibition of the accumulation of [3H]IP induced by 9 mM (Ca2+) et PHD 401 leads to an 82% inhibition under the same conditions and the compound PHD 263, for its part, inhibits the same response by 64%.", "By contrast, the compounds PHD 346 and PHD 348 exhibit a calcimimetic activity since they activate the production of [3H]IP.", "TABLE 2 Antagonist or agonist activity of the compounds according to the invention on the accumulation of tritiated inositol phosphates [3H]IP which is induced by 9 mM Ca2+.", "Accumulation of [3H]IP Compounds at 10 μM % relative to the control Control 100 PHD 346 110 ± 6 PHD 348 119 ± 12 PHD 350 74 ± 2 PHD 349 64 ± 5 PHD 408 56 ± 4 PHD 397 43 ± 1 PHD 404 39 ± 7 PHD 263 37 ± 5 PHD 406 28 ± 1 PHD 403 21 ± 1 PHD 401 18 ± 3 PHD 511 75 ± 10 AK 115 100 ± 4 AK 116 40 ± 5 AK 117 42 ± 5 Specificity of the Activity of the Molecules According to the Invention To assess the specificity of the antagonist activity of the molecules according to the invention, the effect of one of them, PHD 263, was studied under various calcium conditions, in the presence or otherwise of ATP on CHO(WT*) and CHO(CaSR) cells.", "The CHO(WT*) cells were transfected with the plasmid alone and do not express CaSR.", "The accumulation of tritiated inositol phosphates is expressed as a percentage of the basal level observed in the presence of 2 mM Ca2+ (100) in the CHO(WT*) or CHO(CaSR) cells.", "The molecules according to the invention, used alone at a concentration of 10 μM, such as PHD 263, lead to little or no accumulation of [3H]IP in the control CHO(WT*) cells or the CHO(CaSR) cells (124±10%) which suggests a weak nonspecific activity in these cells independent of the presence of CaSR.", "PHD 263 (10 μM) has the same effect on the [3H]IP response in the CHO(WT*) cells in the presence of 2 or 4 mM calcium, indicating an absence of a nonspecific effect of the compound linked to the variation of [Ca2+]e. The PHD 263 (10 μM) effect adds to that of ATP in these CHO(WT) cells; it does not therefore inhibit the [3H]IP response induced by another receptor coupled to the phospholipase C pathway." ] ]

[ [ "Short chain dehydrogenases/reductases(sdr)", "The present invention relates to a method for identifying or verifying members of the short chain dehydrogenase (SDR) family, to a method for providing modulators for members of the SDR family and to the preparation of pharmaceutical agents using these modulators." ], [ "1.A method for identifying or verifying members of the short chain dehydrogenase (SDR) family comprising the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family.", "2.The method according to claim 1, further comprising a step (e) ranking of the positive SDR candidates obtained according to the number of amino acids matching with motifs MT1, MT2, MT3, MT4 and MT5.3.The method according to claim 1, wherein in step (b) the target sequence is compared with core SDR motifs selected from (i) MT1:TGxxxGxG, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at position 90-110 relative to MT1, (iv) MT4:S(11-52:x)YxxxK and (v) MT5:PG, and wherein in step (c) positive SDR candidates are determined containing (i) at least the core SDR motifs MT1 and MT4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5.4.The method according to claim 1, wherein in step (c) positive SDR candidates are determined containing (i) at least the core SDR motifs MV1, MV4 and one of MT2, MT3 and MT5 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5.5.The method according to claim 1, wherein in step (c) positive SDR candidates are determined containing (i) the core SDR motifs MV1, MV4, MT2 and MT3 or MV1, MV4, MT2 and MT5 or MV1, MV4, MT2, MT3 and MT5.6.The method according to claim 1, wherein positive SDR candidates are determined containing the core SDR motifs MV1, MV4, MT2, MT3 and MT5.7.The method according to claim 1, wherein in step (c) positive candidates are determined containing at least 9 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4, and MT5.8.The method according to claim 1, wherein MT2 is defined as NNAG.", "9.The method according to claim 1, wherein MV4 is derived from the motif MT′4:S(11-52:x)YxASK by replacement of 0-2 amino acids.", "10.The method according to claim 9, wherein in step (c) positive candidates are determined containing at least 9 of the 16 amino acids contained in the core motifs used.", "11.The method according to claim 1, wherein MT2 and/or MT5 are extended for identifying or verifying FabG_SDRs, wherein MTy2:VxVNNAG, wherein V can be replaced and MTy5:PGFI, wherein F and/or I are used as search motif.", "12.The method according to claim 1, further comprising one or more of the following further steps: (i) three-dimensional structure comparison and (ii) biological function analysis.", "13.A member of the short-chain dehydrogenase (SDR) family identified with the method according to claim 1.14.The SDR according to claim 13, wherein it is selected from the SDRs shown in Tables 1-5.15.A method for providing modulators for members of the short chain dehydrogenase (SDR) family comprising the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family and (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family.", "16.The method according to claim 15, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) Mv4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family.", "17.The method according to claim 15, wherein in step (b) a protein sequence alignment with known SDR sequences is performed for pre-selecting possible modulators.", "18.A method for evaluation of lead-candidates for possible modulators of a member of the SDR family comprising the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) ranking the target sequences according to the number of amino acids matching with the core SDR motifs used and (c) deriving lead-candidates from metabolites of evolutionary related SDR enzymes.", "19.The method according to claim 18, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV 1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family.", "20.A method for providing a pharmaceutical agent comprising the steps (a) providing tone or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family and (c) formulating said modulators as pharmaceutical agent.", "21.The method according to claim 20, wherein step (a) comprises the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifying positive SDR candidates as belonging to the SDR family.", "22.The method according to claim 20, wherein step (b) comprises the steps (a) providing one or more target sequences of members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family and (b) providing modulators, which enhance or inhibit the activity of the members of the short chain dehydrogenase family.", "23.The method according to claim 20, wherein a modulator is provided, which enhances the activity of the members of the short chain dehydrogenase family.", "24.The method according to claim 20, wherein a modulator is provided, which inhibits the activity of the members of the short chain dehydrogenase family.", "25.The method according to claim 20, wherein the validation of a modulator or a function of a SDR enzyme found with an algorithm using core SDR motifs is performed with biochemical methods.", "26.The method according to claim 20, wherein expressed sequence tags and gene sequence comparison are used to provide a function of the member of the short chain dehydrogenase family, which has been identified or verified with an algorithm using core SDR motifs.", "27.The method according to claim 20, wherein a modulator or a function of an SDR enzyme found with an algorithm using core SDR motifs is validated high throughput function screening for function identification, UHTS for lead compounds, molecular homology modelling, substrate docking simulations, tissue expression, cDNA arrays or analysis of disease in animal or in vitro model systems.", "28.The method according to claim 20, wherein a human SDR enzyme is provided and the pharmaceutical agent is applied for therapeutic or diagnostic purposes.", "29.The method according to claim 28, wherein the human SDR enzyme is selected from the human SDRs shown in Table 1 or 2.30.The method according to claim 20, wherein an SDR from a pathogen and/or a fungi is provided to obtain a high specific pharmaceutical agent.", "31.The method according to claim 30, wherein the SDR is selected from the SDRs shown in Table 3, 4 or 5.32.The method according to claim 20, wherein an SDR enzyme with high homology is provided, which constitutes an essential enzyme.", "33.The method according to claim 20, wherein an SDR enzyme with low homology or high divergence between different species is provided, which allows for a species specific modulation.", "34.A pharmaceutical agent obtainable by a method according to claim 20.35.The pharmaceutical agent according to claim 34 for the prophylaxis, treatment and/or diagnosis of diseases.", "36.The pharmaceutical agent according to claim 34, which is a fungicide or antibiotic.", "37.A method for detection of clinically relevant polymorphisms or single nucleotide polymorphisms comprising the steps (a) providing one or more target sequences or members of the short chain dehydrogenase family based on an algorithm using core SDR motifs for searching members of the SDR family, (b) ranking the members of the short chain dehydrogenase family according to the number of amino acids matching with the core SDR motifs applied, and (c) comparing evolutionary patterns within the SDR enzymes.", "38.The method according to claim 37, wherein disease mechanisms are characterised; 39.The method according to claim 37, wherein metabolisms of xenobiotics are characterised.", "40.The method according to claim 37, wherein structure-function relationships are identified and/or substrates of SDR members with unknown function are identified.", "41.The method according to claim 20, wherein a pharmaceutical agent for affecting immune regulation is provided by developing a modulator for 17β HSD type 3, 17β HSD type 7, 17β HSD type 8, 17β HSD type 10, 11β HSD-1, CR1, UDP glucose epimerase, SDR_SRL, AF067174, AF151840, AF151844, AF0078850, Fvt-1, HEP-27, DKFZ_ORF, WWOX_ORF, or CR3.42.The method according to claim 20, wherein a pharmaceutical agent for affecting autoimmunity is provided by developing a modulator for 17β HSD-3, 17β HSD-8, 11β HSD-1, AF057034, U89717, CR1, AF0078850, HEP-27, or CR-3.43.The method according to claim 20, wherein a pharmaceutical agent for wound healing or partial recovery is provided by developing a modulator for 17β HSD-3, 17β HSD-8, 11β HSD-1, U89717, CR1, AF0078850, HEP-27, or CR-3.44.The method according to claim 20, wherein a pharmaceutical agent for treatment of leukemia is provided by developing modulators for 17-β HSD-10 or Fvt-1.45.The method according to claim 20, wherein a pharmaceutical agent for apoptosis regulation is provided by developing a modulator for 17β HSD-10, U89717, SDR_SRL; or for providing a pharmaceutical agent for affecting immune response by providing a modulator for AF016509, or providing a pharmaceutical agent for the treatment of cancer by providing modulators for AF016509, or providing a pharmaceutical agent for affecting cell growth by providing a modulator for U89717, or providing a pharmaceutical agent for the treatment of lung carcinoma by providing a modulator for SDR_SRL, or providing a pharmaceutical agent for the regulation of inflammation or vasculitis by providing a modulator for DKFZ_ORF." ], [ "<SOH> TECHNICAL BACKGROUND <EOH>The short chain dehydrogenase/reductase (SDR) protein family (H. Jörnvall et al., Biochemistry 34 (1995), 6003 - 6013 ) is an old conserved protein family, the members of which show a residue identity level of only 20-30%.", "However, it has been found that the three-dimensional structure of members of the SDR family are highly similar, determining their functions and affiliation to the SDR family (U. Oppermann et al., Enzymology and Molecular Biology of Carbonyl Metabolism 6, Weiner et al.", "eds., Plenum Press, New York (1996), p. 403-415).", "While initially only two structures of SDR enzymes restricted to bacterial and insect enzymes have been discovered, rapid progress on the knowledge of short chain dehydrogenases/reductases resulted in an increasing number of structures, which could be assigned to the SDR family.", "Currently, about 1.600 putative members are known, from which up to 100 may be derived from human, such as hydroxysteroid dehydrogenases (HSD).", "An approach to identify SDR proteins is described in W. N. Grundy et al., Biochemical and Biophysical Research Communications 231 (1997) 760-766 and in T. L. Bailey et al., J. Steroid Biochem.", "Molec.", "Biol.", "62 (1) (1997) 29-44.Therein homologies are searched for via a hidden Markov model, i.e.", "a self-training model, and thus classified to a certain protein family.", "A classification based on the function is not made in these models.", "Since the SDR enzymes are involved in various metabolitic pathways and show different activities, such as oxidoreductases, lyases, or epimerases and, as discussed above, show only a low identity of 20-30%, it has been difficult, to assign new members unambiguously to the SDR family and to find modulators therefor.", "However, since HSD and other SDR play a critical role in higher vertebrates, it is desirable to discover further members of the SDR family and establish modulators for known and new SDR enzymes.", "It was therefore an object of the present invention to provide an algorithm which allows for the identification or verification of SDR family members with high confidence levels.", "It was a further object of the invention to provide an algorithm which provides a search hierarchy with various levels.", "It was another object of the present invention to provide modulators for SDR family members.", "Still another object of the invention was to provide pharmaceutical agents based on members of the SDR family." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention relates to a method for identifying or verifying members of the short chain dehydrogenase (SDR) family based on an algorithm using core SDR motifs for searching members of the SDR family.", "Further, the present invention relates to a method for providing modulators for such members of the short chain dehydrogenase (SDR) family, which enhance or inhibit the activity therefrom as well as a method for providing a pharmaceutical agent using modulators for members of the SDR family.", "In particular the present invention provides a combination of the steps (i) screening databases to search and find SDR sequences, (ii) store the data on an appropriate medium, rank and validate the hits and (iii) using the SDR sequences found to develop new drugs.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "The present invention relates to a method for identifying or verifying members of the short chain dehydrogenase (SDR) family, to identified SDRs, to a method for providing modulators for members of the SDR family and to the preparation of pharmaceutical agents using these modulators.", "TECHNICAL BACKGROUND The short chain dehydrogenase/reductase (SDR) protein family (H. Jörnvall et al., Biochemistry 34 (1995), 6003-6013) is an old conserved protein family, the members of which show a residue identity level of only 20-30%.", "However, it has been found that the three-dimensional structure of members of the SDR family are highly similar, determining their functions and affiliation to the SDR family (U. Oppermann et al., Enzymology and Molecular Biology of Carbonyl Metabolism 6, Weiner et al.", "eds., Plenum Press, New York (1996), p. 403-415).", "While initially only two structures of SDR enzymes restricted to bacterial and insect enzymes have been discovered, rapid progress on the knowledge of short chain dehydrogenases/reductases resulted in an increasing number of structures, which could be assigned to the SDR family.", "Currently, about 1.600 putative members are known, from which up to 100 may be derived from human, such as hydroxysteroid dehydrogenases (HSD).", "An approach to identify SDR proteins is described in W. N. Grundy et al., Biochemical and Biophysical Research Communications 231 (1997) 760-766 and in T. L. Bailey et al., J. Steroid Biochem.", "Molec.", "Biol.", "62 (1) (1997) 29-44.Therein homologies are searched for via a hidden Markov model, i.e.", "a self-training model, and thus classified to a certain protein family.", "A classification based on the function is not made in these models.", "Since the SDR enzymes are involved in various metabolitic pathways and show different activities, such as oxidoreductases, lyases, or epimerases and, as discussed above, show only a low identity of 20-30%, it has been difficult, to assign new members unambiguously to the SDR family and to find modulators therefor.", "However, since HSD and other SDR play a critical role in higher vertebrates, it is desirable to discover further members of the SDR family and establish modulators for known and new SDR enzymes.", "It was therefore an object of the present invention to provide an algorithm which allows for the identification or verification of SDR family members with high confidence levels.", "It was a further object of the invention to provide an algorithm which provides a search hierarchy with various levels.", "It was another object of the present invention to provide modulators for SDR family members.", "Still another object of the invention was to provide pharmaceutical agents based on members of the SDR family.", "SUMMARY OF THE INVENTION The present invention relates to a method for identifying or verifying members of the short chain dehydrogenase (SDR) family based on an algorithm using core SDR motifs for searching members of the SDR family.", "Further, the present invention relates to a method for providing modulators for such members of the short chain dehydrogenase (SDR) family, which enhance or inhibit the activity therefrom as well as a method for providing a pharmaceutical agent using modulators for members of the SDR family.", "In particular the present invention provides a combination of the steps (i) screening databases to search and find SDR sequences, (ii) store the data on an appropriate medium, rank and validate the hits and (iii) using the SDR sequences found to develop new drugs.", "DETAILED DESCRIPTION OF THE INVENTION Members of the SDR protein family have a common core sequence, which is about 250-350, preferably about 260-290 and in particular about 270 amino acids in length.", "SDR proteins can have extensions at the N-terminus and/or at the C-terminus.", "Typically, these extensions have a length of 20 to several hundred, in particular up to 500 amino acids.", "These extensions can be membrane anchors or other signals or they can constitute completely distinct protein domains.", "Therefore, according to the invention it is primarily searched for SDR core domains, the rest of the protein being analysed only later on.", "In a first embodiment the invention provides a method for identifying or verifying members of the short chain dehydrogenase (SDR) family comprising the steps (a) providing a target sequence of molecules to be classified, (b) comparing said target sequence with core SDR motifs selected from (i) MV1 being derived from the motif MT1:TGxxxGxG by replacement of 0 to 2 amino acids, (ii) MT2:NN(0-2:x)AG, (iii) MT3:N, located at a position 90-110 relative to MT1, (iv) MV4 being derived from the motif MT4:S(11-52:x)YxxxK by replacement of 0-2 amino acids and (v) MT5:PG, (c) determining positive SDR candidates containing (i) at least the core SDR motifs MV1 and MV4 and (ii) at least 7 of the 14 amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5 and (d) classifiying positive SDR candidates as belonging to the SDR family.", "It has been found in many SDR proteins that several motifs of the SDR core domain often occur in combination.", "However, it is not obligatory that all SDR core motifs are present for a protein to be an SDR enzyme.", "Since SDR proteins may lack one or several of the core SDR motifs, they may not be found by simple comparison of the complete SDR core domains.", "Within the SDR core the following functional motifs frequently are found.", "The motifs are given in order from N-terminus to C-terminus assigning a position number 0 to the start of the first motif MT1, which of course need not be the start of the complete SDR protein.", "MT1:TGxxxGxG (circa position 0-7); MT2:NNAG (circa position 75-78); MT3:N (circa position 100); MT4:S-Y-K (circa positions 128/142/146) and MT5:PG (circa position 170/171).", "Using these motifs, the algorithm according to the invention has been developed, which allows for an assignment of target sequences to be an SDR sequence with a confidence level of more than 95%, in particular ore than 98%.", "By relying on motifs of the core SDR region positive hits due to indentity in non significant regions can be excluded.", "It is essential for the present invention that the core SDR motifs were selected because of their functional meaning and not only because of homology comparisions.", "The SDR motifs used form essential parts of nucleotide co-factor binding region (Rossman-fold) and the active site of members of the SDR family.", "The motifs MT1 and MT2 represent components of the co-factor binding site.", "A particular co-factor of SDR enzymes is NAD(P)(H).", "The motif MT3 represents a contact to the active site and the motif MT4 a part of the active site.", "The motif MT5 is of functional importance due to its proximity to the co-factor.", "Thus core SDR motifs are motifs which are essential for the functionality of the SDRs.", "For detecting members of the short chain dehydrogenase (SDR) family in the method according to the invention it is therefore essential that functional aspects are considered, wherein enzymatically active SDRs are detected and not only sequences which exhibit a certain homology to other SDRs at functionally irrelevant positions.", "Contrary to prior art algorithms, according to the invention those amino acids are taken into account which are essential for the function.", "A minimum amount of the amino acids selected thus enables a maximum amount of targets due to the divergence of the SDR family, wherein the detection of erroneously positive targets is basically excluded because of the connection between function and structure.", "This way the target specificity can be considerably improved over algorithms, such as neuronal networks, which are based on homology comparisons (cf.", "J.", "A Gerlt et al., Genome Biology, 1 (5) (2000), Reviews 0005.1-0005.10).", "In addition, further functional information can be easily included in order to screen for functional deficits, such as screening for an associated disease mutations or individualized drug metabolism.", "While the individual proteins assigned to the SDR family using the algorithm of the invention may have identities of only 30% or less, they show a very similar three-dimensional structure.", "It is important for the correct formation of the desired three-dimensional SDR structure that motifs 1 to 5 are present in the above listed succession.", "For the description of the motifs the single letter amino acid code is used.", "x denotes a variable amino acid, selected preferably from the 20 naturally occuring amino acids.", "NN(0-2:x)AG means that 0, 1 or 2 amino acids can be positioned between amino acids N and A.", "S(11-52:x)YxxxK means that from 11 to 52 amino acids are positioned between S and Y and 3 amino acids are positioned between Y and K. A replacement of 0-2 amino acids refers to a replacement of any of the amino acids given (including x), whereby preferably the explicitly named amino acids are replaced.", "A replacement includes deletion of the amino acid or a substitution of the amino acid by another amino acid selected preferably from the 20 naturally occuring amino acids.", "The replacement of 1 or 2 amino acids results in a fuzzy logic including also sequences, in which the motifs are not 100% conserved.", "A strategy combining sequence and structure information is also disclosed by L. Yu et al., Protein Science 7 (1998), 2499-2510.In a preferred embodiment of the invention MT2 is defined to be NNAG (i.e.", "without any amino acids x between NN and AG), but with possible replacement of 1-3 amino acids.", "The motif MT3:N is located at position 90-110, preferably at position 95-105 and in particular at position 100 relative to the start of the motif MT1.In a particularly preferred embodiment of the invention the second part of motif MT4 is defined to be the pattern YxASK with possible replacement of up to 3 of these residues.", "In this preferred embodiment the range of possible scores is extended from 0-14 up to 0-16.In this embodiment positive candidates have a score of at least 7, preferably at least 9, more preferably at least 11 and most preferably at least 13.Preferably the SDR motifs are located in the order given from the N-terminus to the C-terminus for a sequence to be classified as SDR sequence.", "The positions given in brackets above may be shifted by amino acid insertions or deletions within the sequence analyzed.", "Preferably the motifs are found within ±50, more preferably ±20 positions, in particular ±10 positions and most preferably ±5 positions, from the values given.", "A target sequence is classified as belonging to the SDR family according to the invention, if it contains at least the core SDR motifs MV1 and MV4 and at least 7 of the 14 explicitly named amino acids contained in the motifs MT1, MT2, MT3, MT4 and MT5.The confidence level can be controlled by varying the amount of matching amino acids, which have to be present in the target sequence.", "Therefore, if a high confidence level, e.g.", ">98%, more preferably >99% is desired, it may be preferable to classify target sequences as positive SDR candidates, only if they contain at least 9 of the 14 amino acids, or even at least 11 or at least 12 of the 14 amino acids contained in the motifs MT1-MT5.Setting the score at a value of at least 13 results in the detection of exclusively sequences, which are an SDR with a confidence level of almost 100%, e.g.", ">99.8%.", "In a preferred implementation of the method of the invention a file is provided containing a set of protein amino acid sequences, the input set.", "One sequence is taken from the input set, the query sequence.", "The implementation then passes the query sequence to the algorithm, which examines it for occurences of some or all of MT1-5 in the arrangements allowed.", "The algorithm returns a list of the best possible combinations of occurences.", "If the matches contain more than a specified number of amino acids from MT1-5, they are assigned as hits.", "In a particularly preferred embodiment the method of the invention is as follows: The algorithm first searches the whole sequence for instances of the first motif MT1, allowing for up to two replacements as described.", "Each possible MT1 match is then taken as the origin for searches for the motifs MT2 to MT5 whose positions are defined relative to the position of MT1.A data structure based on each position of MT1 is created, which will be used to store the positions of other motifs relative to this MT1.For a given MT1 match at position P, the preferred position of the motif MT2 is P+75.According to the rules MT2 is preferably at position (P+75)+/−50, more preferably (P+75)+/−20.This defines a window on the sequence within which instances of the motif MT2 are searched for, including any variants of MT2 with up to three replacements.", "Since the size of the window affects the time taken to search and the quality of the matches found, the preferred implementation allows the window sizes to be specified for each search.", "Any possible matches within the window are added to the result data structure as children of the current MT1.The procedure is then repeated for instances of MT3, where the window is (P+100)+/−50, more preferably (P+100)+/−20, or any other specified window size.", "Again, any results found within the allowed window are added to the result data structure as children of the current MT1.The same procedure is then followed for MT4, with a window (P+128)+/−50, more preferably (P+128)+/−20, or any other specified window size.", "In this case the window only specifies the position of the Serine residue of MT4, and once a candidate Serine has been found at position PS (and added to the result structure as a child of the current MT1), it defines a window PS+(11-52), within which instances of the second part of MT4 are searched for, allowing for replacements.", "Any candidates are added to the result structure as children of the current Serine match.", "Since MT4 allows replacements, and those replacements could include replacements of the Serine, the implementation additonally searches for the second part for MT4 in cases where the Serine is not found.", "In this case, a virtual window composed of all of the possible positions of the (missing) Serine, offset by the PS+(11-52), is constructed [PS+(11-52)+/−20, i.e.", "the range P+128+(11-20) to P+128+(52+20)], or likewise for any specified window size.", "If any instances of the second part are found they are added to the result data structure as children of the current MT1.The procedure is then repeated for instances of MT5, where the window is (P+170)+/−50, more preferably (P+170)+/−20, or any other specified window size.", "Again, any results found within the allowed window are added to the result data structure as children of the current MT1.At this stage the implementation holds in memory a tree-structured data structure where the possible matches with the specified pattern correspond to depth-first traversals of the tree.", "The implementation enumerates the possible combinations of the full or partial motifs, adds up a score calculated from the number of residues in the motifs which were actually matched, and discards the instances where the overlapping windows have given rise to motifs where the ordering is not MT1-MT2-MT3-MT4-MT5.Any combination with a score equal to the maximum score found is kept and added to a list, and it is this list with its score, the motifs found, and the position in the sequence of each amino acid matched which is returned as the result at this stage of the implementation.", "The preferred implementation includes significant enhancements, in particular: MT2 is defined to be NNAG without the presence of 1 or 2 amino acid insertions between the NN and AG parts.", "The implemenation allows for replacement of 1-3 of the residues, and will continue to search for other motifs even if no instance of MT2 is found.", "The second part of MT4 is defined to be the pattern Y*ASK instead of Y**K, but again the implementation allows replacement of up to 3 of these residues.", "This makes the range of possible scores 0-16 instead of 0-14.The absence of motif MT4 is not used to discard SDR candidates, but the effect on the overall score of its absence (5 out of a possible 16 matches) is significant in excluding matches which do not contain it and additionally the presence of the active site MT4 tyrosine is indicated for each result, as a significant indicator of possible SDR catalytic activity.", "In a further preferred embodiment an enlargement or optimization, of the algorithm is performed also on human extended SDRs.", "Thus it is taken into account that compared to the other SDRs often only a motif MTx1 (TGxxGxxG) as well as a motif MTx4 (YxxxK) is present, wherein MTx1 is a variant of TGxxxGxG.", "For determining human extended SDRs with this enlarged algorithm motifs 2, 3 and 5 can even be missing.", "In a particularly preferred embodiment the algorithm according to the invention comprises the import of a data set, e.g.", "from data bases, organizing the data set by using the method according to the invention, ranking the SDR hits and further analyzing and managing the data of the detected hits, such as a cross-linking to data bases, to BLAST or to other tools.", "Subject matter of the invention is also a data carrier, particularly a diskette containing the method according to the invention and particularly the above described algorithm.", "Whereas the method according to the invention itself already has a very high specificity and reliability in the selection of SDR candidates, the SDR candidates detected can be subjected to further evaluation criteria.", "These criteria are e.g.", "comparing the 3D-structure of the candidates detected with the 3D-structure of known SDR proteins or a standardized 3D-structure, which is derived from SDR candidates identified by the method according to the invention.", "Thus, in a further preferred embodiment of the invention the polypeptides classified as positive SDR candidates in the method according to the invention are subjected to another evaluation step in view of their three-dimensional structure in order to further improve the selectivity and specificity of the method.", "Thus it is possible to use known three-dimensional structures of SDR family members (cf.", "e.g.", "H. Jörnvall, Biochemistry 34 (1995), 6003-6013; U. Oppermann et al., Adv.", "Exp.", "Meth.", "Biol.", "414 (1997), 403-415 or J. Benach et al., J. Mol.", "Biol.", "282 (1998), 383-399).", "However, it is also possible to determine the three-dimensional structures of the SDR candidates detected with the method according to the invention and to prepare a common comparative three-dimensional structure therefrom.", "This way it can be examined, e.g.", "whether the positive SDR candidates exhibit the co-factor binding site typical of SDRs.", "A further criteria may be the presence of amino acid Y at position 152±20, particularly ±10.Further, it is possible to compare the amino acids sequences detected with known SDR sequences, e.g.", "via an alignment.", "After the sequences have been classified as SDRs it is also possible to search for further domains, e.g.", "membrane domains in order to thus classify them to a certain type of tissue.", "An important subgroup of SDRs are FabGs, which are derived from pathogens and which can be identified via the method according to the invention.", "Since FabGs are often strongly degenerated and thus exhibit a relatively low score (e.g.", "9 or more) in the method according to the invention, it can be advantageous to examine possible FabG-SDR candidates in a second step in view of the presence of the following motif variations: MTy2:VxVNNAG, wherein V can be replaced particularly by I, as well as MTy5:PGFI, wherein F and/or I can be missing.", "A list of FabG proteins which were identified by the method according to the invention is shown in Table 4.FabGs are involved in the lipid metabolism of bacteria and are particularly suitable for the development of antibiotics.", "A further group of SDRs which can be identified by the method according to the invention are bacterial SDRs.", "Bacterial SDRs detected with the algorithm according to the invention are shown in Table 3.Further, it is possible to detect production enzymes as well as thermostable enzymes with the method according to the invention.", "In a most preferred embodiment, the so-called SDR Finder, the method according to the invention is based on the implementation of functional data both on the three-dimensional structure and on the biological function (NADP(H)-dependend enzymes).", "The implementation is hierarchically structured according to the smallest common denominator having a functional meaning.", "Contrary to known tools not motifs, but SDR candidates are searched for and thus also for those having a very low homology or hardly conserved core motifs, respectively.", "The search for SDR candidates according to the invention enables a considerably higher specificity.", "The SDR candidates detected are of biologically functional relevance.", "At the same time a greater number of hits is found due to the use of the smallest common denominator.", "Further, it is possible to establish a ranking with the algorithm according to the invention, to export the data in different formats and to selectively search for species.", "Thus, the SDR_Finder represents an “all-in-one” analysis solution including various obtainable possibilites, particularly the woldwide web.", "The implementation of hyperlinks to NCBI, EMBL and their tools (e.g.", "Blast, ClustalW, PfaM, PDB, Medline, OMIN) represents an “in silico” analysis/drug development software of modular structure which is particularly developed for SDR.", "Further modules which can be connected thereto are the examination of three-dimensional structures, the determination of active centres and the substrate docking simulation.", "The latter can also be implemented directly into the SDR_Finder and allow direct access, e.g.", "to 3D-databases and chemical libraries via the worldwide web.", "In a preferred embodiment the SDR_Finder is equipped with fuzzy logic.", "In addition, experimental data can be used, e.g.", "to evaluate the exchange of one amino acid in a motif regarding the functional consequences.", "This is of importance both for the individual adjustment of therapies and the evaluation of pathological problems or for the development of diagnostics, respectively.", "Moreover, it is possible to enlarge the algorithm subgroup-specifically, as is shown herein for the FabG SDRs.", "The method according to the invention can be used to verify sequences, which are already classified as (putative) SDR sequences, e.g.", "by automatic alignment (BLAST), to belong to the SDR family or not.", "Further, it can be used to search for and find new members of the SDR family or to search for and find new isoforms of SDR proteins.", "Therefore, the method of the invention provides additional information with regard to known sequences as well as to novel sequences.", "From the knowledge that a target sequence belongs to the SDR family as well as from the information obtained from the ranking findings about substrates and functions can be obtained.", "An important selection criteria thereby is the drugability of the SDR candidates detected.", "The method according according to the invention can be used to detect e.g.", "human SDRs (human extended SDRs), animal SDRs, particularly mammalian SDRs, but also bacterial SDRs, FabG SDRs, fungi SDRs, SDRs of pathogens, SDRs of parasites, e.g.", "plant parasites.", "The SDR proteins classified with the algorithm according to the invention thus can serve as platform for novel drug development.", "Human SDR proteins can particularly serve as starting point for the treatment of diseases or malfunctions of the body, whereas bacterial SDRs particularly provide a starting point for the development of novel antibiotics.", "Further, respecticve SDRs can serve for the development of antimicotica, pesticides, herbicides etc.", ".", ".", ".", "While the algorithm of the present invention preferably is used to search for protein sequences, it is also possible the convert the motifs given into nucleic acid sequences and screen nucleic acid databases.", "A method to convert amino acid sequences into nucleic acid sequences while considering the degeneration of the genetic code is e.g.", "given from H. Jörnvall, FEBS Letters 456 (1999), 85-88.A search on the nucleic acid level can preferably be used to preselect sequences, which are then confirmed by an alignment in the protein level.", "For the search on nucleic acid level these protein sequences are preferably converted to DNA sequences in particular cDNA sequences and used for the detection of further SDR candidates via a fuzzy logic or a hidden Markov model or via neuronal networks.", "The method of the invention therefore also provides a tool for preselection of SDR candidates on the genomic level.", "Preferably a ranking of the positive SDR candidate is performed e.g.", "according to the number of amino acids matching with motifs MT1-MT5.This way a hierarchy and/or an evolutionary relationship of the obtained SDR candidates can be obtained.", "In a particularly preferred embodiment the target sequences classified as positive SDR candidates contain at least the core SDR motifs MT1 and MT4.By hierarchically classifying the verification of the individual core SDR motifs several levels to detect SDR proteins can be obtained.", "By using the algorithm according to the invention the search for SDR candidates and consequently the development of pharmaceuticals can be decisively enhanced.", "So far for the production of pharmaceuticals in vitro tissue cultures were admixed with different substrates.", "From cultures, wherein a certain substrate was converted, the target protein was isolated.", "According to the invention, this step and thus the knowledge of a substrate for the development of inhibitors or for the development of pharmaceuticals is not necessary.", "Moreover, starting from the sequence found a modulator, in particular an inhibitor or activator can be derived.", "This modulator can e.g.", "be derived from known modulators of other, in particular of related SDR proteins, suitable substrates, related functions and tissue distribution for 17β HSD isoforms are described e.g.", "by H. Peltoketo et al., J. Molecular Endocrinology 23 (1999), 1-11.Further, it is possible to derive a modulator from the 3D-structure of the SDR sequence.", "Such a 3D-structure can be obtained experimentally, e.g.", "by X-ray chrystallography or by computer based calculations, e.g.", "ab initio, force field, or rule based methods.", "Further, by inhibiting the active site of the SDR protein the function thereof can be determined.", "The searching for SDR family members and ranking is also applicable to evaluate lead-candidates for possible inhibitors or modifiers of a specific enzyme.", "Leads may be derived from metabolites of evolutionary closely related or very distant enzymes from other species, if the same metabolite may not be found in the respective target organism.", "The evolutionary relationship of SDRs and their distinction from MDRs (medium chain dehydrogenase) is e.g.", "described by H. Jörnvall et al., FEBS Letters 445 (1999), 261-264 and AKRs (T. M. Penning, Endocrine Rev.", "18(3) (1997) 281-305).", "SDR enzymes are often involved in intermediary metabolisms, as well as in hormone and mediator metabolisms.", "Substrates of known SDR proteins include e.g.", "steroids, such as estrone/estradiol, cortisone/cortisol and testosterone/3a-androstenediol.", "Thus, after classifying a sequence as SDR sequence functional tests for steroid substrates result in higher hit rates.", "Further substrates of SDR proteins are UDP-glucose, UDP-N-actetylglucosamine, sepiapterin, dihydropteridine, R-3-OH-butyrate, dienoyl CoA, trans-Enoyl CoA, fatty acids, L-3-OH-acyl CoA.", "These substrates are particularly converted of SDR enzymes, which are involved in the intermediary metabolism.", "Further substrates of SDR proteins, particularly of SDR enyzmes, which are involved in hormone, mediator and xenobiotic metabolisms, are several hydroxy steroids, e.g.", "3-beta-hydroxysteroids, 11-beta-hydroxy steroids or 17-beta-hydroxy steroids as well as prostaglandines and retinoides.", "Further, searching SDRs, ranking and comparing evolutionary patterns can also be used to detect clinically relevant polymorphisms and/or single nucleotide polymorphisms (SNPs).", "This approach can be used to characterize diseased mechanisms as well as metabolism of xenobiotics, e.g.", "drug metabolism.", "The identification of SDR members, ranking and comparing evolutionary patterns also allows for the identification of structure-function relationships.", "These structure-function relationsships are a key for identification of substrates of ORFs with unknown functions.", "Within a lead oriented characterization first binding of a positive SDR candidate is evaluated.", "Starting from the binding a modulator, e.g.", "an inhibitor or activator, can be developed.", "Useful information for developing an inhibitor can be obtained from protein sequence alignment of full-length sequences, e.g.", "by comparison with known SDRs.", "Further, valuable information can be obtained from expressed sequence tags (EST) and gene sequence comparison.", "The procedure using the algorithm according to the invention allows for a great reduction of possible modulator candidates to be analysed and practically excludes target sequences, which are not SDR sequences.", "Therefore, an analysis of the functions in vitro or in vivo can be performed with much less effort than in the state of the art due to the reduced number of compounds to be tested.", "While in the methods according of the state of the art often the substrate must be known, this knowledge is not essential for developing modulators or/and drugs according to the invention.", "It is even possible to derive possible substrates in a subsequent step from the functions of the SDR enzymes found according to the invention.", "Ligands can be derived according to the procedure described by G. R. Lenz et al., DDT, 5(4) (2000), 145-156.The validation of the potential SDRs found according to the algorithm of the invention, which can be used as new targets for drug development, can then be performed by experimental biochemical methods, such as high-throughput function screening for function identification, ultra high-throughput screening for lead compounds, transfection assays, knock out experiments, microarrays, tissue expression, cDNA arrays or analysis of disease in animal or in vitro model systems.", "However, it is also possible to use virtual methods using e.g.", "computers for validation of the new targets, e.g.", "by molecular homology modelling or substrate docking simulations.", "Suitable strategies include e.g.", "gene expression of an identified SDR protein to obtain the protein molecule and subsequently performing biological functional assays and observe the behaviour of the cell.", "Alternatively, the 3D-structure may be derived from the SDR sequence and an inhibitor for the active site provided.", "Using the inhibitor the function of the SDR within an organism can be evaluated.", "Small weight inhibitors for SDR enzymes, which can be used as starting point for developing new or modified inhibitors, in particular inhibitors for newly identified SDR enzymes include: 1) Steroidal-based inhibitors like steroid carboxylates, acrylates, enolates 3,4- and 16,17-fused ring pyrazoles, 3 alpha, 17-beta or 20-beta-spiro-oxiranes as well as steroidal spirolactones, progestins, ursodexycholate, synthetic analogs of estrone sulfate and estrone-3-amino derivatives.", "2) Inhibitors based on flavonoides and dihydropterin derivatives.", "3) Inhibitors based on polyphenols and derivatives of 2,3-dihydroxy-1-naphthoic acids likegossypol (1,1′,6,6′,7,7′-hexahydroxy-5-5′-diisopropyl-3,3′-dimethyl-2,2′-binaphthalene-8,8′-dicarbaldehyde).", "4) Inhibitors based on glycyrrhizin (3beta,20beta)-29-hydroxy-11,29-dioxoolean-12-en-3-yl 2-O-beta-D glucopyranuronosyl-alpha-D-glucopyranosiduronic acid) and components of enzymatically hydrolysed licorice extract like 3-O-beta-D-glucoronopyranosyl-24-hydroxy-18beta-glycyrrhetinic acid, 3-O-beta-D-glucur-onopyranosyl-18beta-glycyrrhetinic acid and 3-O-beta-D-glucuronopyranosyl-18beta-liquiritic acid, monoglycosylated derivatives of glycyrrhizin as well as carbenoxolone.", "5) Pharmaceutically acceptable salts of the above mentioned molecules such as alkali metal (e.g.", "sodium), alkaline earth metal (e.g.", "magnesium) or ammonium as well as salts of organic carboxylic acids, such as acetic, citric, oxalic, lactic, tartaric, malic, isothionic, lactobionic, ascorbic and succinic acids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-tolysulfonic acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric, and sulfamic acids.", "Further candidates for inhibitors are chalcones (cf.", "Life Sci 68 (7) (2001) 751-761) as well as phytoestrogens (cf.", "Life Sci 66 (14) (2000) 1281-1291) and frenolicin and its derivatives.", "Further, inhibitors can be derived from 3D-structures of the SDRs found, confirmed; identified or verified with the method of this invention, as is described e.g.", "by Liao et al., Structure, Vol.", "9 (2001) 19-27.Since SDR enzymes, in particular human SDR enzymes have been found to be involved in many pathways of the body, they are outstanding targets for developing new drugs.", "In particular human SDR enzymes have been found to be involved in intermediary metabolism, lipid mediator/hormone metabolism or xenobiotic phase I metabolism.", "On the other hand, SDR enzymes often constitute pathogenic factors causing diseases.", "Thus, e.g.", "the AME syndrome is associated with 11β HSD-2, bile acid metabolism is associated with 3β HSD, polycystic kidney disease is associated with Ke6(17β HSD-8) and Alzheimer's disease is associated with ERAB(17-β HSD-10).", "Further diseases which can be effected by influencing, modulating or inhibiting SDRs comprise e.g.", "DHPR deficiency, phenylketonuria, dienoyl CoA reductase deficiency, galactosemia III, tetrahydrobiopterine deficiency, adrenal hyperplasia, adrenogenital syndrome, 11-oxoreductase deficiency, apparent mineralocorticoid excess syndrome, ovarian/breast cancer, male pyseudohermophroditism, Zellweger syndrome, pregnancy/ovarian cancer, polycystic kidney disease, Alzheimer's disease, retinits punctata albescens, retinitis pigmentosa, Down's syndrome, arterial hypertension, oncogenes, follicuolar lymphoma, hepatocarcinogenesis, aging related hormone deficiencies and immunity in general.", "Since many of the SDR enzyme are bidirectional (reversible oxidoreaction) depending on the environment, it is also possible to provide a means for selectively enhance one of the enzymatic reaction, i.e.", "oxidation or reduction or to reverse the action observed.", "Thus, providing new SDR sequences and modulators therefor, as described above, allows for the preparation of drugs or pharmaceutical agents, which can be used to control many different diseases.", "In particular drugs for treatment of cancer, e.g.", "breast cancer or prostate cancer, obesity, diabetes, fertility, osteoporosis, glucose metabolism, or conditions related to aging can be prepared.", "Further applications include steroid resistance, in particular ostrogen resistance and glucocorticoid resistance.", "Further, SDR proteins and in particular hydroxy steroid dehydrogenases (HSDs) are outstanding targets for tissue-specific modulation of hormone-dependent or sensitive diseases, e.g.", "cancer, in particular prostate or breast cancer.", "The present invention is in particular useful for providing a pharmaceutical agent for affecting immune regulation is provided by developing a modulator for 17β HSD type 3, 17β HSD type 7, 17β HSD type 8, 17β HSD type 10, 11β HSD-1, CR1, UDP glucose epimerase, SDR_SRL, AF067174, AF151840, AF151844, AF0078850, Fvt-1, HEP-27, DKFZ_ORF, WWOX_ORF, or CR3, a pharmaceutical agent for affecting autoimmunity is provided by developing a modulator for 17β HSD-3, 17β HSD-8, 11β HSD-1, AF057034, U89717, CR1, AF0078850, HEP-27, or CR-3, a pharmaceutical agent for wound healing or partial recovery is provided by developing a modulator for 17β HSD-3, 171 HSD-8, 11β HSD-1, U89717, CR1, AF0078850, HEP-27, or CR-3, a pharmaceutical agent for treatment of leukemia is provided by developing modulators for 17-β HSD-10 or Fvt-1 or a pharmaceutical agent for apoptosis regulation is provided by developing a modulator for 17β HSD-10, U89717, SDR_SRL; or for providing a pharmaceutical agent for affecting immune response by providing a modulator for AF016509, or providing a pharmaceutical agent for the treatement of cancer by providing modulators for AF016509, or providing a pharmaceutial agent for affecting cell growth by providing a modulator for U89717, or providing a pharmaceutical agent for the treatment of lung carcinoma by providing a modulator for SDR_SRL, or providing a pharmaceutical agent for the regulation of inflammation or vasculitis by providing a modulator for DKFZ_ORF.", "The SDR candidates detected according to the invention can be used particularly for the production of inhibitors, such as antibodies on protein level or antisense on nucleic acid level.", "Moreover, it is possible to provide diagnostica by using the SDR candidates detected according to the invention, e.g.", "in order to show a malfunction.", "An important aspect of the present invention in view of the development of new drugs for the diagnosis and/or treatment of a disease is that the inventive approach aims on a target family, i.e.", "SDRs and not on a specific disease.", "This allows for the development of a number of drugs, which all influence the same target family.", "By this approach the amount of experiments, effort and money necessary to develop a new drug can be significantly reduced, since many results can be used parallel for further members of the same target family leading to further new drugs for different medical applications.", "Further this approach allows for affecting a target which is known or suspected to be highly relevant for a person's health.", "In contrast to the classical approach wherein starting from a disease a suitable target must identified, this time and effort consuming procedure is not necessary with the inventive approach.", "The invention is further elucidated by the following figures wherein FIG.", "1 represents the search engine for SDR candiates; The target sequence is compared to the specified core SDR motif, preferably in order from the N-terminus to the C-terminus.", "FIG.", "2 shows flow charts for the preferred implementation of the algorithm.", "FIG.", "2a shows a flow chart for data processing, while FIG.", "2b shows a flow chart for the algorithm.", "FIG.", "3 depicts the development of pharmaceuticals on the basis of the SDR search according to the invention; The combination of virtual screening and classifying sequences to belong to the SDR family with the development of new drugs, as provided herein, is an efficient novel drug development strategy.", "By using the search results of the virtual SDR search new targets are obtained, from which drugs can be derived by various procedures.", "FIG.", "4 shows an alignment of human SDRs.", "39 human SDR proteins were found in a database using the algorithm according to the invention.", "Throughout the various SDR proteins highly conserved amino acids are underlaid in grey.", "As can be seen from this figure the motifs selected for the algorithm of the invention are present in most of the human SDRs.", "Tab.", "1 Table 1 shows human and/or vertebrate SDRs detection with the algorithm according to the invention.", "The detected SDRs are also subject matter of this invention.", "Further, Table 1 includes an EST search for each SDR detected, with which the corresponding function and localization in tissue can be found or localized.", "Tab.2 Table 2 shows mouse SDRs detected with the method according to the invention and the results of EST searches by using these mouse SDRs in human tissue.", "Thus using SDRs of various species, e.g.", "mammals, allows for localization and identification of new SDRs, in particular human SDRs on a genomic level.", "A preselection and/or identification of the SDR employed can be performed with the method according to the invention.", "Tab.", "3 Table 3 shows in bacterial SDRs which were detected with the method according to the invention.", "Such bacterial SDRs are particularly suitable for the development of novel antibiotics.", "Tab.", "4 Table 4 shows FabG_ proteins, i.e.", "an SDR subgroup.", "It is possible with the method according to the invention specifically identify desired subgroups by selection of further criteria in a second search step.", "Tab.", "5 Table 5 shows SDRs from different fungi." ] ]

[ [ "Method and system for creating marketplace visibility and administering freight shipments using fuzzy commodity transportation instruments", "A utility for creating a real-time bid-ask transportation marketplace where all relevant information may be viewed and acted upon is disclosed.", "See FIG.", "2.", "Users of the present invention tender shipments and offer capacity, which are analyzed and entered into transportation instruments.", "Contracts obligate the shipper to make a load available and the carrier to transport the load at a given time for a given price.", "Shipments may be managed throughout their entire life cycle using software tools that interact with the bid-ask marketplace." ], [ "1.A method of brokering transportation transactions, comprising: receiving into a staging area a plurality of dissimilar bids for shipping goods; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting and aggregating said shipping bids into a set of first fuzzy commodities; sorting and aggregating said carrier offers into a set of second fuzzy commodities; selecting matching sets of said first and second fuzzy commodities in said staging area to create transportation instruments; and creating underlying contracts to support the trading of said transportation instruments.", "2.The brokering method of claim 1, further comprising: facilitating the administration of the underlying contracts by brokers or third party logistic providers.", "3.The brokering method of claim 1, further comprises trading of said transportation instruments within spot, forward, shorthaul, series or derivative markets.", "4.The brokering method of claim 1, wherein said trading step further comprises: measuring the objective performance of shippers and carriers and using that information in selecting a trade.", "5.The brokering method of claim 4, wherein said ratings are used anonymously.", "6.The brokering method of claim 1, wherein said trading step further comprises: evaluating the subjective performance of shippers and carriers and using that information in selecting a trade.", "7.The brokering method of claim 6, wherein said ratings are used anonymously.", "8.The brokering method of claim 1, further comprising: maintaining said bids or offers to remain open in the market for a predetermined time period; and automatically removing said bids or offers at the end of that time period.", "9.The brokering method of claim 1, further comprising: maintaining said contingent bids or contingent offers to be open across multiple modes, lanes or markets of transportation, whereby upon first acceptance at a specific mode, lane or market; removing the remaining contingent bids or contingent offers across all other modes, lanes and markets.", "10.The brokering method of claim 1, further comprising: removing open contingent bids or offers upon achieving an objective performance criterion.", "11.The brokering method of claim 1, further comprising: using a Prioritized Scheduled Push (PSP) for updating one or more links either when a client submits a request to the server or periodically without any direct request from the client.", "12.The brokering method of claim 1, further comprising the offer of contingent capacity for a multi-leg or backhaul; and coordinating said multi-leg or backhaul with said shipper(s) and carrier.", "13.The brokering method of claim 10, wherein the booking of one leg causes the offer prices of remaining legs to be set to a different value.", "14.The brokering method of claim 1, wherein multiple compatible partial loads are combined in booking an offered truck.", "15.The brokering method of claim 1, further comprising: importing and exporting groups of bids or offers from Comma Separated Variable files or equivalents into and from said staging area, thereby allowing information from said staging area to be administered in a Comma Separated Variable file or spreadsheet format.", "16.A method of matching a tendered shipment to offered conveyances, comprising: receiving into a staging area a tendered shipment; receiving into said staging area a plurality of dissimilar offers from carriers; sorting said shipment into a first fuzzy commodity; sorting and aggregating said carrier offers into a second fuzzy commodity; selecting one or more offers from the matching sets of said first and second fuzzy commodities in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipment.", "17.A method of matching an offered conveyance to tendered shipments, comprising: receiving into a staging area an offered conveyance; receiving into said staging area a plurality of dissimilar tendered shipments; sorting said offered conveyance into a first fuzzy commodity; sorting and aggregating said tendered shipments into a second fuzzy commodity; selecting one or more shipments from the matching sets of said first and second fuzzy commodities in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipments.", "18.A computer system for brokering a plurality of freight-shipments and carrier capacity, comprising: marketplace means for establishing a bid-ask (offer) marketplace including shipper bids and carrier offers, wherein said bids and offers are measured by mode, market, and lane and optionally accessorial services.", "19.The computer system of claim 18, further comprising: display means for displaying a marketplace summary.", "20.The computer system of claim 18, further comprising: display means for displaying market details in a bid-ask marketplace.", "21.The computer system of claim 18, further comprising: display means for displaying most recent trades and trade volume 22.The computer system of claim 18, further comprising: acceptance means for enabling a customer to indicate acceptance of bids or offers.", "23.The computer system of claim 18, further comprising: notification means for notifying one or more parties to a transaction.", "24.The computer system of claim 18, further comprising: tracking/tracing means for determining the current location of a specific freight shipment.", "25.The computer system of claim 18, further comprising: alert means for communicating fulfillment problems corresponding to a specific freight shipment.", "26.The computer system of claim 18, further comprising: administration means for mitigating fulfillment problems corresponding to a specific freight shipment.", "27.The computer system of claim 18, further comprising: trading of transportation within spot, forward, shorthaul, series or derivative markets.", "28.The computer system of claim 18, further comprising: maintaining said bids or offers to remain open in the market for a predetermined time period; and automatically removing said bids or offers at the end of that time period.", "29.The computer system of claim 18, further comprising: maintaining said contingent bids or contingent offers to be open across multiple modes, lanes or markets of transportation, whereby upon first acceptance at a specific mode, lane or market; removing the remaining contingent bids or contingent offers across all other modes, lanes and markets.", "30.The computer system of claim 18, further comprising: removing open contingent bids or offers upon achieving an objective performance criterion.", "31.The computer system of claim 18, further comprising: using a Prioritized Scheduled Push (PSP) for updating one or more links either when a client submits a request to the server or periodically without any direct request from the client.", "32.The computer system of claim 18, further comprising the offer of contingent capacity for a multi-leg or backhaul; and coordinating said multi-leg or backhaul with said shipper(s) and carrier.", "33.The computer system of claim 32, wherein the booking of one leg causes the offer prices of remaining legs to be set to a different value.", "34.The computer system of claim 18, wherein multiple compatible partial loads are combined in booking an offered truck.", "35.The computer system of claim 18, further comprising: importing and exporting groups of bids or offers from Comma Separated Variable files or equivalents into and from said staging area, thereby allowing information from said staging area to be administered in a Comma Separated Variable file or spreadsheet format.", "36.The computer system of claim 18, further comprising a method of matching a tendered shipment to offered conveyances, comprising: receiving into a staging area a tendered shipment; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting said shipment into a first fuzzy commodity; sorting and aggregating said carrier offers into a second fuzzy commodity; selecting one or more offers from the matching sets of said first and second fuzzy commodity in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipment.", "37.The computer system of claim 18, further comprising a method of matching an offered conveyance to tendered shipments, comprising: receiving into a staging area an offered conveyance; receiving into said staging area a plurality of dissimilar tendered shipments; sorting said offered conveyance into a first fuzzy commodity; sorting and aggregating said tendered shipments into a second fuzzy commodity; selecting one or more shipments from the matching sets of said first and second fuzzy commodity in said staging area based upon a set of objective and subjective criteria; and creating an underlying contract to support said transport of the shipments.", "38.A computer system for trading transportation futures, comprising: receiving into a staging area a plurality of dissimilar bids for shipping goods; receiving into said staging area a plurality of dissimilar offers for transporting goods; sorting said shipping bids into a set of first futures; sorting and aggregating said carrier offers into a set of second futures; selecting matching sets of said first and second futures in said staging area to create a bid-ask marketplace for transportation future instruments; and creating underlying contracts to support the trading of said transportation future instruments.", "39.A computer system for trading transportation options, comprising: receiving into a staging area a plurality of dissimilar bids for options on shipping goods; receiving into said staging area a plurality of dissimilar offers on options for transporting goods; sorting said shipping bids into a set of first options; sorting and aggregating said carrier offers into a set of second options; selecting matching sets of said first and second options in said staging area to create a bid-ask marketplace for transportation option instruments; and creating underlying contracts to support the trading of said transportation option instruments.", "40.A computer system for trading transportation options on futures, comprising: receiving into a staging area a plurality of dissimilar bids for options on futures for shipping goods; receiving into said staging area a plurality of dissimilar offers on options on futures for transporting goods; sorting said shipping bids into a set of first options on futures; sorting and aggregating said carrier offers into a set of second options on futures; selecting matching sets of said first and second options on futures in said staging area to create a bid-ask marketplace for transportation option on future instruments; creating underlying contracts to support the trading of said option on futures transportation instruments, and bi-directional communication links coupled said computer system to the futures and options computer systems to create price consistency and to facilitate inter-market trading to manage risk taken in a position resulting from a trade in either market.", "41.A method of calculating a standardized transportation line haul rate per mile, comprising: receiving into a staging area transportation data for a shipment; calculating standardized route miles from the zip codes of all stops in transit including origin and final destination and allowable practical routes for the type of cargo transported; calculating the line haul price from the total price less standardized charges for provided accessorials; and calculating the standardized line haul rate per mile by dividing the line haul price by the standardized route miles.", "42.A computer system for calculating historical market data on transportation, comprising: means for receiving into a staging area a plurality of completed shipment transportation data, and; software program to calculate the standardized line haul rate per mile for each completed shipment.", "43.The computer system of claim 42, further comprising: software program to sort and aggregate all shipments by lane, mode, market and date of shipment.", "44.The computer system of claim 42, further comprising: means for receiving into a staging area a plurality of shipment requests and offered capacity transportation data; and software program to calculate the standardized line haul rate per mile for each tendered shipment or offered capacity.", "45.The computer system of claim 42, further comprising: means for requesting the display of data sorted by lane, mode, market and date(s) of shipment.", "46.The computer system of claim 42, further comprising: display means for displaying the requested data.", "47.The computer system of claim 42, further comprising: output means for transferring the data to another computer system for further use." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates to a method and apparatus to arrange for and manage freight shipments.", "Users of the present invention tender shipments and offer capacity, which are analyzed and entered into transportation instruments.", "These instruments are maintained in a real-time bid-ask marketplace where all relevant information may be viewed and acted upon by users.", "Users are shown a listing of available counterparties to a desired transaction, and upon agreeing to a set of terms, create a contractual obligation to perform according to the terms of the instrument.", "Contractual obligations may also be exchanged and sold amongst users.", "Shipments may be managed throughout their entire life cycle using software tools that interact with the bid-ask marketplace.", "2.Background of the Art Today transportation brokers and/or third party logistics companies (“3PLs”) manage shipments on behalf of many shippers and carriers.", "Most transportation brokers, in a manner that is congruent to the thinking of brokers in the financial industry perhaps twenty years ago, believe that it is in their best economic interest to inhibit the visibility of transportation markets.", "They reason that they can maintain large spreads (i.e.", "their commissions) between the price that the shipper is wiling to pay and the fees paid to the carrier by not “commoditizing” transportation.", "Electronic trading within the financial community has indeed reduced spreads; however, the increase in sales volumes has resulted in overall increased profitability.", "Today there are other different ways for freight carriers and shippers to reach agreement.", "Competitive electronic marketplaces employ bulletin boards, static listings of available loads and capacity, and auctions.", "Shippers or carriers put loads or capacity out for bid and rule based exchanges utilize uniform rules and conditions to facilitate automated matching and services.", "Examples include DAT (a bulletin board/negotiating service), logistics.com (an auction service), and NTE (a form of a transportation exchange).", "Today's transportation agreements vary in relative “strength” from highly precise and enforceable dedicated contract carriage, to annual contracts and spot-market agreements with very loose terms and conditions.", "However, each suffers from drawbacks.", "Dedicated contract carriage and annual contracts are each respectively cumbersome to implement, and often require months of negotiation.", "The $950 billion transportation logistics industry represents about 10% of U.S. GDP.", "It is highly fragmented with limited market visibility and largely absent or dysfunctional information technology.", "Most business is conducted via telephone and FAX.", "Both shippers and carriers require user-friendly reliable market access and real-time information to provide the quality-of-service that their customers demand.", "Although there are a large number of transportation web sites, none of them effectively meets the objectives of customers, shippers, and carriers.", "Customers need accurate market data prior to making a decision on transportation, and wish to reduce their uncertainty in the marketplace.", "Furthermore, carriers need the ability to increase the probability of finding a backhaul.", "Shippers, meanwhile, want to lock in capacity for future anticipated needs.", "Finally, shippers also want the cost savings associated with collaborating with other shippers without having to identify or negotiate an agreement with every other shipper.", "Carriers want to predetermine their workload to minimize the cost of asset relocation.", "Carriers also need the ability to lower costs by offering a resource to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers.", "Shippers also need the ability to lower costs by offering a shipment to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers.", "To achieve these and other goals could require the cooperation of a competitor.", "To this end, some systems offer “collaborative logistics” in which dosed communities are formed to gain market efficiency.", "However, these systems are not real-time, and cannot process contingent orders, and they require the cooperation of the members of the community to share proprietary information.", "Often, members of the community are competitors of one another and are unwilling to compromise their competitive advantages to participate in the community.", "Also, these systems over-emphasize virtual world models at the expense of real-world operating environments in which equipment breaks down and there are delivery delays.", "Further, members who participate in these dosed systems often lack the best operating and dispatch people, because these people have migrated to better paying jobs with carrier or 3PLs for whom transportation is the core competency.", "Most other systems cover spot markets that represent only 20% of the for-hire truckload transportation market.", "Contract carriage represents about 80% of the for-hire truckload transportation market; thus, most other systems are aimed at the smaller market segment.", "Also, most other systems do not allow the hedging of price and availability risk by participating in forward or series purchases.", "Such systems thus entirely lack risk management.", "Third party logistics providers (3PLs) work on behalf of their customers i.e.", "shippers to both improve the reliability of transportation and minimize its cost.", "They do this by recommending shipping policy, selecting carriers to transport loads, and managing the entire life cycle of shipments.", "Unlike the financial industry in which perhaps more than 99.9% of all trades “clear” without incident, in transportation perhaps only 95% of all shipments are transported without the intervention of a “transportation expert” to remedy problems.", "When a transportation problem occurs, the 3PL provides a service to their customer and alleviates the problem.", "However, many other systems (some even proudly) do not allow the participation of brokers or 3PL companies to enable their users to avoid having to pay brokerage fees that are typically in the range of 8% to 12% of the total cost of transportation.", "The brokerage fees, which are proclaimed by these sites to be “recoverable” by using their system, are currently paid to the broker or 3PL who provide transportation management services using, for the most, part the inefficient technology of the “FAX and telephone” age.", "The cost of these services may be reduced considerably by using more advanced technology and competition.", "It is important to remember that many of these intermediaries, in addition to matching a shipper and carrier, provide valuable transportation management services and have and will try to protect their well-established customer relationships.", "Thus, these systems disintermediate existing players.", "As a result, they only penetrate a small piece of a well-entrenched market.", "Shippers and carriers need to improve their profitability by reducing the number of empty backhauls, delayed or lost shipments and warehouse bottlenecks, and the amount of effort required to manage core carrier relationships efficiently.", "Typically, when transportation managers have goods “ready-to-go” they send faxes or make multiple calls to their brokers at their 3PLs 100 , as shown in FIG.", "1 .", "This process specifies the shipment and usually states what the shipper is willing to pay.", "The transportation manager is unable to “see the market”; i.e.", "they do not know the current spot price that other shippers are willing to pay or that carriers are willing to accept, or the availability of trucks.", "The brokers then send faxes or make multiple calls to dispatchers 101 at their carriers to check the price and availability of transportation to fill their need.", "The transportation manager does not participate in this process.", "After the shipment is booked, the broker must convey this information back to the shipper 103 and verify that the carrier has adequate insurance in force.", "This slow and people intensive process enables a broker to manage only 5 to 10 shipments a day.", "The above unsophisticated approach results from the fact that the current transportation industry contracting process was created in the “fax and telephone” age.", "Such a contracting process is antiquated, particularly when compared to the prevailing practices in the financial industry in which all participants are able to electronically view real-time markets and immediately execute orders when they see opportunities.", "Shippers and carriers cannot effectively manage risk using current transportation practices—most annual contracts are in reality just rate agreements that do not have firm commitments of shipments or trucks and only represent rates and other possible terms and conditions." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In one aspect, the present invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into said staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating said shipping bids into a set of first fuzzy commodities, sorting and aggregating said carrier offers into a set of second fuzzy commodities, selecting matching sets of said first and second commodities in said staging area to create transportation instruments; and creating underlying contracts to support the trading of the transportation instruments.", "In another aspect, the invention provides a computer system for brokering a plurality of freight-shipments and carrier capacity, including marketplace means for establishing a bid-ask (offer) marketplace including shipper bids and carrier offers, wherein the bids and offers are measured by mode, market, and lane and optionally accessorial services.", "In yet another aspect, the invention provides a computer system for trading transportation futures, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting the shipping bids into a set of first futures, sorting and aggregating the carrier offers into a set of second futures, selecting matching sets of the first and second futures in the staging area to create a bid-ask marketplace for transportation future instruments, and creating underlying contracts to support the trading of the transportation future instruments.", "In yet another aspect, the invention provides a computer system for trading transportation options on futures, including receiving into a staging area a plurality of dissimilar bids for options on futures for shipping goods, receiving into the staging area a plurality of dissimilar offers on options on futures for transporting goods, sorting the shipping bids into a set of first options on futures, sorting and aggregating the carrier offers into a set of second options on futures, selecting matching sets of the first and second options on futures in the staging area to create a bid-ask marketplace for transportation option on future instruments, creating underlying contracts to support the trading of the option on futures transportation instruments, and bi-directional communication links coupled the computer system to the futures and options computer systems to create price consistency and to facilitate inter-market trading to manage risk taken in a position resulting from a trade in either market.", "In yet another aspect, the invention provides a method of calculating a standardized transportation line haul rate per mile, including receiving into a staging area transportation data for a shipment, calculating standardized route miles from the zip codes of all stops in transit including origin and final destination and allowable practical routes for the type of cargo transported, calculating the line haul price from the total price less standardized charges for provided accessorials; and calculating the standardized line haul rate per mile by dividing the line haul price by the standardized route miles.", "In yet another aspect, the invention provides a computer system for calculating historical market data on transportation, including a means for receiving into a staging area a plurality of completed shipment transportation data, and a software program to calculate the standardized line haul rate per mile for each completed shipment.", "In yet another aspect, the invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating the shipping bids into a set of first fuzzy commodities, sorting and aggregating the carrier offers into a set of second fuzzy commodities, selecting matching sets of the first and second fuzzy commodities in the staging area to create transportation instruments, and creating underlying contracts to support the trading of the transportation instruments." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates to a method and apparatus to arrange for and manage freight shipments.", "Users of the present invention tender shipments and offer capacity, which are analyzed and entered into transportation instruments.", "These instruments are maintained in a real-time bid-ask marketplace where all relevant information may be viewed and acted upon by users.", "Users are shown a listing of available counterparties to a desired transaction, and upon agreeing to a set of terms, create a contractual obligation to perform according to the terms of the instrument.", "Contractual obligations may also be exchanged and sold amongst users.", "Shipments may be managed throughout their entire life cycle using software tools that interact with the bid-ask marketplace.", "2.Background of the Art Today transportation brokers and/or third party logistics companies (“3PLs”) manage shipments on behalf of many shippers and carriers.", "Most transportation brokers, in a manner that is congruent to the thinking of brokers in the financial industry perhaps twenty years ago, believe that it is in their best economic interest to inhibit the visibility of transportation markets.", "They reason that they can maintain large spreads (i.e.", "their commissions) between the price that the shipper is wiling to pay and the fees paid to the carrier by not “commoditizing” transportation.", "Electronic trading within the financial community has indeed reduced spreads; however, the increase in sales volumes has resulted in overall increased profitability.", "Today there are other different ways for freight carriers and shippers to reach agreement.", "Competitive electronic marketplaces employ bulletin boards, static listings of available loads and capacity, and auctions.", "Shippers or carriers put loads or capacity out for bid and rule based exchanges utilize uniform rules and conditions to facilitate automated matching and services.", "Examples include DAT (a bulletin board/negotiating service), logistics.com (an auction service), and NTE (a form of a transportation exchange).", "Today's transportation agreements vary in relative “strength” from highly precise and enforceable dedicated contract carriage, to annual contracts and spot-market agreements with very loose terms and conditions.", "However, each suffers from drawbacks.", "Dedicated contract carriage and annual contracts are each respectively cumbersome to implement, and often require months of negotiation.", "The $950 billion transportation logistics industry represents about 10% of U.S. GDP.", "It is highly fragmented with limited market visibility and largely absent or dysfunctional information technology.", "Most business is conducted via telephone and FAX.", "Both shippers and carriers require user-friendly reliable market access and real-time information to provide the quality-of-service that their customers demand.", "Although there are a large number of transportation web sites, none of them effectively meets the objectives of customers, shippers, and carriers.", "Customers need accurate market data prior to making a decision on transportation, and wish to reduce their uncertainty in the marketplace.", "Furthermore, carriers need the ability to increase the probability of finding a backhaul.", "Shippers, meanwhile, want to lock in capacity for future anticipated needs.", "Finally, shippers also want the cost savings associated with collaborating with other shippers without having to identify or negotiate an agreement with every other shipper.", "Carriers want to predetermine their workload to minimize the cost of asset relocation.", "Carriers also need the ability to lower costs by offering a resource to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers.", "Shippers also need the ability to lower costs by offering a shipment to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers.", "To achieve these and other goals could require the cooperation of a competitor.", "To this end, some systems offer “collaborative logistics” in which dosed communities are formed to gain market efficiency.", "However, these systems are not real-time, and cannot process contingent orders, and they require the cooperation of the members of the community to share proprietary information.", "Often, members of the community are competitors of one another and are unwilling to compromise their competitive advantages to participate in the community.", "Also, these systems over-emphasize virtual world models at the expense of real-world operating environments in which equipment breaks down and there are delivery delays.", "Further, members who participate in these dosed systems often lack the best operating and dispatch people, because these people have migrated to better paying jobs with carrier or 3PLs for whom transportation is the core competency.", "Most other systems cover spot markets that represent only 20% of the for-hire truckload transportation market.", "Contract carriage represents about 80% of the for-hire truckload transportation market; thus, most other systems are aimed at the smaller market segment.", "Also, most other systems do not allow the hedging of price and availability risk by participating in forward or series purchases.", "Such systems thus entirely lack risk management.", "Third party logistics providers (3PLs) work on behalf of their customers i.e.", "shippers to both improve the reliability of transportation and minimize its cost.", "They do this by recommending shipping policy, selecting carriers to transport loads, and managing the entire life cycle of shipments.", "Unlike the financial industry in which perhaps more than 99.9% of all trades “clear” without incident, in transportation perhaps only 95% of all shipments are transported without the intervention of a “transportation expert” to remedy problems.", "When a transportation problem occurs, the 3PL provides a service to their customer and alleviates the problem.", "However, many other systems (some even proudly) do not allow the participation of brokers or 3PL companies to enable their users to avoid having to pay brokerage fees that are typically in the range of 8% to 12% of the total cost of transportation.", "The brokerage fees, which are proclaimed by these sites to be “recoverable” by using their system, are currently paid to the broker or 3PL who provide transportation management services using, for the most, part the inefficient technology of the “FAX and telephone” age.", "The cost of these services may be reduced considerably by using more advanced technology and competition.", "It is important to remember that many of these intermediaries, in addition to matching a shipper and carrier, provide valuable transportation management services and have and will try to protect their well-established customer relationships.", "Thus, these systems disintermediate existing players.", "As a result, they only penetrate a small piece of a well-entrenched market.", "Shippers and carriers need to improve their profitability by reducing the number of empty backhauls, delayed or lost shipments and warehouse bottlenecks, and the amount of effort required to manage core carrier relationships efficiently.", "Typically, when transportation managers have goods “ready-to-go” they send faxes or make multiple calls to their brokers at their 3PLs 100, as shown in FIG.", "1.This process specifies the shipment and usually states what the shipper is willing to pay.", "The transportation manager is unable to “see the market”; i.e.", "they do not know the current spot price that other shippers are willing to pay or that carriers are willing to accept, or the availability of trucks.", "The brokers then send faxes or make multiple calls to dispatchers 101 at their carriers to check the price and availability of transportation to fill their need.", "The transportation manager does not participate in this process.", "After the shipment is booked, the broker must convey this information back to the shipper 103 and verify that the carrier has adequate insurance in force.", "This slow and people intensive process enables a broker to manage only 5 to 10 shipments a day.", "The above unsophisticated approach results from the fact that the current transportation industry contracting process was created in the “fax and telephone” age.", "Such a contracting process is antiquated, particularly when compared to the prevailing practices in the financial industry in which all participants are able to electronically view real-time markets and immediately execute orders when they see opportunities.", "Shippers and carriers cannot effectively manage risk using current transportation practices—most annual contracts are in reality just rate agreements that do not have firm commitments of shipments or trucks and only represent rates and other possible terms and conditions.", "SUMMARY OF THE INVENTION In one aspect, the present invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into said staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating said shipping bids into a set of first fuzzy commodities, sorting and aggregating said carrier offers into a set of second fuzzy commodities, selecting matching sets of said first and second commodities in said staging area to create transportation instruments; and creating underlying contracts to support the trading of the transportation instruments.", "In another aspect, the invention provides a computer system for brokering a plurality of freight-shipments and carrier capacity, including marketplace means for establishing a bid-ask (offer) marketplace including shipper bids and carrier offers, wherein the bids and offers are measured by mode, market, and lane and optionally accessorial services.", "In yet another aspect, the invention provides a computer system for trading transportation futures, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting the shipping bids into a set of first futures, sorting and aggregating the carrier offers into a set of second futures, selecting matching sets of the first and second futures in the staging area to create a bid-ask marketplace for transportation future instruments, and creating underlying contracts to support the trading of the transportation future instruments.", "In yet another aspect, the invention provides a computer system for trading transportation options on futures, including receiving into a staging area a plurality of dissimilar bids for options on futures for shipping goods, receiving into the staging area a plurality of dissimilar offers on options on futures for transporting goods, sorting the shipping bids into a set of first options on futures, sorting and aggregating the carrier offers into a set of second options on futures, selecting matching sets of the first and second options on futures in the staging area to create a bid-ask marketplace for transportation option on future instruments, creating underlying contracts to support the trading of the option on futures transportation instruments, and bi-directional communication links coupled the computer system to the futures and options computer systems to create price consistency and to facilitate inter-market trading to manage risk taken in a position resulting from a trade in either market.", "In yet another aspect, the invention provides a method of calculating a standardized transportation line haul rate per mile, including receiving into a staging area transportation data for a shipment, calculating standardized route miles from the zip codes of all stops in transit including origin and final destination and allowable practical routes for the type of cargo transported, calculating the line haul price from the total price less standardized charges for provided accessorials; and calculating the standardized line haul rate per mile by dividing the line haul price by the standardized route miles.", "In yet another aspect, the invention provides a computer system for calculating historical market data on transportation, including a means for receiving into a staging area a plurality of completed shipment transportation data, and a software program to calculate the standardized line haul rate per mile for each completed shipment.", "In yet another aspect, the invention provides a method of brokering transportation transactions, including receiving into a staging area a plurality of dissimilar bids for shipping goods, receiving into the staging area a plurality of dissimilar offers for transporting goods, sorting and aggregating the shipping bids into a set of first fuzzy commodities, sorting and aggregating the carrier offers into a set of second fuzzy commodities, selecting matching sets of the first and second fuzzy commodities in the staging area to create transportation instruments, and creating underlying contracts to support the trading of the transportation instruments.", "BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other features and advantages of the invention will become more apparent from the detailed description of the exemplary embodiments of the invention given below with reference to the accompanying drawings.", "FIG.", "1 shows the “FAX and telephone” implementation of conventional transportation order processing.", "FIG.", "2 shows the online computer system for creating transportation instruments of the present invention.", "FIG.", "3 graphically illustrates the meaning of the “lane miles” of the present invention.", "FIG.", "4 graphically illustrates the meaning of the “route miles” of the present invention, both with and without a Stop In Transit.", "FIG.", "5 shows a summary of contingent order processing of the present invention for the case where the probability of finding a backhaul to a single destination is 10%.", "FIG.", "6 shows the type of roles various users of the present invention fulfills.", "FIG.", "7 shows the basic components of a link in a graphical navigation bar of the present invention, including a title bar and a content area.", "FIG.", "8 illustrates a fill page TrantisLink graphical navigation bar containing six links.", "FIG.", "9 shows the TrantisLink main screen and its five logical areas.", "FIG.", "10 shows an expanded view of a TrantisLink watchlist.", "FIG.", "11 shows an expanded view of the market details behind a transportation instrument in a TrantisLink watchlist.", "FIG.", "12 shows the use of anonymous ratings in the market details.", "FIG.", "13 shows an open order summary list of the present invention.", "FIG.", "14 shows how an open order is be modified within the present invention.", "FIG.", "15 shows a list of incomplete bookings of the present invention.", "FIG.", "16 shows the tracing and tracking booking status of the present invention.", "FIG.", "17 shows the tracking and tracing booking details of the present invention.", "FIG.", "18 shows tracking and tracing booking problems of the present invention.", "FIG.", "19 shows tracking and tracing report a problem of the present invention.", "FIG.", "20 shows tracking and tracing problem submitted of the present invention.", "FIG.", "21 shows an alert list of the present invention.", "FIG.", "22 shows find a truck data entry of the present invention.", "FIG.", "23 shows find a shipment data entry of the present invention.", "FIG.", "24 shows tendering a shipment data entry of the present invention.", "FIG.", "25 shows offering a truck data entry of the present invention.", "FIG.", "26 shows contingent offer of a truck of the present invention.", "FIG.", "27 shows completed shipments data entry of the present invention.", "FIG.", "28 shows lane history data request and response of the present invention.", "FIG.", "29 shows user preferences of the present invention.", "FIG.", "30 shows network administration data entry of the present invention.", "FIG.", "31 shows company administration data entry of the present invention.", "FIG.", "32 shows single company administration data entry of the present invention.", "FIG.", "33 shows the display of trading exposures of the present invention.", "FIG.", "34 illustrates using series and forward contracts to hedge price and availability risks of the present invention.", "FIG.", "35 illustrates a private network utilizing the present invention run by a sponsor (super shipper or 3PL).", "FIG.", "36 illustrates two private networks run by two different sponsors.", "FIG.", "37 shows how some distressed loads on one network is matched on another of the networks shown in FIG.", "36 within the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Overview Traditionally, technological change in the transportation industry has been in a top-down fashion from the largest companies to smallest companies.", "The Internet has turned this upside-down.", "Now, more technologically agile companies with a customer focus and strong backing drive the market.", "The philosophy behind the present invention is that all parties in the transaction process must benefit for the model to operate effectively; unlike companies that disintermediate the industry, the present invention has developed various solutions that allow customers to become more profitable.", "The present invention hereafter referred to as TrantisLinkSM, approaches the transportation logistics market using a unique business model that extends the financial commodity market model to include the commercial realities of transportation.", "The underlying premise is that transportation is managed as a type of “fizzy commodity” which is defined as a physical good having a large number of attributes that is standardized or classified using the TrantisLink methodology described hereinafter such that it is the object of commercial transactions.", "Consider a typical commodity, e.g.", "99.99% gold, which is rigorously defined and therefore traded as a commodity.", "In transportation, all shipments are not created equal; they have different cargos, different pickup and delivery locations and dates, and different required accessorial services.", "The large number of different possible shipments would normally preclude their being traded as a rigorously defined commodity.", "However, within commercially reasonable limits, trucks and shipments are fungible; e.g.", "one dry van may be substituted for another, and one load may be substituted for another.", "Hence using the TrantisLink business methodology, the line-haul parts of multiple shipments are separated out, sorted and aggregated into a fuzzy commodity bid-ask marketplace.", "TrantisLink creates a broader coverage of markets and executable real-time data through “transportation instruments” backed by binding contracts between the shipper and carrier that obligate the shipper to make a load available and the carrier to transport the load at a given time for an agreed-upon price.", "These transportation instruments support the aggregation of the line haul parts of shipments while preserving the essential differences in each shipment.", "Hence participants may trade the line haul and mode with other similar line hauls and modes.", "For example, when a transportation manager has goods “ready-to-go” that manager uses TrantisLink to first access and then evaluate the current market condition.", "The manager views the current price and availability of transportation capacity in real-time and then determine whether they want to delay shipping one day to save perhaps 15 to 20 cents/mile.", "The total 1999 market for motor carrier transportation in the United States was $450 billion.", "For-hire truckload transportation was $110 billion.", "The average number of for-hire truckload shipments per day in the United States is about 500,000.Of these, 100,000 are in the spot-market, and 400,000 are in the annual and dedicated contract marketplaces.", "Also, the trucking industry in the U.S. is highly fragmented.", "According to Armstrong & Associates, the 50 largest carriers combined have $18 billion in revenue, which is less than 20% of the total market.", "The largest company, Schneider National, has annual revenue of slightly more than $3 billion.", "There are over 300,000 trucking companies; only 20,000 carriers have nine or more trucks; the average trucking company has only seven trucks.", "TrantisLink and Third Party Logistics Providers (3PLs) To overcome the fragmentation referred to above, TrantisLink provides online computer support system 105 to accomplish all of the above functionalities using the Internet as a communication medium, as shown in FIG.", "2.The TrantisLink is deployed in a hardware platform such as an Internet server system, but can also be deployed within a LAN/WAN environment.", "In any case, the following detailed description will focus more on the software implementation of the present invention.", "TrantisLink also allows all of the 3PLs to set and collect their own management fees in a competitive environment subject to a minimum fee set by TrantisLink rules that may from time to time be changed.", "Additionally, whenever a problem occurs, the managing 3PL will use the provided software tools to workout the problem on behalf of their damaged customer by finding an appropriate solution in the marketplace.", "Additionally, virtual 3PLs are those 3PLs that actively manage transportation for shippers via web sites.", "While some have spot-market exchanges, their focus is on managing shippers' transportation requirements; thus the TrantisLink marketplace is just a part of their model.", "It will be advantageous for these companies to participate in TrantisLink forward and series marketplaces.", "Speculators can trade transportation contracts based on transportation instruments to gain profit without the intention of actually transporting cargo.", "They are also likely to participate in the forward and series marketplaces.", "Managing 3PLs will also provide advisory services and liquidity to TrantisLink and in return receive: (i) Assignment of new customers when shippers who do not have a prior existing relationship with a 3PL first join the market; (ii) The right to use the TrantisLink system as a “private” system handling only their business transactions; and (iii) Equity in Trantis in proportion to their provision of transaction volume and participation in the TrantisLink Advisory Board.", "Thus, TrantisLink embraces rather than disintermediates third party logistics providers.", "As shown, the Managing 3PLs actively participate in this B2B electronic transportation market.", "Their presence increases market confidence in the TrantisLink system.", "Additionally, TrantisLink may be used by other 3PLs that wish to offer their customers' shipments and carrier capacity to a wider range of counterparties than is currently accessible to them.", "Although TrantisLink will not assign this group of 3PLs new customers to manage, their participation will increase the liquidity of the market.", "Transportation Instruments TrantisLink creates a standardized marketplace because it creates and uses fungible transportation instruments (i.e.", "one shipment may be substituted for another and one truck may be substituted for another in fulfilling an obligation) to aggregate shipping demand and carrier capacity.", "Transportation instruments are based on underlying contracts between a shipper and a carrier that obligate the shipper to make a load available and the carrier to transport the load to its destination.", "To create transportation instruments, individual shipments and trucks are separated out, sorted and aggregated without including the specific details which are necessary for delivery.", "The standardized transportation instruments are characterized by mode (i.e.", "type of transport), market (e.g.", "spot, forward, or series of pickups and their corresponding dates), and lane (i.e.", "zone of origin and destination).", "TrantisLink aggregates multiple shipments sharing common elements from among these parameters and presents the best market prices and available shipments and capacity to the marketplace.", "This standardization will dramatically change the market by permitting shippers and carriers to see the current market and to hedge against future price and availability fluctuations.", "In addition, TrantisLink rules provide for enforceable underlying contracts to support future performance.", "These changes replace the vagaries of current contract market practices.", "As stated, transportation instruments are defined by mode, market, and lane, further described below.", "Additionally, each transportation instrument may have a bid and ask price and an amount available at those prices.", "Mode TrantisLink supports many different modes of transportation.", "Available modes include but are not necessarily limited to dry van, refrigerated/temperature controlled, flatbed/specialized, liquid bulk, dry bulk, and intermodal (combination).", "Offered shipments may be contingently tendered simultaneously to more than one mode.", "Market Markets are described by the intended date(s) of execution.", "The system currently supports four types of markets; i.e.", "spot, shorthaul, forward, and series, and is extendible to support other types of derivative markets (e.g.", "futures, options, etc.).", "A spot market is created when a load is tendered or a truck is offered for pickup on a single predetermined day usually in the near future (e.g.", "today, tomorrow, day after).", "For example; a load tendered for next Monday is a spot market.", "A pickup on the last day of this month is a spot market.", "A pickup scheduled for the first of the month four months ahead is also a (not likely to occur) spot market.", "A shorthaul market is created when a load is tendered or a truck is offered for pickup and delivery on a single predetermined day usually in the near future (e.g.", "today, tomorrow, day after).", "For example, a load tendered for pickup and delivery next Monday is a shorthaul market.", "A pickup and delivery on the last day of this month is a shorthaul market.", "A pickup and delivery scheduled for the first of the month four months ahead is also a (not likely to occur) shorthaul market.", "A forward market is created when a load is tendered or a truck is offered for a single pickup within a single predetermined time period other than a day (e.g.", "week or month).", "For example, a single pickup scheduled for this month, a pickup during the third week in July, and another one for anytime in the month of August are all forward markets.", "A series market is created when loads are tendered or trucks are offered for multiple pickups on more than one predetermined day, week, or month.", "The most common types of series markets are intended to compete with annual contracts.", "For example, pickups scheduled for every Monday during the next month, the first week of each of the next six months, or one day (to be agreed upon ahead of time) of each week for the third quarter are each series markets.", "Forward and series markets specified for weeks or months are not directly deliverable and must be either converted into a spot or shorthaul market prior to delivery or cash settled.", "According to the applicable TrantisLink rules, the shipper selects the method for execution and related pickup and delivery dates and must give adequate prior notice (e.g.", "at least three days for forward contracts).", "The system also supports the possibility of pickup day-of-the-week price modifications.", "Lanes The entire United States is divided into a number of zones (i.e.", "service areas or regions).", "A zone is defined by a combination of states, cities, and/or zip codes.", "A zone may have any name; for example, the “Chicago” zone is defined by a series of zip codes that for commercial reasons includes the city of Milwaukee that is about 90 miles from Chicago.", "A lane is completely defined by a zone of origination and zone of destination.", "For example, the Chicago to LA lane has Chicago as its zone of origination and LA as its zone of destination.", "The number of “lane miles” in a lane is specified as the number of “standard” highway miles from the “center” of the zone of origination 102 to the “center” of the zone of destination 104, as shown in FIG.", "3.An extended price for a basic shipment is found by multiplying the rate per mile by the number of lane miles.", "Lane miles are frequently used in historical comparisons.", "The number of “route miles” for a shipment is specified as the number of “standard” highway miles from the pickup location to the destination location.", "This is illustrated in FIG.", "4 both with and without a Stop In Transit 106.Note that route miles may be larger or smaller than lane miles; however, on longer trips they should be almost the same.", "Out-of-route miles are the difference between the route miles without a stop-in-transit and the route miles with a stop-in-transit.", "TrantisLink may limit the maximum allowable number of stops-in-transit and/or out-of-route miles.", "According to the rules of TrantisLink, accessorial route miles may be added when the final delivery location 108 is remotely located from the center of the zone of destination 110.Rates, Prices, and Quantities The rates and prices quoted in a transportation instrument are usually expressed in net amount available to a carrier; i.e.", "the quoted extended price is the total price paid by the shipper less the management fee taken by the 3PL.", "The following example shows how a shipment may be tendered as either a gross rate per mile or a gross price: Lane miles: 900 3PL fee: $125 (set by the 3PL managing the shipment) Proceed in One of Two Columns (Method 1) (Method 2) Enter: Gross Price Gross Rate per Mile $1,035 $1.15 Calculate: Net Price to Carrier Extended Gross Price $910 $1,035 (=1,035 − 125) (=900 × 1.15) Calculate: Net Rate Per Mile to Carrier Net Price to Carrier $1.01 $910 (=910/900) (=1035 − 125) In either case the market would be displayed as paying $ 1.01/route mile and a total extended price of $ 910 to the carrier.", "The number of trucks required or available are expressed as integer multiples of ¼ of a truck.", "The most frequent size used is a single truck.", "However, multiple truck shipments may be offered or accepted within a single entry.", "The less than a truck load (“LTL”) minimum amount of 0.25TL may be used to support cross company load consolidation.", "The following example illustrates a transportation instrument: LANE MODE MARKET RATE QTY TOT LAST VOL LA-CHI VAN 4/11 1.10- 5 × 7 8 × 12 1.15 27 1.15 This instrument represents the aggregate supply and demand of all transportation in dry vans from the LA zone to the Chicago zone on April 11th.", "The highest basic bid price for shipments is $1.10 per mile and five trucks are available at that rate.", "The lowest basic ask price for trucks is $1.15 per mile and seven trucks are available at that rate.", "The total number of shipments bid is eight, which means that three (8-5) are tendered at rates less than $1.10 per mile.", "The total number of trucks offered in 12, which means that five (12-7) are available at rates more than $1.15 per mile.", "The last trade (i.e.", "booking) was at a rate of $1.15 per mile and 27 dry van shipments have been booked for LA to Chicago on this day.", "Since there are 2,000 lane miles between the center of the LA zone and the center of the Chicago zone, the most that a shipper is currently willing to pay for transport between the two centers is a basic fee of $2,200 a truckload; similarly the least a carrier is asking for is a basic fee of $2,300 per truckload.", "The above example is for illustrative purposes only, and it should be noted that it is necessary to supply additional information including actual route miles to determine the total price for a specific shipment prior to its transport.", "TrantisLink is reliable because 3PLs stand behind booked shipments from inception through final delivery.", "Even though the system is highly automated to support the activities of its users, TrantisLink relies upon 3PL staff to apply their knowledge of the transportation industry to manage operational problems that frequently occur on a day-to-day basis in transportation.", "The TrantisLink marketplace and software tools greatly facilitate this process.", "In the context of TrantisLink, a 3PL or one of its customers manages each shipment.", "The act of management involves an obligation on the part of the 3PL to use commercially reasonable efforts to ensure that the contracted terms of the shipment are met.", "For example, in the event that a truck with a load breaks down on the road, the 3PL (or carrier) must attempt to locate another truck to complete the shipment under the agreed terms.", "TrantisLink functions in real-time to support optimization and collaborative logistics on behalf of both shippers and carriers.", "Currently, many 3PLs have negotiated and work under fixed rate annual contracts with both shippers and carriers.", "Most of these contracts do not include an enforceable minimum number of shipments (or trucks) over their terms and require 3PLs to satisfy shipper demand in the spot market.", "Hence, 3PLs need to maintain the cooperation of carriers to make spot capacity available whenever the demand for trucks is high and the spot rate exceeds the negotiated and agreed upon rate in the annual contract.", "This system of maintaining “relationships” or informal “favors” is often supported by either overpayment of the annual rate and/or indirect payments of another nature.", "TrantisLink uses its real-time functionality to support contingent order processing of four types, time period, multi-mode, multi-destination, and strategy.", "Shippers may be willing to tender a load for a limited period of time.", "Unless otherwise specified, all orders are considered Good-Till-Canceled.", "Shipments tendered for a limited time period are automatically removed by the TrantisLink system when the time period is reached.", "Shippers may be willing to use more than one mode of transportation to transport a load.", "A shipment may be tendered at one rate for expedited two-day delivery by truck, a lower rate for normal three-day delivery by truck, or at a still lower rate for six-day delivery by intermodal.", "All of these bids are simultaneously entered into the real-time market.", "When any one of these loads is booked, the TrantisLink system immediately removes all of the remaining contingent bids for the shipment.", "Similarly, carriers may desire to have a truck at a location carry a load to one of many different destinations.", "All of these offers are simultaneously entered into the real-time market.", "When any one of these truck offers is booked, the TrantisLink system immediately removes all of the remaining contingent offers for the truck.", "The benefit of real-time contingent order processing may be extended to include executing an operational strategy.", "Consider the case of a shipper with a large number of distribution centers stocking a large number of stores using dedicated assets (e.g.", "trucks on annual lease).", "Most head-haul runs are efficiently scheduled and the trucks are substantially filled; most backhauls are not efficiently filled, many are even empty.", "The shipper may increase their operational efficiency by attempting to get a backhaul shipment from another shipper in the vicinity of the store where the truck is making a delivery.", "The probability of finding a backhaul to a single destination is low; for example perhaps 10%.", "As shown in FIG.", "5, the probability of successfully finding a backhaul increases as the number of possible return destinations increases.", "However, to maintain proper operation the shipper must ensure that each of their distribution centers has a minimum number of trucks available each day.", "TrantisLink is capable of executing this operational strategy, specifically, removing all contingent truck offers that would be capable of depleting the number of trucks returning to a given distribution center below its minimum allowable value.", "The TrantisLink system uses a minimum unit of transportation equal to ¼ of a truck to support partial load commerce or cross-shipper load consolidation.", "The following example illustrates partial load commerce: A driver picks up a shipment and notices that the truck is only about ⅔rds full.", "Just before departing the dock he logs in and informs TrantisLink that he has accepted the load and offers a ¼ truck for all possible lanes with pickups and destinations along the route that would allow him to maintain his original schedule.", "The driver also defines the type of load to ensure that any subsequent partial load(s) will be compatible with the initial load on the truck.", "The system notifies the driver of all currently available partial loads that he may book.", "The driver first ensures that the subsequent partial load will be compatible with the initial load on the truck and then accepts one of these loads and modifies his route to accommodate the additional freight.", "When there are no current “matches” the system displays the passive offer of the ¼ truck such that another shipper might manually accept it.", "The driver is electronically notified of any acceptance of his offer and must verify its compatibility with the initial load.", "The system will support multi-trip end-to-end optimization by allowing a carrier to enter different rates for each leg of a trip and an average rate for the entire trip.", "For example, a carrier offers to go one way for $1.35 per mile and $1.15 per mile for an end-to-end round trip.", "The system will initially enter $1.35 for both the front and back haul legs.", "When one of these legs is booked, the system will immediately change the rate of the unfilled leg to $0.95.TrantisLink also supports the booking of short-haul and/or drayage shipments at flat charges according to the TrantisLink rules that may from time to time be changed.", "A transportation instrument is defined for each service area in which any load may be transported from any pickup location within the service area at a fixed price that is determined by the market.", "However, it is to be emphasized that shorthaul or drayage imply that: The fixed price depends on the anticipated amount of time to complete the obligation and not on the actual number of route miles; and Accessorial charges may be added to the fixed price to determine the total price paid to the carrier.", "Proper Disposition (Completion) of Transportation Contracts Transportation contracts are completed once all of their contractual obligations have been met.", "They may be transferred to another suitable counterparty; i.e.", "the obligation may at any commercially reasonable time prior to a scheduled pickup be transferred at its then current market price to another suitable TrantisLink participant.", "This process requires the current owner of one part of the obligation to find a wiling counterparty to accept the transfer for a price.", "Potential counterparties may want to apply their own subjective ratings before accepting the transfer.", "Contracts may also be cancelled subject to an agreed upon penalty.", "Completion implies that an individual shipment(s), characterized by additional shipment details unique to each shipment, has been delivered.", "Shipment details include (1) precise pickup and destination locations, (2) number and location of stops-in-transit, (3) whether driver loading and/or unloading are required, etc., which modify the basic description of the transportation instrument within predefined limits according to the TrantisLink rules.", "Failure implies that transportation of a shipment does not take place.", "The transportation contract matures and the party causing the failure is responsible for paying a financial penalty according to the rules of the exchange.", "The managing 3PL is expected to mitigate the cost of any damages.", "Types of TrantisLink Participants This section presents the types of businesses that participate in TrantisLink, the roles that they play, how their performance is rated by the TrantisLink system, and their insurance coverage.", "These include 3PLs, managing 3PLs, insurance companies and agents, shippers; super-shippers; carriers, mega-carriers, truckstop operators, ancillary service companies, factor receivable banks, insurance companies and agents, and financial institutions.", "Managing 3PLs are 3PLs that have agreed to accept additional assigned TrantisLink responsibilities and benefits.", "Shippers are suppliers of a small amount of transportation demand.", "Super-shippers are suppliers of significant transportation demand.", "It is very likely that super-shippers will require a high-speed interface into their own internal computer systems.", "Carriers are suppliers of a small amount of transportation capacity.", "Mega-carriers are suppliers of significant transportation capacity.", "It is very likely that mega-carriers will require a high-speed interface into their own internal computer systems.", "Truckstop operators provide roadside TrantisLink access for carriers.", "Ancillary service companies are providers of various services to the transportation industry.", "Factor receivable banks are providers of financial services to shippers and carriers.", "Insurance companies and agents are providers of cargo, liability, and other types of insurance coverage and information pertinent to the transportation industry.", "Financial institutions are consumers of transportation information.", "These institutions may, under contract with TrantisLink, redistribute that information.", "Roles of TrantisLink Participants Each firm type may participate in TrantisLink activities as described as FIG.", "6.The definitions of each type of role are supplied below.", "Offer: place loads or transportation capacity into the market.", "A data entry task that specifies only the basic or the complete specification of a load or available truck.", "Accept: book transportation capacity or loads that are in the market.", "Track and Trace: monitor the entire life cycle of shipments.", "Remotely located users follow the shipment from booking through completion.", "Manage Shipments: monitor and control the entire life cycle of shipments.", "View Only: view current prices and capacity in the real-time market or historical prices and capacity over time.", "Sell Ancillary Products and Services: provide products and services to participants.", "Account administration: create sub-accounts and users, assign rights, maintain insurance records, and set credit limits.", "Account supervision: monitor and/or supervise orders.", "System administration: monitor and maintain the proper operation of the system.", "Customer Relationship Management: assist customers in their use of TrantisLink's ancillary information Ratings and Information TrantisLink supports several types of performance ratings and information on insurance coverage.", "These include surveys of shipper perception of the performance of carriers, individual perception of the performance of counterparties, objective performance of shippers, carriers, and 3PLs as measured by the TrantisLink system, and private transaction flags.", "Data from http://www.carrierrankings.com is used to enable shippers to select transportation alternatives for their company.", "Shippers contribute confidential surveys on eight rating criteria (i.e.", "overall performance, operating personnel, administration, information technology, equipment, on-time performance, cost, and safety and compliance) for thousands of carriers.", "The aggregated ratings of all surveys are used to create a ranking and letter grade for each carrier within each mode.", "Shippers may apply their own “importance” weights to each rating criterion and calculate their own custom rankings.", "Additionally, each shipper, carrier and 3PL is able to assign their own letter grades to individual counterparties.", "They may rate a preferred counterparty as “A”, a good provider as “B”, and someone they do not want to use as “F”.", "These custom created grades are then displayed to the user for their private use prior to the complete identification of the counterparty in a transaction.", "Furthermore, the TrantisLink system monitors the actions of all parties to a transaction and rates their objective on-line performance by logging events such as availability and timely arrival of booked loads and trucks, prompt entry of tracking information, cargo damage claims, payment of fees, etc.", "Finally, one or more loads and/or trucks are transferred between the public network and private network of a firm by using a form of “rating” to set/unset a private transaction flag.", "The TrantisLink system connects with insurance providers to obtain and then display information on the current in-force coverage held by shippers and carriers.", "Extending a Basic Transportation Contract to Support Transport Additional information must be added to a basic transportation contract to make it possible to transport a load and calculate the total price of the shipment.", "The following example illustrates some of the information that must be added to qualify a basic transportation contract for delivery: Lane: LA to Chicago Lane miles: 2,000 Basic rate: $1.15 per mile Extended basic price: $2,300 Pickup: Location: Address in LA: ZIP-90021 Accessorial: Loading Fee: NC Stop in Transit: Location: Address in Denver: Accessorial: Stop Fee: $50 Final Destination: Location: Address in Chicago: ZIP-60601 Accessorial: Unloading Fee: $100 Calculation of Total Price: Total Price = (Rate Per Mile × Route Miles) + Accessorail Charges Route miles: 2,315 Mileage Fee: $1.15 × 2,315 $2,662.25 Total Accessorial Fees: $150.00 Total Price: $2,812.25 Average Rate/Route Mile: Payment to Carrier Total Price: $2,812.25 Transaction Fee to $10.00 (set by TrantisLink) TrantisLink: (note: volume discounts may apply) Net Amount to Carrier: $2,802.25 Average Rate/Route $1.2105 Mile: Shipment States Shipments are monitored and controlled throughout their entire life cycle from offering through transit and delivery to payment.", "TrantisLink processes user input to implement this functionality and assigns a revised current state (note: in this context, “state” means status and not geographical location) to each shipment as it is being transported.", "A shipment will be in one and only one of the following ten states: 1) Open A shipment is “open” when it is in the market and not yet accepted.", "2) Cancelled A shipment is “cancelled” when it is in the market and the user who originally placed the shipment (or their supervisor) takes it out of the market without the possibility of later re-entry.", "A cancelled shipment may not be accepted.", "3) Subject A shipment is “subject” when it is in the market and the user who originally placed the shipment (or their supervisor) takes it out of the market and saves it for possible later re-entry.", "A subject shipment may not be accepted.", "The user who originally placed the shipment may cancel a subject shipment.", "4) Booked—Not Confirmed A shipment is “booked—not confirmed” when a party (i.e.", "the “active” party) has accepted it, and the (passive) counterparties have not yet been notified.", "A shipment can be accepted and a passive party may not yet have been notified if they are offline.", "TrantisLink system will attempt to notify the passive side using alternative methods specified in the counterparty user profile (e.g.", "paging, fax, automated voice mail, etc.).", "TrantisLink may (using commercially acceptable rules) set a commercially reasonable time limit that a shipment may remain in the booked—not confirmed status.", "5) Booked—Confirmed A shipment is “booked—confirmed” when the active party has accepted it and all of the passive parties have been notified.", "6) In Transit A shipment is “in transit” after the shipment has been loaded at the pickup location and the carrier has departed the loading dock.", "7) Delivered A shipment is “delivered” after the shipment has been unloaded at the destination location and a delivery receipt has been given.", "8) Completed A shipment is “completed” after the shipment has been received and the carrier has been paid.", "The TrantisLink system supports computerized monitoring of payments whether or not payment is effected through TrantisLink.", "9) Claim-in-Progress A completed shipment may at some time in the future have a damage claim.", "TrantisLink system supports computerized monitoring of damage claims.", "10) Failed A 3PL or the original user who placed part of a shipment may apply TrantisLink, rules to fail a shipment from any of the above states prior to it being in transit.", "For example, a shipper may have a shipment in the booked—confirmed status and realize that it no longer requires transportation.", "This may be due to cargo not ready, consignee unable to accept shipment, etc.", "The shipper (or their 3PL) fails the shipment.", "TrantisLink rules are applied to all parties to the shipment who have an incentive through their respective performance ratings to mitigate the effects of any failure.", "A shipment state may be modified according to TrantisLink rules.", "Open shipments may be cancelled or made subject without obligation before they are matched.", "Subject shipments may be resubmitted or modified and then resubmitted or cancelled.", "Users may modify (or cancel) shipments in a “booked—not confirmed” or “booked—confirmed” status.", "These modifications may incur a financial penalty and require effort by the Managing 3PL to mitigate any damages.", "For example, a shipper accepts vans from three different carriers.", "Two of the carriers are online and their notification of the shipment is logged by the system.", "The third carrier is offline.", "The shipper then cancels the shipment.", "A managing 3PL will workout this cancellation according to TrantisLink rules.", "This could mean finding alternative shipments that are acceptable to the carriers and/or having the shipper pay a financial penalty to the carriers.", "The TrantisLink system supports automatic notification of alerts and events via messages to the user conveyed by a variety of technologies including wireless, voice telephone, and web.", "For example, an owner operator may be notified by telephone that he has received a shipment booking, or affected parties will be notified of a change in shipment status.", "The TrantisLink Graphical User Interface TrantisLink information displays are intended to present information in a timely and useful manner with an absolute minimum of required user interaction.", "This is accomplished through the use of a “graphical navigation bar” and “prioritized scheduled push” of information from the server to the client, as shown in FIG.", "7.A standard navigation bar consists of a number of links displayed in text form.", "The basic components of a link in a graphical navigation bar include a title bar 112 and a content area 114, as shown in FIG.", "7.These are usually presented as a vertical list on the left side of the page or a horizontal list at either the top or bottom of the page.", "This methodology implies that the user understands the functionality underlying the URL of each link from its text.", "Some designers prefer to replace some of the text links with buttons that contain a small graphic or picture to hint at the underlying functionality of the link.", "A graphical navigation bar extends this paradigm still further by displaying a small amount of the underlying information behind each link.", "While this uses more area on the viewed page, it gives visual feedback that a request has been properly processed and frequently obviates the need to click through to the link.", "The basic components of a link in a graphical navigation bar include a title bar 112 and a content area 114, as shown in FIG.", "7.The title bar 112 has a title and may have one or more action buttons.", "Clicking an action button causes the action to occur.", "This may change the data in the current link, change the data in a different link, or may cause the display of new links or pages.", "For example, clicking on the button labeled “action 1” closes the link while clicking on the button labeled “action 2” replaces the current page with a new page with or without a graphical navigation bar.", "The content area 114 contains graphical and or text data.", "Clicking on a graphic (e.g.", "chart or picture) causes an action to occur.", "Clicking on one of the four column headings shown in FIG.", "7 causes the redisplay of the content area sorted by the contents of that column.", "Clicking on a data element, such as data element 116, causes an action such as trading or the further display of additional information.", "FIG.", "8 illustrates an exemplary TrantisLink graphical navigation bar containing six links, defined as follows.", "1.Workspace Selection Tools A set of software tools to select which transportation data entry tool or applet to load in the workspace.", "2.Real-time Market Watchlist Selection Tools A set of software tools to select which set of transportation instruments to display as a real-time marketplace watchlist.", "3.Real-time Marketplace Watchlist A display of the basic market parameters of a number of transportation instruments.", "Selecting a transportation instrument in a watchlist causes the display of additional information in the Workspace link regarding the underlying details of the marketplace for that transportation instrument to facilitate the trading of that instrument.", "Unlike other websites, TrantisLink does not display shipments or capacity that are not currently available or cannot be acted upon.", "4.Open Orders A display of a summary of the last few open orders (shipments or capacity whether or not in the currently displayed real-time market).", "When the user places an order it is displayed at the top of this link to give the user visual confirmation that the system has placed the open order into the marketplace.", "When the order is booked or cancelled it is removed from this link to give the user visual confirmation that the system has fulfilled the request and removed it from the marketplace.", "Selecting an open order displays additional information on open orders in the Workspace link.", "5.Tracking and Tracing A display of a summary of the last few orders that have been booked indicating their current status and additional information that may be used in managing the shipment.", "When the user books a shipment or truck it is displayed at the top of this link to give the user visual confirmation that the system has booked the order and removed it from the marketplace.", "When the order is completed it is removed from this link to give the user visual confirmation that the system has fulfilled the request.", "Selecting a shipment in the tracking and tracing link causes the display of additional information on the tracking and tracing of shipments and trucks in the Workspace link.", "6.Workspace A data entry or applet link facilitates the display and entry of basic market parameters for a transportation instrument.", "Typical functionality includes but is not limited to Find a Truck, Find a Shipment, Offer a Truck, Tender a Shipment, Lane History, Preferences, etc.", "Additionally, the TrantisLink system uses Prioritized Scheduled Push (PSP) to maintain the real-time nature of information displayed within the client browser.", "When the client submits a request to a network server, the server responds by updating the contents of one or more links.", "The server stores information and periodically, perhaps once every few seconds, updates the contents of all links on the client without any request from the client.", "The server is able to prioritize the order in which links are updated and match its signaling to the bandwidth of the communication channel.", "Ancillary Services and Products of TrantisLink This section presents a brief summary of ancillary services and products provided by TrantisLink.", "TrantisLink software is also used to perform industry standard validation of insurance coverage.", "In many cases this includes an interface to the underwriting insurance company.", "Also, using TrantisLink, insurance agents provide information on insurance to shippers and/or carriers.", "TrantisLink software is also used to sell transportation cargo and liability insurance.", "TrantisLink software is also used to facilitate and track payments.", "This includes an interface to one or more factor receivable banks.", "TrantisLink software is also used to facilitate and trade insurance claims.", "In many cases this includes an interface to the underwriting insurance company.", "Managing 3PLs and 3PLs provide services to their shippers and carriers including but not limited to cash advances, fuel purchase information, etc.", "Furthermore, banks use TrantisLink to provide the factoring of payment receivables.", "Spot and historical prices, shipping demand, and transportation capacity data are available as optional data products.", "TrantisLink supports the use of both EDI message structures and XML aggregation and syndication (note: aggregation is the process of collecting data from disparate sources; syndication is the process of distributing data to disparate channels) to support data interchange within the transportation community.", "It should be noted that the present invention could implement any current technology that provides the latest and most effective distribution and promotion of data and user interactivity.", "Specifically, the TrantisLink system supports order entry and monitoring by importing and exporting .CSV files (comma separated variable, e.g.", "MS Excel Spreadsheets) in a pre-defined format.", "Exported files are used by a legacy system or any other type of known database to generate standard reports.", "Subjective evaluation of the performance of carriers developed by survey of shippers and background information on carriers will be made available through a websites such as www.CarrierRankings.com.", "The TrantisLink system may be linked into major Enterprise Resource Planning (“ERP”) and major Customer Relationship Management .", "(“CRM”) systems.", "The TrantisLink user base is initially within the United States, Mexico, and Canada.", "Any transport must have either an initial pickup location or a final delivery location within the continental United States.", "TrantisLink will be operational 24 hours a day seven days a week absent any exceptional maintenance.", "Effect of Disconnection Users may specify preferences for open order processing when they either intentionally logout or are unintentionally disconnected.", "They may choose different preferences for each.", "Upon disconnection they may: 1.Remain active in the market The system keeps the orders in the open state but marks the user connectivity as “none”.", "While open, an aggressor may act upon the order.", "A booked shipment will make a status transition from “open” to “booked—not confirmed” since connectivity is “none”.", "When the user next logs in their connectivity is marked as “connected”, they are notified that their open shipment has been accepted, and the shipment will make a status transition from “booked—not confirmed” to “booked—confirmed”.", "2.Be removed from the market and open orders be marked subject The system makes a status transition from “open” to “subject” and marks the user connectivity as “none”.", "The order may not be booked.", "When the user next logs in their connectivity is marked as “connected” and the order that was “open” continues to be indicated as “subject”.", "The user must resubmit the order to change its state to open 3.Be removed from the market and open orders be marked cancelled The system makes a status transition from “open” to “deleted” and marks the user connectivity as “none”.", "The shipment may not be booked.", "When the user next logs in their connectivity is marked as “connected” and the order, which was “open”, is now unavailable on the system.", "The user must re-enter all of the parameters of the order and then resubmit the order to change its state to “open”.", "Account Maintenance The “TrantisLink System Account” is the fixed top-level account.", "AU other firms are one level down in the hierarchy as sub-accounts of the top-level firm.", "Any account at this level can add a virtually unlimited number of sub-accounts at one further level down.", "All of these child sub-accounts are treated as subordinates of the parent firm immediately above them, which is ultimately responsible for all of their TrantisLink-related activity.", "All accounts are created only as a result of an application process.", "The parent firm is solely responsible for creating, suspending, removing and managing the activities of any child sub-account it might want to create.", "TrantisLink assigns and manages requirements including insurance and credit limits for all top-level (firm) accounts.", "A credit limit specifies a maximum dollar value of open shipments that an account and all of their sub-accounts may have within the system.", "A firm, in turn, is free to assign and manage credit limits to all of its sub-accounts.", "The TrantisLink system ensures that an order may not be placed with an account (or sub-account) if it violates a credit limit of this account or a total credit limit of a parent account.", "Security Features of the TrantisLink System TrantisLink encrypts all information transmission.", "All TrantisLink applications require the user to enter a valid user ID and password set to access the TrantisLink system.", "TrantisLink maintains the privacy of all account information pursuant to its privacy policy.", "The system allows each user to have only a single access to the system at a time.", "If the user enters a valid login and password for an account that is already active from a second GUI he will receive an error message that access is denied because the account is already logged on.", "The TrantisLink system software has primary site redundancy for all critical components.", "It is operated as part of a multi-site duster with fully operational servers located within at least two sites.", "Complete failure of one site may reduce throughput without taking the system out of service.", "The TrantisLink system is able to create and manage offsite backups of all processed data in an industry standard format.", "Description of Various TrantisLink Displays The TrantisLink main screen, shown in FIG.", "9, is divided into five logical areas: an action button bar 118; a watchlist 120; a workspace 122; an open orders summary display 124; and a tracking and tracing summary display 126.1) Action Button Bar 118 Action buttons give users access to various transactional procedures.", "For example, the leftmost button, “Alerts” 128, allows the user to cause a list of received alerts to be displayed in the workspace 122.2) Watchlist (Market Summary Display) 120 FIG.", "10 is an isolation of the watchlist of FIG.", "9.Spot Instruments: The spot instruments shown in FIG.", "10 represent the aggregation of all dry van TrantisLink shipments and capacity from the “Chicago” zone to the “Dallas” zone that are currently available on August 6th, 7th and 8th.", "The highest shipper bid price on August 6th 130 is $1.20 a mile and three shipments 132 are ready to go at that rate.", "The lowest carrier offer price 134 is $1.25 a mile and one truck 136 is ready to go at that rate.", "There are a total of six shipper bids 138; i.e.", "there are three other shipper bids at less than the best price in the stack of bids.", "There are a total of two carrier offers 140; i.e.", "there is one other carrier offer at higher than the best price in the stack of offers.", "The last booking 142 was at $1.20 a mile and there were 3 bookings since the market opened 144.Forward Instruments: The forward instruments also shown in FIG.", "10 represent the same aggregation of all dry van TrantisLink shipments and capacity from the “Chicago” zone to “Dallas” zone currently available for a pickup during September or October.", "The bid price for September is $1.30 and there are 4 shipments available at that price and in total.", "There are no current offers of capacity.", "The last forward booking was at $1.30 a mile and one booking was made since the market opened.", "Series Instruments: The series instruments shown in FIG.", "10 represent the same aggregation for the same lane available for periodic pickups during the 4th quarter of 2001 and the 1st quarter of 2002.It is assumed that the quantities are daily shipments and trucks since no “modifiers” are given in the instrument name.", "TrantisLink uses the prefix “W” to change to weekly quantities, and the prefix “M” to change to monthly quantities.", "The parameter values have the same interpretation as spot and forward instruments.", "Shorthaul Instruments: The shorthaul instruments shown in FIG.", "10 represent the aggregation of all dry van TrantisLink shipments and capacity within the “Chicago” zone on August 6th, 7th and 8th.", "The booking consists of any reasonable number of pickups and deliveries on the same day as long as the driven returns to the terminal on the same day.", "No shipments are bid for any of the days.", "The lowest carrier offer price for the day is $400 for the single truck available on the sixth.", "The best price for the day on the seventh is $350 and ten trucks are available at that price.", "There are five additional trucks offered at poorer prices on the seventh.", "No shorthaul bookings have occurred since the market opened.", "3) Workspace (Market Detail Display) 122 As shown in FIG.", "11, clicking on a transportation instrument in the Watchlist causes the following Market Detail Display to be shown and automatically updated in real-time in the workspace 122.Each workspace row summarizes the details (i.e.", "the “trees”) of one or more similar (fuzzy) shipments or trucks.", "The rates and prices are expressed in net amounts available to a carrier; the price is the sum of the line haul charges plus accessorials for services such as extra stops-in-transit, loading and unloading, extra waiting time, etc.", "All fees are subtracted from the gross fee prior to its display.", "The information is presented in order of increasing rate per mile from top to bottom.", "Hence, the first row is the lowest rate that any shipper is willing to pay; and bottom row is the highest rate any carrier is asking.", "In FIG.", "11, available shipments are shown above the dashed line 146; where each row displays: Ratings A series of ratings for the shipper or in this case the abbreviated name of the shipper Miles Total number of route miles including all stops-in-transit (in some cases the miles are “To Be Determined” because the detailed shipping information is not yet available).", "To Car Total amount to be paid to the carrier including all accessorials (in some cases the amount to be paid is “Not Available” because the detailed shipping information has not yet been entered).", "RPM Line haul Rate Per Mile TL Number of trucks required SIT Number of stops-in-transit L/U Loading/Unloading required FDD Unusual first delivery date TT Carrier must have TrantisTracker capability PX Pallet exchange required 53′ 53 foot dry van required HAZ Hazardous cargo In FIG.", "11, available trucks are shown below the dashed line 146; where each row displays: Ratings A series of ratings for the carrier or in this case the abbreviated name of the carrier MinRev Minimum total revenue that the carrier wants for the trip RPM Line haul Rate Per Mile TL Number of trucks required SIT Maximum allowable number of stops-in-transit L/U Loading/Unloading will be provide TEAM Carrier has “team” drivers to support expedited delivery TT Carrier supports TrantisTracker capability PX Carrier will provide pallet exchange 53′ 53 foot dry van available HAZ Hazardous cargo FIG.", "12 illustrates how marketplace anonymity is maintained by replacing the names of shippers and carriers by “ratings”.", "The column previous used to display the name are replaced by up to three different types of ratings.", "The first column 148 with heading MY represents the user's subjective opinion of the shipper or carrier.", "The second column 150 with heading TR represents an objective TrantisLink rating based on the accumulated experience of all users on the system with a particular shipper or carrier.", "The third column 152 with heading CR represents the subjective opinion of all shippers as surveyed in www.carrierrankings.com 4) Open Orders Summary Display 124 Current unfilled market orders tendering shipments and offering trucks are presented.", "The user clicks on the heading or a specific order to display a complete summary list of open orders 154 in the Workspace as shown in FIG.", "13.Users click on one or more column headings 156 to sort the list by that column.", "Users may then modify 158, cancel 160 or “unhold” 162 the open order.", "Editing the order causes the Workspace display 122 to be replaced with a Tender Shipment display 164 as shown in FIG.", "14 with all original unmodified shipment parameters automatically preloaded.", "5) Tracking and Tracing Summary display 126 A brief summary of currently active or incomplete bookings is presented.", "The user click on the heading or a specific booking to display a complete list of incomplete bookings 166 as shown in FIG.", "15.Users click on one or more column headings 168 to sort the list by that column.", "Users then check the status of a booking by clicking the Status button 170, or display details of a booking by clicking the Booking Details button 172, or report events and/or problems by clicking the Problems button 174, or re-tender a shipment by clicking the Re-tender button 176.FIG.", "16 shows the Tracking and Tracing Booking Status display 178.FIG.", "17 shows the Tracking and Tracing Booking Details display 180.FIG.", "18 shows the Tracking and Tracing Booking Problems display 182 which are used to report and update problems and status.", "Clicking the Add button 184 causes the display of the Report a Problem data entry window 186 as shown in FIG.", "19.The problem is submitted by clicking the Submit button 188 after entering the text describing the problem and characterizing its severity causing the display shown in FIG.", "20.All counterparties to the booking receive automatic alerts 190 as shown in FIG.", "21.This window is displayed by clicking the Alerts button 128.As shown in FIG.", "22, clicking the Find-A-Truck button 192 displays the Find A Truck data entry window 194 which may be automatically preloaded with previously used parameters.", "As shown in FIG.", "23, clicking the Find A Shipment button 196 displays the Find A Shipment data entry window 198 which may be automatically preloaded with previously used parameters.", "As shown in FIG.", "24, clicking the Tender A Shipment button 200 displays the Tender A Shipment data entry window 202 which may be automatically preloaded with previously used parameters.", "Users may use the Clone Existing Shipment button 204, Import List of Shipments button 206, or change customer button 208.Shipments may be “basically” or “completely” tendered.", "Basically tendered shipments do not contain all of the shipment parameters that would be necessary for transporting the cargo.", "For example, a basically tendered shipment may not contain the specific origin and destination addresses.", "A completely tendered shipment contains all the details necessary to transport the cargo.", "As shown in FIG.", "25, clicking the Offer A Truck button 210 displays the Offer A Truck data entry window 212 which may be automatically preloaded with previously used parameters.", "Users may use the Clone Existing Offer button 214, Import List of Offers button 216, or change customer button 218.Trucks may be “singly” or “multi” offered.", "Basically offered trucks have a single destination zone.", "A multi-offered truck has more than one possible destination and rate per mile.", "These destinations and rates are entered in area 220 and submitted by clicking the Submit button 222.The TrantisLink system responds as shown in FIG.", "26 by displaying the Offer Truck Results window 224, modifying the Watchlist 120 to reflect the four contingent offer prices 226, 228,230 and 232 and quantities 234, 236, 238 and 240, and modifying the Open Orders Summary Display 124 to reflect the contingent truck offers 242, 244, 246 and 248 (off screen).", "As shown in FIG.", "27, clicking the Completed Shipments button 250 displays the Completed Shipments data entry window 252 which may be automatically preloaded with previously used parameters.", "As shown in FIG.", "28, clicking the Lane History button 254 displays the Lane History Selection and Display window 256.Users select the mode 258, time period 260 and lane 262.Clicking Retrieve Charts 264, Print Charts 266 or Save Data to File 268 retrieves the corresponding lane history data 270.Shown are the daily range for rate per mile 272, net truck demand 274 and number of shipments 276.As shown in FIG.", "29, clicking the Prefs button 278 displays the Personal Settings data entry window 280.Logging into the Network Administration Site results in the display shown in FIG.", "30.The user may click Company Administration 282 or Company Exposures 284 buttons.", "As shown in FIG.", "31 the user may view and maintain accounts and users for companies.", "The user may search for 286, create 288, or select and administer a company 290 (for example Jack Trucks).", "As shown in FIG.", "32, the user may administer users 292, administer accounts 294, view and update the company profile 296 or set or view the company's trading exposure 298.As shown in FIG.", "33, the user may view the number of problems 300, bookings 302, open orders 304, current exposure 306, remaining exposure 308, and exposure limits 310 for each company or the entire network.", "Thus, TrantisLink enables customers to plan with a far greater degree of confidence than before because TrantisLink provides Best Available rates (shipper bids and carrier offers), total number of currently available shipments and trucks, last trade and daily trade volume, depth of the market including shipment details such as accessorials and special shipment requirements, alerts to transportation problems, tracking and tracing of your loads in transit, completed shipment reports, and even historical rates with supply and demand data.", "Using Series and Forward Contracts to Hedge Price and Availability Risk The current practice within the transportation industry is for large shippers to select “core carriers” usually on an annual basis by sending candidate carriers a copy of last year's annual freight bills and asking them to provide a single annual rate per mile for each type of shipment in a given lane.", "The carriers process the annual freight bills to determine the anticipated usage on a monthly basis and then calculate an average rate for the year.", "A simpler and more effective methodology of selecting “core carriers” is illustrated in FIG.", "34.This process does not require candidate carriers to analyze previous year freight bills to predict monthly usage.", "Rather, the shipper determines their anticipated usage and bids-out a given number of annual series contracts 314, quarterly series contracts 316 and monthly forward contracts 318 Candidate carriers either accept the bid or respond by offering capacity in each of the instruments.", "Either the shipper or carrier finally book a contract and make a firm commitment to transport goods with their newly selected “core carrier”.", "It is possible to book more than one “core carrier” for a partial amount of each contract.", "Actual usage 320 usually closely approximates anticipated usage 312.Buying in the spot market 322 fills any shortfall differences.", "Should actual usage fall below the number of contracts in effect at a given time the shipper sells in the spot market 322.Processing External Market Data to Obtain Clean Lane History Rate Per Mile Prices It is necessary to use standardized accessorial charges to normalize shipment data received from external trading networks.", "Consider for example two offers to perform the same 1,000 mile shipment: Carrier Rate Per Mile Line Haul Accessorials Total Price A $1.20 $1,200 $0 $1,200 B $1.00 $1,000 $200 $1,200 Carrier A has the policy of not charging for the provided accessorials; carrier B has the policy of charging for the accessorials but giving a lower rate per mile.", "Using the total price and applying a standard price for the provided accessorials results in dean lane history rate per mile price data.", "In this example the standard price for the provided accessorials is $100 resulting in a dean rate per mile of ($1,200-$100)/1,000 miles equals $1.10/mile.", "Private Networks and Collaborative Logistics FIG.", "35 illustrates a private network run by a sponsor (super shipper or 3PL) who has authorized access for their shippers (S), carriers (C), consignees (R—receivers of goods) and dispatchers (D).", "Authorized shippers and dispatchers tender loads (L) or find a truck.", "Authorized carriers and dispatchers offer trucks (T) or find a load.", "Shippers, carriers and receivers may be authorized to have access to more than one private network; dispatchers usually have access to only one private network.", "The intersection (i.e.", "shaded area) of the tendered loads (L) and offered trucks (T) represents those shipments that have been booked on the private network.", "The un-shaded areas represent distressed loads and trucks.", "The word distressed is commonly used in the transportation industry to mean unmatched situations and does not convey the more usual English meaning of low quality or undesirable.", "To alleviate the above distress, TrantisLink will also support anonymous collaborative logistics.", "As the number of interconnected private networks increases a point is reached where the multiply connected private networks will become in effect a public marketplace supervised by multiple sponsors or 3PLs.", "These supervisors provide human intervention to alleviate problems that may occur in the marketplace during the life cycle of a transaction.", "Collaborative logistics is the ability of multiple participants to share information for mutual benefit.", "TrantisLink provides anonymous collaborative logistics as explained below.", "FIG.", "36 illustrates two private networks A and B run by two different sponsors, where the two networks are entirely isolated from each other.", "When private networks are isolated from one another as in FIG.", "36, i.e.", "participants on one network do not have access to information on the other network, so that distressed loads on one network may not be matched to distressed trucks on the other network.", "Thus, collaboration between the two networks can not be achieved without interconnecting the two networks.", "However, the advantage of TrantisLink collaborative logistics is that participants do not have to know or enter into agreements with their competitors to gain the benefits of collaboration.", "Unlike other methodologies, TrantisLink collaborative logistics is truly anonymous.", "This is because private networks are but a first step toward public networks.", "As an Application Service Provider (ASP), TrantisLink provides the unique availability to isolate or interconnect multiple private networks on one physical system.", "TrantisLink provides collaborative logistics opportunities by interconnecting its private and public networks.", "Collaborative logistics, the ability of multiple participants to share information for mutual benefit, occurs when one or more private networks are interconnected such that distressed loads and trucks on one network are matched to distressed loads and trucks on the other network.", "This is illustrated in FIG.", "37, which shows how some of the distressed loads (L) on Network A are matched to some of the distressed trucks (T) on Network B, and vice versa.", "TrantisLink provides private networks to TrantisLink, sponsors to allow them to offer their existing and new customers (shippers, carriers, and consignees) state-of-the-art easily integrated information-technology at an affordable price.", "TrantisLink Revenue Model TrantisLink revenue is derived from a combination of transaction fees, license fees for systems and motor carrier market data, and ancillary services.", "Transaction fees for booking and/or tracking-and-tracing are deducted from shipper payments.", "This payment methodology is common to the motor carrier transportation industry.", "Network sponsors pay license fees for private network systems.", "Subscribers to motor carrier market data pay monthly fees on an annual basis.", "These are explained in more detail organized by product segment below.", "The monthly fee for use of TrantisLink is initially capped.", "Also, there are no additional fees after a predetermined number of transactions in a month.", "Cap levels will be increased as product adoption occurs.", "The minimum monthly fee is the larger of a per user fee or per private network fee.", "Also, each subscriber can pay a set fee per month on an annual basis for the ability to view (but not download) historical and real-time motor carrier transportation market data.", "Each subscriber can also pay a set fee per month on an annual basis for the ability to view and/or download historical and real-time motor carrier transportation market data.", "Subscribers will be prohibited contractually from redistributing the data outside of their own company.", "Advantages of TrantisLinkSM Managing transportation is currently a labor-intensive process.", "TrantisLinkSM enables its users to use contingent order processing to facilitate connecting shippers and carriers.", "For example, a carrier offers a truck into the marketplace from a single origin but with multiple possible destinations.", "When one of the offers is accepted all of the other contingent offers are immediately removed.", "TrantisLink supports shippers and carriers connected simultaneously to multiple private networks and the public network at one time.", "This enables them to conduct all of their business with multiple vendors using a single TrantisLink interface, rather than having to use several different types of systems that are not interconnected.", "Need: There is an existing marketplace need for customers to receive and respond to shipment exception alerts.", "Solution: TrantisLink automatically generates and monitors real-time exception alerts.", "Need: 3PLs desire to provide improved services at lower cost.", "Solution: The functionality of TrantisLink allows each broker or 3PL to manage five to ten times the number of simultaneous loads than are handled by outdated FAX and telephone technology.", "Need: Customers need to improve their order processing efficiency and become more profitable.", "Solution: TrantisLink users dick on a market summary line, which will then display the underlying details of loads and available trucks.", "The relevant parameters for each load and truck are presented in a one-line summary.", "Users then click on any column heading to sort (rather than scroll through) the list of opportunities and evaluate them.", "Booking a selected load or truck is as simple as clicking the “book” button.", "The system then immediately notifies all affected parties.", "Need: Easy integration into legacy systems for both shippers and carriers.", "Solution: Many users have legacy applications that prepare lists of orders that they want to submit to TrantisLink.", "Accordingly, TrantisLink system supports order entry and monitoring by importing and exporting CSV files (e.g.", "MS Excel Spreadsheets) in a pre-defined format.", "The exported files may be used by the legacy system to generate standard reports.", "Shippers tender shipments onto TrantisLink and provide one side of the trades on the system.", "TrantisLink shippers now have an alternative to the annual paper-based carrier “contracting” process.", "In addition, TrantisLink provides increased supply-chain visibility and the ability to optimize their transportation networks.", "Carriers utilize TrantisLink to secure shipments from shippers and better utilize their assets.", "TrantisLink carriers benefit from the ability to offer trucks, reduced empty miles, and rules that provide stronger contracts than currently available.", "When the shipment is booked the carrier is immediately notified and both sides have a contractual obligation to perform.", "The 3PL (either internal or external) receives an alert that the shipment was tendered and relies upon software tools to monitor the normal or exception handling of the order.", "When exceptions occur the 3PL broker is able to use TrantisLink software to correct the situation.", "The broker receives notification when insurance is already in place.", "In most circumstance the broker does not have to use the FAX or telephone.", "Hence, brokers are able to manage five to ten times the number of loads per day.", "Need: Customers need accurate market data prior to making a decision on transportation.", "Solution: TrantisLink provides one-line summaries of transportation marketplaces sorted by lane, type of equipment, date, best prices and number of available loads and trucks.", "Users may also view historical price, demand and daily load data.", "Need: Customers want the ability to reduce their uncertainty in the marketplace.", "Shippers want to lock in capacity for future anticipated needs.", "Carriers want to predetermine their workload to minimize the cost of asset relocation.", "Solution: TrantisLink provides these capabilities by supporting forward and/or series purchases of shipments and trucks.", "Need: Shippers want the cost savings associated with collaborating with others without having to identify who they are and then taking on the hassle of negotiating an agreement with every other shipper.", "Solution: TrantisLink, automatically identifies collaborative opportunities, checks to ensure that all of each shipper's requirements are satisfied, and then books the transaction without the need to give up any identifying information or any human intervention.", "Need: Customers need the ability to lower costs by offering a resource to more than one counterparty at a time; and, when the first counterparty accepts the offer, to have the system automatically remove all of the remaining offers.", "Solution: TrantisLink performs this in real time; it is called contingent order processing.", "Need: Customers need the ability to increase the probability of finding a backhaul.", "Solution: TrantisLink uses contingent order processing to increase the probability of finding a backhaul.", "TrantisLink offers “depth of market” displays including one-line summaries of each shipment's details including standard accessorials.", "TrantisLink possesses automated search functions to “find a truck” or “find a shipment”.", "TrantisLink also possesses the ability to buy and sell transportation over a period of time in the future to hedge price and availability risk.", "TrantisLink improves on the current industry practice of annual rate-only agreements without any minimum volume commitments by creating transportation instruments that are backed by enforceable contracts.", "Furthermore, TrantisLink offers tracking-and-tracing of all shipments regardless of whether they were booked on a TrantisLink network.", "This enables shippers and receivers of goods to manage their terminals efficiently even when using more than one carrier.", "In addition to spot transactions, TrantisLink provides forward and series contracts, a very significant advance over the current standard practice of incomplete annual pricing agreements that do not obligate the parties to minimum volume commitments.", "Finally, TrantisLink's market visibility, immediate transaction execution and fulfillment alerts allow each dispatcher to process more shipments.", "TrantisLink provides real-time and historical information on pricing, availability and fulfillment problems.", "The transportation instruments created with TrantisLink are backed by enforceable contracts specifying price, pick-up and delivery.", "TrantisLink offers experienced transportation experts service real-time fulfillment alerts using advanced software tools.", "TrantisLink also enables shippers and carriers to hedge risk by committing to future obligations.", "TrantisLink participants may trade-out of their obligations whenever their anticipated needs are not realized.", "While the invention has been described and illustrated with reference to specific exemplary embodiments, it should be understood that many modifications and substitutions could be made without departing from the spirit and scope of the invention.", "Accordingly, the invention is not to be considered as limited by the foregoing description but is only limited by the scope of the appended claims.", "What is claimed is:" ] ]

[ [ "Sampling device and sampling method", "A sampling device (1) comprises a sampling container (4) for reception of a sample volume and a connecting piece (7) adapted to be connected to a fitting (2) mounted on a processing installation.", "The connecting piece (7) has a sample passage which is provided with a stationary valve port (12) and a corresponding valve member (14) which is displaceable between a closed and an open position.", "The stationary valve part (12) and the displaceable valve member (14) are integrated in the sampling container (4).", "The valve member (14) is spring-loaded towards its closed position and adapted to be automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2)." ], [ "1.A sampling device (1) comprising a sampling container (4) for reception of a sample volume and a connecting piece (7) adapted to be connected to a fitting (2) mounted on a vessel (3) or pipe of a processing installation or the like, the connecting piece (7) having a sample passage which is provided with a stationary valve part (12) and a corresponding valve member (14) which is displaceable between a closed position, in which it abuts the stationary valve part (12) and closes the sample passage, and an open position, in which the sample passage is open, whereby the valve member (14) is spring-loaded towards its closed position and adapted to be automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2), characterized in that the stationary valve part (12) or the valve member (14) is formed as a conical face forming part of a wall surrounding the inside volume of the sampling container (4), and in that the other one of said stationary valve part (12) and said valve member, (14) has the form of a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container.", "2.A sampling device according to claim 1, characterized in that the connecting piece (7) is adapted to be displaced in a longitudinal direction of the container (4) during at least part of the operation of connecting it to the fitting (2), and in that the displaceable valve member (14) is adapted to abut an edge (20) of the fitting (2) during at least part of said displacement.", "3.A sampling device according to any one of the preceding claims, characterized in that the connecting piece (7) is adapted to be screwed onto the fitting (2).", "4.A sampling device according to any one of the preceding claims, characterized in that the sampling container (4) comprises an outer container (5) and an inner container (6), the inner container (6) being arranged displaceably in a longitudinal direction of the outer container (5), in that the outer container (5) at a first end is formed integrally with the connecting piece (7) and at a second end is provided with a bottom (10), in that the inner container (6) at a first end is formed integrally with an annular sealing surface (15), thereby forming the displaceable valve member (14), and at a second end has a bottom (16) in which a spindle passage (17) is provided, in that a spindle (11) has a first end which is provided with the stationary valve part (12) and a second end which is fixed to the bottom (10) of the outer container (5), and in that the spindle passage (17) is arranged tightly around and displaceably along the spindle (11).", "5.A sampling device according to claim 4, characterized in that the outer length of the outer container (5) is between 100 mm and 300 mm, in that the outer diameter of the outer container (5) is between ½ and ⅔ of the outer length of the outer container (5), and in that the smallest diameter of the annular sealing surface (15) of the inner container (6) is between ¼ and ⅔ of the outer diameter of the outer container (5).", "6.A sampling device according to claim 4 or 5, characterized in that the outer container (5) and the inner container (6) are formed from Plexiglass, glass, tempered glass or the like, and in that the stationary valve part (12) is formed from polytetrafluoroethylene or the like.", "7.A sampling device according to any one of the preceding claims, characterized in that the sampling device (1) comprises a fitting (2) mating the connecting piece (7) and having an installation end adapted to be installed onto the vessel or pipe of said processing installation or the like, and in that the fitting (2) has a tubular part (24) provided with an inner shielding meter (25) adapted to shield the stationary valve part (12) in the connected state of the connecting piece (7) and the fitting (2).", "8.A sampling device according to claim 7, characterized in that the tubular part (24) of the fitting (2) is provided with a covering member (31) adapted to cover the displaceable valve member (14) in the connected state of the connecting piece (7) and the fitting (2).", "9.A sampling device according to claim 8, characterized in that the shielding member (25) is adapted to seal against the covering member (31) in the disconnected state of the connecting piece (7) and the fitting (2).", "10.A sampling device according to claim 8 or 9, characterized in that a circumferential contour (29) of the shielding member (25) fits a circumferential contour (30) of the stationary valve part (12), in that the shielding member (25) is adapted to be displaced with the stationary valve part (12) against a spring-load during at least part of the operation of connecting the connecting piece (7) to the fitting (2), in that a circumferential contour (32) of the covering member (31) fits a circumferential contour (33) of the displaceable valve member (14), in that the covering member (31) is fixedly mounted in the fitting (2), in that the circumferential contour (29) of the shielding member (25) fits the circumferential contour (32) of the covering member (31), and in that the circumferential contour (30) of the stationary valve part (12) fits the circumferential contour (33) of the displaceable valve member (14).", "11.A sampling device according to any one of the claims 8 to 10, characterized in that the shielding member (25) has a conical sealing surface (34) corresponding to a conical sealing surface (35) on the covering member (31).", "12.A sampling device according to any one of the claims 8 to 11, characterized in that the shielding member (25) is spring-loaded against the covering member (31) by means of a spring (28) located in a tube element (25) in which a spindle of the shielding member (25) is guided, in that the tube element (26) is guided axially in the tubular part (24) of the fitting (2), and in that the tube element (26) is fixed in a plate (27) adapted to abut an edge (38) of a flange (37) on the vessel or pipe of the processing installation or the like, in the mounted state of the fitting on said flange.", "13.A sampling device according to any one of the claims 7 to 12, characterized in that the fitting comprises a shut-off valve (22).", "14.A sampling device according to any one of the claims 9 to 13, characterized in that the sampling device (1) comprises a nozzle (41) adapted to be connected to the fitting (2) and having an internal projection (44) adapted to keep the shielding member (25) displaced against the spring-load in the connected state of the nozzle (41) to the fitting (2), and in that the nozzle (41) has a tube connection (43) for supply or discharge of cleaning or washing fluid.", "15.A sampling method for taking a sample of a product from a processing installation or the like, comprising the steps of connecting a connecting piece (7) of a sampling device (1) to a fitting (2) mounted on a vessel (3) or pipe of the processing installation or the like, displacing a valve member (14) in a sample passage of the connecting piece (7) from a closed position, in which it abuts a stationary valve part (12) and thereby closes the sample passage, to an open position, in which the sample passage is open, allowing product to pass from the processing installation or the like, through the sample passage, and into a sampling container (4) of the sampling device (1), displacing the valve member (14) from its open position to its closed position, and disconnecting the connecting piece (7) from the fitting (2), whereby the valve member (14) in displaced from its closed position to its open position against a spring-load, and whereby the valve member (14) is automatically displaced to its open position upon connection of the connecting piece (7) with the fitting (2), characterized by that, in the open position of the valve member (14), the product passes through a conical annular passage formed between a conical face forming part of a wall surrounding the inside volume of the sampling container (4) and a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container.", "16.A sampling method according to claim 15, characterized by that the valve member (14) is displaced to its open position by means of a covering member (31) fixedly mounted in the fitting (2) and abutting the valve member (14) during at least part of the operation of connecting the connecting piece (7) to the fitting (2), whereby a circumferential contour (32) of the covering member (31) fits a circumferential contour (33) of the valve member (14), by that a shielding member (25) mounted displaceably in the fitting (2) abuts the stationary valve part (12), whereby a circumferential contour (29) of the shielding member (25) fits a circumferential contour (30) of the stationary valve part (12), and whereby the shielding member (25) is displaced against a spring-load during at least part of the operation of connecting the connecting piece (7) to the fitting (2), and by that, after disconnecting the connecting piece (7) from the fitting (2), the circumferential contour (29) of the shielding member (25) fits the circumferential contour (32) of the covering member (31), and the circumferential contour (30) of the stationary valve part (12) fits the circumferential contour (33) of the displaceable valve member (14).", "17.A sampling method according to claim 15 or 16, characterized by that a shut-off valve (22) of the fitting (2) is opened and subsequently closed in the fully connected state of the connecting piece (7) and the fitting (2) in order to allow the product to pass through the fitting (2).", "18.A sampling method according to claim 16, characterized by that, in the disconnected state of the connecting piece (7) and the fitting (2), a nozzle (41) is connected to the fitting (2), whereby an internal projection (44) in the nozzle (41) keeps the shielding member (25) displaced against the spring-load in the connected state of the nozzle (41) to the fitting (2), and by that a cleaning or washing fluid is supplied to or discharged from the fitting through the nozzle (41)." ], [ "The present invention relates to a sampling device comprising a sampling container for reception of a sample volume and a connecting piece adapted to be connected to a fitting mounted on a vessel or pipe of a processing installation or the like, the connecting piece having a sample passage which is provided with a stationary valve part and a corresponding valve member which is displaceable between a closed position, in which it abuts the stationary valve part and closes the sample passage, and an open position, in which the sample passage is open, whereby the valve member is spring-loaded towards its closed position and adapted to be automatically displaced to its open position upon connection of the connecting piece with the fitting.", "EP 0141.940 codesponding to U.S. Pat.", "No.", "4,580,452 discloses a sampling container being connected through a pipe to a manually operated sampling valve which way be screwed into a fitting mounted on a conduit forming part of a chemical plant installation.", "The fitting is also provided with a manually operated valve in order to close the outlet from the conduit when the sampling valve is not connected to the fitting.", "To take a sample, firstly the sampling valve must be screwed into the fitting, the two valves must be opened and subsequently closed, and the sampling valve must be disconnected from the fitting.", "Obviously, this procedure is time-consuming, and furthermore there is a risk of forgetting to close one of the valves after having taken a sample, whereby possibly hazardous product could escape from the sampling system.", "Additionally, the configuration of the sampling container and its connected valve is awkward to handle and susceptible to damages if dropped, which may result in product spillage.", "Furthermore, the device is suitable for the sampling of fluids only, as products such as powder or granules would clog up the passages through the valves and the pipe.", "DE 40 34 700 describes a dual-valve system for the taking of a fluid sample from a pipe system.", "A first valve member in the form of a truncated cone is arranged in a pipe rotatably about an axis perpendicular to the direction of flow in the pipe and has a through passage in-line with the pipe opening when set to its open position.", "In this open position, a sample may be taken from the pipe through a second valve arranged in a connection piece inserted in the lower side of the truncated cone.", "After having taken a sample, both valves are closed, and the internal product-contaminated surfaces which are situated between the two closed valves are cleaned by means of a spray device.", "However, this procedure is most cumbersome and time-consuming.", "Furthermore, the system also has the disadvantage that the second valve must be closed manually in order to prevent spillage of the product sampled.", "The sampling container protrudes radially from the second valve and makes the device awkward in use.", "DE 43 01 174 discloses a sampling valve having a semi-cylindrical valve member arranged rotatably in a bore extending tangentially to the inner surface of a pipe wall.", "An external sampling container is screwed into a fitting which is mounted on the outside of the pipe wall and is in fluid connection with the bore through a passage.", "When the valve member is in its open position, a fluid may pass from the pipe to the sampling container.", "After removing the sampling container from the fitting, the container is open to the surroundings and consequently product may be spilled.", "DE 197 35 586 shows a sampling valve having an outlet opening through the wall of a pipe and a corresponding valve member which by means of a spindle is manually operable from the outside of the pipe.", "A sampling container for the reception of a fluid may be held under the outlet opening when the valve is opened.", "U.S. Pat.", "No.", "4,689,306 describes a device for sterile sampling from a fermenter.", "The device comprises a sample collector having a normally closed valve mounted on its neck inside a valve housing and a sampler having a normally closed valve, also located in a valve housing, and being positioned on the fermenter.", "To take a sample, the neck of the sample collector is positioned against the sampler, whereby rods of said valves press against each other, thereby opening the valves and allowing product to enter the sample collector through the valve housings.", "U.S. Pat.", "No.", "4,699,356 describes a fluid sampling valve, within the body of which there is a central bore interconnecting a sample end and a vessel end, thereby allowing fluid communication through the valve.", "Within the central bore, there is a cylindrical valve stem having at one end proximate to the sample end a conically-faced head being urged toward a sealing ring in the sample end by a spring, thereby closing the central bore against the surroundings.", "By inserting a hollow cylindrical sampling probe through said sealing ring, the conically-faced valve head is lifted from the sealing ring, and fluid communication is provided through a hole in the wall of the hollow sampling probe to the fluid sample container.", "U.S. Pat.", "No.", "4,150,575 describes a fluid sampling system comprising a fluid coupling and a sampler in which the coupling is included.", "The fluid coupling comprises a coupling valve that includes an annular valve seat axially slidable within a valve housing of the fluid coupling.", "The seat has a conical central opening, within which a valve closure member is slidable and includes a tapered head shaped and dimensioned to fit tightly within the seat.", "In order to take a sample, the coupling is connected to a coupling receiver comprising a receiver housing and a plug which form a fluid tight seal in a manner similar to that of the coupling valve.", "Upon connection, the valves open, and fluid can then enter a cross-bore in the closure member of the valve leading to a small diameter axial bore by which it exits from the opposite end of the coupling.", "The sampling device according to the invention is characterized in that the stationary valve part or the valve member is formed as a conical face forming part of a wall surrounding the inside volume of the sampling container, and in that the other one of said stationary valve part and said valve member has the form of a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container.", "Because the integrated design ensures a short and unimpeded path for the product to be sampled, the sampling device according to the present invention is very suitable for the sampling of powders, granules and the like, as well as any type of fluid or flowable product.", "The sampling container will always be automatically closed by means of the valve member upon disconnection of the container from the fitting, and product spillage through the connection piece may be hindered, even if the container should be dropped.", "Furthermore, the integrated design of the stationary valve part and the valve member in the sampling container provides for a more robust construction and a device which is easier and therefore safer to handle.", "Consequently, also the risk of dropping the device is reduced.", "The displaceable valve member has a conical sealing surface corresponding to a conical sealing surface on the stationary valve part.", "This ensures a good sealing effect.", "In a simple and therefore reliable embodiment, the connecting piece is adapted to be displaced in a longitudinal direction of the container during at least part of the operation of connecting it to the fitting, and the displaceable valve member is adapted to abut an edge of the fitting during at least part of said displacement.", "The valve member will then be displaced in the sampling container to its open position as a result of the displacement of the connecting piece in the direction against the fitting.", "In a preferred embodiment, the connecting piece is adapted to be screwed onto the fitting.", "This ensures a strong connection between the connecting piece and the fitting.", "The device may in this way be designed explosion proof, for instance in order to be able to withstand an internal pressure of 10 bar In an advantageous embodiment, the sampling container comprises an outer container and an inner container, the inner container is arranged displaceably in a longitudinal direction of the outer container, the outer container is at a first end formed integrally with the connecting piece and is at a second end provided with a bottom, the inner container is at a first end formed integrally with an annular sealing surface, thereby forming the displaceable valve member, and has at a second end a bottom in which a spindle passage is provided, a spindle has a first end which is provided with the stationary valve part and a second end which is fixed to the bottom of the outer container, and the spindle passage is arranged tightly around and displaceably along the spindle.", "By the provision of two containers arranged one inside the other, an even more robust construction is achieved, especially in terms of the ability to withstand a high internal pressure, but also considering the risk of dropping the container.", "Furthermore, the device is simple to manufacture because very few components are required.", "In an embodiment, the outer length of the outer container is between 100 mm and 300 mm, the outer diameter of the outer container is between ½ and ⅔ of the outer length of the outer container, and the smallest diameter of the annular sealing surface of the inner container is between ¼ and ⅔ of the outer diameter of the outer container.", "In an advantageous embodiment, the outer container and the inner container are formed from Plexiglass (registered trademark), glass, tempered glass or the like, and the stationary valve part is formed from polytetrafluoroethylene or the like.", "This configuration allows visual inspection of the product sample through the Plexiglass or glass, and a good sealing effect is obtained between Plexiglass or glass and polytetrafluoroethylene.", "In a further embodiment, the sampling device comprises a fitting mating the connecting piece and having an installation end adapted to be installed onto the vessel or pipe of said processing installation or the like, and the fitting has a tubular part provided with an inner shielding member adapted to shield the stationary valve part in the connected state of the connecting piece and the fitting.", "Thereby the outward surface of the stationary valve part is shielded against the sample product during taking of the sample and consequently this surface will be free from product after removal of the sampling container from the fitting.", "This may be an advantage especially in case of hazardous products.", "Further, the product flow may be guided and thereby facilitated by the shielding member.", "In a further embodiment, the tubular part of the fitting is provided with a covering member adapted to cover the displaceable valve member in the connected state of the connecting piece and the fitting.", "Thereby also the displaceable valve member will be free from product after removal of the sampling container from the fitting and the cleanliness of the device is further improved.", "In a further embodiment, the shielding member is adapted to seal against the covering member in the disconnected state of the connecting piece and the fitting.", "This prevents product from leaking from the fitting after removal of the sampling container from the fitting.", "Especially in case of toxic products, this may be an advantage.", "In a further embodiment, a circumferential contour of the shielding member fits a circumferential contour of the stationary valve part, the shielding member is adapted to be displaced with the stationary valve part against a spring-load during at least part of the operation of connecting the connecting piece to the fitting, a circumferential contour of the covering member fits a circumferential contour of the displaceable valve member, the covering member is fixedly mounted in the fitting, the circumferential contour of the shielding member fits the circumferential contour of the covering member, and the circumferential contour of the stationary valve part fits the circumferential contour of the displaceable valve member.", "Thereby a product sample may be taken out from a processing installation in a fully contained way, so that, after disconnection of the sampling container from the fitting, substantially no product will leak to the surroundings of the fitting and the sampling container.", "In this way, the operator will practically not be exposed to the product sampled.", "The shielding member may have a conical sealing surface corresponding to a conical sealing surface on the covering member.", "This ensures improved sealing effect.", "In a further embodiment, the shielding member is spring-loaded against the covering member by means of a spring located in a tube element in which a spindle of the shielding member is guided, the tube element is guided axially in the tubular part of the fitting, and the tube element is fixed in a plate adapted to abut an edge of a flange on the vessel or pipe of the processing installation or the like, in the mounted state of the fitting on said flange.", "Thereby the shielding member will be automatically spring-loaded against the covering member upon installation of the fitting on the flange.", "In an advantageous embodiment, the fitting comprises a shut-off valve.", "This may especially be advantageous if no shielding and covering members are provided in the fitting, or if both high and low pressures may occur in the vessel or pipe of the processing installation.", "In the latter case the shut-off valve ensures that the shielding member is not lifted from the covering member as a result of a pressure difference between the internal of the processing system and the exterior surroundings.", "In a further embodiment, the sampling device comprises a nozzle adapted to be connected to the fitting and having an internal projection adapted to keep the shielding member displaced against the spring-load in the connected state of the nozzle to the fitting, and the nozzle has a tube connection for supply or discharge of cleaning or washing fluid.", "Thereby the interior of the fitting may be cleaned or washed after the taking of a sample.", "The present invention also relates to a sampling method for taking a sample of a product from a processing installation or the like, comprising the steps of connecting a connecting piece of a sampling device to a fitting mounted on a vessel or pipe of the processing installation or the like, displacing a valve member in a sample passage of the connecting piece from a closed position, in which it closes the sample passage, to an open position, in which the sample passage is open, allowing product to pass from the processing installation or the like, through the sample passage, and into a sampling container of the sampling device, displacing the valve member from its open position to its closed position, and disconnecting the connecting piece from the fitting, whereby the valve member is displaced from its closed position to its open position against a spring-load, and whereby the valve member is automatically displaced to its open position upon connection of the connecting piece with the fitting.", "The sampling method is characterized by that, in the open position of the valve member, the product passes trough a conical annular passage formed between a conical face forming part of a wall surrounding the inside volume of the sampling container and a truncated cone carried by a spindle extending coaxially through the inside volume of the sampling container.", "Thereby the above-mentioned advantages are obtained.", "In a further embodiment of the sampling method, the valve member is displaced to its open position by means of a covering member fixedly mounted in the fitting and abutting the valve member during at least part of the operation of connecting the connecting piece to the fitting, whereby a circumferential contour of the covering member fits a circumferential contour of the valve member, by that a shielding member mounted displaceably in the fitting abuts the stationary valve part, whereby a circumferential contour of the shielding member fits a circumferential contour of the stationary valve part, and whereby the shielding member is displaced against a spring-load during at least part of the operation of connecting the connecting piece to the fitting, and by that, after disconnecting the connecting piece from the fitting, the circumferential contour of the shielding member fits the circumferential contour of the covering member, and the circumferential contour of the stationary valve part fits the circumferential contour of the displaceable valve member.", "Thereby a sample may be taken out in a fully contained way as explained above.", "In a further embodiment of the sampling method, a shut-off valve of the fitting is opened and subsequently closed in the fully connected state of the connecting piece and the fitting in order to allow the product to pass through the fitting.", "Thereby the above-mentioned advantages are obtained.", "In a further embodiment of the sampling method, in the disconnected state of the connecting piece and the fitting, a nozzle is connected to the fitting, whereby an internal projection in the nozzle keeps the shielding member displaced against the spring-load in the connected state of the nozzle to the fitting and a cleaning or washing fluid is supplied to or discharged from the fitting through the nozzle.", "Thereby the above-mentioned advantages are obtained.", "The invention will be described in more detail below by means of examples of embodiments with reference to the schematic drawing, in which FIG.", "1 is a partially sectional view of a sampling device according to the invention, connected to a fitting, FIG.", "2 is a sectional view of the sampling device in FIG.", "1, after disconnection from the fitting, FIGS.", "3 and 4 are sectional views of other embodiments of the sampling device, FIG.", "5 is a sectional view of a flange and a fitting for connection to the sampling device, before mounting the fitting on the flange, FIG.", "6 to 8 are sectional views of a further embodiment of the sampling device, in the connected state of the connecting piece on the fitting, in the partially disconnected state, and in the fully disconnected state, respectively, FIG.", "9 to 11 are sectional views of a nozzle of the sampling device, in the connected state of the nozzle on the fitting, in the partially disconnected state, and in the fully disconnected state, respectively.", "FIG.", "1 shows a sampling device 1 according to the invention, connected to a fitting 2 mounted on a vessel 3 of a processing installation of which is shown only part of a wall.", "The sampling device 1 has sampling container 4 comprising an outer cylindrical container 5 and an inner cylindrical container 6 arranged concentric inside the outer container 5 so that it is displaceable in the axial direction of the latter.", "The sampling device 1 and the method of sampling according to the invention may be used in all industries, e.g.", "such as pharmaceutical, biotechnological and chemical, for all kinds of processing equipment or storage containers as well as pipe systems, and for all kinds of products, e.g.", "such as powders, granules, liquid products and gaseous products, as well as any possible combination of products.", "For powders and granules, the processing installation to take samples from may e.g.", "be fluid bed equipment, granulation equipment, agglomeration equipment, coating equipment, layering equipment, extrusion equipment, spray drying equipment, packaging equipment etc.", "Furthermore, heavy, sticky or viscous products or products having limited flow capabilities may be sampled by means of the device and the sampling method according to the invention.", "At a first end, the outer container 5 is formed integrally with a connecting piece 7 in the form of an internal thread 8 which in FIG.", "1 has been screwed onto an outer thread 9 of the fitting 2.At a second end, the outer container 5 has a bottom 10, to which a spindle 11 is fixed rigidly and from which the spindle 11 extends coaxially in the outer container 5 to the area of the first end of the outer container, where a stationary valve part 12 in the form of a truncated cone is formed integrally with the spindle 11 and concentric with this.", "The stationary valve part 12 has a conical sealing surface 13 facing the interior of the sampling container 4.At a first end, the inner container 6 is formed integrally with a displaceable valve member 14 in the form of a conical sealing surface 15 facing away from the interior of the sampling container 4 and corresponding to the conical sealing surface 13 of the stationary valve part 12.At a second end, the inner container 6 has a bottom 16 in which is formed a concentric spindle passage 17 sealed displaceably against the spindle 11 by means of an O-ring 18.Between the bottom 16 of the inner container 6 and the bottom 10 of the outer container 5 is arranged a compression spring 19 around the spindle 11, thereby spring-loading the inner container 6 with its integral valve member 14 towards a closed position of the displaceable valve member 14, in which position the conical sealing surface 15 of the valve member 14 abuts the conical sealing surface 13 of the stationary valve part 12, as shown in FIG.", "2 where the sampling container 4 has been disconnected from the fitting 2.In the connected state of the connecting piece 7 to the fitting 2, as shown in FIG.", "1, the valve member 14 formed on the internal container 6 abuts an edge 20 of the fitting 2, whereby the valve member 14 is maintained in its open position displaced against the load of the compression spring 19 so that a passage 21 is open between the displaceable valve member 14 and the stationary valve part 12.In order to take a sample from the vessel 3, the closed sampling container 4 shown in FIG.", "2 is screwed onto the fitting 2 whereby the displaceable valve member 14 is displaced to its open position shown in FIG.", "1.Subsequently, a shut-off valve 22 comprised by the fitting 2 is operated manually by means of a handle 23 so that product may pass from the interior of the vessel 3 to an internal tubular part 24 of the fitting 2 and into the open sampling container 4.The open position of the handle 23 is indicated by means of dash-dot lines.", "The outer and inner containers 5, 6 may advantageously be formed from Plexiglass (registered trademark), glass, tempered glass or the like, whereby the amount of product entering the sampling container 4 may be observed visually.", "After having taken an appropriate sample, the shut-off valve 22 is closed and the sampling container 4 is screwed off the fitting 2 whereby the valve member 14 is displaced to its closed position as shown in FIG.", "2.Subsequently, the sampling container 4 may be handled without risk of product leakage and possibly transported before the sample is poured out of the container 4.In order to pour out the product contained in the sampling container 4, the container may be screwed on a suitable laboratory equipment having a part fitting the connecting piece 7 similar to the fitting 2, i.e.", "having an outer thread similar to the thread 9 and an edge similar to the edge 20 adapted to abut the displaceable valve member 14 and thereby maintain this in its open position.", "FIG.", "3 shows another embodiment of the sampling device according to the invention.", "The embodiment differs from the one shown in FIG.", "1 in that the fitting 2 is provided with an internal shielding member 25 which is guided in a tube element 26 fixed in a plate 27 which is guided and axially displaceable in the tubular part 24 of the fitting 2.By means of a compression spring 28 located in the tube element 26, the shielding member 25 is spring-loaded to abut the stationary valve part 12 upon connection of the connecting piece 7 to the fitting 2.The shielding member 25 has a circumferential contour 29 fitting a circumferential contour 30 of the stationary valve part 12.FIG.", "6 to a show another embodiment of the sampling device 1.The embodiment differs from the one shown in FIG.", "1 in that the fitting 2 furthermore is provided with both a shielding member 25 and a covering member 31 formed integrally with the tubular part 24 of the fitting 2.The covering member 31 has a circumferential contour 32 fitting both a circumferential contour 33 of the displaceable valve member 14 and a circumferential contour 29 of the shielding member 25.The stationary valve part 12 has a circumferential contour 30 fitting both the circumferential contour 33 of the displaceable valve member 14 and the circumferential contour 29 of the shielding member 25.Furthermore, the shielding member 25 has a conical sealing surface 34 corresponding to a conical sealing surface 35 of the covering member 31.FIG.", "6 shows the connected state of the connecting piece 7 of the sampling container 4 on the fitting 2, in which state the displaceable valve member 14 and the shielding member 25 are in their open positions.", "FIG.", "7 shows a partly disconnected state of the connecting piece 7 and the fitting 2, in which state both the displaceable valve member 14 and the shielding member 25 are in their closed positions and the thread 8 of the connecting piece 7 is engaging the thread 9 of the fitting 2 in order to secure tight connection between the sampling container 4 and the fitting 2.FIG.", "8 shows the disconnected state of the connecting piece 7 and the fitting 2, in which state the displaceable valve member 14 and the shielding member 25 are in their closed positions.", "With this embodiment it is possible to take a sample in a fully contained way so that the surroundings and the operator are practically not exposed to the product sampled.", "After having taken the sample, the sampling container 4 may, as explained above, be transported to and screwed on a suitable laboratory equipment whereby the product sampled may be transferred to said equipment, also in a fully a contained way.", "In a typical execution, the outer container 5 has an outer length of approximately 130 mm, an outer diameter of approximately 75 mm and a wall thickness of approximately 10 mm and the inner container 6 has an inner diameter of approximately 40 mm and an inner length between its bottom 16 and its annular sealing surface 15 of approximately 65 mm.", "In this execution, the length of the internal thread 8 of the outer container 5 is approximately 18 mm and the outer length of the fitting 2 is approximately 53 mm.", "The diameter of the circumferential contour 32 of the covering member 31 fitting the circumferential contour 29 of the shielding member 25 is approximately 34 mm.", "In another typical execution, said dimensions have been multiplied by two.", "The dimensions and the materials may also typically be chosen so as to ensure that the device is explosion proof for a required pressure.", "The embodiments of the sampling device 1 shown in FIGS.", "1 to 3 and 6 to 8 are especially suitable if the pressure in the vessel or pipe of the processing installation to be sampled from is below atmospheric, as substantially the same pressure will be present in the sampling container 4 after having taken the sample.", "Consequently, the pressure difference between the surroundings and the internal of the sampling container 4 will assist the spring 19 in keeping the valve member 14 in its closed position after taking the sample and disconnecting the container 4 from the fitting 2.FIG.", "5 shows the fitting 2 of the sampling device 1 in FIGS.", "6 to 8, before mounting a flange 36 of the fitting 2 on a flange 37 of the processing installation.", "As indicated by means of dash-dot lines, the plate 27 is displaceable axially in the tubular part 24 of the fitting 2 and upon connection of the flange parts 36, 37, an edge 38 of the vessel of the processing installation will abut the plate 27, thereby displacing the plate to the position indicated by means of the dash-dot lines, whereby the compression spring 28 will be compressed and thereby preload the shielding member 25 against the covering member 31.FIG.", "4 shows still another embodiment of the sampling device 1 according to the invention.", "In this embodiment parts similar to parts in the embodiment in FIG.", "6 to 8 are indicated with similar reference numbers.", "The sampling container 4 has an annular stationary valve part 12 formed integrally around its inlet opening.", "The displaceable valve member 14 is by means of a spindle 39 guided in a tube element 40 at the bottom of the sampling container 4.The fitting 2 is provided with a spring-loaded annular displaceable shielding member 25 and a conical fixed covering member 31.This embodiment also permits the taking of samples in a fully contained way and is especially suitable if the pressure in the vessel or pipe of the processing installation to be sampled from is above atmospheric, as substantially the same pressure will be present in the sampling container 4 after having taken the sample.", "Consequently, the pressure difference between the surroundings and the internal of the sampling container 4 will assist the spring 19 in keeping the valve member 14 in its closed position after taking the sample and disconnecting the container 4 from the fitting 2.This will prevent even a small amount of product in leaking upon disconnection of the sampling container 4 from the fitting 2.The embodiments shown in FIGS.", "4 and 6 to 8 may be used with or without the shut-off valve 22 shown in FIG.", "1, depending on the situation, for instance whether the pressure in the installation to be sampled from is constantly above or below atmospheric or may change.", "FIGS.", "9 to 11 show an embodiment of the sampling device 1 comprising a nozzle 41 comprising a connecting piece 42 which may be connected with the fitting 2 and which has a tube connection 43 for the supply of or discharge of cleaning or washing fluid to or from the fitting 2.The tube connection 43 comprises an internal projection 44 which in the connected state of the nozzle 41 on the fitting 2 maintains the shielding member 25 in its open position, thereby allowing cleaning or washing fluid to pass in or out through the fitting 2.FIG.", "9 shows the connected state, FIG.", "10 shows a partly connected state, in which the connecting piece 42 is mounted on the fitting 2, but in which the tube connection 43 is not fully connected to the connecting piece 42 so that the shielding member 25 is still in its closed position, and FIG.", "11 shows the fully disconnected state." ] ]

[ [ "Method of identification and quantification of biological molecules and apparatus therefore", "A method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair is disclosed.", "The method comprises interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair." ], [ "1-105.", "(canceled) 106.A method for detecting binding between all least one first member of a binding pair and at least one corresponding second member of the binding pair, the method comprising: interacting a solid support auto which said at least one first member of the binding pair is immobilized and arrayed with said at least one corresponding second member of the binding pair, wherein said at least one corresponding second member of the binding pair is at least one of: (a) directly tagged; and (b) indirectly tagged; with a heavy atom; and determining a spatial distribution of said heavy atom over a surface of the solid support so as to detect binding between said al least one first member of the binding pair and said at least one corresponding second member of the binding pair.", "107.The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support has a dynamic range of linearity of at least four orders-of-magnitude.", "108.The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a sensitivity of detection equal to at least 1 in 10 binding events.", "109.The method of claim 108, wherein said sensitivity is equal to at least 1 in 5 binding events.", "110.The method of claim 109, wherein said sensitivity is about 1 in 1 binding events.", "111.The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 20.112.The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 50.113.The method of claim 106, wherein said determining the spatial distribution of said heavy atom over said surface of the solid support is at a signal-to-noise ratio of more than 80.114.The method of claim 106, wherein said binding pair is selected from the group consisting of antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme.", "115.The method of claim 106, wherein said determining the spatial distribution for said heavy atom over said surface of the solid support is by particle scattering.", "116.The method of claim 106, wherein said determining the spatial distribution for said heavy atom over said surface of the solid support is by electron scattering.", "117.The method of claim 106, wherein said heavy atom is selected from the group consisting of gold, silver and iron.", "118.A method for detecting binding between at least one first member of a binding pair and at least one corresponding second member of the binding pair, the method comprising: interacting a solid support onto which said at least one first member of the binding pair is immobilized and arrayed with said at least one corresponding second member of the binding pair; and determining a spatial distribution of said at least one corresponding second member of the binding hair at a signal-to-noise ratio of more than 10.119.The method of claim 118, wherein said at least one first member comprises at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "120.The method of claim 118, wherein said at least one second member comprises at least one macromolecule of a known identity selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates.", "121.A method for at least one of identifying and quantifying at least one biological molecule in a sample, the method comprising; contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between said at least one biological molecule and said macromolecules of known identities; and detecting a spatial distribution of said at least one biological molecule over a surface of said microarray at a sensitivity equal to at least 1 in 10 binding events so as to provide at least one of an identification and a quantification of the least one biological molecule in the sample.", "122.The method of claim 121, wherein said at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "123.The method of claim 121, wherein said macromolecules of known identities are selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates.", "124.The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray has a dynamic range of linearity of at least four orders-of-magnitude.", "125.The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray has a signal-to-noise ratio of more than 20.126.The method of claim 121, wherein said detecting the spatial distribution of said at least one biological molecule over said surface of said microarray is by directly or indirectly tagging said at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of said at least one heavy atom.", "127.The method of claim 121, wherein the signal-to-noise ratio is greater thin 20.128.A method for at least one of identifying and quantifying at least one biological molecule in a sample, the method comprising: attaching biological molecules present in the sample to a solid support; contacting said solid support with at least one macromolecule of a known identity under conditions so as to allow binding between said at least one biological molecule and said at least one macromolecule of known identity; and detecting a level of binding between said at least one biological molecule and said at least one macromolecule of known identity by directly or indirectly tagging said at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of said at least one heavy atom, thereby providing at least one of an identification and a quantification of the least one biological molecule in the sample.", "129.The method of claim 128, wherein said at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "130.The method of claim 128, wherein said at least one macromolecule of known identity is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "131.The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity has a dynamic range of linearity of at least four orders-of-magnitude.", "132.The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity is at a sensitivity equal to at least 1 in 10 binding events.", "133.The method of claim 128, wherein said detecting the level of binding between said at least one biological molecule and said at least one macromolecule of known identity is at a signal-to-noise ratio of more than 20.134.A method for at least one of identifying and quantifying biological molecules in a preparation, the method comprising: localizing and tagging the biological molecules in the preparation; preparing the preparation for vacuum; loading the preparation into the specimen chamber of an electron beam device; irradiating the preparation with an electron beam, thus obtaining an image of the tags; and analyzing the image of the biological molecules by image analysis software so as to provide at least one of an identification and a quantification of the biological molecules in said preparation.", "135.A method for at least one of identifying and quantifying biological molecule in a preparation, the method comprising: localizing and tagging the biological molecules in the preparation; lading the preparation into the specimen clamber of an electron beam device; irradiating the preparation with an electron beam, thus obtaining an image of the tags; and analyzing the image of the biological molecules by image analysis software so as to provide at least one of an identification and a quantification of the biological molecules in sail preparation.", "136.An apparatus for analysis of at least two samples comprising biological molecules, comprising: an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from said electron source on the surface of a first sample of said at least two samples; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a second sample of said at least two samples in one chamber while simultaneously inspecting said first sample; at least one electron detector; a measuring system for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive said second sample and to provide at least one degree of motion to said second sample while the first sample is being scanned; and an image analysis system for carrying, out image analysis of the molecules of the first and second samples." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>In the past, genes, proteins, carbohydrates and cells where mainly studied at isolation, greatly limiting the ability to elucidate a realistic picture of the complex array of biochemical processes taking place in living cells.", "Genomics, proteomics, glycomics and cellomics techniques, which evolved in this sequence during the last decade, aim at analyzing biochemical processes, as complex as these may be, in a more integrated fashion, aiming at looking at all or substantially all of the biochemical changes, large scale changes, as well as minute changes, that take place in living cells under various conditions.", "Presently, genomics, proteomics, glycomics and cellomics rely on several technologies that are insufficiently quantitative, insufficiently sensitive and are characterized by a relatively low signal-to-noise (S/N) ratio and as such fail to provide a complete insight of cell function.", "These include the nowadays routine technology of nucleic acid microarrays (e.g., DNA microarrays, also referred to in the art as DNA chips) for analyzing nucleic acid molecules, such as DNA and RNA; the emerging technology of protein (e.g., antigen or antibody) microarrays (also known as protein chips); the recently revived and improved technology of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), both latter techniques serve for analyzing protein molecules; the recently introduced carbohydrate microarrays for the analysis of carbohydrate molecules and various cellomics assays for the analysis of integrated cells.", "DNA microarrays or DNA chips comprise a plurality of DNA strands (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate In a typical assay in which a DNA microarray is used in the analysis of nucleic acids, single stranded molecules (targets or probes, respectively), typically oligonucleotides or cDNA, tagged with fluorescent markers, are interacted with the substrate, resulting in hybridization of targets and probes according to the DNA parity rules.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the sequence of the targets and/or the expression level of a variety of genes.", "The basic limitation associated with the use of nucleic acid microarrays is the ‘flood’ of poor quality data.", "Currently about 90% of the data is insignificant.", "In most cases, weakly expressed genes that can be very important in a biological pathway, are not detected.", "This limitation arises from the poor signal-to-noise ratio (S/N) and insufficient sensitivity of this technique.", "It further leads to poor reproducibility.", "It is difficult to quantify the result of an experiment.", "The results of seemingly identical experiments also vary considerably.", "Furthermore, in many cases, the genes of most importance produce a weak signal that is not at all detected.", "Hence, there is a great need to increase the sensitivity and dynamic range of microarrays and reduce their inherent noise and background levels.", "These challenges are possibly achievable by substantially miniaturizing the microarrays.", "The miniaturization is important since it will provide a possibility to imprint larger portions of the genome on the same array (perhaps even the entire human genome).", "An additional reason for miniaturization is the long period of time required for the target molecules to cover an array by diffusion.", "The smaller the array, the shorter this time is, in a quadratic manner.", "nevertheless, the presently employed analyzing techniques, i.e., the use of fluorescent tags and laser scanning, impose great limitations on further miniaturization, both with respect to spatial resolution and with respect to scanning time.", "An additional type of limitations of the presently employed microarrays arise from the bleaching of the fluorescent tags once analyzed.", "After an array is scanned, it is bleached, meaning that the fluorescent tags emit less than the required intensity of light.", "This property significantly reduces the ability of a user to repeat a measurement of a pre-measured experiment.", "Mainly due to the above limitations, there is no standard by which microarray experiments are performed and/or analyzed, leaving a too large room for personal know-how.", "In many cases, experiments executed in different laboratories cannot be repeated or even compared.", "Hence, it is evident that there is a great need for a microarray detection system whose quantification is limited only by the biology.", "Preferably this system will be based on single molecule detection.", "This system should be free of the limitations associated with fluorescence-based readings and advantageously should have the following properties: (i) high sensitivity; (ii) high S/N ratio; (iii) compatibility with miniaturization of the microarray, and with smaller sample sizes; (iv) it should not bleach, providing the opportunity to rescan a sample or its regions of interest more than once; and (v) it should be able to incorporate assisted hybridization processes (not only diffusion).", "One objective of the present invention is to disclose a microarray, scanning method and system, capable of performing high throughput detection on the level of a single molecule.", "This system is sensitive and reproducible enough to set the industry standard.", "One option that may be considered is the use of a Scanning Force Microscope in the analysis of microarrays.", "However, the inherently low throughput of this system prevents it from being practical.", "The present invention solves the above mentioned limitations by means of scanning electron microscopy.", "An additional limitation, complementary to quantifying genes in microarrays, is the quantitative study of proteomics (the study of proteins).", "Proteins determine many biological processes and are very important to drug discovery and many other applications.", "One tool in proteomics is high resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and the image analysis thereof Electrophoresis is the migration of charged molecules in a solution, in response to an electric field.", "The rate of migration depends on the strength of the field, on the charge, size and shape of the molecules, as well as on the parameters of the medium through which the molecules are moving.", "2D gel electrophoresis is a method to separate molecules that differ in any combination of size or charge.", "The solution is supported by a gel (agarose, polyacrylamide), which prevents undesired migration (convection, diffusion) and sieves the molecules, thus contributing to their separation on the basis of their sizes.", "In the present application, this system is referred to as 2D PAGE.", "The scope is not limited to any particular gel.", "2D PAGE systems typically resolve about 1000 proteins according to their isoelectric propertied through a pH gradient in one direction and thereafter according to their size, in the presence of SDS, in a second direction, perpendicular to the first.", "The abundance of proteins in a cell is within a range from single to millions of molecules.", "There are many proteins in the gel that are not resolved, partly due to the lack of sensitivity of the separation and partly due to the lack of sensitivity of the detection.", "There are two typical phases in protein analysis: (i) separation of the proteins, e.g., via 2D PAGE; and (ii) analyzing the types of the separated proteins, typically by mass spectrometry.", "What is clearly missing is an intermediate stage where the number of proteins in each spot is counted.", "Preferably the counting method will be able to distinguish between the different proteins.", "Preferably it will rely on single molecule detection.", "Currently there is no technology that provides a satisfactory quantitative answer to the issue of how many of the separated proteins exist in each spot.", "An additional question is how many types of proteins exist in each spot.", "Thus, there is a need for a technology that counts the number of proteins before they are inserted in the mass spectrometer.", "The above-mentioned limitations of genomics and proteomics technologies are amplified in the emerging technology of protein microarrays.", "Protein microarrays or protein chips comprise a plurality of proteins (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate.", "In a typical assay in which a protein microarray is used in the analysis of proteins, protein molecules (targets or probes, respectively), typically antigens or antibodies, tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets and/or the level of their expression.", "In many cases, comparative competition assays are performed, where a change in the pattern of fluorescence is indicative of the identity of the targets and/or the level of their expression.", "Protein microarrays will go a long way towards elucidating aspects of cellular functions that DNA chips cannot provide, since measuring mRNA levels alone ignores issues which are of great influence on cellular function, such as, but not limited to, protein lifetime, protein post transnational modifications, etc.", "Protein chips find uses in two major fields: drug discovery and diagnostics.", "In drug discovery, processes such as drug candidate discovery and candidate optimization can be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available.", "In diagnostics, determining titers of viruses and other pathogens, presence, absence or level of cancer and other markers, antibody profiles, etc., could be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available.", "It is apparent that proteomic tools are essential to obtain information that is unavailable when performing analysis on the gene level.", "Expressed genes can be subjected to significant post-translational regulation, and proteins undergo significant post-translational modifications (such as phosphorylation, acetylation, etc.)", "that significantly affect their function at the cellular level.", "In many cases, no correlation exists between the level of a specific messenger RNA and the level/activity of its encoded protein, because, most of the control of the expression of that protein takes place at the post translational phase.", "To this effect, see, for example, S. P. Gygi, et al., Mol.", "Cell.", "Biol.", "19 (1999) 1720-1730).", "Recently it was shown that it is possible to array different proteins, or protein ligands on a microscope slide to study a variety of protein functions (Macbeath et al., Nature 289, 2000).", "The basic challenge is that the proteins bound on surface retain their activity and/or their antigenic epitopes.", "The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other macromolecules, e.g., other proteins, or with small molecules in solution.", "In this study the proteins attached to the slides were probed with fluorescently-labeled proteins.", "Screening for protein-protein interactions, substrates of protein kinases and targets of small molecules was demonstrated.", "It often occurs that the less highly expressed proteins are those that are of most interest since their response to various physiological stimuli is the most interesting and informative.", "Unfortunately, it seems that the low abundance proteins, such as hormones, cytokines, small G-proteins, DNA binding proteins etc., are not easily detected by the present proteomics techniques (S. P. Gygi, Proc.", "Natl.", "Acad.", "Sci.", "USA 97 (2000) 9390-9395).", "Detection on the single or close to single molecule level usually requires painstaking techniques that require tedious sample preparation and imaging and are hard to apply to high throughput methods.", "Carbohydrate microarrays comprise a plurality of carbohydrates (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate.", "In a typical assay in which a carbohydrate microarray is used in the analysis of carbohydrates, molecules such as antibodies directly or indirectly tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets/probes and/or the level of their expression.", "The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays.", "Cell microarrays comprise a plurality of cells immobilized on a surface of a substrate, which cells can be screened for various properties in a living or fixated state.", "The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays.", "There is thus a widely recognized need for, and it would be highly advantageous to have, a technique for the implementation of genomics, proteomics glycomics and cellomics devoid of the above limitations.", "In particular, it would be highly advantageous to have a technique for implementing genomics, proteomics glycomics or cellomics that reaches single molecule detection levels, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple sample preparation, readily applicable for high throughput screening." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide a method for identification and quantification of biological molecules in a sample that overcomes the drawbacks of the existing methods The term biological molecule includes any molecule with biological relevance.", "This includes, but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP GTP etc., drugs, non proteinaceous antigens and any homo- (e.g., protein-protein as example) and hetero- (e.g., drug-protein, DNA-RNA, DNA-protein, etc.)", "complexes, as well as chemically modifications and derivatisations whether naturally occurring or man made, of all these different molecules.", "It is another object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is based on single-molecule detection, has a higher sensitivity and signal-to-noise ratio and broader dynamic range than existing methods.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out without the bleaching that characterizes fluorescence-based detection, thus enabling measuring the same sample several times.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with an Environmental Scanning Electron Microscope (ESEM), thus enabling the investigation of a sample at almost atmospheric pressure.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with a Wafer Inspection Scanning Electron Microscope (WISEM), thus greatly reducing the cost of instrumentation required to implement the method.", "It is still a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out on a miniaturized microarray and allows to imprint larger portions of the human genome, or even the entire human genome, on the same array.", "According to one aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair.", "Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, e.g., i of 5 binding events, most preferably, about 1 of 1 binding events.", "Yet preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably greater than 80.According to another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a dynamic range of linearity of at least four orders-of-magnitude.", "Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than I of 10 binding events, e.g., equals to or greater than I of 5 binding events, optimally the sensitivity is about 1 of 1 binding events.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably, greater than 80.According to still another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, more preferably, the sensitivity equals to about 1 of 1 binding events.", "In a preferred embodiment, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80.According to yet another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80.Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, most preferably, the sensitivity is about 1 of 1 binding events.", "According to further features in preferred embodiments of the invention described below, the binding pair is selected from the group consisting of antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme.", "According to still further features in the described preferred embodiments determining the spatial distribution of the heavy atom over the surface of the solid support is by particle scattering.", "Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is by electron scattering.", "According to still further features in the described preferred embodiments the corresponding second member or members of the binding pair is indirectly tagged with a heavy atom.", "According to still further features in the described preferred embodiments the heavy atom is selected from the group consisting of gold, silver and iron.", "According to an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to yet an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to still further features in the described preferred embodiments the macromolecules of known identities are selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity equals to or greater than 1 of 10 binding events.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events.", "Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events.", "Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to still further features in the described preferred embodiments the at least one macromolecule of known identity is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; preparing the preparate for vacuum; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantity the biological molecules by image analysis software.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantify the biological molecules by image analysis software.", "According to still another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image representing the biological molecules; and analyzing the image to quantify the biological molecules by image analysis software.", "According to still another aspect of the present invention there is provided an apparatus for inspection of a preparate of biological molecules comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned; and means for carrying out image analysis of the molecules on the preparate.", "The biological molecules may be polynucleotides, e.g.", "DNA, cDNA, RNA, clusters, or proteins such as antigens, antibodies.", "The term biological molecules also refers but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP antibodies, GTP, etc., drugs, non proteinaceous antigens and any homo-(protein-protein as example) and hetero- (drug-protein, DNA-RNA, DNA-protein etc.)", "complexes as well as chemically modifications and derivatisations whether naturally occurring or not of all these different molecules.", "The preparate for the polynucleotides may be a microarray, e.g., a DNA chip, and for the proteins may be a 2D PAGE, a protein chip, e.g., an antigen or antibody chip, cell chip, cell preparate, and the like.", "The localization of the biological molecules may be carried out before or after tagging, depending on the type of the biological molecule and of the technique used.", "When the biological molecule in the preparate is a polynucleotide, the localization may be carried out, for example, by hybridization, either to a polynucleotide of known sequence (probe) when the polynucleotide immobilized in the microarray preparate is of unknown sequence (target), or to a polynucleotide of unknown sequence (target) when the polynucleotide immobilized in the microarray preparate is of a known sequence (probe).", "The same is true for protein microarrays, with respect to either antigens and/or antibodies, each of which can serve as a target or probe, and in any case can be immobilized to the microarray or be interacted therewith.", "When the biological molecule in the preparate is a protein, the localization may be carried out, for example, by separating the molecules by one- or two-dimensional electrophoresis, or by attaching the molecules to a blot membrane.", "When the preparate is a 2D PAGE or proteins extracted therefrom, the separation in the gel may be preferably performed on-line under the scanning electron beam.", "Identification of the proteins can be done by mass spectrometry.", "The localization in space may further consist of localizing the molecules by their affiliation to specific biological cells.", "Tagging of the biological molecules such as DNA, RNA and proteins, may be carried out with heavy metals such as silver or gold, for example using colloidal gold or gold clusters, or doping with metal-enriched organic compounds, wherein the metal is, for example, Fe.", "The heavy metal colloids (e.g., gold), preferably of diameter range of 1-200 nm, more preferably, less than 20 nm, create a high intensity back scattered electron signal and, therefore, high image contrast.", "In one embodiment, there is one tag per target molecule.", "More specifically the tagging may be carried out with biotin followed by gold tagged avidin.", "Tagging may also be made with electro-luminescent molecules whereby the electron beam creates a light signal that is detected.", "Tagging may also be done with more than one type of tags to make a distinction between two preparates.", "According to one embodiment multi-labeling or Multi-tagging is achieved.", "This is achieved, for example, by using gold colloids of a plurality of sizes.", "According to another embodiment, multi-labeling is achieved by using a combination of gold colloids and fluorescent labels.", "According to a yet another embodiment, the multi-labeling is achieved by using a plurality of metals that are read by the X-RAY reading apparatus of the SEM, such as Energy Depressive Spectrum and so forth.", "According to one embodiment, the DNA molecules are not tagged and the SEM is sensitive enough to detect density differences between hybridized and non-hybridized regions.", "Direct detection with no tagging enables the identification of an additional variety of substances such as viral particles.", "The preparates are prepared for vacuum by known standard methods that include drying, fixation and coating with a conductive layer such as carbon, to prevent charge accumulation., protection with a membrane and freezing to prevent out-gassing.", "The preparates are examined in a particles beam device, preferably, an electron beam device, namely an electron microscope such as a scanning electron microscope (SEM).", "Presently, most preferably, the preparates are scanned and are analyzed using a wafer inspection SEM (WISEM) typically used in the microelectronics industry.", "The irradiation of the preparate is carried out in such a way as to form sufficient contrast of the electrons that are back scattered from the tags in comparison with those that are emitted/scattered from the background.", "In another embodiment, the SEM system is an environmental scanning electron microscope (ESEM) that works at almost atmospheric pressure, thus minimizing the need to prepare the preparate for vacuum.", "According to a yet another embodiment, the SEM system that allows the proteins to remain in their native wet state and still imaged.", "This embodiment utilizes a device and method that uses membrane partition.", "To this end, see U.S.", "Provisional Patent Application No.", "60/250,879, which is incorporated herein by reference.", "The image analysis may comprise any one of: performing edge detection algorithm to identify the colloids in each region-of-interest (ROI) and counting the colloids; counting fluorescence signals; and identifying X-ray spectrum of each particle for identification by comparison to a reference spectrum.", "The invention further relates to an apparatus for inspection of a preparate of biological molecules according to the above method, the apparatus comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned by the electron beam; and means for carrying out image analysis of the molecules on the preparate.", "In one preferred embodiment, the charged particle beam column of is a microcolumn.", "In a further embodiment, the invention provides a method for the inspection of biological molecules on a preparate using an electron beam, the method comprising localizing the biological molecules in space and tagging them with markers; preparing the preparate for vacuum; taking out the preparate to be analyzed from the preparate cassette; pre-aligning preparate and read preparate number; reading a recipe that contains the information to be detected; loading the preparate on X-Y-T stage (T means tilt) of an electron beam device; aligning the preparate; moving XYT stage to analysis position; positioning the electron beam on the substrate accurately by measuring the position of the substrate; scanning the preparate at low resolution to create a preparate map, while enhancing contrast; determining the regions-of-interest (ROI) spots on the map that should be scanned in a high resolution; scanning the ROIs with the electron beam as the substrate is continuously moving with at least one degree of motion in an x-y plane; detecting electrons emanating from the substrate as a result of previous step and forming an image; enhancing the image contrast; storing both modified and bare image; analyzing the ROIs; and displaying the results.", "The present invention successfully addresses the shortcomings of the presently known configurations by providing a technique for the implementation of genomics, proteomics, glycomics and cellomics that reaches the highest sensitivity of ultimately single molecule detection, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple preparate preparation, readily applicable for high throughput screening." ], [ "FIELD OF THE INVENTION The present invention relates to a method and apparatus for identifying and quantifying molecules present in a sample, in particular by means of irradiating appropriately tagged molecules with a particle beam, such as an electron beam, obtaining an image of the tags and carrying out image analysis.", "The present invention find uses and provides major improvements in the fields of genomics, proteomics, functional proteomics, glycomics and cellomics.", "BACKGROUND OF THE INVENTION In the past, genes, proteins, carbohydrates and cells where mainly studied at isolation, greatly limiting the ability to elucidate a realistic picture of the complex array of biochemical processes taking place in living cells.", "Genomics, proteomics, glycomics and cellomics techniques, which evolved in this sequence during the last decade, aim at analyzing biochemical processes, as complex as these may be, in a more integrated fashion, aiming at looking at all or substantially all of the biochemical changes, large scale changes, as well as minute changes, that take place in living cells under various conditions.", "Presently, genomics, proteomics, glycomics and cellomics rely on several technologies that are insufficiently quantitative, insufficiently sensitive and are characterized by a relatively low signal-to-noise (S/N) ratio and as such fail to provide a complete insight of cell function.", "These include the nowadays routine technology of nucleic acid microarrays (e.g., DNA microarrays, also referred to in the art as DNA chips) for analyzing nucleic acid molecules, such as DNA and RNA; the emerging technology of protein (e.g., antigen or antibody) microarrays (also known as protein chips); the recently revived and improved technology of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), both latter techniques serve for analyzing protein molecules; the recently introduced carbohydrate microarrays for the analysis of carbohydrate molecules and various cellomics assays for the analysis of integrated cells.", "DNA microarrays or DNA chips comprise a plurality of DNA strands (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate In a typical assay in which a DNA microarray is used in the analysis of nucleic acids, single stranded molecules (targets or probes, respectively), typically oligonucleotides or cDNA, tagged with fluorescent markers, are interacted with the substrate, resulting in hybridization of targets and probes according to the DNA parity rules.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the sequence of the targets and/or the expression level of a variety of genes.", "The basic limitation associated with the use of nucleic acid microarrays is the ‘flood’ of poor quality data.", "Currently about 90% of the data is insignificant.", "In most cases, weakly expressed genes that can be very important in a biological pathway, are not detected.", "This limitation arises from the poor signal-to-noise ratio (S/N) and insufficient sensitivity of this technique.", "It further leads to poor reproducibility.", "It is difficult to quantify the result of an experiment.", "The results of seemingly identical experiments also vary considerably.", "Furthermore, in many cases, the genes of most importance produce a weak signal that is not at all detected.", "Hence, there is a great need to increase the sensitivity and dynamic range of microarrays and reduce their inherent noise and background levels.", "These challenges are possibly achievable by substantially miniaturizing the microarrays.", "The miniaturization is important since it will provide a possibility to imprint larger portions of the genome on the same array (perhaps even the entire human genome).", "An additional reason for miniaturization is the long period of time required for the target molecules to cover an array by diffusion.", "The smaller the array, the shorter this time is, in a quadratic manner.", "nevertheless, the presently employed analyzing techniques, i.e., the use of fluorescent tags and laser scanning, impose great limitations on further miniaturization, both with respect to spatial resolution and with respect to scanning time.", "An additional type of limitations of the presently employed microarrays arise from the bleaching of the fluorescent tags once analyzed.", "After an array is scanned, it is bleached, meaning that the fluorescent tags emit less than the required intensity of light.", "This property significantly reduces the ability of a user to repeat a measurement of a pre-measured experiment.", "Mainly due to the above limitations, there is no standard by which microarray experiments are performed and/or analyzed, leaving a too large room for personal know-how.", "In many cases, experiments executed in different laboratories cannot be repeated or even compared.", "Hence, it is evident that there is a great need for a microarray detection system whose quantification is limited only by the biology.", "Preferably this system will be based on single molecule detection.", "This system should be free of the limitations associated with fluorescence-based readings and advantageously should have the following properties: (i) high sensitivity; (ii) high S/N ratio; (iii) compatibility with miniaturization of the microarray, and with smaller sample sizes; (iv) it should not bleach, providing the opportunity to rescan a sample or its regions of interest more than once; and (v) it should be able to incorporate assisted hybridization processes (not only diffusion).", "One objective of the present invention is to disclose a microarray, scanning method and system, capable of performing high throughput detection on the level of a single molecule.", "This system is sensitive and reproducible enough to set the industry standard.", "One option that may be considered is the use of a Scanning Force Microscope in the analysis of microarrays.", "However, the inherently low throughput of this system prevents it from being practical.", "The present invention solves the above mentioned limitations by means of scanning electron microscopy.", "An additional limitation, complementary to quantifying genes in microarrays, is the quantitative study of proteomics (the study of proteins).", "Proteins determine many biological processes and are very important to drug discovery and many other applications.", "One tool in proteomics is high resolution two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and the image analysis thereof Electrophoresis is the migration of charged molecules in a solution, in response to an electric field.", "The rate of migration depends on the strength of the field, on the charge, size and shape of the molecules, as well as on the parameters of the medium through which the molecules are moving.", "2D gel electrophoresis is a method to separate molecules that differ in any combination of size or charge.", "The solution is supported by a gel (agarose, polyacrylamide), which prevents undesired migration (convection, diffusion) and sieves the molecules, thus contributing to their separation on the basis of their sizes.", "In the present application, this system is referred to as 2D PAGE.", "The scope is not limited to any particular gel.", "2D PAGE systems typically resolve about 1000 proteins according to their isoelectric propertied through a pH gradient in one direction and thereafter according to their size, in the presence of SDS, in a second direction, perpendicular to the first.", "The abundance of proteins in a cell is within a range from single to millions of molecules.", "There are many proteins in the gel that are not resolved, partly due to the lack of sensitivity of the separation and partly due to the lack of sensitivity of the detection.", "There are two typical phases in protein analysis: (i) separation of the proteins, e.g., via 2D PAGE; and (ii) analyzing the types of the separated proteins, typically by mass spectrometry.", "What is clearly missing is an intermediate stage where the number of proteins in each spot is counted.", "Preferably the counting method will be able to distinguish between the different proteins.", "Preferably it will rely on single molecule detection.", "Currently there is no technology that provides a satisfactory quantitative answer to the issue of how many of the separated proteins exist in each spot.", "An additional question is how many types of proteins exist in each spot.", "Thus, there is a need for a technology that counts the number of proteins before they are inserted in the mass spectrometer.", "The above-mentioned limitations of genomics and proteomics technologies are amplified in the emerging technology of protein microarrays.", "Protein microarrays or protein chips comprise a plurality of proteins (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate.", "In a typical assay in which a protein microarray is used in the analysis of proteins, protein molecules (targets or probes, respectively), typically antigens or antibodies, tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets and/or the level of their expression.", "In many cases, comparative competition assays are performed, where a change in the pattern of fluorescence is indicative of the identity of the targets and/or the level of their expression.", "Protein microarrays will go a long way towards elucidating aspects of cellular functions that DNA chips cannot provide, since measuring mRNA levels alone ignores issues which are of great influence on cellular function, such as, but not limited to, protein lifetime, protein post transnational modifications, etc.", "Protein chips find uses in two major fields: drug discovery and diagnostics.", "In drug discovery, processes such as drug candidate discovery and candidate optimization can be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available.", "In diagnostics, determining titers of viruses and other pathogens, presence, absence or level of cancer and other markers, antibody profiles, etc., could be greatly assisted should highly sensitive and reliable protein chips and analysis methods were available.", "It is apparent that proteomic tools are essential to obtain information that is unavailable when performing analysis on the gene level.", "Expressed genes can be subjected to significant post-translational regulation, and proteins undergo significant post-translational modifications (such as phosphorylation, acetylation, etc.)", "that significantly affect their function at the cellular level.", "In many cases, no correlation exists between the level of a specific messenger RNA and the level/activity of its encoded protein, because, most of the control of the expression of that protein takes place at the post translational phase.", "To this effect, see, for example, S. P. Gygi, et al., Mol.", "Cell.", "Biol.", "19 (1999) 1720-1730).", "Recently it was shown that it is possible to array different proteins, or protein ligands on a microscope slide to study a variety of protein functions (Macbeath et al., Nature 289, 2000).", "The basic challenge is that the proteins bound on surface retain their activity and/or their antigenic epitopes.", "The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other macromolecules, e.g., other proteins, or with small molecules in solution.", "In this study the proteins attached to the slides were probed with fluorescently-labeled proteins.", "Screening for protein-protein interactions, substrates of protein kinases and targets of small molecules was demonstrated.", "It often occurs that the less highly expressed proteins are those that are of most interest since their response to various physiological stimuli is the most interesting and informative.", "Unfortunately, it seems that the low abundance proteins, such as hormones, cytokines, small G-proteins, DNA binding proteins etc., are not easily detected by the present proteomics techniques (S. P. Gygi, Proc.", "Natl.", "Acad.", "Sci.", "USA 97 (2000) 9390-9395).", "Detection on the single or close to single molecule level usually requires painstaking techniques that require tedious sample preparation and imaging and are hard to apply to high throughput methods.", "Carbohydrate microarrays comprise a plurality of carbohydrates (probes or targets) immobilized on a surface of a substrate, where probes or targets of known identity are located in known and hence addressable locations over the surface of the substrate.", "In a typical assay in which a carbohydrate microarray is used in the analysis of carbohydrates, molecules such as antibodies directly or indirectly tagged with fluorescent markers, are interacted with the substrate, resulting in binding of targets and probes according to their identity.", "Following appropriate washes, the chip is scanned, typically with a laser-scanner, which excites the fluorescent tags (where present) and reads the emitted light.", "Depending on the application, the pattern of fluorescence over the surface of the chip provides information on the identity of the targets/probes and/or the level of their expression.", "The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays.", "Cell microarrays comprise a plurality of cells immobilized on a surface of a substrate, which cells can be screened for various properties in a living or fixated state.", "The limitations described hereinabove with respect to nucleic acid and protein microarrays clearly apply also to carbohydrate microarrays.", "There is thus a widely recognized need for, and it would be highly advantageous to have, a technique for the implementation of genomics, proteomics glycomics and cellomics devoid of the above limitations.", "In particular, it would be highly advantageous to have a technique for implementing genomics, proteomics glycomics or cellomics that reaches single molecule detection levels, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple sample preparation, readily applicable for high throughput screening.", "SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for identification and quantification of biological molecules in a sample that overcomes the drawbacks of the existing methods The term biological molecule includes any molecule with biological relevance.", "This includes, but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP GTP etc., drugs, non proteinaceous antigens and any homo- (e.g., protein-protein as example) and hetero- (e.g., drug-protein, DNA-RNA, DNA-protein, etc.)", "complexes, as well as chemically modifications and derivatisations whether naturally occurring or man made, of all these different molecules.", "It is another object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is based on single-molecule detection, has a higher sensitivity and signal-to-noise ratio and broader dynamic range than existing methods.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out without the bleaching that characterizes fluorescence-based detection, thus enabling measuring the same sample several times.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with an Environmental Scanning Electron Microscope (ESEM), thus enabling the investigation of a sample at almost atmospheric pressure.", "It is a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out with a Wafer Inspection Scanning Electron Microscope (WISEM), thus greatly reducing the cost of instrumentation required to implement the method.", "It is still a further object of the present invention to provide such a method for identification and quantification of biological molecules in a sample that is carried out on a miniaturized microarray and allows to imprint larger portions of the human genome, or even the entire human genome, on the same array.", "According to one aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair, the corresponding second member or members of the binding pair being directly or indirectly tagged with a heavy atom; and determining a spatial distribution of the heavy atom over a surface of the solid support, thereby detecting the binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair.", "Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, e.g., i of 5 binding events, most preferably, about 1 of 1 binding events.", "Yet preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably greater than 80.According to another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a dynamic range of linearity of at least four orders-of-magnitude.", "Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Still preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than I of 10 binding events, e.g., equals to or greater than I of 5 binding events, optimally the sensitivity is about 1 of 1 binding events.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably greater than 50, more preferably, greater than 80.According to still another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, more preferably, the sensitivity equals to about 1 of 1 binding events.", "In a preferred embodiment, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80.According to yet another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair, the method comprising interacting a solid support onto which the first member or members of the binding pair being immobilized and arrayed with the corresponding second member or members of the binding pair; and determining a spatial distribution of the second member or members of the binding pair at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80.Preferably, the corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom, whereas determining the spatial distribution of the second member or members of the binding pair is by determining a spatial distribution of the heavy atom over the surface of the solid support.", "Still preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a dynamic range of linearity of at least four orders-of-magnitude.", "Yet preferably, determining the spatial distribution of the second member or members of the binding pair over the surface of the solid support is at a sensitivity of detection equals to or greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, most preferably, the sensitivity is about 1 of 1 binding events.", "According to further features in preferred embodiments of the invention described below, the binding pair is selected from the group consisting of antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme.", "According to still further features in the described preferred embodiments determining the spatial distribution of the heavy atom over the surface of the solid support is by particle scattering.", "Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is by electron scattering.", "According to still further features in the described preferred embodiments the corresponding second member or members of the binding pair is indirectly tagged with a heavy atom.", "According to still further features in the described preferred embodiments the heavy atom is selected from the group consisting of gold, silver and iron.", "According to an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to yet an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities; and detecting a spatial distribution of the at least one biological molecule over a surface of the microarray by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a sensitivity equals to or greater than 1 of 10 binding events.", "Yet preferably, detecting the spatial distribution of the at least one biological molecule over the surface of the microarray is at a signal-to-noise ratio greater than 20.According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to still further features in the described preferred embodiments the macromolecules of known identities are selected from the group consisting of proteins, glycoproteins, nucleic acids and carbohydrates.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a dynamic range of linearity of at least four orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity equals to or greater than 1 of 10 binding events.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a sensitivity equals to or greater than 1 of 10 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity at a signal-to-noise ratio greater than 20, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events.", "Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom.", "According to still a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample, the method comprising attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity; and detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "Preferably, detecting the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a dynamic range of linearity of at least four orders-of-magnitude.", "Still preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a sensitivity greater than or equals to 1 of 10 binding events.", "Yet preferably, detecting a level of binding between the at least one biological molecule and the at least one macromolecule of known identity is at a signal-to-noise ratio greater than 20.According to further features in preferred embodiments of the invention described below, the at least one biological molecule is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to still further features in the described preferred embodiments the at least one macromolecule of known identity is selected from the group consisting of a protein, a glycoprotein, a nucleic acid and a carbohydrate.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; preparing the preparate for vacuum; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantity the biological molecules by image analysis software.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing and tagging the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image of the tags; and analyzing the image to quantify the biological molecules by image analysis software.", "According to still another aspect of the present invention there is provided a method of identifying and/or quantifying biological molecules in a preparate, the method comprising localizing the biological molecules in the preparate; loading the preparate into the specimen chamber of an electron beam device; irradiating the preparate with an electron beam, thus obtaining an image representing the biological molecules; and analyzing the image to quantify the biological molecules by image analysis software.", "According to still another aspect of the present invention there is provided an apparatus for inspection of a preparate of biological molecules comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned; and means for carrying out image analysis of the molecules on the preparate.", "The biological molecules may be polynucleotides, e.g.", "DNA, cDNA, RNA, clusters, or proteins such as antigens, antibodies.", "The term biological molecules also refers but is not limited to: polysaccharides, small chemical molecules such as lipids, peptides, hormones and other messengers, ATP antibodies, GTP, etc., drugs, non proteinaceous antigens and any homo-(protein-protein as example) and hetero- (drug-protein, DNA-RNA, DNA-protein etc.)", "complexes as well as chemically modifications and derivatisations whether naturally occurring or not of all these different molecules.", "The preparate for the polynucleotides may be a microarray, e.g., a DNA chip, and for the proteins may be a 2D PAGE, a protein chip, e.g., an antigen or antibody chip, cell chip, cell preparate, and the like.", "The localization of the biological molecules may be carried out before or after tagging, depending on the type of the biological molecule and of the technique used.", "When the biological molecule in the preparate is a polynucleotide, the localization may be carried out, for example, by hybridization, either to a polynucleotide of known sequence (probe) when the polynucleotide immobilized in the microarray preparate is of unknown sequence (target), or to a polynucleotide of unknown sequence (target) when the polynucleotide immobilized in the microarray preparate is of a known sequence (probe).", "The same is true for protein microarrays, with respect to either antigens and/or antibodies, each of which can serve as a target or probe, and in any case can be immobilized to the microarray or be interacted therewith.", "When the biological molecule in the preparate is a protein, the localization may be carried out, for example, by separating the molecules by one- or two-dimensional electrophoresis, or by attaching the molecules to a blot membrane.", "When the preparate is a 2D PAGE or proteins extracted therefrom, the separation in the gel may be preferably performed on-line under the scanning electron beam.", "Identification of the proteins can be done by mass spectrometry.", "The localization in space may further consist of localizing the molecules by their affiliation to specific biological cells.", "Tagging of the biological molecules such as DNA, RNA and proteins, may be carried out with heavy metals such as silver or gold, for example using colloidal gold or gold clusters, or doping with metal-enriched organic compounds, wherein the metal is, for example, Fe.", "The heavy metal colloids (e.g., gold), preferably of diameter range of 1-200 nm, more preferably, less than 20 nm, create a high intensity back scattered electron signal and, therefore, high image contrast.", "In one embodiment, there is one tag per target molecule.", "More specifically the tagging may be carried out with biotin followed by gold tagged avidin.", "Tagging may also be made with electro-luminescent molecules whereby the electron beam creates a light signal that is detected.", "Tagging may also be done with more than one type of tags to make a distinction between two preparates.", "According to one embodiment multi-labeling or Multi-tagging is achieved.", "This is achieved, for example, by using gold colloids of a plurality of sizes.", "According to another embodiment, multi-labeling is achieved by using a combination of gold colloids and fluorescent labels.", "According to a yet another embodiment, the multi-labeling is achieved by using a plurality of metals that are read by the X-RAY reading apparatus of the SEM, such as Energy Depressive Spectrum and so forth.", "According to one embodiment, the DNA molecules are not tagged and the SEM is sensitive enough to detect density differences between hybridized and non-hybridized regions.", "Direct detection with no tagging enables the identification of an additional variety of substances such as viral particles.", "The preparates are prepared for vacuum by known standard methods that include drying, fixation and coating with a conductive layer such as carbon, to prevent charge accumulation., protection with a membrane and freezing to prevent out-gassing.", "The preparates are examined in a particles beam device, preferably, an electron beam device, namely an electron microscope such as a scanning electron microscope (SEM).", "Presently, most preferably, the preparates are scanned and are analyzed using a wafer inspection SEM (WISEM) typically used in the microelectronics industry.", "The irradiation of the preparate is carried out in such a way as to form sufficient contrast of the electrons that are back scattered from the tags in comparison with those that are emitted/scattered from the background.", "In another embodiment, the SEM system is an environmental scanning electron microscope (ESEM) that works at almost atmospheric pressure, thus minimizing the need to prepare the preparate for vacuum.", "According to a yet another embodiment, the SEM system that allows the proteins to remain in their native wet state and still imaged.", "This embodiment utilizes a device and method that uses membrane partition.", "To this end, see U.S.", "Provisional Patent Application No.", "60/250,879, which is incorporated herein by reference.", "The image analysis may comprise any one of: performing edge detection algorithm to identify the colloids in each region-of-interest (ROI) and counting the colloids; counting fluorescence signals; and identifying X-ray spectrum of each particle for identification by comparison to a reference spectrum.", "The invention further relates to an apparatus for inspection of a preparate of biological molecules according to the above method, the apparatus comprising an electron source to provide an electron beam; a charged particle beam column to deliver and scan an electron beam from the electron source on the surface of the preparate; a vacuum system including a first and a second chamber in each of which pressurization can be performed independently to permit loading or unloading of a first preparate in one chamber while simultaneously inspecting a second preparate; at least one electron detector; means for measuring X-ray spectrum; a continuously moving x-y stage disposed to receive the preparate and to provide at least one degree of motion to the preparate while the preparate is being scanned by the electron beam; and means for carrying out image analysis of the molecules on the preparate.", "In one preferred embodiment, the charged particle beam column of is a microcolumn.", "In a further embodiment, the invention provides a method for the inspection of biological molecules on a preparate using an electron beam, the method comprising localizing the biological molecules in space and tagging them with markers; preparing the preparate for vacuum; taking out the preparate to be analyzed from the preparate cassette; pre-aligning preparate and read preparate number; reading a recipe that contains the information to be detected; loading the preparate on X-Y-T stage (T means tilt) of an electron beam device; aligning the preparate; moving XYT stage to analysis position; positioning the electron beam on the substrate accurately by measuring the position of the substrate; scanning the preparate at low resolution to create a preparate map, while enhancing contrast; determining the regions-of-interest (ROI) spots on the map that should be scanned in a high resolution; scanning the ROIs with the electron beam as the substrate is continuously moving with at least one degree of motion in an x-y plane; detecting electrons emanating from the substrate as a result of previous step and forming an image; enhancing the image contrast; storing both modified and bare image; analyzing the ROIs; and displaying the results.", "The present invention successfully addresses the shortcomings of the presently known configurations by providing a technique for the implementation of genomics, proteomics, glycomics and cellomics that reaches the highest sensitivity of ultimately single molecule detection, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple preparate preparation, readily applicable for high throughput screening.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings.", "With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention.", "In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.", "In the drawings: FIG.", "1 is a flow-chart illustrating steps of a method according to the teachings of the present invention.", "FIG.", "2 is a general diagram showing a longitudinal cross-section of an SEM used as a preparate analysis apparatus according to the present invention.", "FIG.", "3 is a generalized diagram showing a cross-section of a scanning electron microscope according to an aspect of the present invention.", "FIG.", "4 shows an apparatus that combines an ESEM with a SEM according to the present invention.", "FIG.", "5 presents another embodiment of the present invention, whereby the detection is made by exciting light photons (electro-luminescence).", "FIG.", "6 a SEM device having a microcolumns array for use in the method of the present invention.", "FIG.", "7 is a gel chamber for use in the method of the present invention.", "FIG.", "8 demonstrates tagged proteins immobilized on a surface according to the present invention.", "FIG.", "9 is an image showing gold conjugate proteins as imaged in SEM without image processing according to the present invention.", "FIG.", "10 is a bar graph and a table demonstrating signal (S) to background (B) ratios (defined as (S-B)/B) for STP20, STP40 and Cy3-STP probes in the BSA-biotin—gold/Cy3-streptavidin detection system.", "Only last five dilutions of BSA-biotin are shown.", "FIG.", "11 is a blow-up of FIG.", "10, where only last three dilutions of BSA-biotin are shown.", "FIG.", "12 is a bar graph demonstrating signal (S) to background (B) ratios (defined as (S-B)/B) for STP20 and Cy3-STP probes.", "The data for Cy3-STP probes was obtained by averaging over results of 4 independent slides.", "FIG.", "13 is a graph demonstrating the estimated detection abilities presented as the number of biotinylated BSA molecules detected with STP20 probe versus the total number of biotinylated BSA molecules present in a spot on the slide.", "The number of biotinylated BSA molecules conjugated to the slide is approximated by calculating the number of molecules being able to attach to the surface.", "As shown in the calculations below, this is at most 10% of the total molecules contained in a 10 ml drop that was spotted on the slide.", "This is the upper limit to this number, since most likely less then 10% of the molecules contained in the drop indeed conjugated to the glass surface.", "The number of molecules detected is given by the number of gold colloids detected.", "This number was calculated by extrapolating from the average number of gold colloids counted in a single SEM frame (see, FIG.", "14) to the area of the whole drop.", "The dashed line represents a linear fit.", "From the slope of the fit and the efficiency of attachment of biotinylated BSA to the slide it arises that the detection ability if of between 1:1 and 1:4, hence, the upper limit of detection (sensitivity) was reached, over an unprecedented dynamic range.", "FIG.", "14 is a backscattered electrons image demonstrating single molecule detection using 20 nm gold colloids according to the present invention.", "The high quality is achieved using backscattered electrons in accordance with the teachings of the present invention.", "The number of gold colloids can easily and accurately be quantified manually or via using a simple image analysis software.", "FIG.", "15 is a graph demonstrating signal (S) to background (noise, B) ratios (defined as (S-B)/B) for STP40 probe in the BSA-hapten—biotinylated antibody—gold streptavidin detection system of the present invention employing different dilutions of the biotinylated antibody.", "DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a method and apparatus useful in the implementation of genomics, proteomics, glycomics and cellomics.", "In particular, the method and apparatus of the present invention allows highest sensitivity of ultimately single molecule detection, yields high signal-to-noise ratios, and demonstrates a broad dynamic range, while, at the same time, retaining simple sample preparation, readily applicable for high throughput screening.", "According to one aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair.", "The method according to this aspect of the invention is implemented by interacting a solid support onto which the first member or members of the binding pair are immobilized and arrayed with the corresponding second member or members of the binding pair.", "The corresponding second member or members of the binding pair are directly or indirectly tagged with a heavy atom.", "Thereafter, a spatial distribution of the heavy atom over a surface of the solid support is determined, thereby binding between the first member or members of the binding pair and the corresponding second member or members of the binding pair is detected.", "According to another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair.", "The method according to this aspect of the invention is implemented by interacting a solid support onto which the first member or members of the binding pair is immobilized and arrayed with the corresponding second member or members of the binding pair.", "Thereafter, the spatial distribution of the second member or members of the binding pair is determined at a dynamic range of linearity of at least four, preferably at least five, more preferably at least six, still preferably at least seven or at least eight orders-of-magnitude.", "According to still another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair.", "The method according to this aspect of the invention is implemented by interacting a solid support onto which the first member or members of the binding pair are immobilized and arrayed with the corresponding second member or members of the binding pair.", "Thereafter, the spatial distribution of the second member or members of the binding pair is determined at a sensitivity of detection which equals to or is greater than 1 of 10 binding events, preferably, the sensitivity equals to or greater than 1 of 5 binding events, more preferably, the sensitivity equals to about 1 of 1 binding events.", "According to yet another aspect of the present invention there is provided a method of detecting binding between first member or members of a binding pair and corresponding second member or members of the binding pair.", "The method according to this aspect of the invention is implemented by interacting a solid support onto which the first member or members of the binding pair are immobilized and arrayed with the corresponding second member or members of the binding pair.", "Thereafter, the spatial distribution of the second member or members of the binding pair at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80, still preferably greater than 100.The first and second members of the binding pair according to the present invention can be of any biochemical or chemical nature and serve any physiological or therapeutic function.", "First and second members of binding pair according to the present invention, include, for example, antigen-antibody, antibody-antigen, hapten-antibody, antibody-hapten, nucleic acid-complementary nucleic acid, nucleic acid-substantially complementary nucleic acid, ligand-receptor, receptor-ligand, enzyme-substrate, substrate-enzyme, enzyme-inhibitor and inhibitor-enzyme.", "As used herein, the term “antigen” includes molecules having at least one epitope recognized by an antibody.", "Such a molecule can be, for example, a protein or a part thereof, a carbohydrate or a part thereof or any natural or man made chemical.", "As used herein, the term “hapten” relates to a molecule or a portion of a macromolecule to which an antibody may specifically bind.", "As used herein, the term “antibody”, includes polyclonal antibody, monoclonal antibody, fragment of an antibody, single chain antibody and a chimeric antibody.", "The source of the antibody can be from the serum of an immuned animal, a serum of a patient or produced by immortalized cells, such as hybridomas or virus infected antibody producing cells.", "As used herein, the term “nucleic acid” includes natural nucleic acids such as DNA and RNA, either derived from nature or synthetically prepared, as well as analog nucleic acids capable of base pairing with natural nucleic acids.", "As used herein, the phrase “complementary nucleic acid” refers to a nucleic acid as this term is defined above having a sequence of nucleobases, each of which matches a corresponding nucleobase in another nucleic acid according to the base parity rules.", "As used herein, the phrase “substantially complementary nucleic acid” refers to a nucleic acid having a sequence of nucleobases, most of which (e.g., above 50%, preferably above 60%, more preferably, above 70%, still preferably above 80%, yet preferably, above 90%) match corresponding nucleobases in another nucleic acid according to the base parity rules.", "As used herein the term “ligand” includes natural or man made molecules or macromolecules which are capable of binding to a receptor.", "A ligand can be, for example, a protein, a nucleic acid, a carbohydrate or a small molecule, including for example lipids, steroids, etc.", "The ligand can act as an agonist or antagonist when it binds the receptor.", "As such, a ligand can be a drug, a hormone, etc.", "As used herein the term “receptor” includes macromolecules that bind ligands.", "Such macromolecules may for example be proteinaceous, soluble or anchored to a membrane.", "As used herein the term “enzyme” refers to a proteinaceous macromolecule having catalytic activity with respect to one or more substrates.", "As used herein the term “substrates” refers to any kind of molecule which undergoes faster catalysis in the presence of an enzyme.", "As used herein the term “inhibitor” includes any molecule capable of reversibly or irreversibly slow down catalysis.", "As such, an inhibitor can be a drug.", "According to the present invention determining the spatial distribution of a heavy atom over the surface of the solid support is by particle scattering.", "Preferably, determining the spatial distribution of the heavy atom over the surface of the solid support is by electron scattering.", "Any and all devices capable of producing a particle beam and recording scattered particles are suitable for implementing the present invention.", "Examples of such devices are described in more detail hereinafter.", "The corresponding second member or members of the binding pair used in accordance with the present invention can be either directly or indirectly tagged with a heavy atom.", "Preferably, the corresponding second member or members of the binding pair used in accordance with the present invention is indirectly tagged with a heavy atom.", "It will be appreciated by one of skills in the art that indirectly tagging the corresponding second member or members of the binding pair provides an element of universality to the method of the present invention.", "The heavy atom can be any atom capable of scattering particles better than the atoms making organic molecules, such as C, H, O, N, S and P. Suitable heavy atoms include gold, silver and iron, which are frequently used in electron microscopy.", "Other heavy atoms, such as osmium and platinum may also be considered.", "Methods are known to link such heavy atoms to a variety of organic molecules.", "For example, gold can covalently bind through an SH group which is natural to proteins and can be introduced in other molecules such as nucleic acids and polysaccharides.", "Other heavy atoms can be trapped in suitable chelators, which can be linked to a variety of macromolecules using methods well known in the art.", "According to an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities Thereafter, the spatial distribution of the at least one biological molecule over a surface of the microarray is determined at a dynamic range of linearity of at least four, preferably at least five, more preferably at least six, still preferably at least seven or at least eight orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities.", "Thereafter the spatial distribution of the at least one biological molecule over a surface of the microarray is detected at a sensitivity which equals to or is greater than 1 of 10 binding events, preferably, equals to or is greater than 1 of 5 binding events, more preferably, equals to about 1 of 1 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to yet an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities.", "Thereafter the spatial distribution of the at least one biological molecule over a surface of the microarray is detected at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80, still preferably greater than 100, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to still an additional aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by contacting the sample with a microarray presenting an addressable array of macromolecules of known identities under conditions so as to allow binding between the at least one biological molecule and the macromolecules of known identities.", "Thereafter, the spatial distribution of the at least one biological molecule over a surface of the microarray is detected by directly or indirectly tagging the at least one biological molecule with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity.", "Thereafter, the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is determined at a dynamic range of linearity of at least four, preferably at least five, more preferably at least six, still preferably at least seven or at least eight orders-of-magnitude, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to still another aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity.", "Thereafter the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is detected at a sensitivity a sensitivity which equals to or is greater than 1 of 10 binding events, preferably, equals to or is greater than 1 of 5 binding events, more preferably, equals to about 1 of 1 binding events, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity.", "Thereafter, the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is detected at a signal-to-noise ratio greater than 20, preferably, greater than 50, more preferably, greater than 80, still preferably greater than 100, thereby identifying and/or quantifying the least one biological molecule in the sample.", "According to still a further aspect of the present invention there is provided a method of identifying and/or quantifying at least one biological molecule in a sample.", "The method according to this aspect of the invention is implemented by attaching biological molecules present in the sample to a solid support; contacting the solid support with at least one macromolecule of a known identity under conditions so as to allow binding between the at least one biological molecule and the at least one macromolecule of known identity.", "Thereafter the level of binding between the at least one biological molecule and the at least one macromolecule of known identity is detected by directly or indirectly tagging the at least one macromolecule of known identity with at least one heavy atom and obtaining a particle scattering image of a spatial distribution of the at least one heavy atom, thereby identifying and/or quantifying the least one biological molecule in the sample.", "A method for the identification and quantification of biological molecules on a preparate such as a microarray or a 2D-gel according to preferred embodiments of the present invention includes the following steps (i) localization (the biological molecules are separated in space); (ii) tagging with markers (this step occurs before or after the localization, depending on the tagging, separation technology and/or preparate); (iii) scanning the tags; (iv) identifying or quantifying the tags; (iv) interpreting the number of molecules in each location from the quantity of tags and relating the results to the biological problem that is studied.", "According to the present invention, it is preferable to perform step (iii) with a scanning electron microscope (using appropriate tags in step (ii)).", "This provides a detection method that operates at high sensitivity, broad dynamic range and low background.", "In one embodiment of the invention, the method is carried out as illustrated schematically in the flow-chart of FIG.", "1, and comprises the following steps (1)-(9) (marked as 111-119 in FIG.", "1): 1.Localizing the biomolecules in space.", "2.Tagging the biomolecules with markers.", "3.Preparing the preparate for vacuum.", "4.Loading the preparate on X-Y-T stage (T means tilt) to analysis position.", "5.Scanning the preparate at low resolution to identify the regions of interest (ROIs).", "6.Scanning the ROIs with an electron beam at a high resolution.", "7.Enhancing the image contrast.", "8.Analyzing the ROIs.", "9.Displaying results.", "The step of localizing the biomolecules in space comprises any of the following steps or combinations thereof: A.", "Binding the biological molecules to a known or unknown immobilized array of molecules.", "B.", "Separating the biomolecules with one- or two-dimensional electrophoresis (gels).", "C. Attaching the biomolecules to a membrane (blot).", "D. Observing the biomolecules' affiliation to a specific cell.", "E. Separating the biomolecules by chromatography.", "F. Separating the biomolecules in a flow system.", "The step of tagging the biomolecules with markers can be done before or after the above described stages, depending on the case.", "When the biomolecules are DNA molecules, the tagging is preferably done before the spatial separation, while for proteins in the 2D-gel or microarrays the tagging is preferably done after the spatial separation.", "The tagging may comprises any combination of the following alternatives: A.", "Use of heavy metal colloids (e.g., gold, silver), preferably of a diameter range of 1-200 nm, whereby the colloids create a high intensity back scattered electron signal and, therefore, high image contrast.", "B. Doping the biological molecule with a high atomic number substance, to create a contrast sufficiently strong under an electron beam.", "Preferably the doping substance is an organic compound that contains iron.", "C. A fluorescent electro-luminescent signal whereby the electron beam creates a light signal that is detected.", "Such marking has the advantage of high resolution electron beam inspection with optical reading.", "Tagging with more than one type of tags to make a distinction between two preparates or different molecules is also possible.", "In the case of colloids, the tags may comprise different materials or of different sizes.", "The material analysis can be done with EDS (Energy Dispersive Spectroscopy) yielding an X-ray spectrum.", "The differentiation on the basis of size is done by image analysis.", "For preparing the preparate for vacuum standard methods are used that include: A. Fixation e.g., with formaldehyde.", "B.", "Coating with a conductive layer e.g., carbon.", "C. Freezing to prevent out-gassing.", "The preparation of the preparate for vacuum can be minimal when using an Environmental Scanning Electron Microscope (ESEM), as described further below.", "After loading the preparate in the specimen chamber of an electron microscope, scanning of the preparate is carried out by reading at low resolution either by the electron beam or with a combined optical microscope, including optical fiber where access is indirect.", "In this way, the regions of interest (ROIs) are identified and are then scanned at a higher or highest resolution.", "The scanning of the ROIs with an electron beam at high resolution can be in one or two dimensions.", "The scanning in one dimension can be synchronous with the movement of the preparate in the second horizontal dimension.", "To enhance the image contrast, algorithms well-known in the art can be used such as, for example, by calculating the histogram of pixel values and expanding it over the dynamic range of pixel values (typically 2-255).", "The analysis of the ROIs comprises any one of the following steps: A.", "Performing edge detection algorithm to identify the colloids in each ROI and counting said colloids.", "B.", "Counting electro-luminescent signals.", "C. Identifying X-ray spectrum of each particle for identification by comparison to a reference spectrum.", "D. Counting the signals by image analysis.", "Display of the results may include presenting the number of molecules in each ROI and the spatial map of the ROIs with indication of the different types of molecules.", "Reference is now made to FIG.", "2 which is a general diagram showing a longitudinal cross-section of a SEM 1 used as a preparate analysis apparatus according to the present invention.", "Instruments similar to SEM 1 are used for inspection of semiconductor wafers, as described, for example, in U.S. Pat.", "Nos.", "6,072,178, 5,644,132, 5,502,306, 4,618,938, 4,609,809 and 4,618,938, the contents of which are herein incorporated by reference as if fully disclosed herein.", "A primary electron beam 12, travels through a vacuum path to reach a preparate 20.The electrons are emitted from an electron gun 3, powered by a gun power supply which is electronically controlled as indicated by 7.The beam is focused by a condenser lens 4 and an objective lens 5, to form a focal point on preparate 20.The beam is diverted by a deflector 6 that scans the preparate in one or two dimensions.", "The preparate emits secondary electrons (SE) 14, back scattered electrons (BSE) 16 and characteristic X-rays 8.The characteristic X-rays are subjected to energy analysis to form and X-ray spectrum via an EDS.", "The BSE are detected by the BSE detector 18.SE 14 are detected by the SE detector 13.X-rays are detected by the EDS detector 150.The analog data is acquired and analyzed in a computer schematically represented by box 101.Optical microscope assisted with optical fibers 104 is used for low resolution inspection.", "A preparate that comprises biological molecules is fed via a tray 102 into the SEM.", "The preparate compartment 103 is divided to two sections in each of which pressurization can be performed independently to permit loading or unloading of a preparate in one chamber (e.g., 103A) while simultaneously inspecting a second preparate (in e.g., 103B).", "Each of chambers 103A and 103B are large enough to contain a 12″ (30 cm) diameter preparate 8.A stage 9 drives the preparate under the scanning electron beam 12.The image is formed from the detected electron signal and is digitized for image processing.", "Preparate 20 contains biological molecules that are separated in space.", "According to one embodiment of the present invention, preparate 20 comprises a hybridized DNA microarray, as detailed further below.", "According to another embodiment of the present invention, the preparate comprises a 2D PAGE as also detailed below.", "According to still another embodiment of the present invention, the preparate comprises a protein microarray, a carbohydrate microarray or a cell microarray interacted with any probe or target.", "Reference is now made to FIG.", "3, a generalized diagram showing a cross-section of a scanning electron microscope according to one aspect of the present invention.", "Parts that are the same as those shown in the previous Figure are given the same reference numerals and are not described again, except as necessary for an understanding of the present embodiment.", "According to this aspect of the present invention preparate 20 comprises a DNA microarray 70, after hybridization.", "The substrate 72 is standard, e.g., glass, nitrocellulose membrane, nylon filter, filter paper, other substrates that are used in Southern, western or northern blots or in microarrays.", "The substrate can be silicon, glass, filter paper or any other material that is convenient for microarray fabrication.", "The miss-matched and perfect-match target-probes are marked by 26A and 26B, respectively.", "The technique of gold tags (discussed further below) is used.", "In order to prevent charge accumulation, the microarray is coated by a thin layer of a conductive material, schematically shown by the dashed line of 73.Preferably the coating material is carbon.", "Preferably the thickness range is 50 to 600 A.", "In order to make the preparate vacuum compatible, it is fixed by standard electron microscopy methods, preferably by using formaldehyde.", "An alternative method to increase the compatibility between the microarray 70 and the vacuum is by using a cooling stage 74, preferably based on the Peltier effect.", "The stage is used to cool the microarray and thereby reduce the out-gassing.", "The specific microarray preparate comprises domains of different probes 26 as shown in FIG.", "3 by way of a non-limiting example.", "A domain of base structure TTGC 26A (SEQ ID NO:1), is shown as a representation of a miss-match.", "A domain of ATGC 26B (SEQ ID NO:2) represents perfectly matched probes.", "The probes are hybridized with the target molecules 27.A plurality of tags 28 (e.g., gold colloids) is attached to the target molecules.", "In a preferred embodiment, there is one tag per target.", "The advantage of gold is that it creates a strong signal of back-scattered electrons due to its high atomic weight and corresponding high back-scattered electrons coefficient.", "This allows a high contrast, high resolution image at low current and exposure time, thus minimizing the radiation damage to the preparate.", "It is possible to obtain commercial gold colloids as small as I nm (for example, from Nanoprobes, Inc., 95 Horse Block Road, Yaphank, N.Y. 11980-9710, USA).", "In order to get good correlation between the number of gold particles and the number of hybridized DNA, it is important that the number of colloids per target will be constant, preferably one tag per probe.", "The binding between tags and targets should be stable at a temperature of 50° C., which is typical for hybridization.", "According to a yet another embodiment of the present invention, the tags are iron-rich molecules, as in the technology used by Clinical Micro Sensors, Inc. (126 West Del Mar Blvd, Pasadena, Calif. 91105, USA).", "The iron produces a sufficient contrast for the BSE signal.", "The SEM/gold tags combination along with the ability to drive the preparate mechanically enables quantitative microarray detection.", "According to the present invention, the colloids are counted from the acquired digital image.", "Then, the contrast of the image is enhanced using a contrast enhancement algorithm.", "Then a pattern recognition algorithm identifies the colloids, for example by edge detection.", "Subsequently, the colloids are counted.", "This provides a quantitative measure of even the weakly expressed genes.", "A preferred reading strategy that reduces the reading time is to first scan the entire preparate and identify the regions of interest and then rescan the regions of interest to the desired quality.", "The ability of SEM to work at resolutions that vary from 10 microns down to about 1 nm, provides an ability to perform single molecule detection on a very wide dynamic range.", "The present invention is advantageous over existing methods since it is based on single-molecule detection.", "This means that the sensitivity and dynamic range are considerably higher than in the presently used fluorescence based methods.", "One advantage of single molecule detection scheme is the fact that it is compatible with miniaturization of a microarray.", "The miniaturization is desired since one would like to pack compactly as many molecules as possible on the same chip.", "The following table summarizes a comparison of performance of the single molecule detection method of the present invention and the alternative fluorescence technology.", "A further advantage that relates to the miniaturization is the ability to use smaller preparates.", "In many cases only a limited amount of sample is available.", "Exponential amplification methods such as PCR may alter the results in an uncontrolled manner.", "The advantages of a sensitive system that can detect smaller preparates are clear.", "TABLE 1 Fluorescence Electron Beam Detection Comparison: Detection Detection Resolution (pixel size) [unit] ˜10 μm <10 nm to ˜10 μm Scan Time [min] ˜18 ˜ comparable (depends on number and size of ROIs) 40 min to scan a whole microarray at a 100 nm resolution Sensitivity ˜105 1 [molecules/100 μm2] (Number of molecules per micron squere required for a signal) S/N ˜3-4 Much better, e.g., 20-100 Dynamic range of linearity ˜103 ˜108 Can rescan preparates N Y (no bleaching) Compatible with small N Y preparates (reducing PCR steps) Compatible with miniaturized N Y arrays In another embodiment, the method of the present invention can be carried out at almost atmospheric pressure, for example using an Environmental Scanning Electron Microscope (ESEM), a commercial SEM that works at elevated pressures.", "Further information on ESEM and how it works can be found in Enviromnental Scanning Electron Microscopy, Philips Electron Optics, Eindhoven, The Netherlands (Robert Johnson Assoc.", "El Dorado Hills, Calif. 1996) as well as in U.S. Pat.", "Nos.", "5,250,808, 5,362,964 and 5,412,21 1, the contents of which are hereby incorporated by reference as if filly disclosed herein.", "According to a yet another embodiment of the present invention, the ESEM is used for the inspection of the microarray of FIG.", "2., without the preparation for vacuum.", "In a preferred embodiment, the microarray will be cooled by cooling plate 74, to reduce the vapor pressure.", "The main advantage of the ESEM is the ability to study topography in an elevated pressure, utilizing a prior art secondary electrons (SE) detector.", "The advantage of the ESEM is that the hybridization can be detected simply by measuring the density at each site.", "Thus, as mentioned below, according to one embodiment, the microarray will be analyzed without tagging the targets.", "According to another embodiment of the present invention, the ESEM, or its specimen compartment, will be used instead of the SEM disclosed in FIG.", "2.The lower vacuum and the cooling relaxes the steps needed to prepare the preparate for vacuum.", "For example, when the ESEM is used, it is possible to inspect 2D PAGEs and microarrays without fixation.", "An example of an apparatus that combined the ESEM with the SEM disclosed above is given in FIG.", "4.The SEM 60 contains a pressure limiting aperture 63 that distinguishes between specimen compartment 103 and the column.", "In order to protect the back scattered electrons detector, it is enclosed in a protective chamber 61, the front window of chamber 62, is a membrane transparent to electrons that can hold the pressure difference between the evacuated medium near the detector and the gas.", "The secondary electrons are detected by the prior art ESEM SE detector.", "According to a yet another embodiment of the present invention the targets are not tagged at-all.", "The SEM is sufficiently sensitive to detect density differences between hybridized and non-hybridized regions in nucleic acid microarrays and, similarly, interacted vs. non-interacted molecules in other types of microarrays, including protein and carbohydrate microarrays.", "A difficulty in using an electron beam 12 for biomolecules is that it may damage the preparate.", "The damage to DNA, for example, from a beam of electrons is described in “Measurement of DNA damage by electrons with energies between 25 and 4000 eV”, Folkard et al., Int J. Radiat.", "Biol.", "64(6) pp 651-658 (1993).", "The choice of parameters should be safely below the damage.", "The use of gold allows one to use small probe currents, in the range of 10 pA, and fast scanning, typically 10 or more frames per second, thus minimizing the radiation absorbed by the DNA.", "In the areas where gold is present, it is expected that most of the interaction will be with the heavy gold atoms and not with the biomolecules.", "The main danger of radiation is the formation of free radicals in the water.", "Hence, according to a preferred embodiment of the present invention, a chemical that reduces the formation of free radicals but does not damage the biomolecules is added to the microarray after hybridization/interaction and before inspection.", "Further information on these chemicals can be found in Siddiqi M. A. and Bothe E., Radiation Research, Vol.", "112, pp 449-463 (1987), the contents of which are herein incorporated by reference.", "Preferably, the microarray preparate is situated on a stage 21 that can be moved by a servo motor 22.This arrangement drives the preparate under the electron beam and effectively increases the scanned area.", "The motor may incorporate an electrical vacuum feed-through 23.Such a motor is commercially available from Nanomotion, Ltd. (Mordot HaCarmel Industrial Park, PO BOX 223, Yokneam, 20692, Israel).", "Alternatively, the stage can be moved by a conventional mechanical feed through.", "According to yet another embodiment of the present invention motor 22 is situated on an arm that drives it into the vacuum chamber via a load lock.", "Such an arrangement improves the automation and elevates the throughput of the system.", "According to a preferred embodiment of the present invention, the preparate is driven along one axis (marked by X) and the electrons beam scans the microarray along the perpendicular axis (marked by Y).", "Due to the low signal-to-noise ratio inherent to microarrays, protein microarrays in particular, as well as to other methods currently used to detect biological species, it is desirable to use comparative, rather than absolute measures.", "In the commonly used fluorescence-based microarrays, this is done on the basis of different dye colors.", "According to a yet another embodiment of the present invention, this is done in the SEM by means of an X-ray spectrum analysis.", "The X-ray is detected by an EDS detector.", "The X-ray photons and the detector are marked in FIG.", "2 by 8 and 150, respectively.", "The characteristic X-ray spectrum of the tags serves for comparative study.", "As the colloids are counted, their spectrum is acquired, analyzed and compared to a reference spectrum.", "This method allows comparative study of more than one tag since the number of possible X-ray spectrums is not limited.", "Reference is now made to FIG.", "5, that shows a yet another embodiment of the present invention, whereby the detection is made by exciting light photons (electro-luminescence).", "The target molecules are tagged with luminescent molecules 41, in a similar fashion to the prior art fluorescent tagging.", "According to the present invention, the luminescent tags 41 are excited by the electron beam 12 and/or the excited SE.", "The light beam 42 is guided to a photomultiplier (PMT), by means of a light guide 43 (e.g., made of PMMA).", "The amplified light signal produced by the PMT is transformed to an electrical signal at the SEM detector.", "The device allows inspection of light emitted from the fluorescent DNA molecules, at a resolution below 100 nm.", "A further development that will increase throughput is parallel inspection with microcolumns.", "The microcolumns are miniature scanning electron microscopes that are produced by integrated silicon processes.", "Due to their size, the microcolumns can operate in parallel, considerably reducing the scanning time and the bulkiness of a SEM based system.", "Further details of the microcolumns are given in A. D. Feinerman and Crewe “Miniature Electron Optics”, Advances in Imaging and Electron Physics, Vol.", "102, 187 (1998) as well as U.S. Pat.", "No.", "5,122,663, the contents of which are hereby incorporated by reference as if fully disclosed herein.", "Reference is now made to FIG.", "6.According to the present invention, a plurality of microcolumns arranged in an array 80 is used for analysis of genes or proteins.", "Electrical wiring 81 controls the electron beam and lead the information from the detector to the data processing system.", "The beam of electrons scans the preparate of tagged nucleic acid, proteins, carbohydrates or cells in a microarray and/or 2D-gel, as appropriate.", "According to one embodiment of the present invention, the biological preparate can be coated with a conducting material e.g., carbon, as shown in 83.Alternatively, the preparate may be protected in a close chamber, as shown at 84 and the electrons will travel through the membrane.", "According to an embodiment of the present invention, the SEM is used for the analysis of proteins in a 2D PAGE.", "According to this embodiment, after counting, the preparate is prepared for mass spectrometry.", "According to this embodiment, the separation of proteins is done in a 2D PAGE.", "However, the present invention is compatible with other separation methods, for example, electrophoresis in a fluid or through a membrane (e.g., as in a blot), chromatography (HPLC).", "The typical size of a 2D PAGE is 20×20 cm2.This means that the entire 2D PAGE can easily fit into the standard wafer compartment of a wafer inspection SEM (103 in FIG.", "2.).", "According to the present invention, the preferred tagging method is the attachment of gold colloids.", "This is done with known technologies such as, for example, the one available from British Bio Cell Inc. (Cambridge, GB).", "According to another aspect of the present invention tagging is done by silver staining.", "Since there is no generic tagging that fits all proteins, the type of tagging to be used depends on the biological question that is asked.", "In many cases, the relevant question is whether a known protein exists in a preparate.", "In this case, the specific tagging of this protein, or number of proteins is applied and the desired proteins can be read on a single molecule detection basis.", "In other cases, general tagging, such as or silver staining can be applied.", "According to the present invention, the preparation for vacuum is done as follows: first the tagged molecules (proteins) are driven to the surface by an electric field (shown schematically in FIG.", "7 which is further referred to hereinbelow) and then the proteins are immobilized on the surface.", "According to one embodiment of the present invention, the surface is made of silicon.", "According to another embodiment, the surface is made of glass.", "After the proteins are attached to the surface, the surface is detached from the gel, coated to prevent charge accumulation by the electron beam of the electron microscope and then scanned thereby.", "According to the present invention, the scanning is done by driving the preparate mechanically under the electron beam in a continuous or a ‘step and repeat’ manner.", "The driving is done in correlation with the—scanning.", "According to one aspect of the present invention, the scanning is first done at a low resolution, to identify the ROIs, either automatically via the software or manually.", "Then the preparate is scanned at a higher resolution to count the number of colloids in the significant spots.", "Reference is now made to FIG.", "7.The gel chamber 90 comprises 3 sets of electrodes.", "The electric field that separates the molecules is applied by power supply V1 in the X direction and V3 in the Y direction.", "The attachment to the upper surface is done via V2 (Z direction).", "According to another embodiment of the present invention, the proteins are marked with fluorescent or electro-luminescent molecules, similar to the embodiment disclosed in FIG.", "5 for DNA microarrays.", "According to one embodiment of the present invention, the molecules are tagged before they are separated in the gel or attached to the microarray.", "Typically protein analysis consists of two typical phases: separation or localization in space and identification via mass spectrometry, antibodies, etc.", "What is clearly missing is an intermediate stage where the number of proteins in each spot is counted.", "Preferably the counting method will be able to distinguish between different types of proteins.", "According to the present invention there is disclosed a method of protein analysis that comprises of the following phases: 1.Localization via a 2D PAGE or on a microarray 2.Quantification (in an SEM) 3.Identification (preferably via mass spectrometry, specifically Matrix Assisted Laser Dissociation/Ionization-MALDI, antibodies).", "In another embodiment of the present invention, proteins or protein samples, which can include proteins of a known identity or of an unknown identity, naturally occurring or synthetic, antigens or antibodies, etc., are arrayed over a surface of a microarray and are immobilized thereto and are thereafter interacted with appropriate directly or indirectly tagged macromolecules to generate a preparate suitable for electron microscope inspection.", "Other preparate processing steps are similar to the steps described elsewhere herein.", "In yet another embodiment of the present invention, saccharides or saccharide samples, which can include saccharides of a known identity or of an unknown identity, naturally occurring or synthetic, are arrayed over a surface of a microarray and are immobilized thereto and are thereafter interacted with appropriate directly or indirectly tagged macromolecules to generate a preparate suitable for electron microscope inspection.", "Other preparate processing steps are similar to the steps described elsewhere herein.", "In still another embodiment of the present invention, cells of a known identity or of an unknown identity are arrayed over a surface of a microarray and are immobilized thereto and are thereafter interacted with appropriate directly or indirectly tagged macromolecules to generate a preparate suitable for electron microscope inspection.", "Other preparate processing steps are similar to the steps described elsewhere herein.", "According to the present invention, the disclosed apparatus and method can be used for building databases.", "For example a database that aim at interfacing protein information with DNA mapping and sequence data from genome projects.", "This may also include a file listing all of the information entered for the particular protein.", "An example of such a database, obtained by conventional means is described in: J. E. Celis, FEBS Letters 430, 64-72, 1998 which is incorporated herein by reference.", "According to a preferred embodiment of the present invention a wafer-inspection SEM is used to detect labeled molecules on microarrays.", "Reference is now made to FIG.", "8.The tagged proteins are immobilized on a surface 141.According to a preferred embodiment, the surface 141 is coated with a thin layer of carbon 142.After immobilizing the proteins, the substrate is coated with an additional layer of carbon to prevent charge accumulation in the proteins.", "The substrate is then scanned in the SEM.", "Reference is now made to FIG.", "9, showing gold conjugate proteins as imaged in the SEM.", "The image is shown ‘bare’ without contrast enhancement.", "It can be seen that an image analysis technique can be applied to quantify the number of tags.", "This experiment has been performed with monoclonal antibody (mouse IgG1) 1E10 conjugated to 20 nm gold colloids.", "A silicon substrate was covered with a carbon layer of 150-200 Angstrom.", "The substrate was attached to a conventional SEM aluminum support.", "On the silicon surface was a drop of antigen P277.The drop was dried in a vacuum oven at 40° C. for 20 minutes.", "The gold conjugated antibody was added by putting a drop on the silicon surface, in a way that covered all the surface.", "The antibody added was diluted 1:10.The support was left in a humid chamber for 40 minutes.", "The antibody was washed by dipping the support for a few seconds in PBS (phosphate-buffered saline) a few times and then in double-distilled water.", "As is discussed hereinabove and is further exemplified in the Examples section that follows, the present invention teaches a method that reaches single molecule detection levels, gives high signal-to-noise ratios, and demonstrates a very broad dynamic range, while retaining easy preparate preparation.", "In its preferred embodiment, the method uses gold labeling and electron microscopy, preferably a Wafer-Inspection Scanning Electron Microscope, to probe both protein function arrays and protein detecting arrays and is demonstrated here using the same immobilization chemistry and robotics described by the prior art (G. MacBeath, Science 298 (2000) 1760-1763).", "The reasons gold labeling and SEM scanning demonstrate such abilities are several and include: 1.Significant reduction of noise.", "Unlike fluorescence techniques where auto fluorescence of the glass and the buffers contribute to the noise, in the method of the present invention false positive signals are only due to nonspecific binding of the gold conjugated probe to the slide.", "This is because only signal (back scattered electrons) coming from very heavy atoms such as gold are detected by the SEM detectors.", "2.In small enough dilutions, where the molecules attached to the slide are in distances larger than the diameter of the gold probe, only one gold particle can attach per probed protein or ligand.", "This is because once a gold particle attached, it occludes the molecule on the surface from other gold probes.", "Since the SEM can detect single gold particles, this provides single molecule detection capabilities.", "3.Detection abilities are limited in the lower limit only by false positive signals due to non-specific binding, and in the upper limit by the highest number of colloids able to pack closely in a given area in other words, the upper detection limit can be controlled by the size of the gold colloids chosen as probes.", "This ensures a very broad and highly linear dynamic range.", "Also, detection abilities are not constrained by instrumentation (unlike fluorescent methods where saturation of the photodiodes in light detectors can occur).", "In addition to the increased abilities in sensitivity and dynamic range there are several other advantages over other labeling methods such as fluorescence: 1.There is no bleaching of the signal.", "This means that the preparate can be stored and scanned repeatedly across a long period of time (e.g., in cases that inconsistencies were found in the analysis or new data has accumulated that requires new analysis of an old preparate).", "2.The preparates have a higher reproducibility rate since there is no dependence on the type of buffers and materials used, and there is a weak dependence on the “Hands” preparing the preparate.", "3.In the case of gold tagged proteins, it seems the gold enhances binding selectivity of protein bound to it, possibly because in some cases there are several proteins per gold which can increase the probability of binding.", "Another possibility is that due to the large size of the gold only high affinity and specific interactions survive the stringent washing conditions.", "The potential of the method of the present invention for relatively high throughput is demonstrated in the unrelated semiconductor industry where SEMs are routinely used to scan wafers for microscopic defects and impurities.", "Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.", "Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.", "EXAMPLES Reference is now made to the following examples, which describe experiments aimed at testing the sensitivity (signal-to-noise ratio) and the dynamic range of the method of the present invention.", "To this end, first the universal biotin-tagged avidin detection system was tested comparatively to the prior art, to reveal that under various experimental conditions sensitivity is up to 100 fold improved, whereas the dynamic range is both several orders-of-magnitude broader, with a lower limit reaching the ultimate goal of single molecule detection.", "Then, the universal biotin-tagged avidin detection system was sandwiched to a hapten-antibody detection system and tested for its sensitivity and dynamic range.", "Materials and Experimental Procedures Arraying Proteins on Glass Slides: Proteins were spotted on glass slides presenting aldehyde groups (Telechem International, SuperAldehyde Substrates) using a Biorobotics TAS arrayer.", "Three “flag” proteins (Biotin-BSA at a concentration of 0.1 mg/ml) served as position pointers for each group of 4×4 spots.", "Spotted drops were about 400 μm in diameter and 10 nano-liter (nl) in volume.", "256 protein spots were applied to each spotted area of a slide.", "For fixation via the aldehyde groups, the spotted slides were incubated in a humid chamber for 2-3 hours at ambient temperature.", "When required, spotted areas and/or subareas of a slide were spatially separated by surrounding paraffin lines.", "In order to block free aldehyde groups, the slides were inverted and briefly placed in a solution of 1% BSA in PBS, pH 7.5, and then immediately immersed in a similar fresh solution for 1 hour at room temperature with gentle agitation.", "Following a brief rinse in PBS, the slides were ready for further processing as described below.", "Probing the Slides: Gold conjugated streptavidin (gold-streptavidin) was purchased from British Biocell International.", "Gold colloids were 20 nm (STP20) or 40 nm (STP40) in diameter.", "Gold-streptavidin was spun four times in a cooled centrifuge at 12,000 revolutions-per-minute (RPM) for 20 minutes, resuspended twice in a fresh buffer containing 0.04% Tween20 (Sigma) and 0.1% BSA (w/v); and twice in a fresh buffer containing 20% glycerol, 80% PBS, 0.1% BSA (w/v) and 0.5 M NaCl.", "Final concentration of gold-streptavidin was approximately 15 nM for both STP20 and STP40.Cy3-conjugated streptavidin was purchased from Amersham Pharmacia Biotech and diluted in a solution of 20% glycerol, 80% PBS, 0.1% BSA (w/v) to a concentration of 17 nM.", "Slides were incubated for 4 hours with the gold-streptavidin, or Cy3-streptavidin probes.", "Preparing Gold Probed Slides for Electron Microscopy: All gold-probed slides were immersed in a fixation/dehydration solution (3% paraformaldehyde and 2% glutaraldehyde) for 30 minutes, washed in double distilled water (DDW) for an additional 10 minutes, spun for 5 minutes at 1250 RPM to remove excess buffer, and dried in a vacuum chamber overnight.", "The slides were then cut into 1 inch2 sections that contained all the spotted area, using a diamond glasscutter and attached to electron microscope supports via a carbon double-sided tape.", "In order to avoid charging of the samples, the glass sections were coated with a 200 nm thin carbon coat using an Edward's carbon coater and their edges were colored with a conducting silver paste.", "Scanning the Probed Slides: Fluorescence of the Cy3-probed slides was scanned using a Packard ScanArray 4000 scanner at a 20 μm resolution.", "Intensity was determined by taking the average intensity of the pixels in corresponding spots in all slides and reducing the average intensity of the pixels immediately surrounding the corresponding spots.", "Typically, each data point was based on four individual experiments.", "The gold-probed slides were visualized with a scanning electron microscope (SEM) (Jeol 6400)..", "Images of 48 μm2 (for 40 nm gold colloids) or 12 μm2 (for 20 nm gold colloids, see FIG.", "14) sized frames inside each spot were taken via a back scattered electrons detector.", "This detector can detect only electrons scattered from heavy atoms, and therefore detects only the gold colloids and not any organic, light weight atoms present.", "Gold colloids were counted using the NIH image processing software (shareware downloadable from: http://www.pathsoc.org.uk/wwwboard/messages/214.html).", "Number of gold colloids per spot, were taken to be the average number of gold colloids in corresponding frames multiplied by the number of frames per spot (2.6103 of 48 μm2 sized frames per spot in the case of 40 nm gold colloids; 10.4-103 of 12 μm2 sized frames per spot in the case of 20 nm gold colloids).", "BSA-Biotin—Gold/Cy3-Streptavidin Detection System: BSA and biotin-caproate were purchased from Sigma.", "BSA-biotin conjugate was prepared as follows: BSA (5 mg, 75 nmole) and biotin-caproate, (0.72 mg, 1.9 μmole) were dissolved in ice cold 200 μl DMF and the mixture was left at room temperature for 2 hours.", "The number of biotin molecules per BSA molecule, estimating 50% conjugation efficacy is 12.5 on the average.", "Extensive dialysis was preformed against PBS to remove unconjugated biotin.", "Activity of the BSA-biotin was assayed employing ELISA, using Horse Radish Peroxide conjugated to streptavidin as a probe.", "BSA-biotin, and BSA were dissolved in 40% glycerol, 60% PBS to an initial concentration of 1 mg/ml.", "BSA-biotin was serially diluted 3-fold in 40% glycerol, 60% PBS, pH 7.5, 0.1% BSA (w/v).", "In all dilutions, the total amount of BSA (free BSA+biotinylated BSA) was kept constant at 1 mg/ml.", "BSA-biotin was then spotted on the slides in different concentrations ranging from 1 mg/ml to 100 ng/ml.", "Free BSA, which served as a control was also spotted on the slides.", "Slides were than processed as described above.", "To probe the slides, 40 μl of gold-streptavidin (STP20 or STP40) or Cy3-streptavidin were applied to each printed area and incubated for 4 hours in a humid chamber at ambient temperature.", "Following incubation, the slides were washed 3 times, 3 minutes each time, with PBS supplemented with 0.04% Tween20 (PBS/T).", "Cy3-streptavidin probed slides were additionally rinsed twice in PBS, (3 minutes each rinse), centrifuged for 5 minutes at 1250 RPM to remove excess buffer, and left to dry in a slide box.", "Gold-streptavidin probed slides were further processed for electron microscopy as described above.", "Slides were scanned as described above.", "BSA-Hapten—Biotinylated Antibody—Gold Streptavidin Sandwiched Detection System: BSA-hapten 23.7 [1b (p-nitrobenzyl phosphonate N-glycylglutatarate)] was prepared as described in Tawfik et al.", "Phosphorus and Sulfur, 1993, vol.", "76 123-126.D2.3 antibody was prepared as described in Tawfik et al.", "Proc.", "Natl.", "Acad.", "Sci.", "USA, 1993, vol.", "90 p. 373-377.The affinity constant for hapten 23.7 and D2.3 antibody in solution was determined to be 4 nM by competitive ELISA (Tawfik et al.", "(1997) Eur.", "J. Biochem.", "vol.", "244 p. 619-626) and further by a fluorescence assay (Lindner et al.", "(1999) J. Mol.", "Biol.", "vol.", "285 p. 421-430).", "Biotin-caproate was purchased from Sigma.", "90 μl of biotin-caproate (20 μmole) were dissolved in a solution having a total volume of 1 ml and containing 336 μl D2.3 antibody (1 mg, 6.7 nmole) in PBS and NaHCO3 (1 M; 100 μl).", "The reaction mixture was placed on ice for 3 hours, followed by extensive dialysis against PBS.", "Activity of the D2.3 antibody was assayed with ELISA SA-HRP/GaM HRP.", "BSA-hapten 23.7 was dissolved in 40% glycerol, 60% PBS, pH 7.5 at a concentration of 200 μg/ml and spotted on slides as described above.", "The slides were then further processed as described above.", "The control, non biotinylated D2.3 (5 ng/ml), was dissolved in 20% glycerol, 80% PBS, 0.1% BSA (w/v).", "The biotinylated D2.3 antibody was diluted in a solution containing 20% glycerol, 80% PBS and 0.1% BSA (w/v).", "Dilutions ranged from 50 μg/ml biotinylated D2.3 antibody to 0.5 ng/ml biotinylated D2.3 antibody.", "Twenty μl of each dilution and control were applied to separate sections of the slides.", "Following 2-3 hour incubation in a humid chamber at ambient temperature, the slides were washed 3 timed (3 minutes each wash) with PBS/T.", "To probe the slides, 40 μl of gold-streptavidin were applied to each printed area and incubated for 4 hours in a humid chamber at ambient temperature.", "Following incubation, the slides were washed 3 times for 3 minutes each time in PBS/T.", "Gold-streptavidin probed slides were further processed for electron microscopy as described above.", "All slides were scanned as described above.", "Experimental Results BSA-Biotin—Gold/Cy3-Streplavidin Universal Detection Systems: The results for the BSA-biotin—gold/Cy3-streptavidin universal detection system are shown in FIGS.", "10-14, clearly demonstrating the far superior sensitivity (higher S/N ratios) and dynamic range of the present invention over the prior art in any and all of the experimental conditions employed.", "A few interesting points arise from the results.", "The first is that gold probes (STP20) extend the dynamic range (i.e., the range where signal scales linearly with protein concentration) and the sensitivity of detection by almost 100-fold relative to fluorescence probes (see, FIGS.", "10-12).", "Another result is that 20 nm gold colloids performed far better than 40 nm gold colloids (improving signal-to-noise by about 4-fold, or [R40/R20]2, see FIG.", "10 and 11).", "The latter result indicates that using even smaller gold colloids, e.g., 10 nm and 5 nm colloids, will improve sensitivity and extend the lower limit of the dynamic range by an additional 10-100 fold relative to the fluorescence probe.", "Since the largest number of colloids able to pack closely in a given area limits the upper detection limit, it is expected that decreasing the size of the colloids from 20 nm to 5 nm will further extend the dynamic range in its upper limit by a factor of at least 16 fold.", "Moreover, by careful normalization of measured results, it will be possible to carry out relative measurements of protein samples on the same microarray using distinguishable sizes of colloids.", "In order to approximate the detection abilities of the system, the ratio between the approximated number of BSA-biotin molecules conjugated to the glass surface in one spot, and the number of gold colloids detected in a spot was evaluated.", "To estimate the number of biotin labeled BSA molecules conjugated to the glass in each spot, first the number of aldehyde groups per spot was calculated by multiplying the number of aldehyde groups per cm2 (5·1012 groups per cm2) by the area covered by a drop of a 400 μm in diameter (1.25·10−3 cm2).", "The result is 6·109 aldehyde groups per spot.", "The number of BSA molecules contained in a 10 nl droplet of a 1 mg/ml BSA solution is 9·1010 molecules, which means that a maximal attachment of <10% of the protein molecules in a droplet can be achieved.", "To estimate the number of gold colloids detected in a spot, the average number of gold colloids counted per frame was multiplied by the number of frames contained in the area covered by the drop (see above).", "FIG.", "13 presents the estimated numbers, assuming a maximal attachment of 10%.", "Taking the slope of the linear fit, and taking into consideration that the aldehyde quantification was not done by protein attachment but rather by attachment of small molecules, the real value of protein attachment is probably <10%, hence, the detection is close to 1:1 of all molecules present in a spot.", "In other words, assuming that 10% of the biotin molecules floating in the spotted drop also conjugate successfully to the glass surface via the aldehyde groups thereat, the detection is at worst 1 of every 4, but more likely closer to detecting all biotin molecules.", "This experiment demonstrates that by using the method of the present invention, the lower possible limit of the dynamic range, i.e., every single molecule detection, was reached or nearly reached.", "BSA-Hapten—Biolinylated Antibody—Gold Streptavidin Sandwiched Detection System: To determine the sensitivity to concentration and as a demonstrative application for the method of the present invention a model system based on a sandwich detection system—BSA-hapten—biotinylated antibody—gold/Cy3 streptavidin—was employed.", "In the model system, a hapten conjugated to BSA was spotted on glass slides.", "It was then interacted with different concentrations of a corresponding biotinylated monoclonal antibody.", "The complex BSA-hapten-antibody-biotin was thereafter probed with STP40.The results which are shown in FIG.", "15 reveal that even at concentrations as low as a few tens of picomolars of antibody, detection was successful.", "In order to obtain another approximation of the detection abilities of the method of the present invention, equilibrium equations were used to calculate how many hapten-antibody complexes are expected to form under the experimental conditions employed.", "The affinity constant (K=4 nM) was assumed to be the same as when both hapten and antibody are free in solution, based on 2 independent measurements conducted in Fluorescence and ELISA assays (see methods).", "The efficiency of attachment of the BSA-hapten to the substrate, taking into account the binding capacity of the aldehyde groups was assumed to be <30%.", "Then the ratio of the number of complexes detected by the gold colloids to the maximal number of complexes expected to exist on the glass was calculated.", "A ratio close to 1:10 was obtained, indicating that method detects about one of every ten complexes that are actually formed on the slide.", "In view of the calculations above, it is again anticipated that using smaller gold colloids this ratio will substantially improve.", "It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.", "Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.", "Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art.", "Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.", "All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.", "In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention." ] ]

[ [ "Apparatus for connection of implantable devices to the auditory system", "A connection apparatus for coupling an implantable device, such as a hearing aid transducer, to a middle ear component.", "The connection apparatus includes a first joint member on the implantable device and a second joint member connectable to the middle ear component.", "The first and second joint members are themselves connectable during implantation of the device to couple the device and the middle ear component.", "In one embodiment of the invention, the first and second joint members may form a detachable connection to facilitate removal for repair and or replacement of the device" ], [ "1.A method for coupling an implantable hearing aid transducer to a middle ear component of a patient, the method comprising: providing a first joint member on the transducer; coupling a second joint member to the middle ear component of the patient; and, interconnecting a portion of the first joint member to a portion of the second joint member to form a detachable physical connection between the first and second joint members during implantation of the transducer to couple the transducer to the middle ear component of the patient.", "2.The method of claim 1, comprising: providing a stimulus representative of sound to the middle ear component over the connected first and second joint members.", "3.The method of claim 1, wherein the interconnecting step comprises: inserting a tip portion of one of the first and second joint members into a receiver portion of the other one of the first and second joint members.", "4.", "(canceled) 5.The method of claim 1, wherein the coupling step comprises: forming an interface on the middle ear component; and connecting the second joint member to the interface.", "6.The method of claim 5, wherein the connecting step comprises: placing the second joint member in the interface; and in response to a stimulus, expanding the second joint member outward relative to the interface to apply lateral loading between the second joint member and the interface.", "7.The method of claim 1, the method comprising: exposing the connected first and second joint members to a movement of the middle ear component relative to the transducer; and automatically executing a compensating movement of the first and second joint members to reduce loading pressure on the middle ear component.", "8.The method of claim 7, wherein the step of executing the compensating movement comprises: rotating a ball of one of the first and second joint members in a receiver of the other one of the first and second joint members.", "9.The method of claim 8, wherein the interconnecting step comprises: inserting the ball of the one of the first and second joint members in the receiver of the other one of the first and second joint members.", "10.The method of claim 7, wherein the step of executing the compensating movement comprises: flexing a stem of one of the first and second joint members.", "11.The method of claim 1, wherein the interconnecting step comprises: inserting a tip portion of the first joint member in a sleeve portion of the second joint member; and in response to a stimulus, expanding the tip portion outward to apply lateral loading between the tip portion and the sleeve portion.", "12.The method of claim 1, wherein the interconnecting step comprises: threading the first joint member to the second joint member.", "13.The method of claim 1, the method comprising: disconnecting the first and second joint members; and removing the transducer and the first joint member from the patient.", "14.The method of claim 13, wherein the step of removing comprises: permanently removing the transducer and first joint member from the patient; and leaving the second joint member permanently connected to the ossicles of the patient.", "15.The method of claim 13, the method comprising: subsequent to removing the transducer, repairing the transducer; subsequent to the repairing step, re-implanting the transducer; and reconnecting the first and second joint members.", "16.The method of claim 13, the method comprising: subsequent to removing the transducer, replacing the transducer with a new transducer; providing the first joint member on the new transducer; implanting the new transducer; and interconnecting the portion of the first joint member to the portion of the second joint member during implantation of the new transducer.", "17.A connection apparatus for coupling an implantable hearing aid transducer to a middle ear component of a patient comprising: a first joint member interconnected to a vibratory member of the transducer; a second joint member connectable to the middle ear component of the patient; and means for interconnecting a portion of the first joint member to a portion of the second joint member during implantation of the transducer to provide a detachable physical connection between the interconnected joint members for the transmission of mechanical vibration from the vibratory member to the middle ear component, wherein the means for detachable interconnecting allows for removal of the first joint member independent of removing the second joint member from the middle ear component.", "18.", "(canceled) 19.The connection apparatus of claim 17, wherein the means for interconnecting comprises: a ball on one of the first and second joint members; and a receiver on the other one of the first and second joint members sized and shaped to receive the ball.", "20.The connection apparatus of claim 19, wherein the receiver further comprises: a pair of opposing tangs for application of pressure to open the receiver and uncouple the ball from the receiver.", "21.The connection apparatus of claim 17, wherein the means for interconnecting comprises: a first contact surface on a distal end of the first joint member; and a second contact surface on a distal end of the second joint member, wherein the first and second contact surfaces are adjacently positionable during implantation of the transducer to provide the detachable physical connection.", "22.The connection apparatus of claim 21, comprising: a connector for forming the detachable physical connection between the first and second joint members in the adjacent positional relationship.", "23.The connection apparatus of claim 17, wherein the first and second contact surfaces are magnetized.", "24.The connection apparatus of claim 17, wherein the second joint member comprises a sleeve and the first joint member comprises a stud member including a tip sized and shaped for insertion into the sleeve.", "25.The connection apparatus of claim 17, wherein the means for interconnecting comprises: mating threads on the first and second joint members.", "26.The connection apparatus of claim 17, wherein the first joint member comprises a clamp and the second joint member comprises a stud.", "27.The connection apparatus of claim 17, wherein the interconnected first and second joint members are movable relative to each other to reduce load pressures therebetween." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Implantable hearing aids entail the subcutaneous positioning of some or all of various hearing augmentation componentry on or within a patient's skull, typically at locations proximate the mastoid process.", "Implantable hearing aids may be generally divided into two classes, semi-implantable and fully implantable.", "In a semi-implantable hearing aid, components such as a microphone, signal processor, and transmitter may be externally located to receive, process, and inductively transmit a processed audio signal to implanted components such as a receiver and transducer.", "In a fully-implantable hearing aid, typically all of the components, e.g., the microphone, signal processor, and transducer, are located subcutaneously.", "In either arrangement, a processed audio signal is provided to a transducer to stimulate a component of the auditory system By way of example, one type of implantable transducer includes an electromechanical transducer having a magnetic coil that drives a vibratory actuator.", "The actuator is positioned to mechanically stimulate the ossicles via physical engagement.", "(See e.g., U.S. Pat.", "No.", "5,702,342).", "In this regard, one or more bones of the ossicles are made to mechanically vibrate, causing the vibration to stimulate the cochlea through its natural input, the so-called oval window.", "An example of this transducer is included in the MET™ hearing aid of Otologics, LLC, in which a small electromechanical transducer is used to vibrate the incus (the 2nd of the 3 bones forming the ossicles), and thence produce the perception of sound.", "In this case, the vibratory actuator is coupled to the ossicles during mounting and positioning of the transducer within the patient.", "In one example, such coupling may occur via a small aperture formed in the incus bone.", "As will be appreciated, coupling with the ossicles poses numerous challenges.", "For instance, during positioning of the transducer, it is often difficult for an audiologist or surgeon to determine the extent of the coupling.", "In other words, how well the actuator is attached to the ossicles.", "Additionally, due to the size of the transducer relative to the ossicles, it is difficult to determine if loading exists between the ossicles and transducer.", "In this regard, precise control of the engagement between the actuator of the transducer and the ossicles is of critical importance as the axial vibrations can only be effectively communicated when an appropriate interface or load condition exists between the transducer and the ossicles.", "Overloading or biasing of the actuator can result in damage or degraded performance of the biological aspect (movement of the ossicles) as well as degraded performance of the mechanical aspect (movement of the vibratory member).", "Additionally, an underloaded transducer, e.g., where the actuator is not fully connected to the ossicles, may result in reduced performance of the transducer.", "Another difficulty with such coupling is that in some cases patients can experience a “drop-off” in hearing function after implantation.", "Such a drop off -may be caused by changes in the physical engagement of the actuator, e.g., due to things such as tissue growth, or may be caused by a malfunction of the transducer or other componentry.", "After implantation, however, it is difficult to readily assess the performance and/or adjust an implanted transducer and interconnected componentry.", "For example, in the event of a “drop-off” in hearing function after implantation, it is difficult to determine the cause, e.g., over/under loading of the interface due to tissue growth or some other problem with the hearing aid, without invasive and potentially unnecessary surgery.", "In addition, once coupled for an extended period, the maintenance and/or replacement with a next generation transducer may be difficult." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In view of the foregoing, a primary object of the present invention is to simplify and improve implantation procedures for implantable devices, such as hearing aid transducers.", "Another object of the present invention is to provide a means for achieving a low mechanical bias or no-load interface between transducers and a component of the auditory system, e.g., the ossicles.", "A related object of the present invention is to provide a connection apparatus with the ability to compensate in situ for undesirable interfaces, should one exist, between the middle ear component and a transducer, e.g., loading therebetween.", "Another object of the present invention is to provide a known geometry or uniform interface, on a middle ear component for making a connection with a transducer.", "In the context of the present invention, “in situ,” refers to in its proper position, e.g., in the context of the present transducer, as implanted in a patient and coupled to a middle ear component.", "One or more of the above objectives and additional advantages may be realized by a first aspect of the present invention, which provides a connection apparatus for coupling an implantable device, e.g., a hearing aid transducer, to a middle ear component.", "The connection apparatus includes a first joint member on the transducer, e.g., interconnected to a vibratory member of the transducer, and a second joint member connectable to the middle ear component, e.g., the ossicles.", "The connection apparatus further includes a means for coupling the first and second joint members during implantation of the transducer to provide a communication path between the coupled joint members for the transmission of mechanical energy from the transducer to the ossicles.", "In the context of the subject first aspect, the coupling of the first and second joint members may include an actual connection made therebetween or may include adjacent positioning of the joint members relative to each other to define the communication path.", "Various refinements exist of the features noted in relation to the subject first aspect of the present invention.", "Further features may also be incorporated in the subject first aspect of the present invention as well.", "These refinements and additional features may exist individually or in any combination.", "For instance according to a first feature, the second joint member may be configured to connect to the ossicles of the patient, in a substantially permanent manner.", "The permanent connection may be achieved through an artificial means such as an adhesive, or through bone growth or a fibrous tissue union naturally formed as a function of the body's response to the implantation.", "In some cases, an adhesive or other means may also be utilized to initially secure the joint member to the ossicles until such time as a natural connection via bone or tissue growth forms.", "According to another feature of the subject aspect, the first and second joint members may form a detachable connection.", "The detachable connection between the joint members, in combination with the permanent connection of the second joint member to the ossicles, provides a known geometry for connection and disconnection of a transducer, as well as reconnection of the transducer, such as for repair or replacement.", "In this regard, the second joint member is preferably of a minimal size that provides for a connection with the first joint member.", "This permits the second joint member to remain attached to the ossicles, even following a permanent removal of the transducer and first joint member, without affecting the auditory function.", "In other words, since the Second joint member is relatively small in comparison to a transducer, it is anticipated that the joint member may remain connected to the ossicles, even after permanent removal of the transducer, without substantial affects to the mechanical function of the auditory system.", "The connection between the joint members can be made in any manner as a matter of design choice to define the communication path for transmission of mechanical energy to the ossicles.", "For instance, in one aspect of the connection apparatus, one of the joint members may be a stud including a ball disposed on one end, while the other one of the joint members may be a receiver/pocket that forms a ball joint connection with the stud member.", "Advantageously, such a connection minimizes loading between the ossicles and the transducer by permitting rotational movement between the joint members.", "The ball joint connection may also be further engineered to permit additional compensational movements between the joint members to minimize loading.", "For instance, the receiver/pocket may be connected to the transducer or the ossicles via a stem member.", "The stem member, in turn, may be formed from a flexible material that permits non-permanent deformation in response to compressive forces between the transducer and ossicles.", "In another example according to this characterization, the stem and receiver/pocket may be defined by a pair of flexible structures that displace in a non-permanent manner in response to compressive forces between the transducer and ossicles.", "In another aspect of the connection apparatus, one of the joint members may be a stud member having a planer end for adjacent positioning relative to the other one of the joint members, which also includes a planer end.", "According to this characterization, the joint members are adjacently positionable during implantation to define the communication path.", "In another aspect of the connection apparatus, the joint members may be a pair of studs that are connectable in a detachable manner.", "For instance, in one example, a mechanical connection may be formed using a shape memory alloy to provide the detachable connection.", "In another aspect of the connection apparatus, the first joint member may be a stud member while the second joint member is a sleeve or conduit that is connectable to the ossicles, e.g., in an aperture/interface formed therein.", "According to this characterization, a tip of the stud member is insertable into the sleeve member to make the connection between the joint members.", "Although not necessary according to the present aspect, a material reshapeable in situ may also be disposed within the sleeve between the connected joint members to permit gradual movement of the joint members relative to each other to minimize loading.", "According to this characterization the reshapeable material may be a material that is resistive to high frequency movements to permit efficient acoustic stimulation of the ossicles, but gradually displaceable, e.g., in response to a steady state application of pressure by the ossicles, at body temperature to minimize loading.", "In this regard, the reshapeable material may also be utilized to secure the tip within the sleeve.", "In another example according to this aspect, an adhesive, or other means, such as a threaded connection may also be utilized to secure the tip within the sleeve as a matter of design choice.", "According to a second aspect of the present invention, a method for coupling an implantable device, e.g., a hearing aid transducer, to the ossicles of a patient is provided.", "The method includes the steps of providing a first joint member on the transducer, coupling a second joint member to a middle ear component, and forming a connection between the first and second joint members during implantation of the transducer to couple the transducer to the middle ear component.", "Various refinements exist of the features noted in relation to the subject second aspect of the present invention.", "Further features may also be incorporated into the subject second aspect of the present invention as well.", "These refinements and additional features may exist individually or in any combination.", "For instance according to a first feature of the subject second aspect, the connection between the joint members may be achieved in any manner as a matter of design choice that provides for acoustic stimulation of the middle ear component by the transducer.", "For example, the connection may be a detachable connection, a permanent connection, or an adjacent positioning as described above.", "According to this characterization, the method may further include the step of providing a stimulus representative of sound to the middle ear component over the connected first and second joint members.", "According to another feature of the subject second aspect, the method may include the step of exposing the connected first and second joint members to movement of the middle ear component, and automatically executing a compensating movement of the first and second joint members to reduce loading pressures.", "The step of executing the compensating movement may further include different steps according to the type of connection provided by the first and second joint members.", "For example, the compensating movement may include rotation of a ball relative to a receiver/pocket as described above.", "In another example, the compensating movement may include flexing a stem connected to the receiver/pocket or displacement of a reshapeable material between the interconnected joint members.", "In one aspect of the present method, the method may further include disconnecting the joint members and removing the transducer from the patient.", "The removing step may include the step of permanently removing the transducer, replacing the transducer with a new transducer, and/or repairing the transducer.", "According to this characterization, the method may further include the step of replacing the removed transducer with the repaired or new transducer.", "Those skilled in the art will appreciate numerous other examples of the basic principles of the present invention, namely, providing a connection between a pair of joint members to attach an implantable device to the a middle ear component of a patient.", "Furthermore, additional aspects, advantages and applications of the present invention will be apparent to those skilled in the art upon consideration of the following." ], [ "FIELD OF THE INVENTION The invention is related to the field of hearing aids, and in particular, to connections between implantable hearing aid transducers a component of the auditory system.", "BACKGROUND OF THE INVENTION Implantable hearing aids entail the subcutaneous positioning of some or all of various hearing augmentation componentry on or within a patient's skull, typically at locations proximate the mastoid process.", "Implantable hearing aids may be generally divided into two classes, semi-implantable and fully implantable.", "In a semi-implantable hearing aid, components such as a microphone, signal processor, and transmitter may be externally located to receive, process, and inductively transmit a processed audio signal to implanted components such as a receiver and transducer.", "In a fully-implantable hearing aid, typically all of the components, e.g., the microphone, signal processor, and transducer, are located subcutaneously.", "In either arrangement, a processed audio signal is provided to a transducer to stimulate a component of the auditory system By way of example, one type of implantable transducer includes an electromechanical transducer having a magnetic coil that drives a vibratory actuator.", "The actuator is positioned to mechanically stimulate the ossicles via physical engagement.", "(See e.g., U.S. Pat.", "No.", "5,702,342).", "In this regard, one or more bones of the ossicles are made to mechanically vibrate, causing the vibration to stimulate the cochlea through its natural input, the so-called oval window.", "An example of this transducer is included in the MET™ hearing aid of Otologics, LLC, in which a small electromechanical transducer is used to vibrate the incus (the 2nd of the 3 bones forming the ossicles), and thence produce the perception of sound.", "In this case, the vibratory actuator is coupled to the ossicles during mounting and positioning of the transducer within the patient.", "In one example, such coupling may occur via a small aperture formed in the incus bone.", "As will be appreciated, coupling with the ossicles poses numerous challenges.", "For instance, during positioning of the transducer, it is often difficult for an audiologist or surgeon to determine the extent of the coupling.", "In other words, how well the actuator is attached to the ossicles.", "Additionally, due to the size of the transducer relative to the ossicles, it is difficult to determine if loading exists between the ossicles and transducer.", "In this regard, precise control of the engagement between the actuator of the transducer and the ossicles is of critical importance as the axial vibrations can only be effectively communicated when an appropriate interface or load condition exists between the transducer and the ossicles.", "Overloading or biasing of the actuator can result in damage or degraded performance of the biological aspect (movement of the ossicles) as well as degraded performance of the mechanical aspect (movement of the vibratory member).", "Additionally, an underloaded transducer, e.g., where the actuator is not fully connected to the ossicles, may result in reduced performance of the transducer.", "Another difficulty with such coupling is that in some cases patients can experience a “drop-off” in hearing function after implantation.", "Such a drop off -may be caused by changes in the physical engagement of the actuator, e.g., due to things such as tissue growth, or may be caused by a malfunction of the transducer or other componentry.", "After implantation, however, it is difficult to readily assess the performance and/or adjust an implanted transducer and interconnected componentry.", "For example, in the event of a “drop-off” in hearing function after implantation, it is difficult to determine the cause, e.g., over/under loading of the interface due to tissue growth or some other problem with the hearing aid, without invasive and potentially unnecessary surgery.", "In addition, once coupled for an extended period, the maintenance and/or replacement with a next generation transducer may be difficult.", "SUMMARY OF THE INVENTION In view of the foregoing, a primary object of the present invention is to simplify and improve implantation procedures for implantable devices, such as hearing aid transducers.", "Another object of the present invention is to provide a means for achieving a low mechanical bias or no-load interface between transducers and a component of the auditory system, e.g., the ossicles.", "A related object of the present invention is to provide a connection apparatus with the ability to compensate in situ for undesirable interfaces, should one exist, between the middle ear component and a transducer, e.g., loading therebetween.", "Another object of the present invention is to provide a known geometry or uniform interface, on a middle ear component for making a connection with a transducer.", "In the context of the present invention, “in situ,” refers to in its proper position, e.g., in the context of the present transducer, as implanted in a patient and coupled to a middle ear component.", "One or more of the above objectives and additional advantages may be realized by a first aspect of the present invention, which provides a connection apparatus for coupling an implantable device, e.g., a hearing aid transducer, to a middle ear component.", "The connection apparatus includes a first joint member on the transducer, e.g., interconnected to a vibratory member of the transducer, and a second joint member connectable to the middle ear component, e.g., the ossicles.", "The connection apparatus further includes a means for coupling the first and second joint members during implantation of the transducer to provide a communication path between the coupled joint members for the transmission of mechanical energy from the transducer to the ossicles.", "In the context of the subject first aspect, the coupling of the first and second joint members may include an actual connection made therebetween or may include adjacent positioning of the joint members relative to each other to define the communication path.", "Various refinements exist of the features noted in relation to the subject first aspect of the present invention.", "Further features may also be incorporated in the subject first aspect of the present invention as well.", "These refinements and additional features may exist individually or in any combination.", "For instance according to a first feature, the second joint member may be configured to connect to the ossicles of the patient, in a substantially permanent manner.", "The permanent connection may be achieved through an artificial means such as an adhesive, or through bone growth or a fibrous tissue union naturally formed as a function of the body's response to the implantation.", "In some cases, an adhesive or other means may also be utilized to initially secure the joint member to the ossicles until such time as a natural connection via bone or tissue growth forms.", "According to another feature of the subject aspect, the first and second joint members may form a detachable connection.", "The detachable connection between the joint members, in combination with the permanent connection of the second joint member to the ossicles, provides a known geometry for connection and disconnection of a transducer, as well as reconnection of the transducer, such as for repair or replacement.", "In this regard, the second joint member is preferably of a minimal size that provides for a connection with the first joint member.", "This permits the second joint member to remain attached to the ossicles, even following a permanent removal of the transducer and first joint member, without affecting the auditory function.", "In other words, since the Second joint member is relatively small in comparison to a transducer, it is anticipated that the joint member may remain connected to the ossicles, even after permanent removal of the transducer, without substantial affects to the mechanical function of the auditory system.", "The connection between the joint members can be made in any manner as a matter of design choice to define the communication path for transmission of mechanical energy to the ossicles.", "For instance, in one aspect of the connection apparatus, one of the joint members may be a stud including a ball disposed on one end, while the other one of the joint members may be a receiver/pocket that forms a ball joint connection with the stud member.", "Advantageously, such a connection minimizes loading between the ossicles and the transducer by permitting rotational movement between the joint members.", "The ball joint connection may also be further engineered to permit additional compensational movements between the joint members to minimize loading.", "For instance, the receiver/pocket may be connected to the transducer or the ossicles via a stem member.", "The stem member, in turn, may be formed from a flexible material that permits non-permanent deformation in response to compressive forces between the transducer and ossicles.", "In another example according to this characterization, the stem and receiver/pocket may be defined by a pair of flexible structures that displace in a non-permanent manner in response to compressive forces between the transducer and ossicles.", "In another aspect of the connection apparatus, one of the joint members may be a stud member having a planer end for adjacent positioning relative to the other one of the joint members, which also includes a planer end.", "According to this characterization, the joint members are adjacently positionable during implantation to define the communication path.", "In another aspect of the connection apparatus, the joint members may be a pair of studs that are connectable in a detachable manner.", "For instance, in one example, a mechanical connection may be formed using a shape memory alloy to provide the detachable connection.", "In another aspect of the connection apparatus, the first joint member may be a stud member while the second joint member is a sleeve or conduit that is connectable to the ossicles, e.g., in an aperture/interface formed therein.", "According to this characterization, a tip of the stud member is insertable into the sleeve member to make the connection between the joint members.", "Although not necessary according to the present aspect, a material reshapeable in situ may also be disposed within the sleeve between the connected joint members to permit gradual movement of the joint members relative to each other to minimize loading.", "According to this characterization the reshapeable material may be a material that is resistive to high frequency movements to permit efficient acoustic stimulation of the ossicles, but gradually displaceable, e.g., in response to a steady state application of pressure by the ossicles, at body temperature to minimize loading.", "In this regard, the reshapeable material may also be utilized to secure the tip within the sleeve.", "In another example according to this aspect, an adhesive, or other means, such as a threaded connection may also be utilized to secure the tip within the sleeve as a matter of design choice.", "According to a second aspect of the present invention, a method for coupling an implantable device, e.g., a hearing aid transducer, to the ossicles of a patient is provided.", "The method includes the steps of providing a first joint member on the transducer, coupling a second joint member to a middle ear component, and forming a connection between the first and second joint members during implantation of the transducer to couple the transducer to the middle ear component.", "Various refinements exist of the features noted in relation to the subject second aspect of the present invention.", "Further features may also be incorporated into the subject second aspect of the present invention as well.", "These refinements and additional features may exist individually or in any combination.", "For instance according to a first feature of the subject second aspect, the connection between the joint members may be achieved in any manner as a matter of design choice that provides for acoustic stimulation of the middle ear component by the transducer.", "For example, the connection may be a detachable connection, a permanent connection, or an adjacent positioning as described above.", "According to this characterization, the method may further include the step of providing a stimulus representative of sound to the middle ear component over the connected first and second joint members.", "According to another feature of the subject second aspect, the method may include the step of exposing the connected first and second joint members to movement of the middle ear component, and automatically executing a compensating movement of the first and second joint members to reduce loading pressures.", "The step of executing the compensating movement may further include different steps according to the type of connection provided by the first and second joint members.", "For example, the compensating movement may include rotation of a ball relative to a receiver/pocket as described above.", "In another example, the compensating movement may include flexing a stem connected to the receiver/pocket or displacement of a reshapeable material between the interconnected joint members.", "In one aspect of the present method, the method may further include disconnecting the joint members and removing the transducer from the patient.", "The removing step may include the step of permanently removing the transducer, replacing the transducer with a new transducer, and/or repairing the transducer.", "According to this characterization, the method may further include the step of replacing the removed transducer with the repaired or new transducer.", "Those skilled in the art will appreciate numerous other examples of the basic principles of the present invention, namely, providing a connection between a pair of joint members to attach an implantable device to the a middle ear component of a patient.", "Furthermore, additional aspects, advantages and applications of the present invention will be apparent to those skilled in the art upon consideration of the following.", "BRIEF DESCRIPTION OF THE DRAWINGS FIGS.", "1 and 2 illustrate implantable and external componentry respectively, of a semi-implantable hearing aid system according to the present invention; FIG.", "3 illustrates one embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "4a illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "4b illustrates a flexible stem member of the connection apparatus of FIG.", "4a; FIG.", "5 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "6a illustrates interconnection of a pair of joint members for the connection apparatus of FIG.", "4a; FIG.", "6b illustrates an example of a means for disconnecting the interconnection between the joint member of FIG.", "6; FIG.", "7 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "8 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "9 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "10 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "11 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; FIG.", "12 illustrates another embodiment of a connection apparatus for connecting the implanted componentry of the hearing aid system of FIG.", "1 to the ossicles; and FIG.", "13 illustrates interconnection of a pair of joint members for the connection apparatus of FIG.", "12.DETAILED DESCRIPTION Reference will now be made to the accompanying drawings, which at least assist in illustrating the various pertinent features of the present invention.", "In this regard, the following description of a hearing aid device is presented for purposes of illustration and description.", "Furthermore, the description is not intended to limit the invention to the form disclosed herein.", "Consequently, variations and modifications commensurate with the following teachings, and skill and knowledge of the relevant art, are within the scope of the present invention.", "The embodiments described herein are further intended to explain the best modes known of practicing the invention and to enable others skilled in the art to utilize the invention in such, or other embodiments and with various modifications required by the particular application(s) or use(s) of the present invention.", "Hearing Aid System: FIGS.", "1 and 2 illustrate one application of the present invention.", "The illustrated application comprises a semi-implantable hearing aid system having implanted components shown in FIG.", "1, and external components shown in FIG.", "2.As will be appreciated, the present invention may be employed in conjunction with conventional hearing aids, semi-implantable hearing aids, and fully implantable hearing aids, and therefore the illustrated application is for purposes of illustration and not limitation.", "In the illustrated system, an implanted biocompatible housing 100 is located subcutaneously on a patient's skull.", "The housing 100 includes an RF signal receiver 118 (e.g., comprising a coil element) and a signal processor 104 (e.g., comprising processing circuitry and/or a microprocessor).", "As will become apparent from the following description, various processing logic and/or circuitry may also be included in the housing 100 as a matter of design choice.", "The signal processor 104 is electrically interconnected via wire 106 to an electromechanical transducer 108.Alternatively, however, the transducer 108 may be any type of transducer connectable to a component of the auditory system such as the ossicles 120.The transducer 108 is supportably connected to a positioning system 110, which in turn, is connected to a bone anchor 116 mounted within the patient's mastoid process (e.g., via a hole drilled through the skull).", "The transducer 108 includes a connection apparatus 112 for connecting the transducer 108 to the ossicles 120 of the patient.", "As will become apparent from the following description, the connection apparatus 112-includes first and second joint members that are connectable during implantation of the transducer 108 within the patient.", "In their connected state, the joint members of the connection apparatus 112 provide a communication path for acoustic stimulation of the ossicles 120, e.g., transmission of axial vibrations to the incus 122.Referring to FIG.", "2, the semi-implantable system further includes an external housing 200 comprising a microphone 208 and internally mounted speech signal processing (SSP) unit (not shown).", "The SSP unit is electrically interconnected to an RF signal transmitter 204 (e.g., comprising a coil element).", "The external housing 200 is configured for disposition behind the ear of the patient in alignment with the implanted housing 100.In this regard, the external transmitter 204 and implanted receiver 118 each include magnets, 206 and 102, respectively, to facilitate retentive juxtaposed positioning.", "During normal operation, acoustic signals are received at the microphone 208 and processed by the SSP unit within external housing 200.As will be appreciated, the SSP unit may utilize digital processing to provide frequency shaping, amplification, compression, and other signal conditioning, including conditioning based on patient-specific fitting parameters.", "In turn, the SSP unit provides RF signals to the transmitter 204.Such RF signals may comprise carrier and processed acoustic drive signal portions.", "The external transmitter 204 transcutaneously transmits the RF signals to the implanted receiver 118.As noted, the external transmitter 204 and implanted receiver 118 may each comprise coils for inductive coupling of signals therebetween.", "Upon receipt of the RF signals, the implanted signal processor 104 processes the signals (e.g., via envelope detection circuitry) to provide a processed drive signal via wire 202 to the transducer 108.The drive signals cause the transducer 108 to transmit axial vibrations at acoustic frequencies to the connection apparatus 112 to effect the desired sound sensation via mechanical stimulation of the incus 122 of the patient.", "FIG.", "3 illustrates a broad embodiment of the present connection apparatus 112, namely connection apparatus 300.The connection apparatus 300 is designed to provide a two-part connection between an implantable device, such as transducer 108, and the ossicles 120 of a patient.", "Specifically, the connection apparatus 300 includes a first joint member 302 and a second joint member 304 that are configured to form a mating connection during an implant procedure.", "In this regard, the joint member 302 extends from an end of the transducer 108 that is disposed towered the incus 122, and is operably interconnected to a driver of the transducer 108 such that it is axially vibratable in response to transducer drive signals.", "According to this characterization, the joint member 302 may be a separate structure that is connected to a vibratory member, e.g., vibratory member 112 of the transducer 108, or the vibrataroy member 112 may form the joint member 302.The joint member 304, on the other hand, is connectable to the ossicles 120.In one example, illustrated on FIG.", "3, the joint member 304 is connected to an interface 306 formed on the incus 122.According to this characterization, the interface 306 may be a shallow aperture formed in the incus 122 using an appropriate instrument, such as a laser or hand drill.", "Alternatively, it will be appreciated that the interface 306 may be of any nature that permits connection of the joint member 304 to the incus 122.It should be noted, that while not necessary according to the present connection apparatus 300, the joint member 304 is preferably designed for permanent attachment to the incus 122, e.g., through a mechanical couple.", "According to this characterization, the mechanical couple may be achieved by any appropriate method.", "For instance, in one example the mechanical couple may be achieved through the healing process following the implant, e.g., by bone growth or tissue growth between the joint member 304 and the interface aperture 306.Alternatively, it may be desirable to use another means such as an adhesive or surface discontinuity formed on the joint member 304 to make the connection with the incus 122.Furthermore, it may be desirable to us a combination of methods, such as the use of an adhesive during the implant procedure to temporarily secure the joint member 304 to the incus 122, until such time as the bone growth or tissue growth may form therebetween.", "As will be appreciated by the examples set forth below, the connection between the joint members, 302 and 304, may be made by numerous methods now known and later invented.", "In this regard, however, it is preferable, but not necessary, that the connection between the joint members, 302 and 304, be detachable to achieve the specific advantage of removability of the transducer 108.Further, in this regard, the two-part connection provides a number of other advantages related to the implantation of medical devices in patients.", "For instance, in the present example of the hearing aid transducer 108, the joint member 304 may be utilized as uniform or standard interface on middle ear implants.", "This provides the advantage of accommodating connection of multiple types of transducers to the incus 122.This also facilitates removal and repair of transducers as well as replacement of transducers with next generation transducers.", "It is also anticipated that the two-part configuration of the present connection apparatus will facilitate the initial implantation of the transducer 108 by allowing a surgeon to couple the joint member 304 to the incus 122, prior to mounting the transducer 108 within the skull.", "It is anticipated that the two-part connection will make it easer to properly position and affix a transducer, such as transducer 108 to the incus 122 following connection of the joint member 304.Furthermore, a detachable connection between the joint members, 302 and 304, provides the advantage of removability of the transducer 108, while reducing the potential for damage to the ossicles 120.In this regard, in the event it becomes desirable to permanently remove the transducer 108 from the patient, the joint member 304 may be left permanently attached to the incus 122 without substantially affecting the hearing function.", "In other words, because of its small size, which is on the order of approximately one (1) millimeter, the joint member 304 may be left connected to the incus 122 without affecting operation of the natural mechanical movements of the auditory system.", "Those skilled in the art will appreciate the numerous advantages this provides in relation to implantable devices.", "For instance, the transducer 108 may be removed for repair and or updating with the next generation of transducer technology.", "Further, the transducer 108 may be permanently removed from the patient with reduced risk of damage to the auditory system in the event that future technological advancements make the transducer 108 obsolete.", "According to the above principles, the joint members, 302 and 304, may be any two members that are connectable or positionable relative to each other in a manner that provides for acoustic stimulation of the ossicles 120, with one example being the transmission of axial vibrations representative of sound between the transducer 108 and the incus 122.For instance, as will become apparent from the following description, numerous examples of the joint members, 302 and 304, may be utilized as a matter of design choice, with the different examples each providing unique advantages in their own regard.", "Further, the choice of the type of joint formed by the joint members, 302 and 304, may even be a preference of the surgeon or audiologist performing the implant procedure.", "FIGS.", "4-11 illustrate numerous specific examples of the connection apparatus 300 according to the principles of the present invention.", "As mentioned, however, it is anticipated that numerous other examples of the connection apparatus 300 may be utilized according to the present principles.", "In addition, those skilled in the art will appreciate how the features described below can be combined to form numerous other examples of the present connection apparatus.", "Referring first to FIGS.", "4-6 there is shown one example of the connection apparatus 300, namely connection apparatus 400.The connection apparatus 400 includes a first joint member 402 and a second joint member 404 that form a detachable ball joint connection.", "In this regard, the joint member 404 includes a stud 410 appropriately sized for seating in the interface 306 formed in the incus 122.The stud 410 includes a ball 412 disposed on its distal end to form a detachable connection with the joint member 402.The ball 412 may be a separate structure that is attachable to the stud 410 through an adhesive, weld, solder connection, electrodeposition, or other appropriate biocompatible means.", "Alternatively, the ball 412 may be integrally formed on the distal end of the stud 410 as a single structure.", "Each of the above noted examples provides its own unique advantages and may be utilized as a matter of design choice.", "For instance the latter case provides the advantage of reducing failure of the connection between the ball 412 and the stud 410 following implantation in the patient.", "The separate structure, however, provides the advantage of allowing the ball 412 as well as the transducer 108 to be removed from the patient in the event it is ever desirable to permanently remove the transducer 108.The stud 410 is designed for insertion into the interface 306, such that a fibrous union or bone growth between the stud 410 and interface 306 forms a connection therebetween.", "To facilitate such connection, the stud 410 may be constructed from a ceramic or coated with a ceramic or other suitable material.", "Further, as mentioned above, an adhesive or other connection means, such as a surface discontinuity, may also be utilized during the implantation to facilitate connection of the stud 410 in the interface 306.Alternatively, the stud 410 may be formed from a material such as a shape memory metal that is configured to expand laterally within the interface 306, in response to a stimulus, to couple the stud 410 to the incus 122.In one preferred example, the stimulus may- be the body's temperature such that the coupling is activated upon placement of the stud 410 in the interface 306.It will be appreciated that such loading may be defined in direct relation to the shape memory attributes of the material comprising the stud 410.The ball 412 of the joint member 404 is designed to detachably couple with the joint member 402.In this regard, numerous configurations of the joint member 402 may be utilized to achieve the connection with the ball 412 as a matter of design choice.", "For instance, alternative examples of the joint member 402 are shown are shown in FIGS.", "4, 5, and 6B.", "According to one example of the connection apparatus 400, the joint member 402 includes a receiver 406 disposed on the end of a stem 408.The stem 408 is connected to a distal end 420 of the transducer 108, which as noted above, may be the vibratory member 112.Advantageously, this permits the retrofitting of conventional transducers with the connection apparatus 400 with minimal modification.", "The connection between the stem 408 and end 420 may be made by any appropriate means that permits the transmission of vibrations to the joint member 402 and ultimately the joint member 404.Some examples of the connection include without limitation, adhesives, a weld, electrodeposition, or other appropriate biocompatible connection.", "According to this characterization, the receiver 406 forms a pocket 414 to receive the ball 412, and form the ball Joint connection, illustrated in FIG.", "6a.", "In this regard, the receiver 406 may be constructed in the form of a spring type receiver where the ends, 416 and 418, expand outward as the ball 412 is inserted into the pocket 414, and snap inward around the ball 412 following insertion, to form the connection.", "The ball 412 is then retained in the pocket 414 via the inward pressure applied by the receiver ends, 416 and 418, but is also permitted to rotate relative to the receiver 406.Advantageously, this permits movement of the incus 122 in at least a first dimension, e.g., rotationally along arc (A) relative to the transducer 108.Such movement of the incus 122 may be caused by a variety of circumstances, most notably including swallowing, changes in barometric pressure, e.g., caused by changes in altitude of the patient, tissue growth, and/or swelling after the implantation surgery.", "The rotational movement along arc (A), in turn, reduces loading between the incus 122 and the transducer 108, as movement of the incus 122 is permitted relative to the fixed position of the transducer 108.The stem 408 may be any structure of sufficient rigidity to transmit vibrations to the receiver 406 and ultimately the interconnected joint member 404.For instance, the stem 408 may be a pin, a tube, a wire, etc.", "preferably formed from a biocompatible material including without limitation, titanium, a titanium alloy, platinum, a platinum alloy, or gold-plated stainless steel.", "In one example of the present connection apparatus 400, the stem 408 may be constructed such that it is flexible, as illustrated by the dashed line on FIG.", "4, in response to compressive forces, while at the same time being resistive to high frequency axial movements to permit efficient axial vibration transfer between the connected joint members 402 and 404.According to this characterization, in response to gradual movements of the incus 122 in a second dimension, e.g., in the direction of the transducer 108, the stem 408 is designed to flex to reduce loading between the incus 122 and transducer 108.Similarly, in response to gradual movements of the incus 122 away from the transducer 108, the ends, 416 and 418, of the receiver 406 are designed to expand apart allowing the ball 412 to also move away from the transducer 108 to reduce loading conditions.", "It will be appreciated that such movements of the incus 122 relative to the transducer 108 will inherently generate some loading.", "However, such loading may be reduced by the above design where the stem 408 and/or ends 416 and 418 are configured to flex under a low frequency load, e.g., substantially constant load.", "It will be appreciated that this load may be predetermined and the stem 408 and ends 416 and 418 designed, to permit low frequency compensating movement of the incus 122 but resist higher frequency vibrations for the transmission of sound to the incus 122.Referring to FIG.", "5, another example of the receiver, namely the receiver 500 is shown.", "In this embodiment, the stem 502 and receiver 500 are constructed from two separate structures, 504 and 506.As with the receiver 406, the receiver 500 is designed such that the structures, 504 and 506, are flexible to permit movement of the ossicles 120 relative to the transducer 108 to reduce loading pressures, while also being resistive to sudden axial movements to permit efficient acoustic stimulation of the joint member 404.In this characterization, if the incus 122 moves in the second dimension, e.g., in the direction of the transducer 108, the receiver 500 and stem structures, 504 and 506, are designed to separate or expand apart allowing the ball 412 to also move toward the transducer 108 and reduce loading pressures.", "Similarly, if the incus 122 moves away from the transducer 108, the ends, 416 and 418, of the receiver 500 expand apart allowing the ball 412 to also move away from the transducer 108 to reduce loading pressures.", "As with the above embodiment, the ball 412 and receiver 500 also permit angular movements of the incus 122, via rotation of the ball 412 within the receiver 500, along arc (A).", "Similarly as with the above embodiment, the structures, 504 and 506, are connected to the distal end 420 of the transducer 108 via any appropriate means, with some examples including without limitation, adhesives, welding, electrodeposition, or other appropriate biocompatible connection.", "In another example of the connection apparatus 400, one of the joint members, 402 and 404, may also include structure to facilitate detachment.", "For example, as shown in FIG.", "6A, the joint member 404 includes a pair of opposing tangs 600 and 602 that allow for the application of inward pressure relative to the stem 408 to open the receiver 406 and permit removal of the ball member 412.Advantageously, tangs 600 and 602 permit simple disconnection of the joint members 402 and 404 in a manner that reduces forces on the joint member 404 and incus 122.This in turn reduces the probability of damaging the connection, e.g., fibrous union etc., during disconnection and/or removal of the transducer 108 from the patient.", "Referring to FIG.", "7 there is shown another example of the connection apparatus 400, namely connection apparatus 700.The connection apparatus 700 is similar to the connection apparatus 400 in that it includes a first joint member 702 and a second joint member 704.In contrast, however, the joint members, 702 and 704, form an abutting or adjacent relationship relative to each other for the transmission of mechanical energy therebetween.", "In this regard, the joint members, 702 and 704, include mating planar surfaces, 706 and 708, that are adjacently disposed during implantation of the transducer 108, such that axial vibrations are transferred therebetween.", "As with the stud 410, the joint member 704 is designed for insertion into the interface 306, such that bone growth or a fibrous union between the joint member 704 and interface 306 is formed through tissue healing.", "Alternatively, however, the joint member 704 may be constructed from a material designed to expand laterally within the interface 306 as described above.", "The joint member 702, on the other hand, includes a stem 710, the length of which may be varied as a matter of design choice to facilitate the adjacent positioning the planar surfaces, 706 and 708.The stem 710 is connected to a distal end 420 of the transducer 108, which as noted above, may be the vibratory member 112, thereby providing the advantage of permitting retrofitting of conventional transducers with minimal modification.", "According to this characterization, the stem 710 may be a pin, a tube, a wire, etc.", "preferably formed from a biocompatible material including without limitation, titanium, a titanium alloy, platinum, a platinum alloy, or gold-plated stainless steel.", "In addition, as with the above -embodiment, the stem 710 may be constructed from a material or formed in a manner that permits flexing, as exemplified in FIG.", "4b, while at the same time being resistive to- high frequency axial movements to permit efficient acoustic stimulation of the incus 122.Thus, as with the above embodiment, in response to movement of the incus 122 in the second dimension, e.g., toward the transducer 108, the stem 710 may be reduce loading pressures on the incus 122.In another embodiment, illustrated in FIG.", "8, the planer surfaces, 706 and 708, may be coupled using a detachable coupler 800.In one example of this characterization, the coupler 800 may be constructed from a shape memory alloy, including without limitation, NiTinol (trade name for the standard alloy Nickel-Titanium).", "Such alloys are known for their ability to take on a predetermined shape in response to a stimulus, such as a temperature change, and return to their original shape upon removal of the stimulus.", "Specifically, shape memory alloys, such as NiTinol, undergo a phase transformation when cooled from their high temperature form, Austenite, to their low temperature form, Martensite.", "When such alloys are in the Martensite form, they are easily deformed to a new shape.", "When the alloy is heated, however, they recover their previous shape, hence the name shape memory alloys.", "Advantageously, for alloys such as NiTinol, the temperature at which the alloy returns to its original shape may be adjusted, typically between in the range of 100 degrees Celsius to negative 100 degrees Celsius.", "In yet another embodiment, the ends of the joint members 702 and 704 may be magnetized or formed from materials having a magnetic attraction to facilitate the juxtaposed positioning of the planer surfaces, 706 and 708.Similarly, other means such as an adhesive may also be utilized to facilitate the positioning of the planer surfaces, 706 and 708 as a matter of design choice.", "Referring to FIG.", "9 another example of the connection apparatus 300 is shown, namely connection apparatus 900.In this example, the connection apparatus 900 includes a first joint member 902 and a second joint member 904 that form a connection between the transducer 1.08 and the incus 122.In this example, the joint member 904 is in the form of a conduit or sleeve designed to receive a tip portion 908 of the joint member 902 to make the connection between the joint members 902 and 904.According to this characterization, the joint member 902 is dimensioned such that it may be recessed into the interface 306, such that it forms a sleeve within the interface 306 that provides a uniform interface for the receipt of the tip portion 908 of the joint member 902.As with the above examples, the joint member 904 may be constructed from a ceramic material to facilitate coupling with the interface 306.Alternatively, however, the joint member 904 may be constructed from a shape memory alloy that is conditioned for actuation at bodily temperatures so that the joint member 904 expands outward after surgical placement in the interface 306 to apply lateral loading and secure the same in the interface 306.Such loading may be defined in direct relation to the shape memory attributes of the material comprising the joint member 902.The joint member 902, on the other hand, includes an elongated stud member 906 extending axially from the distal end of the transducer 108.As with the stem 710, the length of the stud 906 may be varied as a matter of design choice to facilitate positioning of the transducer 108 and connection with the joint member 904.It will be appreciated that the joint member 902 may be the transducer vibratory member 112 without modification, to realize the advantages of the present connection apparatus.", "The joint member 902 may be received in the sleeve 904 without additional coupling therebetween.", "Alternatively, however, an adhesive or other attachment means may be utilized to further secure connection between the joint members 902 and 904.In still yet another example, the tip portion 908 of the joint member 902 may be formed from a shape memory metal, as described above, such that it expands outward after surgical placement in the joint member 904 to form a connection with the same.", "In another example of the connection apparatus 900, a reshapeable material 1000, may also be disposed within the sleeve portion of the joint member 904 prior to connection of the joint member 902 as illustrated in FIG.", "10.According to this characterization, the reshapeable material 1000 may serve the dual purpose of retaining the joint member 904 within the sleeve portion of the joint member 904, while permitting gradual movement of the incus 122 relative to the transducer 108 to reduce loading pressures.", "In other words, the reshapeable material 1000 permits the incus 122 to gradually move or positionally adjust relative to the transducer 108 while reducing loading pressures caused by such movements.", "Although it will be appreciated that numerous materials (currently in existence and that will be available in the future) exhibiting the above-described properties may be utilized, some examples of the reshapeable material described herein include without limitation, wax based materials, elastomer based materials, and/or silicon based materials.", "Referring to FIG.", "11, in another example of the connection apparatus 900, the joint members, 902 and 904, may include threads, 1100 and 1102 respectively, for making a threaded connection therebetween.", "In this regard, the threads 1100 may be connected to the threads 1102 by rotation of the entire transducer 108.Alternatively, the transducer 108 may be configured such that the joint member 902 is rotatable independent of the transducer 108 to make the threaded connection.", "In either case, the threaded connection provides the advantage of securing in a detachable manner the joint members 902 and 904.Yet, another advantage of this characterization is that the joint members, 902 and 904, may be threaded together prior to implantation of the transducer 108.Thereafter, the joint members, 902 and 904, are easily disconnectable, even after a fibrous union is formed between the joint member 904 and the interface 306 during the healing process.", "Referring to FIGS.", "12 and 13, another example of the connection apparatus 300 is shown, namely connection apparatus 1200.In this example, the connection apparatus 1200 includes a first joint member 1202 and a second joint member 1204 that form a detachable connection between the transducer 108 and incus 122.According to this characterization, the joint member 1204 is a stud connectable to the incus 122 according to any of the above-described methods.", "The joint member 1202, on the other hand, is in the form of a clamp designed for insertion over the stud 1204 to make the connection.", "In one example of the joint member 1202, the clamp may be a spring that is separated prior to being inserted over the stud 1204, such that it clamps down on the stud 1204, as shown in FIG.", "13, when it is released.", "In another example of the joint member 1202, the clamp may be crimped around the stud 1204 to make the connection of FIG.", "13.In yet another example of the joint member 1202, the clamp may be a shape memory metal, as discussed above, that automatically clamps down on the stud 1204 following insertion over the stud 1204.Those skilled in the art will appreciate variations of the above-described embodiments that fall within the scope of the invention.", "As a result, the invention is not limited to the specific examples and illustrations discussed above, but only by the following claims and their equivalents." ] ]

[ [ "Cell phone bungie cord", "The “Cell Phone Bungle” is a precision made and engineered device to allow the user to avoid damaging or losing their cell phone or other personal items.", "It is made of high quality velcro fasteners to secure the cell phone to the bungle and also secure the bungle to a belt loop, belt, gym bag, purse, etc.", "The bungle is made from vinyl coated memory cord which is considered to be one of the best on the market.", "The Velcro fastener which secures the bungle to the person of article has a chrome plated swivel fastener to avoid a tangled bungle cord.", "It is capable of holding up to one pound and maintaining its adhesive qualities from 0 to 110 degrees.", "This product has been thoroughly tested and inspected by an engineer to guarantee is quality and durability." ], [ "1.I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” is made of the best materials available to me at the time of manufacture.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle's” memory cord to be approximately 3.0″ in length while relaxed and up to 23.0″ in the extended “in use” position.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” to fit or attach to any small item, ie.", "beepers, flash lights, remote controls, hunting items, small tools, wallets.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will hold or secure items up to one pound.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will attach to belt loops, belts, gym bags, purse straps or others items used to secure personal belongings as long as less that 1.25″ in diameter.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will maintain its holding ability in temperatures from 0 degrees to 110 degrees.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” that the holding strap is made from Velcro 4.0, to 10″ in length.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will significantly cut the loss or breakage of the items it is used to protect.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” uses a swivel chrome fastener to prevent knotting of bungle cord.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will STOP THAT DROP creating a significant decline in the loss and damage of cell phones and other small personal items when used as directed.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” will significantly decline the loss and damage of cell phones and other small personal items when used during biking, recreation vehicles, equine events, fishing, boating, on the Job, construction work, motorcycling, etc.", "I, Stewart H. Kemp, do hereby claim that the “Cell Phone Bungle” can relieve the stress of constantly looking for your cell phone or possible loss or damage to your cell phone." ], [ "DRAWINGS Reference Numerals (attached) DETAILED DESCRIPTION OF THE INVENTION—FIGURES I-VI Figure.", "I—Extendable and retractable memory cord covered with multi vinyl coated tubing for memory positioning.", "The vinyl tubing's center is filled with two vinyl coated stranded copper wires to provide strength, size retention, endurance and longevity.", "The memory cord begins with a straight section of the described chord 3.75 to 7.75 inches long.", "This straight section is followed by a 2.375 to 4.625 inch section of extendable and retractable memory cord ending with another straight section of memory cord 0.5 to 1.365 inches in length.", "There is no splice or break in the before described memory cord in its entirety.", "Figure II—The invention is fitted on the 3.75 to 7.75 inch end of cord with two industrial grade rectangular pieces with rounded or square corners of hook and latch material commonly referred to as Velcro, measuring 1.125 to 1.625×1.75 to 2.375 inches.", "The two latch sides of the hook and latch material of attached together with permanent industrial pressure sensitive adhesive.", "The end of the memory referred to above must be inserted into the latch pads exactly 0.625″ before adhering (this 0.625 inch measurement is critical for the safe use of the invention with products chosen by owner).", "Figure III—A stainless steel pin measuring 1-inch long×2 mm in diameter must be placed 0.375 inches in sided the latch pad prior to adding memory cord end and adhering the latch together.", "This is added for consistent drop control and product strength and durability.", "Figure IV—A heavy-duty black colored staple is applied to the latch pad 0.125 inch below the stainless steel pin (figure III) while straddling the memory cord inside the latch pad.", "Figure V—The invention is delivered with two industrial grade rectangular pieces with rounded or square corners of hook and latch material commonly referred to as Velcro, measuring 1.125 to 1.625×1.75 to 2.375 inches.", "These two hook pads are to be attached to the back or the cell phone, other items of the nature as well as personal items.", "This will allow the memory cord to be attached to the item desired.", "Figure VI—The straight end of the memory cord measuring 0.5 to 1.365 inches in length is fitted with a chromed steel swivel head fastener 0.5 inches in length.", "It is attached with a pressure application tool.", "Figure VII—Prior to attached the above described rivet a doubled sided 0.5 inch wide strip of industrial grade hook and latch material 4.75 to 7.125 inches in length is slid onto the memory cord straight end measuring 0.5 to 1.365 inches in length through a 6 mm hole cut into the middle of the hook and latch strap." ] ]

[ [ "Digital conversation piece table", "The new art of my invention pertains to be able to leave messages on your table or any office desk, or at your cubicle.", "An example is like if your manager wants you in her office and she comes to your desk and writes a note on a piece of paper, she would not have to do that).", "She can type it in or leave a message by blocks or typing center that is a dual combination.", "It can be used in all office settings and work settings, and it can also be a novelty in your home for a conversation piece.", "It can be used with the tray inside and it opens up to leave message or type in message on the typing center with a box that is computerized to leave message and be able to use more than one letter." ], [ "1.I claim the Conversation Piece Digital Table will be a message center Where you can leave messages by touching buttons with the alphabet, and Numbers on the buttons and it will electronic on top of the table where Your message will appear.", "Their will be a tray that you can pull out and Leave messages manually on it.", "This tray or table can also be digital And you are able to leave a message on top of the table once typed in.", "Their will be 2 different concepts.", "Specifications: The table will be 36 by 36 mainly list it can be different Sizes and shapes.", "1.Their will be fluorescent lights at the bottom about 4 inches from the The bottom and lights on the side about 4 inches out.", "2.The lights can be different colors.", "3.The table will give artistic decorative effect.", "4.The design is changeable to give varying visual effects: 5.Uniqueness gives it a conversation piece appeal: 6.There are variations from transparent to translucent interest bodies Give optionally variable effects: 7.Although distinctively unique design display, the module may be used also in a conventional coffee table use; 8.Optional variations of nature provide an ongoing challenge to the Home owner or decorator; and can be made to blend in with various Styles of room furniture." ], [ "1.The table be at least 36 by 36 square or different sizes.", "Digital table conversation piece with the alphabet and numbers from 1 to 100 included.", "About 6 inches from the end of the table will be a typing center or you can use blocks for the alphabet and numbers also.", "The rest of the area will be the message area, when leaving a message.", "Their also can be flourescent lights at the bottom to light up the table whan a message is being left.", "DESCRIPTION OF INVENTION Title: Conversation Piece Digital 36 by 36 Made of plexiglass and foam Flourescent lights The alphabet and the numbers 1 through 100 Digital buttons to leave message To be able to move around alphabet and numbers Different colors (red, green, yellow, orange, etc.", "What the table does is plug in the wall and you are able to leave a message if you leave home and want to let someone know where you are or where you are going.", "It will be clear like a box square, any other shapes can be made also.", "It is for novelty, offices and places like pier one and art museums." ] ]

[ [ "Grooved crucible ceramic clay", "The nature of the technical disclosures of my invention is simple and unique.", "A ¼×0.0381 inches groove around a ceramic clay crucible is simple to adapt.", "I invented a mold which has the feature of ¼×0.0381 inches groove around, to make the casting of a grooved ceramic clay crucible feasible.", "This is new in the art of casting because the ceramic clay crucibles used in Goldsmith industry do not have a groove around them.", "The purpose of the groove is to enable anyone who is working in casting precious metals under excessive heat to handle the crucible which is used in the process more efficiently and securely.", "This groove would certainly improve the melting and casting procedures simply because the strap around the newly grooved crucible would be firmly fastened, thus preventing any spills, and at the same time protecting the worker." ], [ "1.A designed Groove for holding a strap around a ceramic clay crucible." ], [ "<SOH> BACKGROUND OF INVENTION <EOH>1.Field of Invention Technically eliminates the hazards of spilling hot precious melted metals.", "Thus giving protection and safety to the one who is handling the molding procedure and at the same time preserving precious melted metals directly in the mold “no waste”.", "2.Background Art Goldsmith industries which include molding had been much the same over the decades.", "Safety first and spilling precious melted metals are my concerns.", "My invention is to solve these problems which are facing those who work in the industry." ], [ "<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The advantages of the invention are: 1.Safety procedures are my primary concerns.", "2.Not wasting precious melted metals when put in a mold.", "3.Proper handling of the whole process.", "Brief description of the several views of the figures; listing of all figures by numbers (e.g.", "FIG.", "1A ) and with corresponding statements explaining what each figure depicts.", "1.FIG.", "1 —A side view of a grooved crucible.", "2.FIG.", "2 —Shows how the strap is fastened firmly around the groove of the crucible.", "3.FIG.", "3 —Illustration of the invention.", "4.FIG.", "4 —Application of the invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "BACKGROUND OF INVENTION 1.Field of Invention Technically eliminates the hazards of spilling hot precious melted metals.", "Thus giving protection and safety to the one who is handling the molding procedure and at the same time preserving precious melted metals directly in the mold “no waste”.", "2.Background Art Goldsmith industries which include molding had been much the same over the decades.", "Safety first and spilling precious melted metals are my concerns.", "My invention is to solve these problems which are facing those who work in the industry.", "BRIEF SUMMARY OF THE INVENTION The advantages of the invention are: 1.Safety procedures are my primary concerns.", "2.Not wasting precious melted metals when put in a mold.", "3.Proper handling of the whole process.", "Brief description of the several views of the figures; listing of all figures by numbers (e.g.", "FIG.", "1A) and with corresponding statements explaining what each figure depicts.", "1.FIG.", "1—A side view of a grooved crucible.", "2.FIG.", "2—Shows how the strap is fastened firmly around the groove of the crucible.", "3.FIG.", "3—Illustration of the invention.", "4.FIG.", "4—Application of the invention.", "DETAILED DESCRIPTION OF THE INVENTION A ceramic clay crucible is used in the molding process of melting precious metals, when excessive heat is directed at the metal by an oxygen gas torch, it is usually held by a strap around the crucible which in turn may slip “no matter how tightened it is”, mainly because of the excessive heat and consequently might fall and cause damage to the person who is handling it and at the same time spill the melted precious metals all over.", "To this end, my invention is to make a ¼×0.0381 inches groove around the crucible (see FIG.", "1A).", "Which will hold the strap around the crucible firmly, safely and properly, and make the molding procedure safe." ] ]

[ [ "2-3-disubstituted quinuclidiness as modulators of monoamine transporters and theraperutic and diagnostic methods based thereon", "The present invention relates to a class of compounds of formula (I) and (II): wherein R1 is hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra, penta substituted aryl or heteroaryl; COOR3; —(CH2)n-aryl; —COO—(CH2)nR3; —(CH2)n—COOR3; —C(O)R3; —C(O)NHR3; or an unsubstituted or substituted oxadiazole; and R2 is hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra, penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; onnthracene; or —(CH2)n-Ph; wherein the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C1-C5 alkyl, C1-C5 alkenyl, H, F, Cl, Br, I, —NO2, NHR, or —OR, R is C1-C7 alkyl; R3 is C1-C5 alkyl, C1-C5 alkenyl, benzyl, substituted aryl or heteroaryl; and n=1-7.", "These compounds are discovered, synthesized and confirmed as potent inhibitors of dopamine (DA), serotonin (5-HT), and norepinephrine inhibitors.", "These compounds are therefore particularly useful in the treatment conditions or diseases wherein modulation of the monoamine neurotransmitter system involving dopamine (DA), serotonin (5-HT), and norepinephrine plays a role." ], [ "1.A compound or a pharmaceutically acceptable salt thereof, wherein the compound is of formulae (I) or (II): wherein R1 is a hydrogen; linear or branched C1-C15 alkyl; C2-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; —(CH2)n-aryl; COOR3; —COO—(CH2)nR3; —(CH2)n—COOR3; —C(O)R3; —C(O)NHR3; or an unsubstituted or substituted oxadiazole; R2 is a hydrogen; linear or branched C1-C15 alkyl; C2-C15 alkenyl; C3-C10 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; anthracene; or —(CH2)n-Ph; and R3 is C1-C5alkyl, C2-C5 alkenyl, benzyl, substituted aryl or heteroaryl; and wherein R1 and R2 are independently selected; n=1-7, the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C1-C5 alkyl, C2-C5 alkenyl, H, F, Cl, Br, I, —NO2, NHR, or —OR, wherein R is C1-C7 alkyl.", "2.A compound according to claim 1, wherein the compound is of formula (I) and is selected from the group consisting of the (±)-; (+)- and (−) isomers.", "3.A method of preparing a compound according to claim 1, wherein the method comprises: (a) preparing a quinuclidinone having a first substituent under Mannich reaction conditions; (b) reacting the product of step (a) to add a second substituent to the quinuclidinone thereby producing the compound.", "4.The method of claim 3, further comprising (c) reducing the compound obtained in step (b) to produce a disubstituted quinuclidine of formula (I).", "5.The method of claim 4, further comprising chiral separation of the product of step (c) to obtain a compound of formula (I) in non-racemic enantiomer form.", "6.The method of claim 5, wherein the chiral separation produces a (+)- enantiomer or (−)- enantimer.", "7.A method of treatment of a condition or disease wherein dopamine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound according to claim 1.8.A method of treatment of a condition or disease wherein serotonin flow plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound according to claim 1.9.A method of treatment of a condition or disease wherein norepinephrine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment a compound according to claim 1.10.A method for the treatment of cocaine abuse in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.11.A method for the treatment of depression in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.12.A method for the treatment of anxiety in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.13.A method for the treatment of an eating disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.14.A method for the treatment of Parkinson's disease in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.15.A method for the treatment of Alcoholism in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.16.A method for the treatment of a neurological disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.17.A method for the treatment of chronic pain in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.18.A method for the treatment of obsessive compulsive disorder in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound according to claim 1.19.A compound according to claim 1, wherein the compound is 2-Butyl-3-phenylquinuclidine.", "20.A compound according to claim 1, wherein the compound is 2-Butyl-3-(4-methylphenyl)quinuclidine.", "21.A compound according to claim 1, wherein the compound is 2-Butyl-3-(4-chlorophenyl)quinuclidine 22.The compound of claim 19, wherein the compound is in substantially pure (+)- or (−)- form.", "23.The compound of claim 20, wherein the compound is in substantially pure (+)- or (−)- form.", "24.A compound according to claim 1, wherein the compound is compound 16 or compound 17 as shown in Table 2.25.The compound of claim 21 in substantially pure (+)- or (−)- form.", "26.A compound according to claim 1, wherein the compound is selected from the compounds listed in Table 2.27.A method of diagnosis of a condition wherein at least one of dopamine, serotonin and norepinephrine flow plays a role, the method comprising contacting a sample of body fluid with a compound according to claim 1, wherein the compound is labeled.", "28.The method of claim 27 wherein the compound is labeled with a radioactive agent.", "29.The method of claim 27, wherein the compound is labeled with a fluorescent agent.", "30.The method of claim 27, wherein the compound is labeled with an electromagnetic moiety.", "31.The method of claim 27, wherein the compound is conjugated to an antibody.", "32.A compound according to claim 1, wherein the compound is labeled with a label selected from the group consisting of a radioactive agent and a fluorescent agent.", "33.A method of treatment of a condition involving an antigen, wherein the method comprises administering to a subject a compound according to claim 1, wherein the compound is conjugated to an antibody that binds to the antigen.", "34.The method of claim 33, wherein the compound of claim 1 is labeled." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Related Applications This application is based on U.S.", "Provisional Application Ser.", "No.", "60/226,581, filed Aug. 21, 2000, the contents of which are hereby incorporated by reference in their entirety.", "2.Field of the Invention The present invention relates to discovery, synthesis and enantiomer separation of compounds 2,3-disubstituted quinuclidines as potent inhibitors for dopamine, serotonin and norepinephrine transporters and therapeutic uses of such compounds.", "3.Summary of the Related Art The specific reuptake of the monoamine neurotransmitters, dopamine (DA), serotonin (5-HT), and norepinephrine (NE) from the synaptic cleft is the primary physiological mechanism for the termination of monoaminergic neurotransmission.", "Blocking the uptake increases synaptic availability of the neurotransmitters, thereby potentiating the signal (Kitayama, S. Dohi, T. Jpn.", "Pharmacol.", "1996, 72, 195-208).", "This has been exploited to develop treatments for a large number of neurological disorders.", "The selective serotonin transporter (SERT) inhibitor, such as fluoxetine (Prozac) is used for the treatment of depression.", "The selective dopamine transporter (DAT) inhibitor, benzotropine, is used clinically for the treatment of Parkinson's disease.", "Other potent and selective DAT inhibitors such as RTI-113 and GBR 12909 are now in clinical trials for the treatment of cocaine abuse.", "Norepinephrine transporter (NET) inhibitors such as desipramine are effective in the treatment of depression.", "The present invention relates to a novel class of compounds, 2,3-disubstituted quinuclidines as potent inhibitors of dopamine, serotonin and norepinephrine transporters and their therapeutic use.", "Potent, long-duration DAT inhibitors with no or little abuse liability themselves can be used for the treatment of cocaine abuse.", "One aspect of the present invention can be used as novel therapeutic agents for the treatment of cocaine abuse.", "Cocaine abuse is one of the greatest concerns of the American public today, and has therefore become a focus of medical, social, and political debate.", "Cocaine is one of the most addictive substances known, and cocaine addicts may lose their ability to function at work or in interpersonal situations.", "Although cocaine potently inhibits the reuptake of both norepinephrine (NE) and serotonin (5-HT), many lines of evidence indicate that its ability to act as a reinforcer stems from its ability to inhibit the reuptake of dopamine (DA) into dopaminergic neurons.", "Cocaine exerts this effect via specific interaction with DA transporter (DAT) proteins (cocaine receptor) located on DA nerve terminals.", "This increase of dopaminergic transmission in the reward mediating brain mesolimbic system is the essence of the dopamine hypothesis for cocaine action.", "However, recent studies have shown that the simultaneous flow of dopamine, serotonin and norepinephrine plays an important role in the molecular mechanisms involved in addiction to cocaine.", "A common molecular aspect to the flow of dopamine, serotonin and norepinephrine involves monoamine transporters.", "Therefore, it would be greatly beneficial if a class of small molecule compounds could be identified or designed to modulate the activity of monoamine transporters, thereby simultaneously modulating the uptake of dopamine serotonin and norepinephrine by monoamine transporters.", "Such novel compounds and therapeutic and diagnostic methods based thereon will be greatly beneficial in the treatment of numerous neurological disorders.", "Of particular interest are lead compounds capable of antagonizing all or some of cocaine action." ], [ "<SOH> SUMMARY AND OBJECTS OF TILE INVENTION <EOH>It is an object of the invention to provide compounds which inhibit abnormal dopamine signaling in the synaptic space in neurons.", "It is another object of the invention to provide compounds which are antagonistic of cocaine.", "Another object of the invention is to provide a method for modulation of brain dopamine flow in a subject in need of such control.", "The method comprises administering to the subject a compound identified according to the above-described method.", "Yet another object of the invention is to provide a method of inhibiting cocaine action in a subject in need of such inhibition comprising administering to the subject a compound identified according to the method described above.", "A still further object of the invention is to provide a method of promoting dopamine reuptake action in a subject in need of such action comprising administering to said subject a compound identified according to the method described above.", "In one aspect, the invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is of formulae (I) or (II): wherein R 1 is a hydrogen; linear or branched C 1 -C 15 alkyl; C 1 -C 15 alkenyl; C 3 -C 6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; —(CH 2 ) n -aryl; COOR 3 ; —COO—(CH 2 ) n R 3 ; —(CH 2 ) n —COOR 3 ; —C(O)R 3 ; —C(O)NHR 3 ; or an unsubstituted or substituted oxadiazole; R 2 is a hydrogen; linear or branched C 1 -C 15 alkyl; C 1 -C 15 alkenyl; C 3 -C 6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; anthracene; or —(CH 2 ) n -Ph; and R 3 is C 1 -C 5 alkyl, C 1 -C 5 alkenyl, benzyl, substituted aryl or heteroaryl; and n=1-7; and wherein the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C 1 -C 5 alkyl, C 1 -C 5 alkenyl, H, F, Cl, Br, I, —NO 2 , NHR, or —OR, wherein R is C 1 -C 7 alkyl.", "The compounds of formula (I) are preferably prepared and isolated in an enantiomeric form selected from the group consisting of the (±)-; (+)- and ( − ) isomers.", "Another aspect of the invention provides a method of preparing a compound according to the invention, wherein the method comprises:(a) preparing a quinuclidinone having a first substituent under Mannich reaction conditions; and (b) reacting the product of step (a) to add a second substituent to the quinuclidinone thereby producing the compound.", "The method of the invention, further comprises (c) reducing the compound obtained in step (b) to produce a disubstituted quinuclidine of formula (I).", "The invention also provides a method of treatment of a condition or disease wherein dopamine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "The invention also provides a method of treatment of a condition or disease wherein serotonin flow plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "The invention also provides a method of treatment of a condition or disease wherein norepinephrine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "One particularly advantageous aspect of the invention provides a method for the treatment of cocaine abuse in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound of formulae (I) or (II) as described above.", "The compounds of the invention are greatly advantageous in the treatment of various neurological disorders that involve the dopamine, serotonin and/or norepinephrine monoamine transmitter reuptake.", "The compounds of the invention are particularly useful in the treatment of condition such as clinical depression, anxiety, Alcoholism, eating disorders and Parkinson's disease.", "The compounds of the invention are also useful in the treatment of chronic pain and obsessive compulsive disorders by modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to a subject a compound according of formulae (I) or (II).", "Preferred compounds according to the invention include 2-Butyl-3-phenylquinuclidine, preferably in substantially pure (±)- enantiomeric form, and 2-Butyl-3-(4-methylphenyl)quinuclidine, preferably in substantially pure (±)- or (+)- enantiomeric form.", "Other preferred compounds of the invention are listed in Table 2.In another aspect, the invention provides a method of diagnosis of a condition wherein modulation at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake plays a role, the method comprising contacting a sample of body fluid with a compound of formulae (I) or (II), wherein the compound is labeled.", "Preferred labeling agents include radioactive agents, fluorescent agents and labeling agents containing a traceable electromagnetic moiety." ], [ "BACKGROUND OF THE INVENTION 1.Related Applications This application is based on U.S.", "Provisional Application Ser.", "No.", "60/226,581, filed Aug. 21, 2000, the contents of which are hereby incorporated by reference in their entirety.", "2.Field of the Invention The present invention relates to discovery, synthesis and enantiomer separation of compounds 2,3-disubstituted quinuclidines as potent inhibitors for dopamine, serotonin and norepinephrine transporters and therapeutic uses of such compounds.", "3.Summary of the Related Art The specific reuptake of the monoamine neurotransmitters, dopamine (DA), serotonin (5-HT), and norepinephrine (NE) from the synaptic cleft is the primary physiological mechanism for the termination of monoaminergic neurotransmission.", "Blocking the uptake increases synaptic availability of the neurotransmitters, thereby potentiating the signal (Kitayama, S. Dohi, T. Jpn.", "Pharmacol.", "1996, 72, 195-208).", "This has been exploited to develop treatments for a large number of neurological disorders.", "The selective serotonin transporter (SERT) inhibitor, such as fluoxetine (Prozac) is used for the treatment of depression.", "The selective dopamine transporter (DAT) inhibitor, benzotropine, is used clinically for the treatment of Parkinson's disease.", "Other potent and selective DAT inhibitors such as RTI-113 and GBR 12909 are now in clinical trials for the treatment of cocaine abuse.", "Norepinephrine transporter (NET) inhibitors such as desipramine are effective in the treatment of depression.", "The present invention relates to a novel class of compounds, 2,3-disubstituted quinuclidines as potent inhibitors of dopamine, serotonin and norepinephrine transporters and their therapeutic use.", "Potent, long-duration DAT inhibitors with no or little abuse liability themselves can be used for the treatment of cocaine abuse.", "One aspect of the present invention can be used as novel therapeutic agents for the treatment of cocaine abuse.", "Cocaine abuse is one of the greatest concerns of the American public today, and has therefore become a focus of medical, social, and political debate.", "Cocaine is one of the most addictive substances known, and cocaine addicts may lose their ability to function at work or in interpersonal situations.", "Although cocaine potently inhibits the reuptake of both norepinephrine (NE) and serotonin (5-HT), many lines of evidence indicate that its ability to act as a reinforcer stems from its ability to inhibit the reuptake of dopamine (DA) into dopaminergic neurons.", "Cocaine exerts this effect via specific interaction with DA transporter (DAT) proteins (cocaine receptor) located on DA nerve terminals.", "This increase of dopaminergic transmission in the reward mediating brain mesolimbic system is the essence of the dopamine hypothesis for cocaine action.", "However, recent studies have shown that the simultaneous flow of dopamine, serotonin and norepinephrine plays an important role in the molecular mechanisms involved in addiction to cocaine.", "A common molecular aspect to the flow of dopamine, serotonin and norepinephrine involves monoamine transporters.", "Therefore, it would be greatly beneficial if a class of small molecule compounds could be identified or designed to modulate the activity of monoamine transporters, thereby simultaneously modulating the uptake of dopamine serotonin and norepinephrine by monoamine transporters.", "Such novel compounds and therapeutic and diagnostic methods based thereon will be greatly beneficial in the treatment of numerous neurological disorders.", "Of particular interest are lead compounds capable of antagonizing all or some of cocaine action.", "SUMMARY AND OBJECTS OF TILE INVENTION It is an object of the invention to provide compounds which inhibit abnormal dopamine signaling in the synaptic space in neurons.", "It is another object of the invention to provide compounds which are antagonistic of cocaine.", "Another object of the invention is to provide a method for modulation of brain dopamine flow in a subject in need of such control.", "The method comprises administering to the subject a compound identified according to the above-described method.", "Yet another object of the invention is to provide a method of inhibiting cocaine action in a subject in need of such inhibition comprising administering to the subject a compound identified according to the method described above.", "A still further object of the invention is to provide a method of promoting dopamine reuptake action in a subject in need of such action comprising administering to said subject a compound identified according to the method described above.", "In one aspect, the invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is of formulae (I) or (II): wherein R1 is a hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; —(CH2)n-aryl; COOR3; —COO—(CH2)nR3; —(CH2)n—COOR3; —C(O)R3; —C(O)NHR3; or an unsubstituted or substituted oxadiazole; R2 is a hydrogen; linear or branched C1-C15 alkyl; C1-C15 alkenyl; C3-C6 cycloalkyl; mono, di, tri, tetra or penta substituted aryl or heteroaryl; unsubstituted or substituted naphthyl; 1,3-Benzodioxole; fluorene; indole; isoquinoline; quinoline; pyridine; pyrimidine; anthracene; or —(CH2)n-Ph; and R3 is C1-C5alkyl, C1-C5 alkenyl, benzyl, substituted aryl or heteroaryl; and n=1-7; and wherein the heteroaryl comprises N, O, or S, the mono or multi substituents on the aryl or heteroaryl are independently C1-C5 alkyl, C1-C5 alkenyl, H, F, Cl, Br, I, —NO2, NHR, or —OR, wherein R is C1-C7 alkyl.", "The compounds of formula (I) are preferably prepared and isolated in an enantiomeric form selected from the group consisting of the (±)-; (+)- and (−) isomers.", "Another aspect of the invention provides a method of preparing a compound according to the invention, wherein the method comprises:(a) preparing a quinuclidinone having a first substituent under Mannich reaction conditions; and (b) reacting the product of step (a) to add a second substituent to the quinuclidinone thereby producing the compound.", "The method of the invention, further comprises (c) reducing the compound obtained in step (b) to produce a disubstituted quinuclidine of formula (I).", "The invention also provides a method of treatment of a condition or disease wherein dopamine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "The invention also provides a method of treatment of a condition or disease wherein serotonin flow plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "The invention also provides a method of treatment of a condition or disease wherein norepinephrine flow in the brain plays a role, wherein the method comprises administering to a subject in need of such treatment an effective amount of a compound of formulae (I) or (II) as described above.", "One particularly advantageous aspect of the invention provides a method for the treatment of cocaine abuse in a subject in need of such treatment, wherein the method comprises modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to said subject a compound of formulae (I) or (II) as described above.", "The compounds of the invention are greatly advantageous in the treatment of various neurological disorders that involve the dopamine, serotonin and/or norepinephrine monoamine transmitter reuptake.", "The compounds of the invention are particularly useful in the treatment of condition such as clinical depression, anxiety, Alcoholism, eating disorders and Parkinson's disease.", "The compounds of the invention are also useful in the treatment of chronic pain and obsessive compulsive disorders by modulating at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake by administering to a subject a compound according of formulae (I) or (II).", "Preferred compounds according to the invention include 2-Butyl-3-phenylquinuclidine, preferably in substantially pure (±)- enantiomeric form, and 2-Butyl-3-(4-methylphenyl)quinuclidine, preferably in substantially pure (±)- or (+)- enantiomeric form.", "Other preferred compounds of the invention are listed in Table 2.In another aspect, the invention provides a method of diagnosis of a condition wherein modulation at least one of dopamine, serotonin and norepinephrine monoamine transmitter reuptake plays a role, the method comprising contacting a sample of body fluid with a compound of formulae (I) or (II), wherein the compound is labeled.", "Preferred labeling agents include radioactive agents, fluorescent agents and labeling agents containing a traceable electromagnetic moiety.", "BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS Table 1 is representative monoamine transporter inhibitors of Formula (I) and their activity at the three monoamine transporter sites.", "Table 2 is representative monoamine transporter inhibitors of Formula (II) and their activity at the three monoamine transporter sites.", "FIG.", "1 is the chemical structures of cocaine, WIN 35065-2, the lead compound (3) and a potent cocaine analog.", "FIG.", "2.is the pharmacophore model used in 3D-database pharmacophore searching, which led to the identification of the lead compound 3.FIG.", "3 is the two possible overlaps between the lead compound 3 (green) and cocaine (yellow) using the three pharmacophore elements defined in FIG.", "2.FIG.", "4 is an alternative overlap between the lead compound 3 (green) and cocaine (1, yellow) using an augmented pharmacophore model.", "FIG.", "5 is the overlaps between the designed analog 12 (green) and cocaine (yellow) (A), and between 12 (green) and WIN 35065-2 (2, yellow) (B).", "FIG.", "6 is the X-ray structure of analog 13.FIG.", "7 shows scheme I which illustrates the synthesis route of compounds with general formulae (I) and (II).", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A lead compound according to the invention is a chemical compound selected for chemical modification to design analog compounds useful in the treatment of a given condition.", "The lead compound can be a known compound or a compound designed de novo.", "A pharmacophore according to the invention is a chemical motif including a number of binding elements.", "The elements are presumed to play a role in the activity of compounds to be identified as a lead compound.", "The pharmacophore will be defined by the chemical nature of the binding elements as well as the geometric arrangement of those elements.", "Basically, our invention is applicable to conditions or diseases where modulation of the monoamine neurotransmitter system involving dopamine (DA), serotonin (5-HT), and norepinephrine, may have beneficial effects or diseases where modulation of the monoamine neurotransmitter system involving dopamine (DA), serotonin (5-HT), and norepinephrine, may have beneficial effects.", "Examples of such conditions include depression anxiety alcoholism chronic pain eating disorder obsessive compulsive disorders cocaine abuse.", "The present invention includes compounds which are rationally designed to control dopamine flow in the brain.", "These compounds can be dopamine transporter inhibitors and/or cocaine antagonists.", "Rational design of the compounds of the present invention includes identifying a mechanism associated with dopamine flow in the brain.", "Information relating to the mechanism is then analyzed such that compound structures having possible activity in interfering with such a mechanism are formulated.", "In particular, structures are synthesized based on “building blocks”, wherein each building block has a feature potentially capable of interfering with a particular mechanism associated with dopamine flow, particularly, a mechanism mediated by dopamine transporter protein (DAT).", "Compounds having different building block combinations are then synthesized and their activity in relation to the identified mechanism tested.", "Such tests are conducted in vitro and/or in vivo.", "The information obtained through such tests is then incorporated in a new cycle of rational drug design.", "The design-synthesis-testing cycle is repeated until a candidate compound having the desired properties for a targeted therapy; e.g.", "dopamine flow control, is obtained.", "The candidate compound is then clinically tested.", "An approach for controlling dopamine flow in the brain for the treatment of cocaine addiction is to design cocaine antagonists which can affect dopamine uptake.", "More specifically, this approach is based on rationally designing compounds which are antagonists of cocaine which reduce or block cocaine binding to DAT.", "Preferably, antagonists are designed which reduce or block cell cocaine binding while leaving other aspects of dopamine transport unaffected.", "The designed antagonists should provide a basis for therapeutic protocols based on the selective control of dopamine transport and thereby control of synaptic signaling without disruption of the normal flow of dopamine in the brain.", "Although both cocaine and dopamine bind to the DAT, recent mutagenesis and pharmacokinetic studies provide evidence that dopamine and cocaine do not share an identical binding site on the DAT.", "Thus, one object of the present invention is to discover molecules that will compete with cocaine at its binding site, yet bind to the DAT in a manner that would not significantly inhibit the transport of dopamine.", "These molecules could potentially function as cocaine antagonists or as partial agonists if they bind in such a way that inhibition of dopamine uptake is incomplete.", "Such compounds would be useful to counter some of the adverse effects of cocaine in cases of cocaine overdose or help maintain patients in cocaine treatment program.", "Recent advances in molecular biology have identified the amino acid sequences of the DAT, but no experimental 3D structures have been obtained for the DAT.", "The lack of experimental structures makes it difficult to employ a structure-based design strategy for the discovery of DAT inhibitors as cocaine antagonists.", "On the other hand, a wealth of SAR data on cocaine analogs and other classes of dopamine transporter inhibitors are available.", "This makes it feasible to derive “putative 3D pharmacophore models”, defined as the representation of crucial chemical structural features and their 3D geometric relationships that are important for the biological activity of interest.", "With the pharmacophore models, one can search large chemical databases to discover compounds whose 3D structures meet the pharmacophore requirements.", "Using a lead compound identified according to the invention, a large number of DA inhibitors were designed and tested.", "Compounds have been identified which exhibit promising cocaine antagonism in our functional assay.", "Identification of a Pharmacophore for Rational Drug Design of Cocaine Antagonists In order to design a pharmacophore representing assumable key features in DAT inhibition, a number of functional groups shared by cocaine and its analog WIN-35065 have been considered.", "The chemical structures of cocaine and WIN-35065 are shown in FIG.", "1.Based on extensive analysis of structure-function relationships of cocaine and its analogs, three binding elements have been identified which are believed to play important roles in the binding and reuptake activities of cocaine and its analogs, (1) an aromatic system at the 3β-position of the tropane ring; (2) a 2β ester group or a small hydrophobic group at this position; and (3) a nitrogen at position 8.The nitrogen at position 8 may be replaced by an oxygen.", "The next step in formulating a pharmacophore based on the above binding elements is to determine the 3D geometric relationships of these binding elements in cocaine and its analogs and incorporating those relationships as geometric parameters, which will define the geometric requirements of the pharmacophore models.", "In order to determine the geometric parameters for the design of a pharmacophore directed to cocaine based compounds, conformational analysis was performed on cocaine and WIN-35065.The X-ray crystal structure of cocaine was used as a starting point for modeling cocaine.", "The initial structure of WIN 35065 was built by replacing the benzoyloxy group with a phenyl group using the QUANTA molecular modeling package.", "The structures of both compounds were minimized, and a systematic conformational search was performed, using the program QUANTA.", "The binding elements described above were represented by a nitrogen atom, a carbonyl oxygen, and an aromatic ring, respectively.", "In determining the geometric requirements of the pharmacophore, three distance parameters were defined: (i) the nitrogen and the oxygen; (ii) the distance between the nitrogen and the geometric center of the aromatic ring; and (iii) the distance between the oxygen and the geometric center of the aromatic ring.", "The ranges for these distance parameters were determined by generating conformational profiles of cocaine and WIN-35065.The ranges were centered around the distance between two binding elements in cocaine and WIN-3 5065 conformations of low energy.", "The conformational profiles were then processed to determine the limits of each range.", "FIG.", "2 shows the chemical structure and distance requirements of the pharmacophore employed in the identification of a lead compound for the design of compounds which can be useful in dopamine flow control, e.g., cocaine antagonists.", "The distance requirements obtained for the pharmacophore of FIG.", "2 are: (i) a distance (d1) between the nitrogen and the oxygen of from 2.2 Å to 4.5 Å; (ii) a distance (d2) between the nitrogen and the geometric center of the aromatic ring of from 5.0 Å to 7.0 Å; and (iii) a distance d3 between the oxygen and the geometric center of the aromatic ring of from 3.4 to 6.1 Å.", "This essentially covers the possible distance span between these atoms in cocaine and WIN 35065.Some margin was allowed for both the lowest distance value (2.6 Å) and largest distance value (4.2 Å).", "The limits of the distance ranges were selected in order to provide a fairly large distance tolerance.", "This stems from the consideration that while the identified lead compound should be based on the general structure of cocaine, for such lead compound to be useful in the design of cocaine antagonists the distance requirements of the pharmacophore should have sufficient flexibility such that compounds having diverse chemical structures can be identified.", "Such a broadly defined pharmacophore allows identification of compounds that not only effectively compete with cocaine binding to the DAT, but also may display different profiles by having a binding mode significantly different from that of cocaine and WIN-35065 compounds.", "3D-Database Pharmacophore Search of the NCI 3D-Databases.", "As discussed above, based upon the molecular modeling studies of cocaine (1) and its WIN analogs such as 2 (WIN 35065-2), the present invention relates to the development of molecules that are designed based in the pharmacophore model described above, which includes three chemical groups believed to play an important role in binding to the DAT.", "It should be noted, however, that since different classes of DAT inhibitors may bind to the DAT with a combination of common and unique binding elements, more than one pharmacophore model may be developed.", "Using the pharmacophore model shown in FIG.", "2, we have searched 3D-databases of approximately 500,000 compounds and identified over 1000 small molecules that met the chemical and 3D geometrical requirements specified in the pharmacophore model.", "To date, testing of 200 potential DAT inhibitors led to the discovery of more than 20 new classes of DAT inhibitors with micromolar to nanomolar potency as measured in [3H]mazindol binding and inhibition of DA reuptake (data not shown).", "Specifically, based on the pharmacophore model shown in FIG.", "2, the chemical structures of the 206,876 “open” compounds in the NCI 3D-database were analyzed with the program Chem-X.", "During the search process, a compound was first examined for the presence of the required binding elements, i.e., a secondary or a tertiary nitrogen, a carbonyl group, and an aromatic ring system.", "If the three binding elements are present in a compound retrieved from the database, the program then investigates whether the compound has a conformation that meets the geometric requirements of the pharmacophore.", "Compounds having at least one conformation that met the distance requirements of the pharmacophore were selected for further processing.", "Up to 3,000,000 conformations were examined for each compound containing the three binding elements which define the pharmacophore.", "Based on the pharmacophore model shown in FIG.", "2, a first group of compounds containing 4094 compounds, i.e., 2% of 206,876, was formed for further processing to identify a lead compound for rational drug design.", "The first step in processing the compounds in the first group involved pruning the first group by eliminating all compounds having a molecular weight greater than 1000.This is to focus the drug design on smaller compounds having a limited number of sites to be modified.", "The group of compounds was further pruned by eliminating compounds wherein the nitrogen atom in the pharmacophore is not capable of accepting a hydrogen bond; e.g., due to the chemical environment of the nitrogen atom in the compound.", "Finally, in order to provide a relatively small number of compounds without sacrificing the structural diversity of the group of compounds obtained through the above two pruning steps, the compounds in the pruned group were distributed in clusters according to structural similarity, each cluster providing a class of compounds represented by one compound which was selected for the next step, i.e., in vitro testing.", "Based on the pruning steps described above, of the 4094 compounds identified according to the pharmacophore requirements, 385 compounds were finally selected for testing in [3H]mazindol and [3H]DA reuptake assays.", "Screening of Compounds in [3H]Mazindol and [3H]DA Assays.", "In the first batch of screening, 70 compounds out of the 385 selected candidates were evaluated in the [3H]mazindol binding assay.", "Thirteen compounds displayed more than 50% inhibition at 10 μM in the [3H]mazindol binding assay.", "An additional 23 compounds showed an inhibitory activity of 30% to 50% at 10 μM and 8 more compounds had an inhibitory activity of 20% to 30% at 10 μM in the [311]mazindol binding assay.", "Overall, 63% of 70 (44/70) compounds showed significant activity at 10 μM in the [3H]mazindol assay.", "These results show that the pharmacophore model used in the 3D pharmacophore search was unexpectedly effective in identifying compounds with diverse chemical structures that can effectively compete with [3H]mazindol binding to the cocaine site on the DAT.", "The group of compounds having DAT binding activity were further tested for their ability to antagonize cocaine's inhibition of [3H]DA uptake.", "Four classes of compounds were found to display significant functional antagonism.", "In selecting lead compounds for rational drug design of novel molecules targeted at interfering with cocaine activity and DA reuptake, several approaches or strategies are adopted.", "One approach is based on the selection of a lead compound displaying relatively high (initial) binding affinity and inhibition of DA reuptake properties.", "The lead compound is then utilized in designing new molecules having binding affinity and DA reuptake properties that are significantly improved compared to the (initial) properties of the lead compound.", "This strategy requires that the lead compound display rather good starting or initial binding properties which are then significantly improved through rational drug design.", "A second approach which is the subject of the present application is centered on the selection of a lead compound based on the degree of variation between the chemical structure of the lead compound and that of cocaine or its analogue employed in formulating the pharmacophore.", "That is, a lead compound having a chemical structure that is significantly different from that of cocaine is selected for further drug design even if the compound does not have a binding properties indicating strong potential in antagonizing cocaine activity as long as the compound displays some activity as cocaine antagonist.", "While designing novel molecules based on this approach may require more extensive research, it is believed that designing molecules having core chemical structures or scaffolds that are vastly different from those of cocaine may provide novel molecules that more potent than those designed based on lead compounds having significant cocaine antagonistic properties but also have a chemical structure that is less dissimilar to that of cocaine or its analogues employed in formulating the pharmacophore utilized in identifying or designing the lead compound.", "The present invention is based on the selection of compound 3, the structure of which is shown on FIG.", "1, which represents a new class of DAT inhibitors with novel structural scaffold.", "Compound 3, which may be classified as 2,3-disubstituted quinuclidine was found to have Ki values of 7270 and 8910 nM in binding affinity and inhibition of DA reuptake, respectively, (Table 1).", "Despite its very weak activity, approximately 30-fold less potent than cocaine, the present invention is based on the hypothesis that Compound 3 may represent a promising lead in the design of a novel class of DAT inhibitors since it has a structural scaffold different from other classes of known DAT inhibitors.", "That is, the present invention is based on designing novel molecules having a chemical structure that includes the core scaffold structure of Compound 3 yet display vastly improved DAT binding affinity and DA uptake properties compared to those of Compound 3.The subject invention is based on the discovery that rationally designed 2,3-disubstituted quinuclidines provide a novel class of dopamine transporter inhibitors.", "As discussed below, molecules according to the present invention having a chemical structure including the core scaffold structure associated with Compound 3 have been synthesized and tested through pharmacological testing.", "The molecules of the invention provide a novel class of quinuclidines that are potent DAT inhibitors.", "Specifically, as discussed below, one quinuclidine compound designed, synthesized and tested according to the invention has shown, in its more active enantimeric form excellent DAT binding and DA reuptake properties as illustrated by Ki values of 14 and 32 nM in binding affinity and inhibition of DA reuptake, respectively.", "The lead compound 3 has two basic nitrogen atoms, one carbonyl group and two equivalent phenyl groups.", "Thus, two different overlaps are possible between Compound 3 and cocaine using the three pharmacophore elements defined in FIG.", "2, i.e.", "a tertiary nitrogen, a carbonyl group and a phenyl ring, as the reference points.", "It was found that lead compound 3 has a fairly good overlap with cocaine with respect to the three crucial pharmacophore elements.", "The lowest root-mean-square deviation (RMSD) values in these two different overlaps (FIGS.", "3 (A) and (B)) between low energy conformations of 3 and the X-ray structure of cocaine (1) are 1.12 and 0.95 Å, respectively, using the four reference points (an nitrogen atom, a carbonyl group and an aromatic ring center).", "Although lead Compound 3 and cocaine have fairly good overlap with respect to the pharmacophore elements defined in FIG.", "2, close examination of the two overlaps between Compound 3 and cocaine (FIG.", "3) showed that there is a large exclusion volume between these two molecules.", "While Compound 3 and cocaine have an overlapping volume of 159 and 174 Å3 with the superposition shown in FIGS.", "3 (A) and (B), respectively, they have an exclusion volume of 212 and 198 Å3, respectively.", "Van der Waals (steric) interaction is perhaps the single most important factor in determining the binding mode of a drug molecule to its receptor.", "Thus, two compounds binding to the same binding site with similar binding modes often have a minimal exclusion volume especially if the binding site is not on the receptor surface.", "Molecular modeling and mutagenesis analysis showed that the binding site of cocaine at the DAT is not located on a surface.", "Therefore, the two overlaps shown in FIG.", "3 may not represent the “true” binding mode of the lead Compound 3 in comparison to that of cocaine.", "In designing more potent molecules based on Compound 3, further overlap between Compound 3 and cocaine was explored.", "One avenue for designing novel molecules based on Compound 3 yet have additional overlap with cocaine is based on the observation that replacement of the ester group in cocaine at position 2 with small alkyl groups results in very potent DAT inhibitors.", "For example, Compound 4 with a butyl group at the 2□ position and a p-Cl-phenyl group at the 3□ position is a highly potent DAT inhibitor with a low nanomolar potency in binding affinity and inhibition of DA reuptake.", "It is believed that the carbonyl group defined in the pharmacophore model in FIG.", "2 can be modified to include alkyl groups.", "With this modified pharmacophore model, it is hypothesized that the small N,N-dimethylmethlyamino group of Compound 3 may mimic the ester group at the 2□ position of cocaine and the 2-hydroxyl-2,2-diphenylacetate group at position 3 may mimic the benzoate group at the 3□ position of cocaine.", "The lowest RMSD value obtained between the low energy conformations of the lead compound 3 and the X-ray structure of cocaine is 0.50 Å, using the nitrogen in the quinuclidine ring in Compound 3 and the nitrogen in the tropane ring in cocaine, and three corresponding atoms at position 2 in the quinuclidine ring and in the tropane ring, and an aromatic ring center in Compound 3 and in cocaine as the reference points.", "The overlap between lead Compound 3 and cocaine is shown in FIG.", "4.As can be seen, a nice overlap was found between these two molecules (FIG.", "4).", "The 2-hydroxy-2,2-diphenylacetate group at position 3 of the quinuclidine ring locates in the same region as the phenyl ester group at the 3□ position of cocaine, and the N,N-dimethylamino group at position 2 of the quinuclidine ring overlaps nicely with the methyl ester group at the 2□ position of cocaine.", "However, the 2-hydroxyl-2,2-diphenylacetate group at position 3 of the quinuclidine ring appeared to be too bulky for achieving optimal potency based upon the structure-activity relationships (SAR) of cocaine and its analogs.", "Indeed, molecular volume calculations showed that with the overlapping manner shown in FIG.", "4, Compound 3 and cocaine have an overlapping volume of 179 Å3 and an exclusion volume of 194 Å3.Although the exclusion volume is only slightly better than that shown in FIG.", "3, it was found that the bulky 2-hydroxyl-2,2-diphenylacetate group accounts for much of this exclusion volume.", "It was shown that in cocaine, replacement of its benzoate group at the 3□ position with a phenyl group resulted in compound 2 (WIN 35065-2) with a binding affinity 4-times better than cocaine at the DAT site.", "Thus, the bulky 2-hydroxy-2,2-diphenylacetate group at position 3 of the quinuclidine ring in 3 may be replaced with a simple phenyl group to improve the overlapping volume and consequently the activity.", "Since a small ester or a simple alkyl group at the 2□ position of cocaine is desirable for high affinity at the DAT site, the N,N-dimthylmethylamino group at position 2 of the quinuclidine ring in 3 may be replaced with a simple alkyl group for achieving potent activity at the DAT site.", "The two substituents at positions 2 and 3 of the quinuclidine ring can be in either trans or cis configurations.", "Molecular modeling showed that analogs with a cis-configuration have a better overlap with cocaine (1) and WIN 35065-2 (2).", "These analyses led to the design of Compound 12, which has a simple butyl group at position 2 and a phenyl group at position 3 with a cis configuration between them.", "A fairly good overlap was found between 12 and cocaine as depicted in FIG.", "5 (A) and the lowest RMSD value was 1.07 Å using the 5 reference points shown in FIG.", "5(A) with the low energy conformations of 12 and the X-ray structure of cocaine.", "Importantly, an excellent overlap was found between 12 and WIN 35065-2 (2), an analog more potent than cocaine, as depicted in FIG.", "4(B) and the lowest RMSD value was 0.30 Å between their low energy conformations using the 5 reference points shown in FIG.", "4(B) for superposition.", "Compound 12 and WIN 35065-2 (2) have an overlapping volume of 179 Å3 and an exclusion volume of 54 Å3, indicating an excellent overlap in terms of their overall shape.", "It is of interest to note that although the locations of the nitrogen atom in 12 and WIN 35065-2 (2) (FIG.", "4(B)) are within 0.1 Å, the orientations of the nitrogen lone pair in these two compounds differ by approximately 60°.", "A previous study indicated that the orientation of the nitrogen lone pair in cocaine and its analogs is important for their selectivity among the three monoamine transporters.", "Taken together, our molecular modeling results suggested that 12 should be a potent DAT inhibitor.", "Synthesis of 12 and other 2,3-disubstituted quinuclidines in racemic form was accomplished using a synthetic procedure as shown in Scheme I.", "Briefly, starting from 3-quinuclidinone (5), 2-methylene-3-quinuclidinone (6) was prepared by using Mannich reaction.", "Reaction of 5 with aq.", "dimethylamine and aq.", "formaldehyde in ethanol, water mixture at 70° C. gave the Mannich base, which on deamination under distillation gave compound 6 in 86% yield.", "Reaction of 6 with allylmagnesium bromide in the presence of CuI.Me2S and Me3SiCl at −78° C. furnished the conjugate addition product 7 in 47% yield along with the 1,2-addition product in 12% yield (structure not shown).", "Aryl Grignard addition was carried out using arylmagnesium bromide in THF at 0° C. to give compound 8, which was subsequently treated with a 1:1 mixture of EtOH and 6N HCl under reflux conditions to give the dehydrated compound 10.Reduction of the double bonds was carried out using standard hydrogenation conditions (Pd/C, H2, EtOH, 60 Psi) to provide compound 12 in near quantitative yield.", "Compound 12 was evaluated as a DAT inhibitor.", "Two intermediates 7 and 8 were also tested to obtain additional information about the SARs of this class of compounds.", "The Ki values of 12 in [3H] mazindol binding and inhibition of DA reuptake are 210 and 237 nM (Table 1), respectively, representing a 31- and 32-fold improvement over the lead compound (3), and is as potent as cocaine, thus confirming our designing strategy.", "Compound 7 did not show any measurable activity at 10 □M in inhibition of DA reuptake (Table 1), suggesting an important role of the phenyl group and/or a detrimental effect of the ketone group at position 3.Compound 8 had a Ki value of 31.2 □M (Table 1), 131-fold less potent than 12, suggesting a detrimental effect of the hydroxyl group at position 3 to the activity at the DAT site.", "Previous studies have shown that an additional substitution to the phenyl ring such as a p-methyl may further improve the potency.", "Thus, compound 13 with an additional p-methyl group should have an improved activity if it can adopt the similar low energy conformation of 12 as shown in FIG.", "4.Molecular modeling showed that 13 has an excellent overlap with WIN 35065-2 (2) and 12 with their low energy conformations.", "Compound 13 in racemic form was synthesized using the same procedure as for 12, as shown in Scheme I and evaluated as a DAT inhibitor.", "It was found that 13 has Ki values of 20 and 49 nM in binding affinity and inhibition of DA reuptake, respectively, representing 365- and 181-fold improvement over the lead compound 3, and 11- and 5-fold improvement over 13 in binding and uptake activities, respectively.", "To confirm the cis-configuration between substituents at positions 2 and 3 in 13 and the molecular modeling results, the X-ray structure of 13 was obtained (FIG.", "5).", "As can be seen, the butyl group at position 2 and the p-methylphenyl at position 3 indeed have the desired cis-configuration.", "Since the binding of cocaine to DAT is stereospecific, it was thus interesting to investigate the stereospecificity of compound 13 in binding to the DAT.", "The enantiomers (+)-13 and (−)-13 were obtained using a semi-preparative chiral HPLC column (Chirex 3018), in which chiral stationary phase (CPS) consists of (S)-Proline and (R)-1-α-Naphthylethylamine covalently bound to a γ-aminopropyl silanized silica gel, and hexane/CH2Cl2/EtOH-TFA (20-1) in 83/15/2 ratio as the eluent.", "The optical rotation of (+)-13 was found to be [α]D=+104 (c 0.5, acetone) and that of (−)-13 was [α]D=−104 (c 0.5, acetone).", "It was found that (−)-13 has Ki values of 14 and 32 nM, while (+)-13 has Ki values of 343 and 354 nM in binding affinity and inhibition of DA reuptake, respectively.", "Hence, (−)-13 is approximately 2-fold more potent than (±)-13 and is 11-fold more potent than its enantiomer (+)-13.In summary, we discovered a lead (3) through 3D-database pharmacophore searching, but its activity was approximately 30-fold less potent than cocaine in binding affinity and inhibition of DA reuptake.", "Molecular modeling-assisted, rational design and chemical modifications led to rapid optimization and the identification of (−)-13 with Ki values of 14 and 32 nM in binding affinity and inhibition of DA reuptake, respectively, representing 519- and 278-fold improvement in binding affinity and inhibition of DA reuptake over the lead compound (3).", "Compound (−)-13 is 17- and 9-times more potent than cocaine in binding affinity and inhibition of DA reuptake.", "Previously, a class of tricyclic tropane analogs (tropaquinuclidines) was designed based upon cocaine and was shown to be potent monoamine transporter inhibitors with activity toward the serotonin and/or norepinephrine transporter.", "Although the quinuclidine ring in 3 (the lead compound), 12 and 13 is imbedded in the tricyclic tropaquinuclidines, the 2,3-disubstituted quinuclidines reported here differ from tropaquinuclidines in their basic ring structures and substitution patterns.", "Preliminary evaluations also showed that 12 and 13 have activity toward the DAT site (data not shown).", "Thus, compound 12 and 13 represent a novel class of potent DAT inhibitors with a basic quinuclidine ring and 2,3-disubstitutents Chemistry: General Methods.", "THF was freshly distilled under nitrogen from sodium benzophenone.", "1H and 13C NMR spectra were obtained with a Varian Unity Inova instrument at 300 and 75.46 MHz, respectively.", "1H chemical shifts (δ) are reported in ppm downfield from internal TMS.", "13C chemical shifts are referenced to CDCl3 (central peak, δ=77.0 ppm).", "Melting points were determined in Pyrex capillaries with a Thomas-Hoover Unimelt apparatus and are uncorrected.", "Mass spectra were measured in the EI mode at an ionization potential of 70 eV.", "TLC was performed on Merck silica gel 60F254 glass plates; column chromatography was performed using Merck silica gel (60-200 mesh).", "The following abbreviations are used: THF=tetrahydrofuran; DCM=dichloromethane; ether=diethyl ether.", "2-Methylene-3-quinuclidinone (6): A solution of 3-quinuclidinone (5), (6.0 g, 48.0 mmol), 40% aqueous dimethylamine (10.0 mL, 72.0 mmol), 37% aqueous formaldehyde (6.0 mL, 72.0 mmol), 20.0 mL of ethanol and 8.0 mL of water was stirred at reflux for one hour, then at 70° C. for 17 hours and allowed to cool to room temperature.", "The solvents and excess reagents were evaporated in vacuo and the oily residue fractionally distilled to provide 5.7 g. (86%) of title compound as a light yellow oil, b. p. 91-92°/7 mm.", "1H NMR (300 MHz, CDCl3) δ 1.90-1.98 (4H, m), 2.51-2.55 (1H, narrow m), 2.87-2.98 (2H, m), 3.03-3.13 (2H, m), 5.18 (1H, s), 5.78 (1H, s); 13C NMR (CDCl3) δ 24.9, 40.3, 48.3, 113.3, 152.3, 204.1.Anal.", "(C7H11NO) C, H, N. 2-But-3-enylquinuclidin-3-one (7): To a solution of CuI.Me2S complex [prepared by the addition of Me2S (0.8 mL, 10.9 mmol) to CuI (1.4 g, 7.3 mmol) at 0° C.] in THF at −78° C. was added 1M solution of allylmagnesium bromide (9.5 mL) and HMPA (2.5 mL, 15.6 mmol) stirred for 20 min.", "To this, a mixture 2-methylene-3-quinuclidinone (6), (1.0 g, 7.3 mmol) and TMS-Cl (1.02 mL, 8.0 mmol) in THF was slowly added and stirred at the same temperature for 2 h., quenched with aq.", "NH4Cl solution.", "The organic layer separated and the aqueous layer extracted with ethyl acetate, and the combined organic layers were dried over Na2SO4 and evaporated to get the crude compound.", "This was purified by column chromatography using ether/acetone/triethylamine in 85:10:5 ratio to afford the title compound as a colorless oil (610 mg, 47%) 1H NMR (300 MHz, CDCl3) δ 1.46-1.59 (1H, m), 1.79-1.93 (5H, m), 2.05-2.23 (2H, m), 2.30-2.35 (1H, m), 2.71-3.11 (5H, two m), 4.90-5.02 (2H, m), 5.68-5.82 (1H, m); 13C NMR (CDCl3) δ 25.3, 26.0, 27.1, 30.4, 39.8, 40.6, 48.5, 68.8, 115.1, 137.5, 221.7; MS m/z (%) 179 (6), 110 (100); Anal.", "(C11H17NO.HCl) C, H, N. General Procedure for the Aryl Grignard addition: To the ketone in dry THF at 0° C. was added the appropriate Grignard reagent (1.1 eq).", "The mixture was stirred at the same temperature for 30 min, quenched with aq.", "NH4Cl, and extracted with ethyl acetate.", "The combined organic extracts were dried (Na2SO4) and concentrated under reduced pressure.", "The resulting crude compound was purified by column chromatography using ether/acetone/triethylamine as eluent to afford the following compounds: 2-But-3-enyl-3-phenylquinuclidin-3-ol (8): colorless thick syrup; yield 70%; 1H NMR (300 MHz, CDCl3) δ 1.34-1.46 (3H, m), 1.48-1.58 (1H, m), 1.81-1.94 (2H, m), 2.05-2.33 (4H, m), 2.64-2.75 (1H, m), 2.87 (2H, broad t, J=8.3 Hz), 3.10-3.20 (1H, m), 3.35-3.42 (1H, m), 4.98-5.07 (2H, m), 5.79-5.93 (1H, m), 7.30 (1H, d, J=7.3 Hz), 7.39 (2H, t, J=7.1 Hz), 7.58 (1H, d, J=7.5 Hz); 13C NMR (CDCl3) δ 21.8, 23.2, 26.1, 31.4, 35.6, 41.1, 48.8, 61.8, 75.1, 114.8, 126.0, 127.2, 128.2, 138.7, 146.2; MS m/z (%) 257 (12), 124 (100); Anal.", "(C17H23NO.HCl) C, H, N. 2-But-3-enyl-3-(4-methylphenyl)quinuclidin-3-ol (9): colorless syrup; yield 74%; 1H NMR (300 MHz, CDCl3) δ1.32-1.54 (4H, m), 1.78-1.89 (2H, m), 2.01-2.28 (4H, two m), 2.34 (3H, s), 2.62-2.71 (1H, m), 2.80-2.86 (2H, m), 3.06-3.17 (1H, m), 3.29-3.34 (1H, m), 4.94-5.05 (2H, m), 5.76-5.89 (1H, m), 7.16 (2H, d, J=8.6 Hz), 7.42 (2H, d, J=6.6 Hz); 13C NMR (CDCl3) δ 21.0, 22.0, 23.5, 26.2, 31.6, 35.8, 41.3, 49.0, 62.1, 75.1, 114.9, 126.1, 129.1, 136.9, 139.0, 143.5; Anal.", "(C18H25NO.HCl) C, H, N. General Procedure for the dehydration: To a solution of hydroxy compound in EtOH, 6N HCl was added, refluxed overnight and cooled to room temperature.", "The reaction mixture was neutralized by slow addition of solid NaHCO3 and extracted with ethyl acetate.", "The combined organic layers were washed with sat.", "NaCl solution, dried (Na2SO4) and concentrated to get the crude compound, which was purified by passing through a silica gel column using acetone/ether as eluent.", "2-But-3-enyl-2,3-didehydro-3-phenylquinuclidine (10): colorless syrup; yield 61%; 1H NMR (300 MHz, CDCl3) δ 1.62-1.79 (4H, m), 2.30-2.42 (4H, m), 2.64-2.73 (2H, m), 2.86-2.92 (1H, narrow m), 3.01-3.10 (2H, m), 4.95-5.08 (2H, m), 5.78-5.90 (1H, m), 7.24-7.29 (3H, m), 7.37 (2H, t, J=7.6 Hz); 13C NMR (CDCl3) δ 29.1, 30.8, 32.2, 38.8, 48.9, 114.5, 126.4, 127.6, 128.1, 138.4, 139.5, 140.2, 146.9; MS m/z (%) 239 (22), 82 (100), Anal.", "(C17H21N.HCl) C, H, N. 2-But-3-enyl-2,3-didehydro-3-(4-methylphenyl)quinuclidine (11): colorless syrup; yield 66%; 1H NMR (300 MHz, CDCl3) δ1.56-1.75 (4H, m), 2.28-2.38 (7H, m), 2.60-2.70 (2H, m), 2.81-2.86 (1H, m), 2.96-3.05 (2H, m), 4.90-5.05 (2H, m), 5.75-5.88 (1H, m), 7.18 (4H, s); 13C NMR (CDCl3) δ 21.2, 29.4, 31.2, 32.4, 34.0, 49.2, 114.6, 127.6, 129.0, 136.1, 136.9, 138.7, 140.1, 147.0; Anal.", "(C18H23N.HCl) C, H, N. General Procedure for the hydrogenation: A mixture of olefin and a catalytic amount of Pd/C in EtOH was hydrogenated under 60 psi of H2 at 25° C. for 24 h. The catalyst was filtered off, and the filtrate was concentrated to give the crude compound as a yellow syrup, which on purification by column chromatography with ether/triethylamine afforded the saturated compound as a colorless thick syrup in quantitative yield.", "2-Butyl-3-phenylquinuclidine (12): colorless syrup; 1H NMR (300 MHz, CDCl3) δ 0.80 (3H, t, J=7.1 Hz), 1.07-1.37 (6H, two m), 1.46-1.54 (1H, m), 1.70-1.76 (2H, m), 2.01-2.10 (2H, m), 2.67-2.78 (1H, m), 2.96-3.05 (1H, m), 3.09-3.29 (4H, m), 7.19-7.34 (5H, m), 13C NMR (CDCl3) δ 14.0, 22.3, 22.7, 26.8, 29.7, 30.2, 30.3, 40.7, 45.4, 49.4, 60.2, 125.5, 127.8, 128.9, 142.9; MS m/z (%) 243 (18), 42 (100); Anal.", "(C17H25N.HCl) C, H, N. 2-Butyl-3-(4-methylphenyl)quinuclidine (13): colorless syrup; 1H NMR (300 MHz, CDCl3) δ 0.77 (3H, t, J=6.8 Hz), 1.02-1.32 (6H, two m), 1.40-1.49 (1H, m), 1.65-1.72 (2H, m), 1.96-2.06 (2H, m), 2.32 (3H, s), 2.64-2.74 (1H, m), 2.89-3.23 (5H, two m), 7.06-7.14 (4H, m); 13C NMR (CDCl3) δ 14.2, 21.1, 22.5, 22.9, 27.2, 29.9, 30.4, 30.6, 40.9, 45.3, 49.7, 60.4, 128.8, 129.0, 135.1, 140.0; MS m/z (%) 257 (29), 42 (100); Anal.", "(C18H27N.HCl) C, H, N. HPLC Separation of (±)-13 The chiral HPLC was performed on a Shimadzu SCL-10A-VP system at a flow rate of 5 mL/min at room temperature and UV detection at 254 and 280 nm.", "The sample for injection was prepared by dissolving racemic compound (5 mg/mL) in mobile phase and the separation was carried out by injecting 30 μL on a 250×10 mm chiral column.", "Pharmacology: [3H]Mazindol Binding Binding assays were conducted as previously described.", "Briefly conventional P2 membrane pellets were prepared by differential centrifugation from rat striatum.", "The P2 pellet was resuspended in Krebs-Ringer-HEPES (KRH) buffer consisting of (in mM): NaCl (125), KCl (4.8), MgSO4 (1.2), CaCl2 (1.3), KH2PO4 (1.2), glucose (5.6), nialamide (0.01), and HEPES (25) (pH 7.4) and centrifuged again.", "Finally, the pellet was resuspended in 30 volumes of buffer, pelleted at 15,000×g and frozen at −80° C. until used.", "The striatal homogenates were thawed by resuspension in the buffer described above at 75-125 □g protein/ml and incubated with [3H]mazindol, with or without competing drugs, for 60 min in a 4° C. cold room.", "Non-specific binding was determined with 30 □M cocaine.", "The bound and free [3H]mazindol were separated by rapid vacuum filtration over Whatman GF/C filters, using a Brandel M24R cell harvester, followed by two washes with 5 ml of cold buffer.", "Radioactivity on the filters was then extracted by allowing to sit over night with 5 ml of scintillant.", "The vials were vortexed and counted.", "IC50 values were determined using the computer program LIGAND.", "Synaptosomal Uptake of [3H]DA The effect of candidate compounds in antagonizing dopamine high-affinity uptake was determined using a method previously employed.", "For [3H]DA uptake, freshly dissected rat striata were homogenized with a Teflon-glass pestle in ice-cold 0.32 M sucrose and centrifuged for 10 min at 1000×g.", "The supernatant was centrifuged at 17,500×g for 20 min.", "This P2 synaptosomal pellet was resuspended in 30 volumes of ice-cold modified KRH buffer.", "An aliquot of the synaptosomal suspension was preincubated with the buffer and drug for 30 min at 37° C., and uptake initiated by the addition of [3H]dopamine (5 nM, final conc.).", "After 5 min, uptake was terminated by adding 5 ml of cold buffer containing glucosamine as a substitute for NaCl and then finally by rapid vacuum filtration over GF-C glass fiber filters, followed by washing with two 5 ml volumes of ice-cold, sodium-free buffer.", "Radioactivity retained on the filters was determined by liquid scintillation spectrometry.", "Specific uptake is defined as that which is sensitive to inhibition by 30 □M cocaine.", "It is identical to that calculated by subtracting the mean of identical tubes incubated at 0° C. IC50 values were determined by a computer assisted, iterative fit to a four-parameter sigmoidal equation (ALLFIT).", "These values were then converted to Ki values according to the Cheng-Prusoff equation assuming classical competitive inhibition.", "Preincubation of the drug and synaptosomes at 37° C. for 30 min was used to approximate equilibrium conditions as necessary to satisfy the requirements of the Cheng-Prusoff equation.", "Molecular Modeling Studies In Vivo Testing of Compound 6 The techniques, procedures, materials and computer programs employed in the experiments discussed herein are extensively described in the article “Discovery of a novel dopamine transporter inhibitor as a potential cocaine antagonist through 3D-data base pharmacophore searching, structure activity relationships and molecular modeling studies”, Wang et al, submitted for publication in the Journal of Medicinal Chemistry.", "The contents of the article and the references cited therein are hereby incorporated by reference in their entirety.", "3D-Database Search The Chem-X program (version July 96), running on a Silicon Graphics Indigo2 R10000, was used to carry out 3D-database pharmacophore searching.", "This program has been used to build the NCI-3D database, and was successfully used to carry out 3D-database pharmacophore searching.", "The primary reason for choosing this program was its ability to generate and search multiple conformations for flexible compounds in the database.", "The problem of multiple conformations for flexible compounds was found to be of utmost importance m building and searching a 3D-database because flexible compounds may be able to adopt a number of different conformations depending on their environment.", "It is often difficult to know precisely which conformation is the biologically active one if a compound can adopt multiple conformations with little energy difference.", "The biologically active conformations may be different for the same compound when it binds to different receptors.", "Therefore, it was decided that the best way to handle this situation is to generate and search multiple conformations for flexible compounds.", "The ability of the Chem-X program to generate and search a large number of conformations for flexible compounds was found to be one key factor for our success in identifying a large number of structurally diverse lead compounds in several projects carried out so far.", "We have found that if only single conformations for flexible compounds are searched, many identified lead compounds would be missed.", "Therefore, multiple conformations for flexible compounds are necessary.", "However, for a flexible compound with more than 10 single bonds, using a step size of 60° in generating conformations, the total number of possible conformations will exceed 60 million.", "In practice, we set 3 million conformations as the maximum number to be examined for any single compound.", "The current version of the NCI 3D database was built using the July 94 version of the Chem-X program.", "It consists of 206,876 “open” compounds.", "Employing the Chem-X program, it is straightforward to search the NCI 3D-database of 206,876 “open” compounds for structures that meet the requirements specified in the pharmacophore models.", "The defined pharmacophore model was built into a pharmacophore query, which included all the specifications as described in the pharmacophore models, such as substructural requirements, and distance and distance ranges between these crucial pharmacophore components.", "The Chem-X program first checked if the compound has a carbonyl group, an aromatic ring, and a nitrogen attached to at least two carbon atoms and one more carbon or hydrogen.", "After a compound passes this sub-structural check, it was subjected to a conformational analysis.", "In this step, conformations were generated and evaluated with regard to geometric requirements specified in the pharmacophore query.", "Compounds, which have at least one conformation satisfying the geometric requirements, were considered as “hits”.", "“Hits” are only considered as potential candidates for biological testing.", "A number of additional criteria were used in the selection of compounds for biological evaluation in order to achieve maximum efficiency in the discovery of lead compounds.", "These criteria include simple chemical structure, small molecule, non-peptidic and chemical structure diversity.", "EXPERIMENTAL SECTION Molecular Modeling Conformational analysis was performed using the conformational analysis module in the QUANTA program.", "Generally, if a compound has fewer than five rotatable single bonds, the grid scan conformational search protocol was employed.", "In this protocol, each rotatable bond was systematically rotated to generate a starting conformation, which was subsequently minimized using the CHARMm program within QUANTA.", "If a compound has more than five rotatable bonds, a random sampling protocol was used to generate conformations.", "Up to 5000 conformations were generated and minimized.", "Energy minimization of each conformation was computed with 5000 iterations or until convergence, defined as an energy gradient of 0.001 kcal mol−1 Å−1 or less.", "An adopted basis Newton-Raphson algorithm, implemented in the CHARMm program, was used for energy minimization.", "A constant dielectric constant (equal to 1) was used throughout all the calculations.", "Upon the completion of conformation generation and energy minimization, the most stable conformation was identified (the global minimum in vacuum).", "It is noted, however, that the lowest energy conformation may not be the bio-active conformation, as was shown previously.", "For this reason, other low energy conformations, typically within 5 kcal/mol of the global minimum were identified.", "Cluster analysis was performed to determine the number of truly unique conformations (clusters), using the cluster analysis module available in the QUANTA program.", "These low energy conformational clusters together are likely to include the bio-active conformations for a compound.", "3D-Database Search The Chem-X program (version July 96), running on a Silicon Graphics Indigo2 R10000, was used to carry out 3D-database pharmacophore searching.", "The primary reason for choosing this program was its ability to generate and search multiple conformations for flexible compounds in the database.", "The problem of multiple conformations for flexible compounds was found to be important in building and searching a 3D-database because flexible compounds may be able to adopt a number of different conformations depending on their environment.", "It is often difficult to know precisely which conformation is the biologically active one if a compound can adopt multiple conformations with little energy difference.", "The biologically active conformations may be different for the same compound when it binds to different receptors.", "Therefore, it was decided that a best way to handle this was to generate and search multiple conformations for flexible compounds.", "The ability of the Chem-X program to generate and search a large number of conformations for flexible compounds was found to be one key factor for our success in identifying a large number of structurally novel, diverse lead compounds in several projects carried out so far.", "We have found that if only single conformations for flexible compounds are searched, many identified lead compounds would be missed.", "Therefore, multiple conformations for flexible compounds are necessary.", "However, for a flexible compound with more than 10 single bonds, using a step size of 60° in generating conformations, the total number of possible conformations will exceed 60 million.", "In practice, we set 3 million conformations as the maximum number to be examined for any single compound.", "Employing the Chem-X program, a total of 4094 compounds were identified as “hits”, i.e.", "compounds that meet the requirements specified in the pharmacophore model (FIG.", "1).", "A number of additional criteria were used in the selection of compounds for biological evaluation in order to achieve maximum efficiency in the discovery of lead compounds.", "These criteria include simple chemical structure, small molecular weight, non-peptidic and chemical structure diversity.", "Con Formational Analysis Conformational analysis was performed using the conformational analysis module in the QUANTA program.", "Generally, if a compound has fewer than five rotatable single bonds, the systematic grid conformational search protocol was employed.", "In this protocol, each rotatable bond was systematically rotated to generate a starting conformation, which was subsequently minimized using the CHARMm program within QUANTA.", "If a compound has more than five rotatable bonds, a random sampling protocol was used to generate conformations.", "Up to 5000 conformations were generated and minimized.", "Energy minimization of each conformation was computed with 5000 iterations or until convergence, defined as an energy gradient of 0.001 kcal mol−1 D−1 or less.", "An adopted basis Newton-Raphson (ABNR) algorithm, implemented in the CHARMm program, was used for energy minimization.", "A constant dielectric constant (equal to 1) was used throughout all the calculations.", "Upon the completion of conformation generation and energy minimization, the most stable conformation will be identified (the global minimum).", "It is noted, however, that the lowest energy conformation may not be the bio-active conformation, as was shown previously.", "For this reason, other low energy conformations, typically within 5 kcal/mol of the global minimum were identified.", "Cluster analysis was performed to determine the number of truly unique conformations (clusters), using the cluster analysis module available in the QUANTA program.", "These low energy conformational clusters together are likely to include the bio-active conformations for a compound.", "Synthesis of Lead Compound 3 and Its Analogs 1H NMR and 13C NMR spectra were obtained with a Varian Unity Inova instrument at 300 and 75.46 MHz, respectively.", "1H chemical shifts (δ) are reported in ppm downfield from internal TMS.", "'3C chemical shifts are referenced to CDCl3 (central peak, δ=77.0 ppm).", "NMR assignments were made with the help of COSY, DEPT, and HETCOR experiments.", "Melting points were determined in Pyrex capillaries with a Thomas-.Hoover Unimelt apparatus and are uncorrected.", "Mass spectra were measured in the El mode at an ionization potential of 70 eV.", "TLC was performed on Merck silica gel 60F254 glass plates; column chromatography was performed using Merck silica gel (60-200 mesh).", "The following abbreviations are used: THF=tetrahydrofuran; DCM=dichloromethane; CH3CN=acetonitrile; ether=diethyl ether.", "General Procedure for the Synthesis of Compounds 3.In Vitro [3HlMazindol Binding Assays.", "For binding assays, caudate nuclei were homogenized using a polytron in 0.32 M sucrose and centrifuged for 10 mm at 1000×g.", "The supernatant was resuspended in cold sucrose and centrifuged at 17,500×g for 20 mm.", "The pellet was resuspended in Krebs-Ringer-HEPES (KRH) buffer consisting of (in mM): NaCl (125), KCl (4.8), MgSO4 (1.2), CaCl2 (1.3), KH2PO4(1.2), glucose (5.6), nialamide (0.01), and HEPES (25) (pH 7.4) and centrifuged again.", "Finally, the pellet was resuspended in 30 volumes of buffer, pelleted at 15,000×g and frozen at −80° C. until used.", "The striatal homogenates were thawed by resuspension in the buffer described above at 75-125 μg protein/ml and incubated with [3H]mazindol, with or without competing drugs, for 60 mm in a 4° C. cold room.", "Non-specific binding was determined with 30 μM cocaine.", "The bound and free [3H]mazindol were separated by rapid vacuum filtration over Whatman GF/C filters, using a Brandel M24R cell harvester, followed by two washes with 5 ml of cold buffer.", "Radioactivity on the filters was then extracted by allowing to sit over night with 5 ml of scintillant.", "The vials were vortexed and counted.", "IC50 values were determined using the computer program LIGAND.", "Synaptosomal Uptake of [3H]Dopamine.", "The effect of candidate compounds in antagonizing dopamine high-affinity uptake was determined using a method previously employed.", "For [3H]DA uptake, dissected rat striata were homogenized with a Teflon-glass pestle in ice-cold 0.32 M sucrose and centrifuged for 10 mm at 1000×g.", "The supernatant was centrifuged at 17,500×g for 20 mm.", "This P2 synaptosomal pellet was resuspended in 30 volumes of ice-cold modified KRH buffer.", "An aliquot of the synaptosomal suspension was preincubated with the buffer and drug for 30 mm at 37° C., and uptake initiated by the addition of [3H]dopamine (5 nM, final conc.).", "After 5 mm, uptake was terminated by adding 5 ml of cold buffer containing glucosamine as a substitute for NaCl and then finally by rapid vacuum filtration over GF-C glass fiber filters, followed by washing with two 5 ml volumes of ice-cold, sodium-free buffer.", "Radioactivity retained on the filters was determined by liquid scintillation spectrometry.", "Specific uptake is defined as that which is sensitive to inhibition by 30 μM cocaine.", "It is identical to that calculated by subtracting the mean of identical tubes incubated at 0° C. [3H]5-HT and [3H]NE uptake were measured in an entirely analogous fashion using synaptosomes prepared from rat midbrain and parietal/occipital cortex, respectively.", "Also, specific uptake of [3H]5-HT and [3H]NE were defined in the presence of 10 uM fluoxetine and 1 uM desipramine, respectively.", "Functional Antagonism Assay First, the effects of approximate IC10 to IC50 concentrations of candidate of compounds on the inhibition of [3H]dopamine uptake by cocaine was determined.", "The IC50 value of cocaine in the presence of antagonist was then compared to the IC50 value of cocaine alone.", "Significant differences in 1C50 values were compared to theoretical IC50 values expected from models of “same site” antagonism.", "IC50 values greater than those expected for “same site” antagonism will be taken as evidence of functional antagonism.", "Compounds demonstration antagonism were tested at lower concentrations to determine their minimum effective concentration.", "This test was performed under the preincubation conditions described above to allow slowly equilibrating compounds to reach equilibrium.", "Further, any artifactual differences in Ki due to differences in temperature, buffer, etc.", "were negated in this assay as binding of cocaine and the putative antagonists to both the cocaine binding site and the transporter occurred under identical conditions.", "This insures that a right shift in the cocaine inhibition curve beyond what is expected for two drugs acting at the same site is a true measure of functional antagonism.", "In Vivo Testing Locomotor Activity Test The test compounds were tested for the locomotor effects using male Swiss Webster mice.", "The potencies and efficacies [not reported] of test compounds to stimulate motor activity were determined and compared with cocaine's effects.", "The mice were placed in acrylic chambers which in turn were placed inside the activity monitors (Truscan, Coulbourn Instruments, Columbus, Ohio) equipped with infrared light sensitive detectors mounted along two perpendicular walls.", "Following 1 hr of habituation to test environment, test compounds, saline or cocaine were injected i.p.", "in a volume of 1 ml/100 g body weight and immediately placed back in the activity monitors.", "The data was recorded for a minimum of two hours.", "Each dose was studied in a minimum of ten mice and each mouse was used only once.", "The dose-effect functions on horizontal distance were constructed after subtracting the saline control group response from the test compound response.", "The 30-min period responses were computed from the 2 hour data.", "The 30-mm period during which the maximal responses would occur will be used for plotting dose-response function.", "Data were analyzed using standard analysis of variance and linear regression techniques.", "ED50 values were determined from data using the linear ascending portion of the dose-effect curves.", "Therapeutic Applications Based on the results obtained with the compounds synthesized to date, it is projected that these compounds will have significant therapeutic applications.", "The 2,3-disubstituted quinuclidines as listed in Tables 1 and 2 were determined to be potent inhibitors for dopamine, serotonin and norepinephrine transporters.", "Furthermore, the selectivity of these compounds can be designed toward on particular monoamine transporter.", "Therefore, these compounds can be used as therapeutic agents for the treatments of a large number of neurological disorders, where blocking the uptake of the neurotransmitters and increasing the availability of the neurotransmitters can have beneficial effects.", "The uses of such agents are well established in the treatment of depression (Brokekkamp, C. L. E.; Leysen, D.; Peeters, B. W. M. M.; Pinder, R. M. J. Med.", "Chem.", "1995, 38, 4615-4633), anxiety (Frances, A.; Manning, D. Marin, D. Kocsis, J.; McKinney, K.; Hall, W.; Klein, M. Psychopharamacol.", "suppl.", "1992, 106, S82-S86), alcoholism (Kranzler, H. R.; Amine, H.; Modesto-Lowe, V.; Oncken, C. Pharmacol.", "Treat.", "Drug.", "Alcohol Depend.", "1999, 22, 401-423), chronic pain (Sullivan, M. J. Reesor, K.; Mikail, S.; Fisher, R. Pain, 1993, 52, 294), eating disorder (Peterson, C. B.; Mitchell, J. E. J. Clin.", "Psychiatry, 1999, 55, 685-697), obsessive compulsive disorders (Brody, A. L. Saxena, S.; Schwartz, J. M.; Stoessel, P. W.; Maidment, K.; Phelps, M. E. Baxter, L. R. Jr. Psychiatr.", "Research, 1998, 84, 1-6), cocaine abuse ((a).", "Singh, S. Chemistry, design, and structure-activity relationship of cocaine antagonists.", "Chemical Reviews, 2000, 100, 925-1024 ref 7 Smith, M. P.; Hoepping, A.; Johnson, K. M.; Trzcinska, M.; Kozikowski, A. P. Dopaminergic agents for the treatment of cocaine abuse.", "Drug Discovery Today, 1999, 7, 322-332), and Parkinson's disease.", "But the present invention is not limited to these areas.", "Basically, the present invention is applicable to a wide range of neurological disorder, conditions or diseases where modulation of the monoamine neurotransmitter system involving dopamine (DA), serotonin (5-HT), and norepinephrine, may have beneficial effects, according to well established art in these areas.", "TABLE 1 Representative monoamine transporter inhibitors of Formula (I) and their activity at the three monoamine transporter sites.", "Ki (nM) [3H] Mazin- DA Name dol Binding Reuptake NE SER R-cocaine 231 ± 22a 274 ± 20 108 ± 4 155 ± 0.4 (1) Lead 7270 ± 400 8910 ± 400 (3) 7 >10000 8 31200 ± 2620 (±)-12 210 ± 17 237 ± 7 136 ± 11 655 ± 21 (±)-13 20 ± 1 49 ± 1 62 ± 5 77 ± 7 (−)-13 14 ± 2 32 ± 2 15 ± 3 26 ± 3 (+)-13 343 ± 16 354 ± 1 164 ± 47 508 ± 22 aMean ± standard error or range of 2-3 experiments, each conducted ussing six concentrations of drug in triplicate.", "TABLE 2 Representative monoamine transporter inhibitors of Formula (II) and their activity at the three monoamine transporter sites.", "Mazindol binding DA NE SER Structure Ki (nM) Ki (nM) Ki (nM) Ki (nM) 14 260 (±) 4 461 (±) 18 163 (±) 16 2070 (±) 230 15 155 (±) 21 186 (±) 16 187 (±) 15 1266 (±) 158 16 14 (±) 1 32 (±) 5 47 (±) 2 74 (±) 2 17 30 (±) 1 57 (±) 4 73 (±) 2 312 (±) 10 The subject therapies will comprise administration of at least one compound or a pharmaceutically accepted salt thereof, according to the invention in an amount sufficient to elicit a therapeutic response, e.g., inhibition of cocaine activity and/or promotion of dopamine reuptake activity in the presence of cocaine.", "The compound may be administered by any pharmaceutically acceptable means, by either systemic or local administration.", "Suitable modes of administration include oral, dermal, e.g., via transdermal patch, inhalation, via infusion, intranasal, rectal, vaginal, topical, and parenteral (e.g., via intraperitoneal, intravenous, intramuscular, subcutaneous, injection).", "Typically, oral administration or administration via injection is preferred.", "The subject compounds may be administered in a single dosage or chronically dependent upon the particular disease, condition of patient, toxicity of compound, and whether this compound is being utilized alone or in combination with other therapies.", "Chronic or repeated administration will likely be preferred based on other chemotherapies.", "The subject compounds will be administered in a pharmaceutically acceptable formulation or composition.", "Examples of such formulations include injectable solutions, tablets, milk, or suspensions, creams, oil-in-water and water-in-oil emulsions, microcapsules and microvesicles.", "These compositions will comprise conventional pharmaceutical excipients and carriers typically used in drug formulations, e.g., water, saline solutions, such as phosphate buffered saline, buffers, and surfactants.", "The subject compounds may be free or entrapped in microcapsules, in colloidal drug delivery systems such as liposomes, microemulsions, and macroemulsions.", "Suitable materials and methods for preparing pharmaceutical formulations are disclosed in Remington's Pharmaceutical Chemistry, 16th Edition, (1980).", "Also, solid formulations containing the subject compounds, such as tablets, and capsule formulations, may be prepared.", "Suitable examples thereof include semipermeable materials of solid hydrophobic polymers containing the subject compound which may be in the form of shaped articles, e.g., films or microcapsules, as well as various other polymers and copolymers known in the art.", "The dosage effective amount of compounds according to the invention will vary depending upon factors including the particular compound, toxicity, and inhibitory activity, the condition treated, and whether the compound is administered alone or with other therapies.", "Typically a dosage effective amount will range from about 0.0001 mg/kg to 1500 mg/kg, more preferably 1 to 1000 mg/kg, more preferably from about 1 to 150 mg/kg of body weight, and most preferably about 5 to 50 mg/kg of body weight.", "The subjects treated will typically comprise mammals and most preferably will be human subjects, e.g., human cocaine addicts.", "The compounds of the invention may be used alone or in combination with other agents.", "Additionally, the compounds may be utilized with other types of treatments to provide combination therapies which may result in synergistic results.", "Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.", "Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.", "While the invention has been described in terms of preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the spirit thereof.", "Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof." ] ]

[ [ "Compact Sewage Secondary Treatment System", "A treatment tank (1) for the secondary treatment of sewage, for providing the processes of aeration, nitrification and denitrification, in a single structure, for which a single, small horsepower, effluent pump (26) is the only moving part.", "In the treatment tank (1), the sewage is subjected to two separate biological treatments, in two separate chambers under different conditions.", "One biological treatment is carried out under anoxic conditions in a pipe coil (3).", "Anoxic conditions are ensured by keeping the pipe coil (3) full at all times; the pipe coil axis is vertical, and the pump forces the fluid flow upwardly through the coil.", "The second biological treatment is carried out under aerobic conditions in a trickle down filter (8).", "In a preferred embodiment, a welded pipe coil (3) used both to provide the anoxic conditions and to provide a tank containing the trickle down filter (8).", "The secondary treatment tank is generally used as part of a raw sewage treatment system, which will include a recycle loop which ensures that even when there is no raw sewage entering the system there is always a flow of liquid through the treatment tank." ], [ "1.A secondary sewage treatment apparatus comprising in combination in sequence: (a) a sewage inflow means; (b) a circulating pump means; (c) a coil of pipe having an inlet and an outlet, the outlet being disposed vertically higher than the inlet; (d) a treatment tank containing a trickle down filter; (e) a collection means to receive the flow of sewage from the trickle down filter; and (f) an effluent outflow means; wherein: (i) the circulating pump means provides a flow of sewage from the sewage inflow means to the pipe coil inlet at a pressure sufficient to provide a flow of sewage at the pipe coil outlet; (ii) the pipe coil outlet is constructed and arranged to pass sewage to the trickle flow filter; (iii) the collection means is constructed and arranged to receive a flow of treated sewage from the trickle down filter; and (iv) the outflow means is constructed and arranged to receive the flow of treated sewage collected by the collection means.", "2.A secondary sewage treatment apparatus according to claim 1 wherein the pipe coil is substantially cylindrical with its axis vertical.", "3.A secondary sewage treatment apparatus according to claims 1 wherein the pipe coil is substantially cylindrical with its axis vertical, and the pipe coil is welded together to provide a cylindrical wall for the treatment tank.", "4.A secondary sewage treatment apparatus according to claim 1 or 2 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is nested inside the treatment tank.", "5.A secondary sewage treatment apparatus according to claim 1 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is nested inside the treatment tank.", "6.A secondary sewage treatment apparatus according to claim 1 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is wound around the outside of the treatment tank.", "7.A secondary sewage treatment apparatus according to claim 2 wherein the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is wound around the outside of the treatment tank.", "8.A secondary sewage treatment apparatus according to claim 1 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means.", "9.A secondary sewage treatment apparatus according to claim 3 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means.", "10.A secondary sewage treatment apparatus according to claim 1 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means.", "11.A secondary sewage treatment apparatus according to claim 3 wherein the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means.", "12.A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate at least one half of the flow of treated sewage.", "13.A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate at least one half of the flow of treated sewage.", "14.A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate about one half of the flow of treated sewage.", "15.A secondary sewage treatment apparatus according to claim 10 wherein the sewage recirculation means is constructed and arranged to recirculate about one half of the flow of treated sewage.", "16.A secondary sewage treatment apparatus according to claim 12 wherein the sewage inflow means includes a mixing tank where inflowing sewage is mixed with re-circulated treated sewage from the outflow means.", "17.A secondary sewage treatment apparatus according to claim 13 wherein the sewage inflow means includes a mixing tank where inflowing sewage is mixed with re-circulated treated sewage from the outflow means.", "18.A secondary sewage treatment apparatus according to claim 1 wherein the effluent outflow means further includes a tank with a hydraulic balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system.", "19.A secondary sewage treatment apparatus according to claim 3 wherein the effluent outflow means also includes a tank with a hydraulic balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system." ], [ "This invention relates to an apparatus for the secondary treatment of moderate flows of sewage effluent.", "It is suitable for the treatment of a sewage effluent flow derived from communities of thirty to one thousand homes.", "It is thus more particularly concerned with an apparatus useable in a communal sewage treatment system to treat sewage.", "In a communal sewage treatment system, the sewage from a number of dwellings or establishments, for example a small town or village, is treated in order to convert the raw sewage into a water effluent that can be safely disposed of into ground water or into a larger body of water such as a stream or lake.", "Communal sewage treatment systems are used in locations where it is not economically feasible to provide a conventional municipal sewage processing system.", "In comparison to the installation of individual septic tank and tile bed systems, a communal system is more economical in land usage, and also permits a higher building density, particularly in locations where wells are required because there is no municipal water supply system.", "The apparatus of this invention will be located to follow a conventional primary sewage treatment system, such as a septic tank, in which insoluble solids, oils and grease are separated from the raw sewage.", "The apparatus of this invention generally will be used as part of a sewage treatment system which will include tankage used to equalize the effluent flow into the treatment system, tankage used to settle out suspended solids after flowing through the treatment apparatus, and at least one pump unit.", "The treatment of secondary sewage generally requires the use of two process, which are generally applied in sequence to the sewage flow.", "Both processes rely on the presence of suitable bacteria.", "The principle process in secondary sewage treatment is the aeration of the secondary sewage in the presence of certain bacteria.", "This process results in nitrification of the effluent.", "There are several known communal sewage treatment systems that oxygenate the secondary sewage by bubbling air into it.", "This is an inefficient method of oxygenation.", "With the exception of so-called trickle filters, the only practicable secondary sewage treatment apparatus that is reasonably compact is a rotating biological contactor (RBC).", "An RBC consists essentially of horizontal tank and a series of discs carried on a horizontal shaft which are partially immersed in the sewage in the partially filled tank.", "The shaft is rotated slowly, thus promoting sewage aeration.", "RBC's have two disadvantages.", "First, the apparatus is both complex, expensive to install and expensive to operate, since it includes many parts which require constant attention and maintenance.", "It thus requires a significant level of skilled supervision.", "Second, the system is relatively inefficient since its ability to aerate the sewage is directly linked to the combined surface area of the series of discs; there are practical limits on just how large these can be and on how large the RBC unit as a whole can be.", "The other process is denitrification, or the reduction of total nitrogen, referred to as Total Kjeldahl Nitrogen.", "This is accomplished in an anoxic environment, so that bacteria, along with a supplied food source, will reduce the nitrites and nitrates present in the sewage, releasing free nitrogen gas.", "In the known treatment systems, denitrification is carried out by turning off the air supply, and stirring the liquid to encourage mixing.", "This is usually done in the same chamber as the oxygenation, with the result that the specific bacteria of the denitrification process, which are different from the oxygenation bacteria, are not allowed to concentrate.", "This invention seeks to provide a secondary sewage treatment apparatus which can be compact, and which can provide the conditions for oxygenation and denitrification separately and more or less independently of each other.", "The apparatus can also be configured to require only one pump to move the sewage flow through it; no other moving parts are required, this minimizing supervision and maintenance requirements.", "In the secondary sewage treatment apparatus of this invention, the sewage is subjected to two separate biological treatments in separate parts of the same apparatus, where the specific bacteria of each process are allowed to colonize and congregate, under separate anoxic and aerobic conditions.", "Aerobic conditions are obtained by the use of a trickle down filter, and anoxic conditions are obtained by pumping the sewage upwardly through a pipe coil, the axis of which is substantially vertical; during operation the coil is always full, thus excluding the presence of air.", "The apparatus of this invention simplifies the sewage treatment process, and does not require sophisticated control equipment.", "In a preferred embodiment, a welded pipe coil is used as the outer cylindrical wall of the trickle down filter unit.", "In practice, the treatment unit of this invention is used as part of a communal sewage treatment system, which will also include suitable tankage, pipe systems and pumps.", "Thus in a first embodiment this invention seeks to provide a secondary sewage treatment apparatus comprising in combination in sequence: (a) a sewage inflow means; (b) a circulating pump means; (c) a coil of pipe having an inlet and an outlet, the outlet being disposed vertically higher than the inlet; (d) a treatment tank containing a trickle down filter; (e) a collection means to receive the flow of sewage from the trickle down filter; and (f) an effluent outflow means; wherein: (i) the circulating pump means provides a flow of sewage from the sewage inflow means to the pipe coil inlet at a pressure sufficient to provide a flow of sewage at the pipe coil outlet; (ii) the pipe coil outlet is constructed and arranged to pass sewage to the trickle flow filter; (iii) the collection means is constructed and arranged to receive a flow of treated sewage from the trickle down filter; and (iv) the outflow means is constructed and arranged to receive the flow of treated sewage collected by the collection means.", "Preferably the pipe coil is substantially cylindrical with its axis vertical More preferably, the pipe coil is substantially cylindrical with its axis vertical, and the pipe coil is welded together to provide a cylindrical wall for the treatment tank.", "Alternatively, the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is nested inside the treatment tank.", "Alternatively, the treatment tank is substantially cylindrical with its axis vertical, and the pipe coil is wound around the outside of the treatment tank.", "Preferably, the sewage outflow means includes means to recirculate at least a proportion of the flow of treated sewage received from the collection means to the sewage inflow means.", "Preferably, the sewage inflow means includes a mixing tank where inflowing sewage is mixed with re-circulated treated sewage from the outflow means.", "Preferably, the effluent outflow means also includes a tank with a hydraulically balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system.", "The invention will now be described with reference to attached drawings in which: FIG.", "1 shows the main features of a preferred embodiment of the treatment tank; FIGS.", "2, and 3 show constructional details of the treatment tank of FIG.", "1; FIGS.", "4 and 5 show alternative constructions for other embodiments of the pipe coil and treatment tank; FIG.", "6 shows schematically a typical complete secondary treatment system incorporating the treatment tank of FIG.", "1; and FIG.", "7 shows a cross section of the tank mounting used in FIG.", "6.In this invention, the way in which the sewage is processed is determined primarily by the arrangement of the pipe coil and the treatment tank.", "The preferred embodiment for these components is shown in FIGS.", "1, 2 3 and 4.Referring first to FIGS.", "1, 2 and 3 the treatment tank shown generally at 1 has a substantially cylindrical wall 2.The cylindrical wall 2 comprises a square section tube 3 wound and welded into a helix to provide both the cylindrical tank wall 2 and the pipe coil.", "The helix is fabricated as a single unit, thus saving on both the space required and apparatus cost.", "Welded pipe coils of this type fabricated in polyethylene are available in several pipe sizes, overall diameters and overall pipe lengths under the trade mark Weholite from KWH Pipe Ltd., of Mississauga, Ontario, Canada.", "If desired, the pipe coil can be fabricated from a material other than polyethylene; polythene is preferred due to its known resistance to degradation over extended periods of time in the presence of sewage.", "At the bottom end of the pipe coil, the sewage inflow pipe 4 receives sewage from a circulating pump (not shown; see FIG.", "6) at a pressure sufficient to overcome the pressure head of the pipe coil 2.A suitable pressure tights seal is used between the inflow pipe 4 and the pipe coil 3 as at 4A.", "The anoxially treated sewage leaves the top end of the pipe coil 2 at 5 through the exit pipe 6, which is also sealed to the pipe coil as at 6A.", "Anoxic conditions are ensured within the pipe coil 3 by the upward flow of sewage which keeps the pipe coil 3 full of liquid at all times.", "The sewage flow from pipe 6 is distributed by a conventional distributor 7 over the top of the trickle down filter 8.The distributor 7 is a conventional perforated plate, which also is conveniently fabricated from polyethylene and welded as at 10 to the inside surface of the pipe coil 2.The trickle down filter 8 is supported by a grating 9, which is conveniently fabricated from fibre reinforced plastic, such as the material commonly known as fiberglass.", "The grating 9 is held in place by a support ring 11.If a polyethylene pipe coil is used, the support ring 12 is conveniently a polyethylene ring welded to the bottom of the polyethylene coil pipe 2 as at 12, 13.Several materials are available for the trickle filter 8; a suitable one is ACCU-PAK (trade mark), grade CF 1900 available from Brentwood Industries, of Reading, Pa., USA.", "As the sewage flows downwardly through the trickle filter, a suitable upward air flow is usually created through natural causes, thus ensuring aerobic conditions for the biological treatment step.", "If the naturally induced air flow is found to be insufficient, a blower means can be used to supplement the natural flow (see FIG.", "6).", "The distributor 9 and the grating 11 are both provided with a sufficient number and size of holes to allow the passage of sewage downwardly through the trickle down filter 8 and to allow a sufficient flow of air upwardly through the trickle down filter 8.The treatment tank 1 is designed and fabricated in such a way that the distributor plate 7 and the support ring 11 act to lock the trickle filter 8 and the grating 9 in place, allowing the whole unit to be laid on its side for shipping.", "It can thus be seen that the treatment tank itself can be fabricated from a single material which is unaffected by raw sewage, such as polyethylene or polyvinyl chloride (PVC), contains no moving parts and needs only a small amount of space.", "It is also contemplated within this invention that the pipe coil can be fabricated as a separate free standing unit, located near to the treatment tank, in an appropriate vertical position to ensure that anoxic conditions are maintained within the coil.", "This arrangement has the disadvantage of requiring approximately twice as much space as the unit of FIG.", "1.Alternatively, the pipe coil 3 can be fabricated separately, and either nested within the tank 2, as shown schematically in FIG.", "4, or wound around the outside of the tank 2, as shown schematically in FIG.", "5.FIG.", "6 shows schematically a typical complete secondary sewage treatment system incorporating the treatment tank of FIG.", "1.In FIG.", "6 the arrows indicate directions of flow within the system, and the line 14 indicates ground level around the system.", "Raw anaerobic sewage enters the system in pipe 20 from a primary treatment unit such as a conventional septic tank, which separates oil, grease, and insolubles such as grit from the raw sewage (not shown).", "The raw sewage enters a flow equalization tank 21.A first submersible effluent pump 22 pumps the raw sewage 23 at a constant rate through pipe 24 to a mixing tank 25.A second submersible pump 26 pumps mixed sewage 25 from the tank 25 to the treatment tank 1, which is constructed as shown in FIG.", "1.The submersible pump 26 develops sufficient pressure at the inlet 4 to the treatment tank 1 to overcome the hydraulic head within the pipe coil 3.The treatment tank 1 is contained within a suitable casing 28 such as a concrete silo, partly for safety and partly for weather protection.", "Inside the casing 28 the treatment tank 1 is supported by a set of benches 29 supported by the base 30 of the casing 28.A sloping floor 31 is provided within the casing 28 which serves to direct the flow of treated sewage from the grating 9 to the sewage outflow pipe 32.The pipe 32 delivers the treated sewage flow, which will also usually contain sloughed off bacterial debris from the trickle down filter 8, to a settling tank 33.The free space 34 around the treatment tank 1 normally ensures a sufficient flow of air through the trickle down filter 8.If it is found that the natural air flow is insufficient, additional air flow can be provided by a suitable blower 35 which feeds air into the casing 28 through the pipe 36 (both shown ghosted in FIG.", "7) into the casing 28.The treated sewage in pipe 32 enters a settling well 37 supported inside the settling 33.Inside the well 37 any biological debris, and any other solid matter in the treated sewage, settles to the bottom part 33A and is periodically removed by the scavenge pump 38.The solids free treated sewage has two pathways out of the settling tank 33; which is used depends upon the amount of raw sewage entering the treatment system in pipe 20.If the pump 22 in the flow equalization tank 21 is pumping raw sewage into the mixing tank 25, then treated sewage will leave the settling tank 33 through pipe 40 for final disposal.", "Treated sewage will also leave the settling tank 33 through pipe 41, through which it is returned to the mixing tank 25.Alternatively, if the pump 22 is not pumping raw sewage into the mixing tank 25, then the liquid level in the settling tank 33 will fall below the weir 39, and the only flow out of the settling tank 33 is through pipe 41 to the mixing tank 25.This arrangement has two advantages.", "First, when the pump 22 is feeding raw sewage, the settling tank acts a flow splitting device, ensuring that only a part of the treated sewage entering in pipe 32 is discharged in pipe 40, and the remainder is returned through pipe 41 to the mixing tank.", "This recycle loop ensures proper treatment of the incoming raw sewage.", "Second, when the pump 22 is not pumping raw sewage into the flow equalization tank, all of the treated sewage in pipe 32 entering the settling tank 33 is returned through pipe 41 and then by pump 26 to the treatment tank 1, thus ensuring that the pipe coil 3 is kept full of liquid, and the trickle down filter 8 is always kept wet.", "This ensures that the bacterial populations in the pipe coil 3 and on the trickle down filter 8 continue to thrive at all times.", "If required, in order to ensure that the correct flow rates are obtained in pipes 40 and 41, suitable valves 40A and 41A are included in pipes 40 and 41 respectively.", "It can thus be seen that the tank 33 together with its associated pipe connections provides a hydraulic balancing means constructed and arranged to maintain a minimum of sewage recirculating through the treatment tank, even when there is no flow of raw sewage into the treatment system.", "The ratio between the flow rates in pipes 40 and 41 is determined by the settings of pumps 22 and 26, and the setting of the control valves 42 and 43.In practice, it has been found that pumps 22 and 26 and valves 42 and 43 should be coordinated so that the flow at B in pipe 27 is at least approximately twice the flow at A in pipe 24.If the flow rate ratio A:B is less than approximately 1:2 then adequate treatment of the raw sewage will not necessarily be obtained.", "The ratio A:B can be as high as 1:4 if desired; it practice it appears that a ratio within the range of from 1:2 to 1:3 is generally sufficient.", "For most applications, a ratio of 1:2 appears to be adequate.", "Since the flow rate of the incoming raw sewage is rarely constant, it is convenient to provide float activated control switches 44, 45 and 46 in the equalization tank 21.Switch 44 is activates a high level alarm, indicating that the level in tank 21 is too high.", "There can be several reasons for this; for example if the raw sewage flow in pipe 20 is more than the system can handle, or if pump 22 has failed.", "Switches 45 and 46 act together: switch 45 turns on pump 22 when there is sufficient raw sewage in tank 21, and switch 46 turns off pump 22 when the liquid level in tank 21 falls below a preset minimum.", "Varying flow rates in pipe 20 are then accommodated by the level difference between switches 44 and 45.In practice, it has been found convenient to install the treatment tank 1 above the surrounding ground level indicated at 14, and the other three tanks 21, 25 and 33 below ground level.", "All of the tanks will also normally be vented as at 47, and provided with an access inspection cover as at 48.The manner in which these units are installed needs to take into account the thermal requirements of the microbiological colonies which are essential to the operation of the treatment tank 1.These normally only work well within a temperature range of from about 15° C. to about 50° C. It may thus be necessary to provide protection against ambient temperatures outside this range.", "It may also be necessary to provide for heating and/or cooling of the air flow through the trickle down filter 8.It has also been found advantageous to use electric submersible self priming pumps for the units 22, 26 and 38." ] ]

[ [ "Lighting system and method", "A lighting system and method (1) is disclosed having a pulse-width modulation (PWM) lighting system and method for use in environments where the PWM ratio is variable, for example though not exclusively, in 42V and/or dual-voltage electrical systems in automotive applications." ], [ "1.A lighting system for driving light bulbs having a lower voltage tolerance than the voltage supply to the system, comprising: a pulse wave modulator converter supplied with a required voltage from a system source voltage supply of the system for regulating an average output signal of the pulse wave modulator converter supplied to a register to selectively connect the output signal of the pulse wave modulator converter to a light bulb selected by the converter supply from a plurality of light bulbs, each light bulb having a lower voltage tolerance than the system voltage, the register and converter synchronised via a clock source, wherein the converter drives a single light bulb at any one time even with a variable PWM mark-space ratio.", "2.A lighting system as claimed in claim 1, wherein the system further comprises a light bulb driver connected between each light bulb and register.", "3.A lighting system as claimed in claim 1 or 2 wherein the pulse wave modulator comprises a comparator with one input receiving a required voltage, and another input receiving the output signal of the converter from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal.", "4.A lighting method for driving light bulbs having a lower voltage tolerance than the voltage supply to the system, comprising the steps or: supplying a required voltage from system source voltage from a system source voltage supply; regulating an average output signal from a pulse wave modulator converter supplied with the system source voltage from the source system supply; supplying the regulated average output signal to a register, the register and converter synchronised via a clock source; and selectively connecting the output signal of the pulse wave modulator converter from the register to a single light bulb at any one time from a plurality of light bulbs, even with a variable PWM mark-space ratio, wherein each light bulb having a lower voltage tolerance than the system voltage supply.", "5.A lighting method as claimed in claim 4, wherein the step of selectively connecting the output signal of the pulse wave modulator converter from the register to a single light bulb further comprises a light bulb driver connected between each light bulb and register.", "6.A lighting method as claimed in claim 4 or 5 wherein the step of regulating an average output signal from the pulse wave modulator converter further comprises comparing at a comparator in the pulse wave modulator converter one input receiving the required voltage, with another input receiving the output signal from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal.", "7.A lighting system substantially as hereinbefore described and with reference to the drawings.", "8.A lighting method substantially as hereinbefore described and with reference to the drawings.", "9.A lighting system as claimed in claim 2 wherein the pulse wave modulator comprises a comparator with one input receiving a required voltage, and another input receiving the output signal of the converter from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal.", "10.A lighting method as claimed in claim 5 wherein the step of regulating an average output signal from the pulse wave modulator converter further comprises comparing at a comparator in the pulse wave modulator converter one input receiving the required voltage, with another input receiving the output signal from a D-type flip-flop that receives the output signal of the comparator, feedback through a resistor and capacitor, for regulating the average output signal." ], [ "<SOH> BACKGROUND OF THE DISCLOSURE <EOH>Lighting of incandescent lamps or light bulbs in, for example, automotive applications typically involves a driving a 12V filament.", "However, developments in the automotive industry have been made to introduce vehicles with 42V electrical systems and dual voltage electrical systems.", "Vehicles with dual voltage electrical systems require two batteries having nominal voltages of 14V and 42V (12V and 36V rated batteries respectively).", "The 12V battery typically has a high amp-hour rating and is used to provide energy to 14V loads such as lighting circuits, which are difficult to implement at higher voltages, and other applications for example driving 12V resistive loads, like small heaters, and 12V inductive loads, like motors, relays, solenoids.", "The 36V battery typically has a high cranking current capability and is coupled to a 42V generator and higher voltage loads, which may include the engine starter motor.", "However, compared with single voltage electrical systems, having an additional power rail such as the 12V power rail in the dual-voltage electrical system increases cost, adds additional weight and reduces efficiency in the system.", "Vehicles with 42V electrical systems require a 36V rated battery that is coupled to a 42V generator, as in the dual voltage electrical system.", "Clearly, in 42V electrical systems all electrical systems, including the lighting system, must be driven by the 36V rated battery.", "There are currently several lighting arrangements for 42V and dual voltage electrical systems.", "A first lighting arrangement that is presently not very practical is to drive incandescent lamps with 42V filaments.", "However, filaments for 42V/36V are too long and fragile.", "The 42V/36V bulb filaments are relatively thin filaments when compared to filaments in 12V bulbs, for example, for the same power at 42V/36V filament of the same diameter would be 9 times the length, or alternatively ⅓ the diameter of a 12V filament.", "43V/36V bulbs are presently unpractical to use because the thin filaments results in a lifetime that would be unacceptably low in the automotive environment.", "Another lighting arrangement that has been proposed for 42V and dual voltage electrical systems is a DC-DC converter to step 42V down to 14V/12V.", "Although this system provides the convenience of 12V bulbs, it is an expensive solution to implement the DC-DC converter and such a system consumes, for example, more than 300 W. For these reasons, using a DC-DC converter is also presently not practical to implement in an automotive environment.", "There are also lighting systems that us PWM for driving lamps.", "Such a system is disclosed in SU909805.PWM systems typically operate at 120 Hz to replicate 50 to 60 Hz AC operation.", "However, especially where multiple lights are switched on together, very high peak currents, which may rupture the bulb filament, are reached in the wiring of the lighting system resulting in excessive heat and electromagnetic interference.", "In 42V applications, for example, when a bulb is first switched on a large current surge flows in a 42V/36V PWM system, which may be three times as large as 12V DC system, which may rupture the bulbs filament.", "For these reasons, such PWM lighting systems are unacceptable in an automotive environment.", "Therefore, there is a need in the art for a cost effective and power efficient way to power a bulb with a lower voltage tolerance, for example 12V or 6-8V, from a higher voltage supply, for example 42V/36V as used in automotive systems and/or 24V as used in truck systems, in an environment wherein the PWM mark-space ratio is variable such as for example in a 42V or dual-voltage electrical automotive systems." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>Embodiments of the invention will now be more fully described, by way of example, with reference to the drawings, of which: FIG.", "1 is a schematic block diagram of lighting apparatus according to a an embodiment of the invention; FIG.", "2 is a schematic block diagram of a lighting apparatus according to an embodiment of the invention; and FIG.", "3 is a graph charting the timing of signals according to an embodiment of the invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates generally to a lighting system and method.", "More specifically, the invention relates to a pulse-width modulation (PWM) lighting apparatus and method, for example though not exclusively, for use in 42V automotive applications wherein the PWM mark-space ratio is variable.", "BACKGROUND OF THE DISCLOSURE Lighting of incandescent lamps or light bulbs in, for example, automotive applications typically involves a driving a 12V filament.", "However, developments in the automotive industry have been made to introduce vehicles with 42V electrical systems and dual voltage electrical systems.", "Vehicles with dual voltage electrical systems require two batteries having nominal voltages of 14V and 42V (12V and 36V rated batteries respectively).", "The 12V battery typically has a high amp-hour rating and is used to provide energy to 14V loads such as lighting circuits, which are difficult to implement at higher voltages, and other applications for example driving 12V resistive loads, like small heaters, and 12V inductive loads, like motors, relays, solenoids.", "The 36V battery typically has a high cranking current capability and is coupled to a 42V generator and higher voltage loads, which may include the engine starter motor.", "However, compared with single voltage electrical systems, having an additional power rail such as the 12V power rail in the dual-voltage electrical system increases cost, adds additional weight and reduces efficiency in the system.", "Vehicles with 42V electrical systems require a 36V rated battery that is coupled to a 42V generator, as in the dual voltage electrical system.", "Clearly, in 42V electrical systems all electrical systems, including the lighting system, must be driven by the 36V rated battery.", "There are currently several lighting arrangements for 42V and dual voltage electrical systems.", "A first lighting arrangement that is presently not very practical is to drive incandescent lamps with 42V filaments.", "However, filaments for 42V/36V are too long and fragile.", "The 42V/36V bulb filaments are relatively thin filaments when compared to filaments in 12V bulbs, for example, for the same power at 42V/36V filament of the same diameter would be 9 times the length, or alternatively ⅓ the diameter of a 12V filament.", "43V/36V bulbs are presently unpractical to use because the thin filaments results in a lifetime that would be unacceptably low in the automotive environment.", "Another lighting arrangement that has been proposed for 42V and dual voltage electrical systems is a DC-DC converter to step 42V down to 14V/12V.", "Although this system provides the convenience of 12V bulbs, it is an expensive solution to implement the DC-DC converter and such a system consumes, for example, more than 300 W. For these reasons, using a DC-DC converter is also presently not practical to implement in an automotive environment.", "There are also lighting systems that us PWM for driving lamps.", "Such a system is disclosed in SU909805.PWM systems typically operate at 120 Hz to replicate 50 to 60 Hz AC operation.", "However, especially where multiple lights are switched on together, very high peak currents, which may rupture the bulb filament, are reached in the wiring of the lighting system resulting in excessive heat and electromagnetic interference.", "In 42V applications, for example, when a bulb is first switched on a large current surge flows in a 42V/36V PWM system, which may be three times as large as 12V DC system, which may rupture the bulbs filament.", "For these reasons, such PWM lighting systems are unacceptable in an automotive environment.", "Therefore, there is a need in the art for a cost effective and power efficient way to power a bulb with a lower voltage tolerance, for example 12V or 6-8V, from a higher voltage supply, for example 42V/36V as used in automotive systems and/or 24V as used in truck systems, in an environment wherein the PWM mark-space ratio is variable such as for example in a 42V or dual-voltage electrical automotive systems.", "STATEMENT OF THE INVENTION In accordance with the present invention there is provided a lighting system as claimed in claim 1, and a lighting method as claimed in claim 4.BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be more fully described, by way of example, with reference to the drawings, of which: FIG.", "1 is a schematic block diagram of lighting apparatus according to a an embodiment of the invention; FIG.", "2 is a schematic block diagram of a lighting apparatus according to an embodiment of the invention; and FIG.", "3 is a graph charting the timing of signals according to an embodiment of the invention.", "DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Referring to FIG.", "1, a lighting system and method 1 of an embodiment of the invention is shown.", "The lighting system 1 may power bulbs with a lower voltage tolerance, for example 12V, from a higher voltage supply, for example 42V, in an environment wherein the PWM mark-space ratio is variable such as for example in a 42V or dual-voltage electrical automotive systems.", "A clocked PWM converter 10 of the lighting system 1 is supplied a system source voltage or required voltage 9, such as 42V/36V DC power supply as used in 42V or dual-voltage electrical automotive systems.", "A clock source 11 supplies a clock signal 12 to the clocked PWM converter 10 and a register 13.The register 13 may be for example an eight-bit shift register.", "The clock source 11 may be for example, a basic 1 KHz oscillator with 1 ms period, prbs giving a random output frequency between 1-2 KHz with 1 ms period, a filter and digitised random or chaotic source giving 1-2 KHz with 1 ms period, and or three oscillators configured, such as 1 KHz, 1.2 KHz and 1.5 KHz that are connected together with AND gates to give a 1 KHz oscillation with variable period.", "The examples of oscillators other than the basic 1 KHz oscillator may generate less electromagnetic interference then the basic 1 KHz oscillator because as the energy is spread over a range of frequencies, rather than all appearing at one frequency and its harmonics.", "The clocked PWM converter 10 provides an output 25 to the register 13.The register 13 then selectively provides a lighting signal to a bulb driver 14-16 that provides a turning on/off signal to a respective bulb or lamp 17-19.Bulb driver 16 and bulb 19 are in dashed lines in FIG.", "1 to show that there may be more than one or two bulbs in the system 1.Of course, it will be appreciated that any type of bulb may be used, and any number of bulbs may be used on this system, from a single bulb to n number of bulbs.", "Optimal electromagnetic interference and low peak currents may be achieved with placement of the bulb loads on outputs based on probability of simultaneous operation and bulb wattage as well as operating voltage range.", "These optimum low levels may be achieved by minimising the current ripple on the 42V power input.", "Bulb driver 14 is also provided with bulb on/off input.", "The bulb drivers limit the inrush current to the bulbs to a value approximately equal to that when the bulbs are powered from the rated DC voltage.", "An example of the clocked PWM converter 10 is shown in FIG.", "2.A required voltage 21 is supplied to an input of comparator 22.The comparator 22 also receives a combined feedback signal in the converter circuit 10 from D-type flip-flop 24 that receives clock signal 12 from clock source 11, and resistor 26, and capacitor 28.On the D-type flip-flop 24, Q should switch between OV and the PWM supply voltage, for example 42V when the lighting system 1 is implemented in a 42V or dual-voltage electrical automotive systems.", "The values of resistor (R) 26 and capacitor (C) 28 are not critical, as long as R*C>> period of clock input 12, but not so large that the comparator output signal 23 becomes noisy.", "The timing diagram of signals in the PWM converter 10 circuit during operation is shown in FIG.", "3.The signal on the top of the diagram is the clock signal 12 generated by clock source 11.The inputs to the comparator, that is required voltage signal 21 and the voltage across capacitor 28 (Vc) signal 29, are shown are shown directly below the clock signal.", "PWM converter output signal 25 and comparator output signal 23 are respectively shown below the comparator input signals 21, 29.If Vc is less than Vreq, as at the start of FIG.", "3, Vc signal 29 increases.", "As soon as Vc signal 29 reaches Vreq 21 at point 30, the output at comparator 22 changes, for example the comparator output signal 23 goes to zero from one, as shown at point 31.At the next leading edge 32 of the clock signal 12, Vc reaches a maximum value at point 33, and the output 25 of the clocked PWM converter changes, for example the converter output signal 25 goes to one from zero, as shown at point 34.After point 33 the Vc signal 29 drops, and at point 35 the Vc signal 29 again reaches Vreq 21, and the comparator output 23 changes value again, for example from zero to one, as shown at point 36.Vc continues to drop from point 35 until the next leading edge 37 of clock signal 12.At this point Vc reaches minimum value, and the PWM converter output signal 25 changes value again, for example from zero to one, as shown at point 39.It will be appreciated that the signal response of the circuit will repeat, that is from point 38 Vc signal 29 will increase again until Vc reaches Vreq 21.With this configuration of lighting system 1, the clocked PWM converter 10 may regulate the average output voltage, so keeping the light output constant as the supply rail changes.", "The clocked PWM converter circuit 10 spreads the bulb switching times so, whenever possible, at most 1 bulb has power at any one instant.", "In this way, electromagnetic interference and resistive power loss are minimised.", "To illustrate this, I*I*R=power loss, (where I=current in bulb when run at 12V and R=resistance), in, for an example, a system with 8 bulbs that are switched on at the same time.", "In conventional systems, this would be worse case scenario because each bulb requires current.", "Thus the power in wiring in the conventional systems is (3*3*8*8*I*I*R)/9, i.e.", "“/9” as only on {fraction (1/9)} time=64*I*I*R. In contrast, in the lighting system 1 of the embodiments described, when the bulbs are switched one at a time, the power loss is 3*3*I*I*R*{fraction (8/9)}, which is 8*I*I*R, that equates to ⅛ the power loss as in the conventional systems.", "Similarly, electromagnetic interference is reduced by a similar ratio in the lighting system 1 of the embodiments described when compared to the conventional systems.", "Similar configurations and embodiments may also be implemented, for example rather than using only one clocked PWM converter 10 as shown, it will be appreciated that in another embodiment multiple clocked PWM converters may be used.", "With such a multiple clocked PWM converter configuration, with the clocks applied in the correct sequence, each bulb in a system having a plurality of bulbs may be individually dimmed via a control input to the converter.", "For example, such that the pulse widths on the bulb drivers 14-16 may be limited to a value below the maximum value, may allow individual bulbs 17-19 to be selectively dimmed.", "One example of achieving this is to combine the signal to the bulb drivers 14-16 from the register 13 with another PWM signal, for example the output of another circuit shown in FIG.", "2 where Vreq is a dimming input signal.", "In another embodiment, to keep the quiescent current low, the whole clocked PWM converter 10 circuit could be switched off, when for example no lights are required.", "However, care must be taken when a bulb is switched on that the circuit starts correctly.", "To ensure that the circuit starts correctly, for example, the outputs of the register 13 may be held reset until the first negative edge is seen on the output signal 25 of the clocked PWM converter 10.Although particular embodiments of the invention have been described above, various other modifications and improvements may be made by a person skilled in the art without departing from the scope of the present invention." ] ]

[ [ "Haemophilus influenzae lipopolysaccharide inner-core oligosaccharide epitopes as vaccines for the prevention of haemophilus influenzae infections", "The present invention relates to a lipopolysaccharide moiety comprising a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extension, and to vaccines obtaines therefrom which are cross-reactive for Haemophilus influenzae strains.", "The invention also relates to defined mutations in the biosynthetic machinery for lipopolysaccharide (LPS;) expression in Haemophilus influenzae useful to obtain the abovementioned moiety.", "The invention also relates to using conjugates of the LPS from the mutant strains so obtained to elicit a heterologous immune response against a wide range of disease-causing H. influenzae strains.", "More specifically, the invention relates to vaccines for prevention of bacterial infections comprising core lipopolysaccharide of Haemophilus influenzae.", "A lipopolysaccharide moiety comprising a conserved triheptosylinner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions." ], [ "1.A lipopolysaccharide moiety consisting essentially of a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions.", "2.A lipopolysaccharide moiety consisting essentially of a triheptosyl inner-core moiety of lipopolysaccharide having the structure I: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, and Kdo is 3-deoxy-D-manno-2-octulosonic acid.", "3.A lipopolysaccharide moiety consisting essentially of a triheptosyl inner-core moiety of lipopolysaccharide having the structure II: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, and P-Petn is pyrophosphoethanolamine.", "4-18.", "(canceled) 19.The lipopolysaccharide moiety of claim 2, wherein HepII is substituted at the 6 position by Petn or a functional equivalent.", "20.The lipopolysaccharide moiety of claim 2, substantially free of oligosaccharide chain extensions which are cross-reactive with mammalian tissue.", "21.The lipopolysaccharide moiety of claim 2, substantially free of oligosaccharide chain extensions which mimic human tissue antigens.", "22.The lipopolysaccharide moiety of claim 2, wherein an O-acyl is substituted at any position within the triheptosyl inner-core moiety thereof.", "23.An immunogenic composition for conferring protection in an animal host against a disease caused by Haemophilus influenzae, comprising the lipopolysaccharide moiety defined in claim 2.24.The immunogenic composition of claim 23 which is a conjugated vaccine.", "25.Use of at least one gene in a biosynthetic pathway for the production of lipopolysaccharide in Haemophilus influenzae to obtain a Haemophilus influenzae strain comprising the lipopolysaccharide moiety defined in claim 2.26.Use of at least one inmunogenic epitope to elicit a functional cross-reactive antibody against Haemophilus influenzae wherein the epitope comprises the lipopolysaccharide moiety defined in claim 2.27.The use of claim 26 wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae.", "28.A functional antibody which is cross-reactive against Haemophilus influenzae and which is elicited by the lipopolysaccharide moiety defined in claim 2.29.A method for the production of a functional cross-reactive antibody against Haemophilus influenzae which comprises: (a) generating antibodies to the lipopolysaccharide moiety defined in claim 2, (b) testing such antibodies against a plurality of Haemophilus influenzae strains, and (c) selecting those antibodies which are cross-reactive.", "30.The method of claim 29, wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae.", "31.A method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition defined in claim 23.32.A method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition defined in claim 24.33.The method of claim 31, wherein the Haemophilus influenzae is non-typeable Haemophilus influenzae.", "34.The method of claim 31, wherein the disease is selected from the group consisting of otitis media, meningitis, pneumonia, and respiratory tract infection.", "35.The method of claim 33, wherein the disease is selected from the group consisting of otitis media, meningitis, pneumonia, and respiratory tract infection." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Haemophilus influenzae is a major cause of disease worldwide.", "Six capsular serotypes (“a” to “f”) and an indeterminate number of acapsular (non-typeable) strains of H. influenzae are recognised.", "Type b capsular strains are associated with invasive diseases, including meningitis and pneumonia, while non-typeable H. influenzae (NTHi) is a primary cause of otitis media in children and respiratory tract infections in adults.", "Otitis media is a common childhood disease which accounts for the highest frequency of paediatric visits in the United States (Stool et al., Pediatr.", "Infect.", "Dis.", "Suppl., 8:S11-S14, 1989).", "The development of a vaccine for NTHi diseases has proved difficult because of a lack of understanding of the antigens that confer protective immunity.", "Efforts in developing a NTHi vaccine have been focused on cell surface antigens such as outer membrane proteins and pili or fimbria (Kyd et al., Infect.", "Immun., 63:2931-2940, 1995; Deich et al., Vaccine Res., 2:31-39, 1995).", "Recent advances in molecular genetics, molecular structure analysis and immunochemistry provide powerful tools which have permitted the identification of carbohydrate: structures as candidate vaccine antigens.", "Gram-negative bacteria have an outer membrane comprised of components including proteins, lipoproteins, phospholipids, and glycolipids.", "The glycolipids comprise primarily endotoxin lipopolysaccharides (LPS).", "LPS are molecules comprised of a) a Lipid A portion which consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages; b) a core polysaccharide which is attached to Lipid A by an eight carbon sugar, Kdo (ketodeoxyoctonate), and heptose, glucose, galactose, and N-acetylglucosamine; and, optionally, c) o-specific side chains comprised of repeating oligosaccharide units which, depending on the genera and.", "species of bacteria, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose (abequose, colitose, tyvelose, paratose, trehalose).", "LPS which lacks repeating O-side chains is sometimes referred to as short chain lipopolysaccharide, or as lipooligosaccharide (LOS).", "In this application, the term lipopolysaccharide (or LPS) includes short chain lipopolysaccharide and lipooligosaccharide (and LOS).", "The major antigenic determinants of gram-negative bacteria are believed to reside in the complex carbohydrate structure of LPS.", "These carbohydrate structures vary significantly, even among different species of the same genus of gram-negative bacteria, primarily because of variations in one or more of the sugar composition, the sequence of oligosaccharides, the linkage between the monomeric units of the oligosaccharides and between the oligosaccharides themselves, and substitutions/modifications of the oligosaccharides (particularly the terminal oligosaccharide).", "For this reason, development of a vaccine having a broad spectrum effect against Haemophilus influenzae (particularly against NTHi) has been unsuccessful.", "LPS is a bacterial component which has potential as a vaccine immunogen because of the antigenic determinants (“epitopes”) residing in its carbohydrate structures.", "However, the chemical nature of LPS detracts from its use in vaccine formulations; i.e., active immunization with LPS is unacceptable due to the inherent toxicity, in some animals, of the Lipid A portion.", "The pathophysiologic effects induced (directly or indirectly) by Lipid A of LPS in the bloodstream include fever, leucopenia, leucocytosis, the Shwartzman.", "reaction, disseminated intravascular coagulation, abortion, and in larger doses, shock and death.", "It has been established that vaccines comprised of capsular polysaccharides are effective at preventing human disease caused by the homologous encapsulated bacteria.", "These carbohydrate antigens are often poorly immunogenic in humans due to a lack of T-cell dependent response.", "However, by conjugating the specific polysaccharide antigen to a suitable protein carrier, the immunogenicity of the carbohydrate antigen can be greatly enhanced in patients who do not respond to the polysaccharide alone.", "Glycoconjugate vaccines based on the specific capsular polysaccharide of type b H. influenzae (Hib), e.g.", "ProHiBit™, and ActHib™, have already proven successful in the control of invasive Hib disease in infants.", "Capsular polysaccharide-protein conjugate Hib vaccines do not provide protection against disease caused by acapsular (non-typeable) strains of H. influenzae (i.e.", "against disease caused by NTHi) because they are only protective against infections caused by H. influenzae strains bearing the type b capsule.", "Lipopolysaccharide (LPS) is a major NTHi cell surface antigen.", "LPS of Haemophilus influenzae has only been found to contain lipid A and oligosaccharide (OS) components.", "Because the lipid A component of LPS is toxic, it must be detoxified prior to conjugation to an immunogenic carrier, as discussed above.", "Barenkamp et al.", "(Pediatr.", "Infect.", "Dis.", "J., 9:333-339, 1990) demonstrated that LOS stimulated the production of bactericidal antibodies directed against NTHi.", "McGehee et al.", "(Am.", "Journal Respir.", "Cell Biol., 1:201-210, 1989) showed that passive immunization of mice with monoclonal antibodies directed against LOS from NTHi enhanced the pulmonary clearance of NTHi.", "Green et al.", "(Vaccines, 94:12S-129, 1994) disclose a NTHi vaccine comprising a conjugate of NTHi oligosaccharide and the mutant nontoxic diphtheria protein CRM.sub.197.The lipid A moiety was removed from LOS by treatment with acid, followed by derivatizing the resulting OS with adipic acid dihydrazide (ADH) and coupling to CRM.sub.197.Despite the showing of Barenkamp et al.", "that LOS stimulated production of bactericidal antibodies against NTHi, the conjugates of Green et al.", "were determined to be poorly immunogenic after injection into mice.", "Moreover, the conjugates did not elicit bactericidal antibodies against NTHi.", "Gu et al.", "(U.S. Pat.", "No.", "6,207,157) is concerned with the detoxification of isolated NTHI LOS by removal of ester-linked fatty acids therefrom, so that it may be made suitable for vaccine preparation.", "However, Gu does not describe any other modifications to or desired chemical attributes of NTHi LOS.", "There is currently no vaccine available to provide broad spectrum protection against infections caused by Haemophilus influenzae .", "Thus, there is a need for a vaccine having broad spectrum efficacy against Haemophilus influenzae , particularly NTHi.", "In order to utilise an antigen for vaccine development, four essential criteria must be fulfilled.", "That is, the immunogenic epitope must be: 1.genetically stable; 2.conserved in all clinically relevant strains across the species; 3.accessible (in vitro and in vivo) to host immune mechanisms; and, 4.able to induce protective antibodies in vivo.", "There is a need to identify LPS carbohydrate epitopes of H. influenzae , particularly NTHi, which satisfy these criteria." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The subject invention is directed at immunity providing B-cell activating molecules derived from H. influenzae lipopolysaccharide (LPS), said molecules comprising one or more epitopes of a conserved inner-core oligosaccharide portion of the lipopolysaccharide.", "The invention discloses a strategy to identify and characterise said epitopes that are representative of LPS expressed by H. influenzae strains across the range of disease causing isolates.", "The invention is also directed at methods for obtaining such epitopes, both synthetically and through genetic engineering techniques.", "Furthermore, the invention relates to methods for preparing conjugates of molecules comprising said epitopes with suitable carriers, optionally in liposome formulations.", "In one aspect, the invention provides a lipopolysaccharide moiety comprising a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions.", "In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [I]: wherein R is hydrogen or phosphoethanoelamine, Glcs is D-glucopyranose, and Kdo is 3-deoxy-D-manno-2-octulosnic acid.", "In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [II]: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, and P-Petn is pyrophosphoethanolamine.", "In another aspect, the invention provides an immunogenic composition for conferring protection in an animal host against a disease caused by Haemophilus influenzae , comprising either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a use of at least one gene in a biosynthetic pathway for the production of lipopolysaccharide in Haemophilus influenzae to obtain a Haemophilus influenzae strain comprising either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a use of at least one immunogenic epitope to elicit a functional cross-reactive antibody against Haemophilus influenzae wherein the epitope comprises either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a functional antibody which is cross-reactive against Haemophilus influenzae and which is elicited by either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a method for the production of a functional cross-reactive antibody against Haemophilus influenzae which comprises: (a) generating antibodies to either of the lipopolysaccharide moieties described above, (b) testing such antibodies against a plurality of Haemophilus influenzae strains, and (c) selecting those antibodies which are cross-reactive.", "In another aspect, the invention provides a method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition described above." ], [ "FIELD OF THE INVENTION The present invention relates to defined mutations in the biosynthetic machinery for lipopolysaccharide (LPS) expression in Haemophilus influenzae.", "The invention also relates to using conjugates of the LPS from the mutant strains so obtained to elicit a heterologous immune response against a wide range of disease-causing H. influenzae strains.", "More specifically, the invention relates to vaccines for prevention of bacterial infections comprising core lipopolysaccharide of Haemophilus influenzae.", "BACKGROUND OF THE INVENTION Haemophilus influenzae is a major cause of disease worldwide.", "Six capsular serotypes (“a” to “f”) and an indeterminate number of acapsular (non-typeable) strains of H. influenzae are recognised.", "Type b capsular strains are associated with invasive diseases, including meningitis and pneumonia, while non-typeable H. influenzae (NTHi) is a primary cause of otitis media in children and respiratory tract infections in adults.", "Otitis media is a common childhood disease which accounts for the highest frequency of paediatric visits in the United States (Stool et al., Pediatr.", "Infect.", "Dis.", "Suppl., 8:S11-S14, 1989).", "The development of a vaccine for NTHi diseases has proved difficult because of a lack of understanding of the antigens that confer protective immunity.", "Efforts in developing a NTHi vaccine have been focused on cell surface antigens such as outer membrane proteins and pili or fimbria (Kyd et al., Infect.", "Immun., 63:2931-2940, 1995; Deich et al., Vaccine Res., 2:31-39, 1995).", "Recent advances in molecular genetics, molecular structure analysis and immunochemistry provide powerful tools which have permitted the identification of carbohydrate: structures as candidate vaccine antigens.", "Gram-negative bacteria have an outer membrane comprised of components including proteins, lipoproteins, phospholipids, and glycolipids.", "The glycolipids comprise primarily endotoxin lipopolysaccharides (LPS).", "LPS are molecules comprised of a) a Lipid A portion which consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages; b) a core polysaccharide which is attached to Lipid A by an eight carbon sugar, Kdo (ketodeoxyoctonate), and heptose, glucose, galactose, and N-acetylglucosamine; and, optionally, c) o-specific side chains comprised of repeating oligosaccharide units which, depending on the genera and.", "species of bacteria, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose (abequose, colitose, tyvelose, paratose, trehalose).", "LPS which lacks repeating O-side chains is sometimes referred to as short chain lipopolysaccharide, or as lipooligosaccharide (LOS).", "In this application, the term lipopolysaccharide (or LPS) includes short chain lipopolysaccharide and lipooligosaccharide (and LOS).", "The major antigenic determinants of gram-negative bacteria are believed to reside in the complex carbohydrate structure of LPS.", "These carbohydrate structures vary significantly, even among different species of the same genus of gram-negative bacteria, primarily because of variations in one or more of the sugar composition, the sequence of oligosaccharides, the linkage between the monomeric units of the oligosaccharides and between the oligosaccharides themselves, and substitutions/modifications of the oligosaccharides (particularly the terminal oligosaccharide).", "For this reason, development of a vaccine having a broad spectrum effect against Haemophilus influenzae (particularly against NTHi) has been unsuccessful.", "LPS is a bacterial component which has potential as a vaccine immunogen because of the antigenic determinants (“epitopes”) residing in its carbohydrate structures.", "However, the chemical nature of LPS detracts from its use in vaccine formulations; i.e., active immunization with LPS is unacceptable due to the inherent toxicity, in some animals, of the Lipid A portion.", "The pathophysiologic effects induced (directly or indirectly) by Lipid A of LPS in the bloodstream include fever, leucopenia, leucocytosis, the Shwartzman.", "reaction, disseminated intravascular coagulation, abortion, and in larger doses, shock and death.", "It has been established that vaccines comprised of capsular polysaccharides are effective at preventing human disease caused by the homologous encapsulated bacteria.", "These carbohydrate antigens are often poorly immunogenic in humans due to a lack of T-cell dependent response.", "However, by conjugating the specific polysaccharide antigen to a suitable protein carrier, the immunogenicity of the carbohydrate antigen can be greatly enhanced in patients who do not respond to the polysaccharide alone.", "Glycoconjugate vaccines based on the specific capsular polysaccharide of type b H. influenzae (Hib), e.g.", "ProHiBit™, and ActHib™, have already proven successful in the control of invasive Hib disease in infants.", "Capsular polysaccharide-protein conjugate Hib vaccines do not provide protection against disease caused by acapsular (non-typeable) strains of H. influenzae (i.e.", "against disease caused by NTHi) because they are only protective against infections caused by H. influenzae strains bearing the type b capsule.", "Lipopolysaccharide (LPS) is a major NTHi cell surface antigen.", "LPS of Haemophilus influenzae has only been found to contain lipid A and oligosaccharide (OS) components.", "Because the lipid A component of LPS is toxic, it must be detoxified prior to conjugation to an immunogenic carrier, as discussed above.", "Barenkamp et al.", "(Pediatr.", "Infect.", "Dis.", "J., 9:333-339, 1990) demonstrated that LOS stimulated the production of bactericidal antibodies directed against NTHi.", "McGehee et al.", "(Am.", "Journal Respir.", "Cell Biol., 1:201-210, 1989) showed that passive immunization of mice with monoclonal antibodies directed against LOS from NTHi enhanced the pulmonary clearance of NTHi.", "Green et al.", "(Vaccines, 94:12S-129, 1994) disclose a NTHi vaccine comprising a conjugate of NTHi oligosaccharide and the mutant nontoxic diphtheria protein CRM.sub.197.The lipid A moiety was removed from LOS by treatment with acid, followed by derivatizing the resulting OS with adipic acid dihydrazide (ADH) and coupling to CRM.sub.197.Despite the showing of Barenkamp et al.", "that LOS stimulated production of bactericidal antibodies against NTHi, the conjugates of Green et al.", "were determined to be poorly immunogenic after injection into mice.", "Moreover, the conjugates did not elicit bactericidal antibodies against NTHi.", "Gu et al.", "(U.S. Pat.", "No.", "6,207,157) is concerned with the detoxification of isolated NTHI LOS by removal of ester-linked fatty acids therefrom, so that it may be made suitable for vaccine preparation.", "However, Gu does not describe any other modifications to or desired chemical attributes of NTHi LOS.", "There is currently no vaccine available to provide broad spectrum protection against infections caused by Haemophilus influenzae.", "Thus, there is a need for a vaccine having broad spectrum efficacy against Haemophilus influenzae, particularly NTHi.", "In order to utilise an antigen for vaccine development, four essential criteria must be fulfilled.", "That is, the immunogenic epitope must be: 1.genetically stable; 2.conserved in all clinically relevant strains across the species; 3.accessible (in vitro and in vivo) to host immune mechanisms; and, 4.able to induce protective antibodies in vivo.", "There is a need to identify LPS carbohydrate epitopes of H. influenzae, particularly NTHi, which satisfy these criteria.", "SUMMARY OF THE INVENTION The subject invention is directed at immunity providing B-cell activating molecules derived from H. influenzae lipopolysaccharide (LPS), said molecules comprising one or more epitopes of a conserved inner-core oligosaccharide portion of the lipopolysaccharide.", "The invention discloses a strategy to identify and characterise said epitopes that are representative of LPS expressed by H. influenzae strains across the range of disease causing isolates.", "The invention is also directed at methods for obtaining such epitopes, both synthetically and through genetic engineering techniques.", "Furthermore, the invention relates to methods for preparing conjugates of molecules comprising said epitopes with suitable carriers, optionally in liposome formulations.", "In one aspect, the invention provides a lipopolysaccharide moiety comprising a conserved triheptosyl inner-core moiety of lipopolysaccharide substantially free of variable outer core oligosaccharide chain extensions.", "In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [I]: wherein R is hydrogen or phosphoethanoelamine, Glcs is D-glucopyranose, and Kdo is 3-deoxy-D-manno-2-octulosnic acid.", "In another aspect, the invention provides a lipopolysaccharide moiety comprising a triheptosyl inner-core moiety of Haemophilus influenzae lipopolysaccharide having the following structure [II]: wherein R is hydrogen or phosphoethanolamine, Glc is D-glucopyranose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, and P-Petn is pyrophosphoethanolamine.", "In another aspect, the invention provides an immunogenic composition for conferring protection in an animal host against a disease caused by Haemophilus influenzae, comprising either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a use of at least one gene in a biosynthetic pathway for the production of lipopolysaccharide in Haemophilus influenzae to obtain a Haemophilus influenzae strain comprising either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a use of at least one immunogenic epitope to elicit a functional cross-reactive antibody against Haemophilus influenzae wherein the epitope comprises either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a functional antibody which is cross-reactive against Haemophilus influenzae and which is elicited by either of the lipopolysaccharide moieties described above.", "In another aspect, the invention provides a method for the production of a functional cross-reactive antibody against Haemophilus influenzae which comprises: (a) generating antibodies to either of the lipopolysaccharide moieties described above, (b) testing such antibodies against a plurality of Haemophilus influenzae strains, and (c) selecting those antibodies which are cross-reactive.", "In another aspect, the invention provides a method of immunizing a host against disease caused by infection with Haemophilus influenzae which comprises administering to the host an immunoeffective amount of the immunogenic composition described above.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 is a structural verification of the high molecular weight form RM118.FIG.", "2 is the electrophoretic gel-migration patterns after T-SDS-PAGE of LPS purified from RM118 wild type and strains mutated in putative glycosyltransferase genes, wherein RM118 corresponds to the wild type LPS and the isogenic mutants are listed by the relevant LPS gene.", "FIG.", "3 is a negative ion ESI-MS of the O-deacylated LPS from the lpsA mutant of H. influenzae RM118 showing doubly and triply charged ions from the major Hexi glycoform (Table 1, structure 3).", "FIG.", "4 is a 1H NMR spectrum of the O-deacylated LPS from the lpsA mutant of H. influenzae RM118 showing the α-anomeric proton region between 5.0 and 6.0 ppm, wherein.", "anomeric resonances corresponding to the 3,4-disubstituted Hep (HepI), 6-PEtn substituted Hep (HepII), terminal HEP (HEPIII) and phosphorylated α-GlcN in the Lipid A region are indicated.", "FIG.", "5 is a capillary electrophoresis analysis of LgtD activity in H. influenzae strain RM118.Panel A, trace 1 is a complete reaction mixture using the 100,000 xg pellet of a sonicate as enzyme source; trace 2 is similar to the reaction mixture in 1, except it is missing UDP-GalNAc; trace 3 is a complete reaction mixture from the mutant RM118:lgtD; trace 4 is similar to trace 3 except it is missing UDP-GalNAc.", "The peak A is an impurity in the FCHASE-PK preparation, peak b is FCHASE-Globotetraose, peak c is FCHASE-PK.", "Panel B, trace 1 is the TLC purified product from a reaction as described for Panel A trace 1.Trace 2 is the same material as trace 1, but treated with β-hexosaminidase.", "FIG.", "6 is a negative ion ESI-MS of the triply charged molecular ion region of the O-deacylated LPS.", "from the lgtF mutant of H. influenzae RM118 wherein peaks arising from the Hex 2 (β-D-Galp-(1→4)-β-D-Glcp), Hex 3 (α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp), and Hex3•HexNAc (β-D-GalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp) are indicated.", "FIG.", "7 is a schematic representation of the structure of LPS from H. influenzae strain RM118 based on the results of the analysis of Risberg et al.", "The proposed site of action in LPS biosynthesis of loci characterised in this study are shown, linked by arrows to the relevant saccharide linkage.", "Phase variable loci are underlined.", "Represented in the LPS structure: KDO, 2-keto-3-deoxyoctulosonic acid; Hep, L-glycero-D-manno-heptose; Glc, D-glucose; Gal, D-galactose; GalNAc, N-acetylgalactosamine; PEtn, phosphoethanolamine; P. phosphate; Pcho, phosphocholine.", "For the heptose residues, listed top to bottom are heptose I, heptose II then heptose III.", "FIG.", "8 is a scheme for conjugation of a H. influenzae strain Rdlic1lpsA LPS-OH to a carrier protein (BSA in the present example).", "FIG.", "9 illustrates the genes involved in chain extension from the LPS inner-core of H. influenzae strain Rd− Genes orf3 and lic2b in the lic2 locus control chain extension from Hep II in type b strains: these genes are missing in strain Rd−.", "Licl controls incorporation of PCho.", "FIG.", "10 is a representation of part of the negative-ion ESI-MS spectrum of O-deacylated LPS from NTHi strain 486.The proposed compositions of the major glycoforms are indicated.", "Sodiated adducts are indicated by an asterisk (*).", "FIG.", "11 is a representation of a 270 MHz 1H NMR spectra of Fr.2 dervived from NTHi 176 (a) and Fr.", "3, (b).", "FIG.", "12 illustrates the structure of the major Hexi LPS glycoform observed in NTHi 285.FIG.", "13 illustrates the postulated structure of the major LPS glycoform in NTHi 1158.FIG.", "14 is a representation of a Biogel P-4 chromatogram of LPS (100 mg) of NTHi 176 after mild acid hydrolysis.", "Indicated are Fr.1 (1 mg), Fr.2 (10 mg) and Fr.3 (6.4 mg).", "FIG.", "15 is a representation of parts of the 500 MHz 2D NOESY spectrum of Fr.2 of NTHi 176.FIG.", "16 illustrates various structures of oligosaccharides derived from NTHi 176 LPS.", "FIG.", "17 illustrates the structure of the major NeuAc containing LPS glycoform of NTHi 486.DETAILED DESCRIPTION OF THE INVENTION Lipopolysaccharide (LPS) is an essential and characteristic surface-exposed antigen of H. influenzae.", "(As discussed above, the terms lipopolysaccharide and LPS as used herein encompass short chain lipopolysaccharide and lipooligosaccharide (LOS).)", "H. influenzae strains express heterogeneous populations of low-molecular-weight LPS which can exhibit extensive antigenic diversity among multiple oligosaccharide epitopes.", "The LPS carbohydrate structures of H. influenzae described by the Applicant herein can provide a source of protective antigens when they are presented to the human immune system in an appropriate fashion, for example, as a protein-conjugate or in a liposome formulation.", "Antibodies against certain NTHi LPS have shown bactericidal activity in vitro (Sun et al.", "Vaccine 18:1264-1272).", "A recent study (Gu et al., U.S. Pat.", "No.", "6,207,157) has indicated that: immunization with LPS-based conjugates can reduce the incidence of NTHi-induced otitis media due to a homologous strain in an animal model.", "LPS proved useful as a vaccine candidate in Applicant's study because, surprisingly, surface expressed carbohydrate antigens were identified which possess oligosaccharide epitopes that are genetically and physiologically stable, that are conserved across the range of clinically relevant strains, and that are accessible to host clearance mechanisms.", "The carbohydrate regions of H. influenzae LPS molecules provide targets for recognition by host immune responses.", "Expression of certain oligosaccharide epitopes is known to contribute to the pathogenesis of H. influenzae infections.", "Determination of structure is crucial to understanding the biology of H. influenzae LPS and its role in bacterial virulence.", "H. influenzae LPS comprises a heterogeneous mixture of molecules consisting of a variable oligosaccharide moiety and a membrane anchoring-Lipid A component (Zamze et al., J. Gen.", "Microbiol., 133:1443-1451, 1987).", "Based on the experiments described herein, a structural model was developed for Haemophilus LPS consisting of a conserved triheptosyl inner-core moiety which is attached via a phosphorylated ketodeoxyoctonoate residue to a lipid A component.", "In every strain investigated by Applicant to date this triheptosyl moiety consists of the following structural element L-α-D-Hepp-(1→2)-L-α-D-Hepp-[β-D-Glcp-(1→4)]-(1→3)-L-α-D-Hepp-(1→5)-Kdo Additionally, in every strain investigated by Applicant to date, the 1,2-linked heptose residue (HepII) is substituted with a phosphoethanolamine moiety at the 6 position.", "Each of the heptose residues within the inner-core region can provide a site for elongation by hexose-containing oligosaccharide chains or for attachment of non-carbohydrate substituents.", "Published data (Masoud et al., Biochem.", "36:2091-2103, 1997; Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999) indicates that the distal heptose residue (HepIII) in this conserved inner-core moiety can be substituted by a β-D-Glcp or β-D-Galp residue at the O-2 position.", "Substitutions are also possible on HepII, specifically by an α-D-glucose or a substituted α-D-glucose at the 3 position.", "The β-D-glucose residue which is 1,4-linked to the proximal heptose residue (HepI) can itself be further substituted by a β-D-glucose, a β-D-galactose, a heptose (including L-glycero-D-manno-heptose and D-glycero-D-manno-heptose), or oligosaccharides thereof.", "Additionally to these sugar residues, oligosaccharide chain extensions can comprise β-D-galactose, β-D-glucosamine, β-D-galactosamine, and β-N-acetlyneuraminic acid (sialic acid).", "The degree of substitution and chain extension from the triheptosyl moiety varies within and between strains (Masoud et al., Biochem.", "36:2091-2103, 1996; Risberg, et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "Phosphate-containing substituents which include free phosphate groups (P), phosphoethanolamine (PEtn), pyrophosphoethanolamine (PPEtn), and phosphocholine (PCho) contribute to the structural variability of these molecules.", "Moreover, ester substituents (including O-acetyl and O-glycyl) also contribute to the structural variability of LPS.", "Other modifications of LPS are possible such as addition of sialic acid residues.", "Sialylated oligosaccharides are commonly found in mammalian tissue and this modification is believed to improve mimicry of human tissue structures.", "Detailed structural studies of LPS from defined mutants of the type b strain RM7004 (Schweda et al., Carbohydr.", "Res.", "246:319-330, 1993; Schweda et al., Carbohydr.", "Res.", "272:213-224, 1995), transformed variants of the type d derived strain RM118 (Risberg et al., Eur.", "J. Biochem.", "243:701-707, 1997; Risberg et al., Eur.", "J. Biochem.", "265:1067-1074, 19,99), and transposon mutants of the type b strain A2 (Phillips et al., Biochem.", "35:5937-5947, 1996) provide further evidence for the presence of a common heptose-containing trisaccharide inner-core moiety in H. influenzae LPS.", "There has been reported the structure of a globotetraose (β-D-GalpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp) containing.", "LPS from H. influenzae strain RM118 (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999), the strain (Rd) for which the complete genome sequence has been determined (Fleischmann et al., Science 269:496-512, 1995).", "In this study, three major populations of LPS glycoforms were identified, all containing a PCho→6)-β-D-Glcp group off the Hep attached to the Kdo moiety (HepI), but differing in the length of the oligosaccharide chains off the distal Hep (HepIII) of the inner-core element.", "In addition to LPS glycoforms expressing a fully assembled globotetraose side-chain, sequentially truncated glycoforms containing globoside (α-D-Galp-1→4)-β-D-Galp-(1→4)-β-D-Glcp) and lactose (β-D-Galp-(1→4)-(β-D-Glcp) have been characterised (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "The availability of the complete genome sequence of H. influenzae strain Rd facilitated a comprehensive study of multiple LPS biosynthetic loci in representative Hi strains.", "Non-typeable strains were obtained from Prof. Eskola as part of a Finnish Otitis Media Cohort Study and are mainly isolates obtained from the inner ear of children.", "These strains are further described in Hood et al., Mol.", "Microbiol.", "33:679-792, 1999.102 NTHi otitis media strains were sent to Richard Goldstein in Boston to be included in a survey of the diversity of over 600 H. influenzae capsulate and NTHi strains, obtained from around the world and over a 35 year period, by ribotyping analysis.", "When a dendrogram was drawn from the results of the ribotyping, the NTHi otitis media isolates were found to be present in almost all of the branches obtained.", "The 25 representative strains were selected from branches spanning the dendrogram and thus represent the known diversity associated with the species H. influenzae.", "Included in the 25 strains are some selected from the same cluster to allow an assessment of the diversity of closely related isolates.", "Many predicted gene functions were correlated with particular steps in the synthesis of the LPS by analysis of LPS structure from the appropriate mutant strains (Hood.", "et al., Mol.", "Microbiol.", "22:951-965).", "The genetic basis for expression of the globotetraose structure has not previously been reported.", "As described in the examples, Applicant employed a structural fingerprinting strategy to determine and compare the structures of LPS obtained from a series of defined-mutants in LPS biosynthetic genes in H. influenzae strain RM118.", "(For a description of strain RM118, see Risberg et al., 1999, supra.)", "By examination of LPS from strains in which specific genes are inactivated, Applicant presents definitive evidence leading to the identification of the glycosyltransferases involved in the biosynthesis of the inner-core region of the LPS molecule.", "Applicant has.", "also identified genes involved in the assembly of sialylated lactose side chain, as further described below.", "Moreover, the transferase functions of genes involved in addition of α-2,3-linked NeuSAc (lic3A), α-1,4-linked Galp (1gtC) and β-1,3-linked GalpNAc (1gtD) to give the globotriose and globotetraose structures, respectively, were unambiguously determined by enzymatic assays with synthetic acceptors.", "This is the first study to identify a genetic blueprint for biosynthesis of a LPS oligosaccharide moiety for a strain of H. influenzae.", "In our investigation of the H. influenzae LPS inner-core, attempts to remove Kdo, the first sugar added to the Lipid A, have repeatedly failed presumably as Kdo is required to complete Lipid A synthesis and is thus likely essential for cell viability.", "The Kdo transferase function of kdta has been demonstrated by complementation experiments in E. coli (White et al., FASEB J.", "12:L44, 1998).", "Applicant's studies indicate that opsX, rfaF and orfH are the genes encoding the enzymes which add the first, second and third heptoses (HepI, HepII and HepIII), respectively, to the Kdo to form the inner-core.", "opsX has some homology to heptosyl transferases of the enteric bacteria (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996) and corresponds to a gene responsible for adding HepI to Kdo.", "The rifaF mutant has a functional opsX gene and its LPS has a single Hep attached to Kdo-Lipid A.", "The rfaF gene of RM118 (Rd) has homology to other heptosyl transferases (Allen et al., J. Bacteriol.", "180:35-40, 1998).", "LPS from RM118orfH which has functional opsX and rfaF genes comprise structures containing two heptose residues.", "The data on structural analyses of LPS from RM118opsX, rfaF and orfH mutants is consistent with that obtained with the same mutations in the type b strains RM153 (also known as strain Eagan) and RM7004 (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "In the type b strains, opsX, rfaF and orfH are proposed as the genes encoding the HepI, HepII and HepIII transferases respectively.", "Applicant has shown that the LPS of H. influenzae has the afore-mentioned triheptosyl inner-core moiety in which each heptose residue provides a point for elongation by oligosaccharide chains (Masoud et al., Biochem.", "36:2091-2103, 1996; Risberg et al., Eur.", "J. Biochem 261:171-180, 1999).", "Each of the genes lpsA, lic2A, lic3A, lgtc, lgtD and lgtF encode glycosyltransferase enzymes involved in oligosaccharide elongation.", "These results indicate that the lpsA gene product plays a role in controlling oligosaccharide chain extension from HepIII.", "A mutation in the lpsA gene affords a truncated LPS in which HepIII is devoid of oligosaccharide chain extensions.", "ESI-MS analysis of the RM118lpsA derived O-deacylated LPS indicated a Pcho-containing glycoform containing a single hexose residue as the major LPS species, confirming that HepI can be substituted in the absence of hexose extension from HepIII.", "The lic2A, lgtC and lgtD mutants which contain a functional lpsA gene are capable of adding a β-D-Glcp residue in a 1,2-linkage to initiate chain extension from HepIII.", "lpsA is a homologue of a gene encoding a glycosyltransferase in Pasteurella haemolytica (Potter et al., FEMS Microbiol.", "Lett.", "129:75-81, 1995), the protein of which has homology to the group of galactosyltransferases typified by Lic2A and LgtB of Haemophilus and Neisseria respectively.", "In strain RM153, it was also found that LPS from a lpsA mutant lacked any chain extension from the third heptose (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "Moreover, Applicant has confirmed that LpsA mutants in several NTHi strains lack chain extensions from HepIII.", "Thus, LpsA is the transferase for the addition of the first sugar to HepIII in H. influenzae LPS biosynthesis.", "The RM118lic2A mutant showed a Pcho-containing Hex2 glycoform as a major LPS species (Structure 4; Table 1) and RM118lgtC, which contains a functional lic2A gene, elaborates LPS containing a lactose side chain at HepIII (Structure 5; Table 1).", "This is consistent with the involvement of the lic2A gene to add the β-D-Galp unit in a 1,4 linkage to the terminal.", "β-D-Glcp residue attached to HepIII.", "Lic2A homologues in the type b strains, RM153 and RM7004 have been shown to be involved in expression of the digalactoside-containing PK epitope (the globoside trisaccharide having the structure α-D-Galp-1→4)-β-D-Galp-(1→4)-β-D-Glcp).", "The role of lic2A in phase variable expression of this epitope has been previously demonstrated (High et al., Mol.", "Microbiol.", "9:1275-1282, 1993).", "Homology comparisons with other data bank sequences support the function of Lic2A as a β-galactosyltransferase.", "Importantly, it has significant homology to the Neisseria LgtB and LgtE proteins, both of which are galactosyltransferases (Wakarchuk et al., J. Biol.", "Chem.", "271:1916-1917, 1996).", "Structural analysis of LPS from an RM118 strain, mutated in lgtC, confirmed the loss of α-D-Galp supporting the α-galactosyltransferase function for this gene.", "A homologue of this gene in N. meningitidis has been demonstrated to be an α-galactosyltransferase (Gotschlich, J. Exp.", "Med.", "180:2181-2190, 1994; White et al., FASEB J.", "12:L44, 1998; Wakarchuk et al., Protein Eng.", "11:295-302, 1998).", "lgtc from H. influenzae has an associated tetranucleotide repeat (5′-GACA-3′) just within the 5′ end of the reading frame (Hood et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 93:11121-11125, 1996) and therefore contributes to the variable phenotype of the oligosaccharide in RM118 LPS.", "Correspondingly, the lgtD mutant and the parent strain which contain a functional lgtC gene are capable of adding an α-D-Galp in a 1,4 linkage to the terminal β-D-Galp of the lactose epitope (Structure 6; Table 1) The function of LgtC was confirmed by demonstrating α-galactosyltransferase activity with the recombinant LgtC protein and a synthetic FCHASE-Lac acceptor.", "It follows that the lgtc gene encodes the specific α-galactosyl-transferase for the synthesis of the α-D-Galp-(1→4)-β-D-Galp of the RM118 Hex4 LPS glycoform.", "The H. influenzae lgtD gene is a homologue of two Neisseria genes, lgtA and lgtD, which add GlcNAc and GalNAc respectively to N. gonorrhoeae LPS (Gotschlich, J. Exp.", "Med.", "180:2181-2190, 1994).", "The lgtA gene product has been emonstrated to be a glycosyltransferase in N. meningitidis (Wakarchuk et al., J. Biol.", "Chem.", "271:1916-1917, 1996) There is significant homology between the Neisseria lgtA and lgtd genes and the best database match of RM118 HIl578 is to the N. gonorrhoeae lgtd gene.", "Enzyme assays with extracts of RM118 and the RM118lgtD mutant established the presence of.", "β-D-GalpNAc transferase activity.", "The parent strain RM118 that contains a functional lgtD gene is capable of elaborating the complete globotetraose unit which is indicative of its role in adding the terminal β-D-GalpNAc.", "The lgtd gene has been investigated previously (called lgtA in Hood et al., Mol.", "Microbiol.", "22:951-965, 1996) and was found not to be present in the type b strains RM153 and RM7004.Correspondingly, the LPS elaborated by strain RM153 does not contain a GalNAc moiety (Masoud et al., Biochem.", "36:2091-2103, 1996).", "Many NTHi strains have been found to contain the LgtD gene and the LPS oligosaccharide side chains of these strains have been found to contain a GalNAc moiety.", "It is noteworthy that, while the glycosyltransferase activities encoded by the genes involving addition of the distal residues of the globotetraose (lgtD) and globoside (lgtC) oligosaccharide side-chains could be confirmed in an enzymatic assay with the appropriate synthetic acceptor, experiments to assay the transferase activity of the genes involved in synthesis of the lactose moiety (lic2A and lpsA) were unsuccessful.", "It is likely that the two latter enzymes have more stringent specificities that require the acceptor sugar to be linked to the inner-core heptose residues, thereby precluding recognition of the simple synthetic FCHASE-glycoside acceptors.", "Characterisation of the initial set of genes and mutant strains available for study of RM118 LPS biosynthesis gave no obvious candidate responsible for the addition of β-D-Glcp unit to HepI.", "Further candidate LPS genes were investigated by searching the strain Rd genome sequence for low homology matches to genes adding hexose sugars to heptose residues in the LPS of other organisms.", "Search sequences included the rfaK and lgtF genes of Neisseria, genes involved in addition of hexose residues to heptose (Kahler et al., J. Bacteriol.", "178:6677-6684, 1996).", "A lgtF homologue was identified in strain RM118 and analysis of the LPS from the strain mutated in this gene was indicative of the role of LgtF in chain extension from HepI.", "The ESI-MS showed molecular ions corresponding to a mixture of glycoforms having chain extensions from HepIII of a triheptosyl inner-core moiety, which lacks PCho→6)-β-D-Glcp at HepI (FIG.", "6).", "The lgtF and lpsA genes are key to the hexose extensions from the heptose containing inner-core unit of RM118 LPS, encoding the glycosyl transferase enzymes responsible for adding the first glycose to HepI and HepIII, respectively.", "The processes of chain extension from both HepI and HepIII appear to be largely independent in the LPS of strain RM118.Mutant strain RM1181psA produces LPS which includes the β-D-Glcp moiety from HepI.", "Strain RM118lgtF produces a heterogeneous LPS containing oligosaccharide extensions from HepIII that include lactose and globotetraose chains.", "In strain RM153, lpsA apparently plays a slightly different role being responsible for the addition of galactose as the sole extension from the third heptose (Hood et al., Mol.", "Microbiol.", ", 22:951-965, 1996).", "Surprisingly, it was determined that in certain non-typeable strains, lpsA can add either glucose or galactose at the O-3 position instead of the O-2 position.", "The heterogeneity of H. influenzae LPS structure must be due in part to intrinsic variation in the biosynthesis of such a complex structure whereby not every molecule will be synthesised to completion.", "However, the majority of variation observed appears to be due to specific LPS biosynthetic genes capable of variable expression (phase variation).", "Structural analysis of LPS derived from wild-type and mutant RM118.strains has allowed Applicant for the first time to confirm the genes involved in the synthesis of an important phase variable epitope of H. influenzae LPS, the digalactoside.", "In strain RM118, Lic2A adds the proximal β-D-Galp and LgtC the terminal α-D-Galp to the digalactoside (α-D-Galp-(1→4)-β-D-Galp) as part of the oligosaccharide extension from HepIII whereas, the same epitope is expressed as the terminal extension from the second heptose in the type b strain RM153 (Masoud et al., Biochem.", "36:2091-2103, 1996).", "Both lic2A and lgtC are phase variable.", "genes, making the expression of the epitope highly variable within and between organisms.", "The digalactoside epitope is expressed in the LPS of many of the NTHi strains disclosed in the present invention as well as in related bacteria, including Neisseria (Virji et al., Microb.", "Pathogen.", "9:441-450, 1990).", "The epitope is potentially immunodominant and is of interest in pathogenesis as its presence offers the potential for molecular mimicry of host structures and can influence the survival of Haemophilus influenzae within experimental systems (Weiser et al., Mol.", "Microbiol.", "30:767-775, 1998; Hood et al., Proc.", "Natl.", "Acad.", "Sci.", "USA.", "93:11121-11125, 1996).", "Sialylation of oligosaccharides is one modification which is believed to improve mimicry of human tissue structures, since sialylated oligosaccharides are commonly found in mammalian tissue.", "A described in the examples, Applicant has identified sialylated LPS in NTHi strains 375, 486 and strain RD, elucidating a role for lic3A in LPS synthesis (Hood et al., Mol.", "Microbiol.", "39:341-350, 2001).", "A survey of 25 NTHi strains representative of the diversity of the species identified sialylated LPS oligosaccharide chain extensions in all but one.", "The mutation of lic3A in some NTHi strains, such as NTHi 486, has been demonstrated to have a major influence upon resistance to the killing effect of normal human serum.", "A comparison of the structures of LPS from NTHi 486 and its lic3A mutant revealed the presence of sialylated glycoforms (sialyl-lactose) only in the parent strain, which points to the importance of Lic3A for serum resistance in this strain.", "Addition of a charged sialic acid residue to LPS in Haemophilus influenzae appears to modulate antigenic mimicry of LPS epitopes.", "In addition to the order and stereochemistry of the sugar residues which constitute the oligosaccharide portion of the LPS molecules, the location, type and frequency of substituents such as P, PEtn and PCho can have a profound affect on LPS structure and biological function.", "The lic1 locus has been shown to be essential for the phase-variable addition of PCho to the H. influenzae LPS molecule (Weiser et al., Infect.", "Immun.", "65:943-950, 1997; Lysenko et al., Mol.", "Microbiol.", "35:234-245, 2000).", "It has been demonstrated that PCho contributes to the resistance of H. influenzae to innate humoral immunity (Weiser et al., Mol.", "Microbiol.", "30:767-775, 1998, Lysenko et al., Mol.", "Microbiol.", "35:234-245, 2000).", "The gene encoding a Kdo kinase, kdkA, responsible for phosphorylation of Kdo, has been identified (White et al., J. Biol.", "Chem.", "274:31391-31400, 1999).", "This gene has previously been investigated by the applicant as orfZ and when mutated was shown to alter bacterial survival in an infantra model of infection (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "The only remaining substituents in the core oligosaccharide,: whose genetic control remains unknown, therefore, are the PEtn residues that are attached to the 6-position of the HepII residue stoichiometrically and sometimes to the phosphate group on Kdo.", "In summary, the genetic blueprint for complete synthesis of the major oligosaccharide chain extensions of Haemophilus influenzae have been elucidated.", "Applicant has investigated in excess of 25 strains representative of the known diversity of Haemophilus influenzae.", "Applicant has confirmed that the triheptosyl inner-core moiety of LPS (labelled HepI-HepIII) is conserved in all strains tested.", "This determination permits the synthesis of suitable oligosaccharides from the inner-core moiety of LPS for use in the preparation of vaccines which afford broad spectrum protection against Haemophilus influenzae, including NTHi.", "It also permits the selection and modification of strains of Haemophilus influenzae which synthesize suitable oligosaccharides from the inner-core moiety of LPS for use in the preparation of vaccines.", "FIG.", "7 summarizes the genes involved in the biosynthesis of the major globotetraose-containing oligosaccharides of LPS in H. influenzae Rd which have been identified.", "Applicant has identified the genes which; are responsible for chain extension from HepI (lgtF) and HepIII (IpsA).", "Furthermore, some strains of Haemophilus influenzae (eg.", "Eagan and RM7004) elaborate LPS which can show chain extension from HepII.", "Applicant has shown that a gene in the lic2 locus (orf3) initiates chain extension from HepII.", "Differential expression of these genes can lead to a manifold of variable oligosaccharide epitopes emanating from the conserved triheptosyl inner-core moiety.", "Through defined mutations in these genes, Applicant can control not only the degree of complexity that a particular Haemophilus influenzae strain expresses, but also the available epitopes on the resulting core region of the LPS.", "Thus, LPS from H. influenzae strains with defined mutations in their biosynthetic machinery provided suitable candidates for developing a broad spectrum LPS-based vaccine.", "Applicant has determined that this strategy is useful for vaccine design because it provides cross-reactivity against a variety of pathogenic strains of Haemophilus influenzae, including non-typeable strains which have hitherto exhibited such LPS variability as to discourage development of vaccines.", "Variation in the substitution patterns at HepII and HepIII in the LPS triheptosyl inner-core leads to alternate inner-core oligosaccharide epitopes.", "The following are examples of useful substitutions and modifications of the inner core region of LPS of Haemophilus influenzae which illustrate the invention.", "These are not intended to be limiting and one of skill in the art will appreciate that other substitutions and modifications are possible in keeping with the teachings of the invention.", "Based on genomic and phenotypic analysis of LPS from a collection of NTHi strains, Applicant has identified, for the first time, Haemophilus influenzae strains expressing LPS with an alternate inner-core structure in which chain extension occurs from O-3 of HepIII instead of O-2.Homologous lpsA genes mediate the specific addition of β-D-Gal or β-D-Glc to the O-2 or O-3 position of HepIII depending on the strain.", "Furthermore, some Haemophilus influenzae strains express LPS in which HepIII is not substituted by oligosaccharide chains.", "These findings are detailed in the examples.", "The results in the examples confirm the presence of a conserved inner-core element in NTHi.", "These results also provide for the first time evidence for a new structural motif, namely an alternate substitution site on HepIII.", "HepIII has earlier been found to be substituted by hexose residues at the O-2-position, and this was found to be the case in NTHi, for example in strains 285 and 1158.In other NTHi strains, for example 176 and 486, this substitution pattern is not observed.", "Instead, substitution occurs at the O-3 position of HepIII.", "Surprisingly, it has been shown that the lpsA gene can add a β-D-Glcp in some strains (strain RD, FIG.", "9) or a β-D-Galp in others (type b, eg strain Eagan) in a 1,2-linkage to initiate chain extension from HepIII.", "Applicant has found that a homologue of lpsA is responsible for addition of a β-D-Glcp residue or a β-D-Galp residue in a 1,3-linkage to HepIII.", "Another useful modification of the inner-core region relates to the PCho epitope.", "This is a common feature on surface structures of pathogens residing in the human respiratory tract, including H. influenzae.", "In H. influenzae strain Rd, PCho is attached to O-6 of a terminal β-D-Glcp residue at HepI (R1=PCho→6)-β-D-Glcp; FIG.", "9).", "In other H. influenzae strains, for example type b strain Eagan, PCho is attached to O-6 of a terminal β-D-Galp residue at HepIII.", "(Structure 1; R3=PCho→6)-β-D-Galp) (Schweda et al.", "Eur.", "J. Biochem.", "267:3902-3913, 2000).", "Both of these substitution patterns have been found in NTHi strains.", "Furthermore, PCho has been found on the terminal β-D-Glcp residue (R2=PCho→6)-α-D-Glcp).", "The expression and phase variation of the PCho epitope on the H. influenzae LPS require the four genes of the lic7 locus (Weiser et al.", "Infec.", "Immun.", "65:943-950, 1997).", "These genes have been found on every NTHi strain investigated.", "Applicant has determined that polymorphism in the licD gene affects the site at which the PCho is added.", "The nature of the substitution pattern to the triheptosyl inner-core moiety is relevant.", "to the way in which inner-core epitopes are presented to the immune system and recognised by monoclonal antibodies.", "For example, Applicant has found that a murine IgG2a monoclonal antibody (L6A9).", "recognises a LPS inner-core oligosaccharide epitope in strains containing a β-D-Galp residue at O-2 of HepIII, but not when the β-D-Galp residue is absent or attached to the O-3 position.", "Structural verification of the high molecular weight form is shown in FIG.", "1.As stated above, key biosynthetic genes involved in.", "H. influenzae LPS expression have been identified.", "This gave Applicant the ability to construct mutant strains of H. influenzae that comprise the conserved inner-core moiety, but not oligosaccharide extensions that mimic human tissues structures.", "Th examples set out the results of this procedure.", "Applicant has found that oligosaccharides containing the conserved triheptosyl inner-core moiety of H. influenzae LPS are immunogenic when formulated as, e.g.", "protein conjugates, using methods known to those skilled in the art (Gu.", "patent, supra).", "For example, Applicant has shown that the immunization of mice with an oligosaccharide-protein conjugate comprised of O-deacylated LPS from Hi strain Rdlic1lpsA conjugated with bovine serum albumum (BSA) produces immune serum which is cross reacitve with a majority of LPS samples from a genetically diverse set of NTHi strains (Table 3).", "O-deacylation of the LPS is achieved by using anhydrous hydrazine.", "This has the effect of solubilizing the LPS, oligosaccharide for the subsequent conjugation reaction with protein and leads to significant detoxification of the Lipid A component (Gu patent, supra).", "The O-deacylated LPS is conjugated via the carboxyl group of the Kdo moiety according to methods known to those skilled in the art (Gu patent, supra).", "Mice were given a final intraperitoneal boost with O-deacylated LPS from Hi strain 1003lic1lpsA (see the examples) Mouse immune sera from this experiment reacted with LPS from NTHi strains which have multiple chain extensions from the inner core.", "Rdlic1lpsA LPS elaborates LPS that is representative of the conserved triheptosyl inner-core moiety, and contains minor glycoforms having lacto-N-neotetraose (LNnT)-containing oligosaccharide chain extensions.", "This is evident from the reactivity with Mab LLA5 (Table 4).", "The Rdlic1lpsA O-deacylated LPS-BSA conjugate also showed reactivity with Mab LLA5 as well as the inner-core Mab, LLA4.NTHi strain 1003 does not elaborate LPS having LNnT containing oligosaccharide chains.", "Applicant determined this by structural, genetic and immunochemical analyses.", "LPS from this strain does not react with LLA5, does not contain the rfb genetic locus.", "The double mutant, 1003lic1lpsA expresses LPS having the conserved inner-core moiety with no chain extensions.", "It is recognized by Mab LLA4.The above shows that the conserved inner-core moiety of the H. influenzae LPS (which is present in Rdlic1lpsA for example) can elicit an immune response that is cross reactive with LPS from strains representative of the diversity of the bacterium.", "Lactose, LNnT and their sialylated analogues are oligosaccharide structures that are found on human tissues.", "To develop a LPS based vaccine, it is therefore preferable to ensure these oligosaccharide extensions are not present in the vaccine formulation.", "The above experiments provide evidence that these oligosaccharide extensions are common throughout H. influenzae strains, and that Applicant has taught the methodology to identify them and to construct strains without them.", "By the same means, other mutant strains of Hi can be constructed with substitutions to or modifications of the conserved triheptosyl inner-core moiety of LPS which result in epitopes which elicit a broad spectrum immune response to Hi, including NTHi, and/or which elicit an improved immune response because of mimicry of, or avoidance of mimicry of animal tissues.", "Vaccination methods for treating or preventing infection in a mammal comprise use of a vaccine formulation of the invention to be administered by any conventional route, particularly to a mucosal (e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.", "Preferred routes depend upon the choice of the vaccine formulation.", "Treatment may be effected in a single dose or repeated at intervals.", "The appropriate dosage depends on various parameters understood by skilled artisans such as the vaccine formulation itself, the route of administration or the condition of the mammal to be vaccinated (weight, age and the like).", "Standard laboratory techniques for preparing and purifying lipopolysaccharides are used in the preparation of lipopolysaccharide therapeutics of the invention.", "For use as a vaccine, a lipopolysaccharide of the invention is formulated according to various methods outlined below.", "Since the Lipid A component of LPS can render it toxic, preferably the LPS immunogen is detoxifed.", "Although the use of hydrazine for detoxification of LPS from NTHi is described herein, the use of any reagent or enzyme capable of removing ester-linked fatty acids from Lipid A is within the scope of the present invention.", "Dried LPS from the selected strain of NTHi is suspended in liquid anhydrous hydrazine at a temperature of between 1° C. and 100° C., preferably between 25° C. and 75° C., more preferably about 37° C., for a period between 1 hour and 24 hours, most preferably for a period of about 2-3 hours.", "After removal of ester-linked fatty acids, DLPS is conjugated to the linker adipic acid dihydrazide (ADH) prior to conjugation to the immunogenic carrier proteins TT or NTHi HMPs.", "Although ADH is the preferred linker, the use of any linker capable of stably and efficiently conjugating dLPS to an immunogenic carrier protein is contemplated.", "The use of linkers is well known in the conjugate vaccine field (see Dick et al., Conjugate Vaccines, J. M. Cruse and R. E.", "Lewis., Jr., eds., Karger, New York, pp.", "48-114.dLPS may be directly covalently bonded to the, carrier.", "This may be accomplished, for example, by using the cross-linking reagent glutaraldehyde.", "However, in a preferred embodiment, DLPS and the carrier are separated by a linker.", "Presence of a linker promotes optimum-immunogenicity of the conjugate and more efficient coupling of the DLPS with the carrier.", "Linkers separate the two antigenic components by chains whose length and flexibility can be adjusted as desired.", "Between the bifunctional sites, the chains can contain a variety of structural features, including heteroatoms and cleavage sites.", "Linkers also permit corresponding increases in translational and rotational characteristics of the antigens, increasing access of the binding sites to soluble antibodies.", "Besides ADH, suitable linkers include, for example, heterodifunctional linkers such as epsilon.", "aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenyl amine.", "Coupling reagents contemplated for use in the present invention include hydroxysuccinimides and carbodiimides.", "Many other linkers and coupling reagents known to those of ordinary skill in the art are also suitable for use in the invention.", "Such compounds are discussed in detail by Dick et al., supra.", "The presence of a carrier increases the immmunogenicity of the polysaccharide.", "In addition, antibodies raised against the carrier are medically beneficial.", "Polymeric immunogenic carriers can be a natural or synthetic material containing a primary and/or secondary amino group, an azido group or a carboxyl group.", "The carrier may be water soluble or insoluble.", "Any one of a variety of immunogenic carrier proteins may be used in the conjugate vaccine of the present invention.", "Such classes of proteins include pili, outer membrane proteins and excreted toxins of pathogenic bacteria, nontoxic or “toxoid” forms of such excreted toxins, nontoxic proteins antigenically similar to bacterial toxins (cross-reacting materials or CRMs) and other proteins.", "Nonlimiting examples of bacterial toxoids contemplated for use in the present invention include tetanus toxin/toxoid, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid and Clostridium perfringens exotoxins/toxoid.", "The toxoid forms of these bacterial toxins is preferred.", "The use of viral proteins (i.e.", "hepatitis B surface/core antigens; rotavirus VP 7 protein.", "and respiratory syncytial virus F and G proteins) is also contemplated.", "CRMs include CRM.sub.197, antigenically equivalent to diphtheria toxin (Pappenheimer et al., Immunochem., 9:891-906, 1972) and CRM3201, a genetically manipulated variant of pertussis toxin (Black et al., Science, 240:656-659, 1988).", "The use of immunogenic carrier proteins from non-mammalian sources including keyhole limpet hemocyanin, horseshoe crab hemocyanin and plant edestin is also within the scope of the invention.", "There are many coupling methods which can be envisioned for dLPS-protein conjugates.", "dLPS can be selectively activated by 1-methyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) -mediated ADH derivatization of the terminal 3-deoxy-D-manno-2-octulosonic acid (Kdo) group of dLPS, followed by EDC-mediated coupling to TT.", "Alternatively, another method for producing the instantconjugates involves cystamine derivatization of dLPS, by, for example, EDC-mediated derivatization, followed by disulfide conjugation to N-succimidyl-3-(2-pyridyldithio) propionate-derivatized protein.", "Other methods well known in the art for effecting conjugation of oligosaccharides to immunogenic carrier proteins are also within the scope of the invention.", "Such methods are described in, for example, U.S. Pat.", "Nos.", "5,153,312 and 5,204,098; EP 0 497 525; and EP 0 245 045.The molar ratio of ADH to dLPS in the reaction mixture is typically between about 10:1 and about 250:1.A molar excess of ADH is used to ensure more efficient coupling and to limit dLPS-dLPS coupling.", "In a preferred embodiment, the molar ratio is between about 50:1 and about 150:1; in a most preferred embodiment, the molar ratio is about 100:1.Similar ratios of AH-dLPS to both TT and HMP in the reaction mixture are also contemplated.", "In a preferred embodiment, one ADH per DLPS is present in the AH-dLPS conjugate.", "In another preferred embodiment, in the final dLPS-carrier protein conjugate, the molar ratio of dLPS to carrier is between about 15 and about 75, preferably between about 25 and about 50.Immunogenicity of the conjugates in both mice and rabbits is enhanced by the use of mohophbsphoryl Lipid A plus trehalose dimycolate (Ribi-700™; Ribi Immunochemical Research, Hamilton, Mont.)", "as an adjuvant.", "Although this adjuvant is not approved for use in humans, the skilled artisan will appreciate that other well known standard adjuvants may be used in the invention, including aluminum compounds (i.e.", "alum), chemically-modified lipopolysaccharide, suspensions of killed Bordetella pertussis, N-acetylmuramyl-L-alanyl-D-glutamine and other adjuvants known to one of ordinary skill in the art.", "The use of aluminum compounds is particularly preferred.", "Such adjuvants are described by Warren et. al.", "(Ann.", "Rev.", "Biochem., 4:369-388, 1986).", "The dLPS-carrier protein conjugates for parenteral administration may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.", "This suspension may be formulated according to methods well known in the art using suitable dispersing or wetting agents and suspending agents.", "The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.", "Suitable diluents include, for example, water, Ringer's solution and isotonic sodium chloride solution.", "In addition, sterile fixed oils may be employed conventionally as a solvent or suspending medium.", "For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.", "In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectable preparations.", "The conjugate vaccine of the invention may be in soluble or microparticular form, or may be incorporated into microspheres or microvesicles, including liposomes.", "Although various routes of vaccine administration including, for example, intramuscular, subcutaneous, intraperitoneal and intraarterial are contemplated, the preferred route is intramuscular administration.", "In a preferred embodiment, the dosage of the conjugate administered will range from about 10 g to about 50 μg.", "In a more preferred embodiment, the amount administered will be between about 20 μg and about 40 μg.", "In a most preferred embodiment, the amount administered is about 25 μg.", "Greater doses may be administered on the basis of body weight.", "The exact dosage can be determined by routine dose/response protocols known to one of ordinary skill in the art.", "The vaccine of the invention may be administered to warm-blooded mammals of any age and are adapted to induce active immunization in young mammals, particularly humans, against otitis media and respiratory infections caused by NTHi.", "As a childhood vaccine, the conjugate is administered at about 2 to 4 months of age.", "Typically, two booster injections of between about 10 μg and about 25 μg are administered at about 2 and again about 13 months after the initial injection.", "Alternatively, three booster injections are given at 2, 4 and 16 months after the initial injection.", "The IgG antibodies elicited by systemic administration of the conjugate vaccine will transfer to local mucosa and inactivate NTHi inoculum on mucosal surfaces (i.e., nasal passages).", "Secretory IgA will also play a role in mucosal immunity if the conjugate vaccine is administered to the mucosa (i.e.", "intranasally).", "Thus, the conjugate vaccine will prevent local, as well as systemic, NTHi infection.", "The examples describe conjugate vaccines using NTHi Rdlic1lpsA.", "Vaccines from other NTHi strains are within the scope of the present invention and are made using the same techniques.", "NTHi strain Rdlic1lpsA.", "Other clinically relevant NTHi strains contemplated as sources of dLPS for generation of a dLPS-carrier conjugate vaccine include strains 9274, 2019, 1479, 5657 and 7502 (type III, II, I, IV and V, respectively), as well as strains from the Finnish Collection referred to herein.", "These strains, as well as strain 2019, are described by Campagnari et al.", "(Infect Immun., 55:882-887, 1987), Patrick et al.", "(Infect Immun., 55:2902-2911, 1987), and Hood et al., (Mol.", "Microbiol 33:679-692, 1999), and are generally available from the research community.", "A multivalent vaccine comprising a mixture of conjugates, each having a dLPS from a different NTHi strain, is also within the scope of the invention.", "A person of ordinary skill in the art will appreciate that LPS from these other clinically relevant strains may be detoxified by removal of fatty acids therefrom, for example as described in Gu et al.", "(U.S. Pat.", "No.", "6,207,157).", "In a preferred embodiment, the DLPS moieties thus obtained are at least about 5,000 fold less toxic than LPS itself.", "In a particularly preferred embodiment, the dLPSs are at least about 10,000 fold less toxic than dLPS.", "Determination of toxicity may be performed, for example, as described in Gu et al.", "(U.S. Pat.", "No.", "6,207,157).", "Live vaccines available in the art include viral vectors such as adenoviruses, alphaviruses and poxviruses as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette-Guerin (BCG), and Streptococcus.", "Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens or not, and their use as oral vaccines are described in WO 92/11361.Preferred routes of administration include all mucosal routes; most preferably, these vectors are administered intranasally or orally.", "Other bacterial strains used as vaccines are described for Shigella flexneri in High et al., EMBO 11:1991, 1992 and Sizemore et al., Science 270:299, 1995; for Streptococcus gordonii in Medaglini et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 92:6868, 1995; and for Bacille Calmette Guerin in Flynn J. L., Cell.", "Mol.", "Biol.", "40 (suppl.", "I):31, 1994 and in WO 88/06626, WO 90/00594, WO 91/13157, WO 92/01796, and WO 92/21376.An alternative formulation utilizes the vaccine in association with agents that assist in cellular uptake.", "Examples of such agents are (i) chemicals that modifycellular permeability, such as bupivacaine (see, e.g., WO94/16737), (ii) liposomes for encapsulation of the lipopolysaccharide, the vaccine, or partially de-acylated LPS, or (iii) cationic lipids 10 or silica, gold, or tungsten microparticles which associate themselves with the lipopolysaccharides.", "Anionic and neutral liposomes are well known in the art (see,.", "e.g., Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including lipopolysaccharide-based vaccines.", "For use in a composition of the invention, in one embodiment, a lipopolysaccharide-based vaccine or derivative thereof is formulated into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune response.", "Cationic lipids are also known in the art.", "Such lipids include Lipofectin™ also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DTAP (1,2-bis(oleyloxy)-3-(trimethylammonio) propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidolbglycyl spermine) and cholesterol derivatives such as DC-Chol (3-β-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol).", "A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Pat.", "No.", "5,283,185, WO 91/15501, WO 95/26356, and U.S. Pat.", "No.", "5,527,928.The route of administration is any conventional route used in the vaccine field.", "As general guidance, a lipopolysaccharide-based vaccine of the invention is administered via a mucosal surface, e.g., an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, and urinary tract surface; or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route.", "The choice of administration route depends upon a number of parameters, such as the formulation that is selected, and the adjuvant associated with the lipopolysaccharide.", "A lipopolysaccharide-based vaccine formulated in association with bupivacaine may be advantageously administered into muscles.", "When a neutral or anionic liposome or a cationic lipid, such as DOTMA or DC-Chol, is used, the formulation can be advantageously injected via intravenous, intranasal (aerosolization), intramuscular, intradermal, and subcutaneous routes.", "A lipopolysaccharide in a naked form can advantageously be administered via the intramuscular, intradermal, or sub-cutaneous routes.", "If a mucosal adjuvant is used, the intranasal or oral route is preferred.", "If a lipid formulation or an aluminum compound is used, the parenteral route is preferred with the sub-cutaneous or intramuscular route being most preferred.", "The choice also depends upon the nature of the vaccine agent.", "Therapeutic or prophylactic efficacy of a vaccine formulation of the invention can be evaluated as described below.", "Another aspect of the invention provides (i) a composition comprising a lipopolysaccharide-based vaccine of the invention together with a diluent or carrier; specifically (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a lipopolysaccharide-based vaccine of the invention; (iii) a method for inducing an immune response against Haemophilus influenzae in a mammal, by administering to the mammal an immunogenically effective amount of a lipopolysaccharide of the invention to elicit a protective immune response to Haemophilus influenzae; and particularly, (iv) a method for preventing and/or treating a Haemophilus influenzae infection, by administering a prophylactic or therapeutic amount of a lipopolysaccharide of the invention to an infected individual.", "Additionally, the invention encompasses the use of a lipopolysaccharide-based vaccine of the invention in the preparation of a medicament for preventing and/or treating Haemophilus influenzae infection.", "As used herein, the composition of the invention contains one or several lipopolysaccharides or derivatives of the invention.", "The composition optionally contains at least one additional Haemophilus influenzae antigen, or a subunit, fragment, homolog, mutant, or derivative thereof.", "In one embodiment, the lipopolysaccharide-based vaccine, composition or treatment is free of adjuvant, specifically adjuvants commonly or specifically used in rodents.", "The lipopolysaccharide-based vaccine, composition or treatment may be used to treat disorders whose symptoms are caused or aggravated at least in part by Haemophilus influenzae infection, and includes such disorders as otitis media, respiratory infection, meningtidis and pneumonia.", "Preferred lipopolysaccharide compositions include those formulated for in vivo administration to an animal, preferably a human, to confer protection or treatment against disease caused by Haemophilus influenzae.", "Also preferred are compositions formulated as a microparticle, capsule, or liposome.", "Alternatively, the vaccine formulation may further contain an adjuvant, preferably an adjuvant appropriate for human or veterinary use and which preferably excludes rodent-specific adjuvants.", "If so, a systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable such as, e.g., QS21, which is described in U.S. Pat.", "No.", "5,057,546.A number of adjuvants are known to those skilled in the art.", "Preferred adjuvants are selected as provided below.", "In one embodiment, adjuvants other than liposomes and the like are also used and are known in the art.", "Adjuvants may protect the antigen from rapid dispersal by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system.", "An appropriate selection can conventionally be made by those skilled in the art, for example, from those described herein.", "Treatment is achieved in a single dose or repeated as necessary at intervals, as can be determined readily by one skilled in the art.", "For example, a priming dose is followed by three booster doses at weekly or monthly intervals.", "An appropriate dose depends on various parameters including the recipient (e.g., adult or infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect (e.g., protection and/or treatment), as can be determined by one skilled in the art.", "In general, a vaccine antigen of the invention is administered by a mucosal route in an amount from about 10 μg to about 500 mg, preferably from about 1 mg to about 200 mg. For the parenteral route of administration, the dose usually does not exceed about 1 mg, preferably about 100 μg.", "Adjuvants useful in any of the vaccine compositions described above are as follows.", "Adjuvants for parenteral administration include aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate.", "The antigen is precipitated with, or adsorbed onto, the aluminum compound according to standard protocols.", "Other adjuvants, such as RIBI (ImmunoChem, Hamilton, Mont.", "), are used in parenteral administration.", "Adjuvants for mucosal administration include bacterial toxins, e.g., the cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof such as a purified preparation of native cholera toxin subunit B (CTB).", "Fragments, homologs, derivatives, and fusions to any of these toxins are also suitable, provided that they retain adjuvant activity.", "Preferably, a mutant having reduced toxicity is used.", "Suitable mutants are described, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/06627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant).", "Additional LT mutants that are used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants.", "Other adjuvants, such as a bacterial monophosphoryl lipid A (MPLA) of, e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, or polylactide glycolide (PLGA) microspheres, may also be used in mucosal administration.", "Adjuvants useful for both mucosal and parenteral administrations include polyphosphazene (WO 95/02415), DC-chol (3-β-(N-(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol; U.S. Pat.", "No.", "5,283,185 and WO 96/14831) and QS-21 (WO 88/09336).", "Any pharmaceutical composition of the invention containing a lipopolysaccharide, or an antibody of the invention, is manufactured in a conventional manner.", "In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline.", "In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.", "Suitable pharmaceutical carriers or diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF (National Formulary).", "The invention also includes methods in which Haemophilus influenzae infection are treated by oral administration of a Haemophilus influenzae lipopolysaccharide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antacid, sucralfate, or a combination thereof.", "Examples of such compounds that can be administered with the vaccine antigen and the adjuvant are antibiotics, including, e.g., macrolides, tetracyclines, and derivatives thereof (specific examples of antibiotics that can be used include azithromycin or doxicyclin or immunomodulators such as cytokines or steroids).", "In addition, compounds containing more than one of the above-listed components coupled together are used.", "The invention also includes compositions for carrying out these methods, i.e., compositions containing a Haemophilus influenzae antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.", "Amounts of the above-listed compounds used in the methods and compositions of the invention are readily determined by one skilled in the art.", "Treatment/immunization schedules are also known and readily designed by one skilled in the art.", "For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen+adjuvant can be administered on days 7, 14, 21, and 28.Throughout this application, various references are referred to in parenthesis to describe more fully the state of the art to which this invention pertains.", "The disclosures of these references are hereby incorporated by reference into the present disclosure.", "Biological Deposits Certain strains of Haemophilus influenzae that are described and referred to herein have been deposited with the International Depositary Authority of Canada (IDAC) located at Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2.The deposits have been made pursuant to the provisions of the Budapest Treaty.", "The deposit information is: Haemophilus influenzae strain Rd lic1 lpsA was deposited on Aug. 25, 2001 under accession number IDAC 250801-1.Haemophilus influenzae strain 1003 lic1 lpsA was deposited on Aug. 25, 2001 under accession number IDAC 250801-2.The invention described and claimed herein is not limited in scope by the biological materials deposited, and the deposited biological material is intended only as an illustration of embodiments of the invention.", "EXAMPLES The following examples illustrate preferred embodiments of aspects of the invention, and are not intended to limit the scope of the invention.", "Example 1 Hi LPS Mutant Strains Bacterial Strains and Culture Conditions.", "The H. influenzae Rd strain was originally obtained from Alexander and Leidy (Alexander et al., J. Exp.", "Med.", "93:345-359, 1951) by Herriot.", "It was given to H. O. Smith who named the strain KW-20 and used it in the genome sequencing project (Fleischmann et al., Science 269:496-512, 1995).", "This same strain obtained from the Smith laboratory has been used by Applicant (RM118).", "The genotypes of mutants derived from this strain are listed in Table 2.H.", "influenzae strains were grown at 37° C. in brain heart infusion (BHI) broth supplemented with haemin (10 μg/ml) and NAD (2 μg/ml).", "For selection after transformation, kanamycin (10 μg/ml) was added to the growth medium.", "Escherichia coli strain DH5α was used to propagate cloned PCR products and gene constructs and was grown at 37° C. in Luria-Bertani (LB) broth supplemented with ampicillin (100 μg/ml) or kanamycin (50 μg/ml) as required (Sambrook et al.", "Molecular cloning; A laboratory manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).", "Identification of LPS Related Genes from the H. influenzae Genome Sequence.", "Putative LPS biosynthetic genes had been previously identified by an in silico search of the H. influenzae genome sequence with heterologous sequences of LPS biosynthetic genes from a wide range of organisms obtained from publicly available data bases (Hood et al.", "Mol.", "Microbiol.", "22:951-965, 1996).", "The RM118lgtF locus (HI0653) was identified by searching the Institute for Genomic Research (TIGR) H. influenzae strain Rd sequence database www.", "tigr.", "org/tdb/CMR/ghi/htmls/SplashPage.", "html for matches with the LgtF protein sequence from N. meningitidis (GenBank Accession No.", "U58765).", "Recombinant DNA Methodology, Cloning and Mutation Restriction endonucleases and DNA modifying enzymes were obtained from Boehringer Mannheim and used according to the manufacturer's instructions.", "Plasmid DNA preparation, Southern blotting and hybridization analysis were performed as described by.", "Sambrook et al., (Molecular cloning; A laboratory manual, 2nd Ed., 1989, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).", "Chromosomal DNA was prepared from Haemophilus by the method described elsewhere (High et al., Mol.", "Microbiol.", "9:1275-1282, 1993).", "Apart from lgtF, putative H. influenzae LPS biosynthetic genes were cloned and mutated as previously reported (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "For the lgtF locus, oligonucleotide primers, lgtFa (5′-TGGTGGTGGGCAAGACGC-3′) and lgtFb (5′-AGCCTGAATTCGACAGCC-3′) were designed from the strain Rd genome sequence to amplify a 1461 bp fragment including HI0653 by the polymerase chain reaction (PCR).", "PCR conditions were for 1 minute periods of denaturation (94° C.), annealing (50° C.) and polymerization (72° C.) for 30 cycles.", "1 μl of PCR product was ligated with 50 ng of plasmid pT7Blue (Novagen) and transformed into E. coli strain DH5α.", "Recombinant plasmids were prepared from transformants then confirmed by restriction endonuclease digestion and sequencing from plasmid specific primers (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "The lgtF gene was inactivated by inserting a kanamycin resistance cassette (released by digestion with EcoR1 from pUC4Kan, Pharmacia) into a MunI restriction site 257 bp inside the 5′ end of HI0653 to give plasmid pDQl.", "Construction of Mutant Strains 2-3 μg of linearised plasmid, containing mutated LPS biosynthetic genes, were used to transform H. influenzae strain RM118 by the MIV procedure (Herriot et al., J. Bacteriol.", "101:517-524, 1970) and transformants were selected on kanamycin.", "To construct strain RM118licl, RM118 was transformed with 5 μg of sheared chromosomal DNA isolated from the corresponding RM153 mutant.", "Strain RM118lic2A was constructed by transformation of RM118 with 1 μg of a PCR product including lic2A and the adjacent gene ksgA amplified from strain RM153lic2A.", "PCR used the primers L2A 5′-CTCCATATTACATAAT-3′ and L2D 5′-AAACACTTAGGCCATACG-3′ under conditions as described above.", "All transformants were checked by re-culturing on appropriate BHI/antibiotic plates then were confirmed as mutants by PCR amplification and/or Southern blotting/hybridisation of endonuclease digested chromosomal DNA.", "1psAlic1 Mutant.", "The licl locus was identified by transfer of a bank of DNA fragments cloned from strain RM7004, which expresses LPS epitopes recognised by monoclonal antibodies (mAb) 4C4, 6A2 and 12D9, into strain RM118 which naturally expresses none of these in epitopes in its LPS.", "A cloned 4.4 kb fragment of DNA conferring reactivity to mAbs 6A2 and 12D9 was sequenced and 4 open reading frames (ORFs) were identified.", "These 4 ORFs were knocked out together by deletion of a 2.7 kb ClaI/EcoRV fragment from the cloned DNA and replacement with a tetracycline resistance cassette from plasmid pHVT1.This construct was used to make a licl mutation in strain RM7004 then strain RM118 by homologous recombination after transformation.", "The lpsA gene was identified from the genome sequence of strain Rd as a homologue of a gene encodinga glycosyltransferase in Pasteurella haemolytica.", "The gene was amplified by the polymerase chain reaction (PCR) using primers designed from the Rd genome sequence.", "The PCR product was cloned into plasmid pT7Blue then the gene was disrupted by digestion with NsiI and insertion of a kanamycin resistance cassette derived from pUC4kan, by digestion with PstI.", "The resulting plasmid was linearised then used to transform strains RM118 and NTHi 1003.The transformants were checked by PCR to confirm mutants.", "The double mutant strains RM118lpsAlic1 (referred to interchangeably as RdlpsAlic1 or Rdlic1lpsA) and 1003psAlic1 (referred to interchangeably as 1003lic1lpsA) were constructed by transforming each of the RM118 and 1003 lpsA mutants with chromosomal DNA derived from the RM118lic1 mutant strain.", "The double mutant strains were confirmed by PCR analysis of, the relevant loci and the backgrounds of the strains were confirmed by restriction enzyme digestion and comparison of the patterns of the DNA fragments on gels with the relevant wild type strains.", "Strains were subsequently checked using a number of mAbs, each specific for particular LPS epitopes.", "In each case the RM118 and 1003 lpsAlic1 double mutant strains had lost reactivity to mAb TEPC-15, an antibody specific for phosphocholine, when compared to wild type.", "Phosphocholine incorporation into H. influenzae LPS is dependent upon the lic1 locus.", "The LPS derived from the mutant strains was also fractionated on polyacrylamide gels and compared to the relevant wild type LPS.", "In each case the double mutant strain had LPS which was truncated when compared to that from wild type strains.", "Analysis of Lipopolysaccharide by Immunoblotting The reactivity of wild-type and mutant strains of H. influenzae strain RM118 to LPS specific monoclonal antibodies was analyzed as described by Roche et al., (FEMS Microbiol.", "Lett.", "120:279-284, 1994).", "Analysis of Lipopolysaccharide by Electrophoresis The patterns of LPS isolated from wild type and mutant strains were determined after fractionation by tricine-SDS-polyacrylamide gel electrophoresis (T-SDS-PAGE) (Lesse et al., J. Immunolog.", "Methods 126:109-117, 1990) as described previously (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "Structural Fingerprinting of Lipopolysaccharides Cells from 10 L batch cultures (10 lots of 1 L) were harvested after overnight growth, LPS was extracted by the hot phenol-water method (Westphal et al., Meth.", "Carbohydr.", "Chem.", "5:83-91, 1965), followed by ethanol precipitation as described by Thibault and Richards, 2000).", "LPS was purified by repeated ultracentrifugation (105 000 g, 4° C., 2×5 h) and samples were analysed as their O-deacylated derivatives (LPS-OH).", "O-deacylation was achieved by mixing the LPS (1-10 mg) with anhydrous hydrazine (0.2-1.0 ml) at 37° C. for 1 h as previously described (Holst et al., Eur.", "J. Biochem.", "214:703-710, 1993) Sugars were identified by gas-liquid chromatography-mass spectrometry (GLC-MS) as their alditol acetates as previously described (Jarosik et al., Infect.", "Immun.", "62:4861-4867, 1994).", "Linkage analysis was accomplished following acetylation of the oligosaccharides with acetic anhydride (0.5 ml) and 4-dimethylaminopyridine (0.5 mg) at room temperature for 24 h. Peracetylated material was then treated with methyl iodide in dimethylsulfoxide in the presence of lithium methylsulfinylmethanide to afford the methylated oligosaccharides which were recovered using a SepPak™ C18 cartridge and subjected to sugar analysis (Blakeney et al, Carbohydr.", "Res.", "140:319-324, 1985).", "The relative proportions of the various alditol acetates and partially methylated alditol acetates obtained in sugar and methylation analyses were measured from the detector response of the GLC-MS and are uncorrected.", "GLC-MS was carried out with a Delsi Di200™ chromatograph equipped with a NERMAG R10-10H™ quadrupole mass spectrometer or with a Varian Iontrap™ system using a DB-5 fused silica capillary column (25 m×0.25 mm×0.25 gm) and a temperature gradient of 160° C. (1 min) →250° C. at 3° C./min.", "Electrospray ionization-mass spectrometry (ESI-MS) was performed on a VG Quattro™ Mass Spectrometer (Micromass, Manchester, UK) in the negative-ion mode.", "Samples were dissolved in water and then mixed in a 1:1 ratio with 50% aqueous acetonitrile containing 1% acetic acid.", "Sample solutions were injected via a syringe pump into a running solvent of H2O:CH3CN (1:1) at a flow rate of 5 μL/min.", "One-dimensional (1D) 1H NMR spectra were recorded at 500 MHz for solutions in deuterium oxide at 22° C., after several lyophylizations with D2O, on a Bruker AMX 500™ spectrometer.", "To enhance spectral resolution, perdeutero-EDTA (2 mM) and perdeutero-SDS (10 mg/ml) were added to the D2O solutions (Risberg et al., Eur.", "J. Biochem.", "243:701-707, 1997).", "Chemical shifts are referenced to the methyl proton resonance (δ; 2.225 ppm) of internal acetone.", "Analysis of Enzymatic Activity from LgtC and LgtD The enzyme encoded by lgtD was assayed with the synthetic acceptor FCHASE-Pk using capillary electrophoresis for detection of the product.", "Capillary electrophoresis was performed essentially as described previously (Wakarchuk et al., J. Biol.", "Chem.", "271:1916-1917, 1996).", "FCHASE-Pk was synthesised from FCHASE-Lac using the Neisseria meningitidis LgtC enzyme as previously described (Wakarchuk et al., Protein Eng.", "11:295-302, 1998).", "The reaction conditions were 0.5 mM acceptor, 1 mM UDP-GalNAc, 50 mM HEPES-NaOH pH 7.0, 10 mM MgCl2, 10 mM MnCl2.The extract was made by sonicating the cells, and then collecting the membrane fraction by centrifugation at 100,000 ×g for 30 minutes.", "Both RM118 and the mutant RM118:lgtD were analysed.", "The small amount of product was isolated by TLC as previously described (Wakarchuk et al., J. Biol.", "Chem.", "271:1916-1917, 1996).", "Because the conversion to product was small, some of the starting material was also isolated with it.", "The recovered mixture was divided into 2 equal parts and then treated with β-hexosaminidase as recommended by the enzyme supplier (NTEB).", "The product of the LgtD reaction was shown to have β-anomeric specificity by digestion with β-hexosaminidase.", "Activity for LgtC was below the limit of detection in extracts of RM118, so the gene was cloned into an expression vector and activity was assayed in E. coli.", "The gene was amplified by PCR (as described above) using primers lgtca 5′ GGG GGG CAT ATG GGA CGG ACT GTC AGT CAG ACA ATG and ltCb 5′ GGG GGG GTC GAC TCA TTA ATT ATC TTT TAT TCT CTT TCT TAATC The gene was then inserted into pCWori plus at the NdeI and SalI sites similar to what was described for lgtC from N. meningitidis (Wakarchuk, et al., Protein Eng.", "11:295-302, 1998).", "Crude sonicated extracts of the recombinant clone were assayed with 1 mM FCHASE-Lac, 1 mM UDP-Gal, 10 mM MnCl2, 5 mM DTT, 50 mM HEPES pH 7.5.The enzyme was shown to be unstable in E. coli and needed to be assayed within a few hours of when the extracts were made (data not shown).", "The product of the enzyme reaction was analyzed by specific glycosidase digestion, mass spectrometry and co-chromatography with authentic FCHASE-Pk (data not shown).", "Construction and Screening of Mutant Strains The set of mutants prepared as above was used to investigate in detail the genetic basis of the biosynthesis of the oligosaccharide portion of LPS from H. influenzae strain RM118, the index strain from which the complete genome sequence was derived.", "Table 2 lists the genes which have been investigated.", "The DNA constructs used to mutate the.", "majority of these genes have been previously reported (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "Each construct consisted of a plasmid vector into which a putative LPS gene, with a kanamycin resistance gene inserted within the 5′ end (first third) of the predicted reading frame, was cloned.", "There was no obvious candidate gene responsible for adding the glucose to the.", "first heptose (HepI).", "Searching the Rd genome sequence data base with the lgtF sequence from Neisseria gave a match (31% identify over 247 amino acids) to reading frame HI0653.lgtF was amplified by PCR from chromosomal DNA of strains RM118 and RM153.The cloned product from strain RM118 was inactivated by insertion of a kanamycin cassette to give plasmid pDQl and used to transform H. influenzae strains.", "lic1 and lic2A are phase variable LPS biosynthetic loci (High et al., Mol.", "Microbiol.", "9:1275-1282, 1993, Weiser, et al., Cell 59:657-665, 1989).", "lic1 has been shown to be involved in the addition of PCho groups to H. influenzae LPS (Weiser, et al.. Infect.", "Immun.", "65:943-950, 1997).", "For each locus, strain RM118 was transformed using the mutated gene constructs (10-105 transformants/μg input DNA).", "For most loci, RM118 was the source of DNA that had been cloned but in several instances DNA derived from strain RM153 was used as donor with no change in transformation efficiency.", "Genes responsible for the synthesis of Kdo, the first sugar added to lipid A (kdsA, kdsb) and the Kdo transferase (kdtA) have been identified from the genome sequence (Fleischmann et al., Science 269:496-512, 1995).", "Attempts to construct strains mutated in the kdta gene, using a variety of plasmid constructs failed to yield any transformants.", "This is similar to findings with type b strains (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996) and isassumed to be due to non-viability of this mutant.", "LPS isolated from RM118 and the isogenic mutant strains was analysed by T-SDS-PAGE (data not shown).", "Strains mutated in genes most likely encoding glycosyltransferases for RM118 oligosaccharide synthesis, and which showed an altered pattern of LPS bands when compared to wild type by T-SDS-PAGE (FIG.", "2), were selected for detailed structural analysis of their LPS as described below.", "A mutant in which the lic1 locus is inactivated was also investigated.", "Structural Characterization of LPS Analysis of LPS from strain RM118 by T-SDS-PAGE showed a heterogeneous pattern of bands corresponding in electrophoretic mobility to populations of low-molecular-mass LPS composed of lipid A and oligosaccharide components differing in the number of attached sugar residues (FIG.", "2).", "Applicant has previously shown that strain RM118 grown under similar conditions expresses populations of LPS containing three to five glycose residues attached to a common inner-core element (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "LPS from strains with mutations in licl, lgtF, and lgtD showed similar complex banding patterns, while those from strains with mutations in lgtC, lic2A, lpsA, orfH, rfaF and opsX gave less complex patterns comprising bands having consecutively faster mobilities consistent with successive sugar deletions.", "Identification of the nature and location of sugar deletions in the LPS samples from mutant strains was achieved by comparative structuralanalysis.", "LPS was extracted from isogenic mutants of strain RM118 after growth in liquid culture.", "Structural fingerprinting was done using ESI-MS and 1D 1H-NMR analysis of O-deacylated LPS (LPS-OH) samples, obtained following treatment with anhydrous hydrazine.", "In addition, glycose and linkage analyses were carried out on intact LPS samples.", "A comparison of the data obtained from mutant strains containing specifically inactivated putative glycosyltransferase genes with that from the structural model for the globotetraose containing RM118 LPS (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999) established the key structural features of the altered LPS glycoforms.", "ESI-MS provides a valuable tool for probing the structural composition of low molecular mass LPS (Gibson et al., J. Bacteriol.", "175:2702-2712, 1993; Masoud et al., Biochem.", "36:2091-2103, 1996; Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999; Risberg et al., Eur.", "J. Biochem.", "243:701-707.1997; Phillips et al., Biochem.", "35:5937-5947, 1996).", "The ESI-MS data obtained in the negative ion mode on the O-deacylated LPS samples is presented in Table 5.The LPS-OH samples from the mutant strains gave data consistent with the presence of oligosaccharides linked via Kdo-4-phosphate to a common O-deacylated lipid A moiety differing in the number of heptose, hexose and phosphate-containing.", "substituents (Phillips et al., Biochem.", "31:4515-4526, 1992; Masoud et al., Biochem.", "36:2091-2103, 1996; Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999; Schweda et al., Carbohydr.", "Res.", "246:319-330, 1993; Schweda et al., Carbohydr.", "Res.", "272:213-224, 1995; Risberg et al., Eur.", "J. Biochem.", "243:701-707, 1997; Phillips et al., Biochem.", "35:5937-5947, 1996).", "opsX Mutant.", "Inactivation of opsx gave rise to deep-rough LPS, which was devoid of heptose or hexose residues, containing only a phosphorylated Kdo attached to the Lipid A moiety (Table 5).", "It has been previously shown that mutation in the opsX gene of H. influenzae type b strain RM153 results in truncation of the LPS between HepI and Kdo (Hood et al., Mol.", "Microbiol.", "22:951-965, 1996).", "MS-MS analysis of that LPS-OH sample by low energy collisional activation of the doubly charged molecular ion (m/z 625) afforded a major fragment ion at m/z 951 (Lipid A-OH) arising from cleavage of the Kdo-β-D-glucosamine bond (data not shown).", "The mass of this fragment ion is consistent with that expected for H. influenzae Lipid A-OH (Helander et al., Eur.", "J. Biochem.", "177:483-492, 1988).", "It is evident that RM118 and RM153 opsX mutants express LPS similar to that from the previously characterized.", "Rdisn (I69) mutant strain (Helander et al., Eur.", "J. Biochem.", "177:483-492, 1988; Preston et al., J. Bacteriol.", "178:396-402, 1996).", "The I69 LPS phenotype arises from a mutation in the heptose biosynthetic gene gmha (Brook et al., J. Bacteriol.", "178:3339-3341, 1996), rendering the mutant strain incapable of adding heptose to its LPS.", "rfaF Mutant.", "1H NMR analysis of LPS-OH from RM118rfaF showed, in addition to the expected 1H resonance from the α-linked glucosamine residue of lipid A, an anomeric proton-resonance (˜5.19 ppm) in the low-field region from a single heptose unit.", "Sugar analysis confirmed the Hep residue.", "to be L-glycero-D-manno heptose.", "Correspondingly, the ESI-MS spectrum was dominated by a single abundant doubly charged ion at m/z 721.6 consistent with the structure, Hep1-Kdo-Lipid A-OH (Table 5).", "orfH Mutant.", "Organisms in which the orfH gene is inactivated gave a mixture of LPS glycoforms, each containing two Hep residues, as evidenced from the ESI-MS data (Table 5).", "In addition to the major population of glycoforms containing an additional Hep residue, ie, Hep2•PEtn0-2•Kdo-Lipid A-OH, compared to RM118rfaF LPS, were species containing a Hex-PCho unit.", "Sugar analysis indicated the presence of D-glucose and the PCho methyl protons gave an intense signal in the 1H NTMR at 3.24 ppm.", "As expected, LPS from this strain reacted with TEPC-15, a PCho specific monoclonal antibody (Mab) (W,Teiser et al., Infect.", "Immun.", "65:943-950, 1997), in immunoblot experiments.", "Linkage analysis revealed the presence of terminal Hep, 3-substituted Hep and 3,4-disubsituted Hep residues (Table 6).", "Based on the structure of the parent strain, this data is consistent with RM118orfH having the capacity to elaborate LPS expressing the two major glycoform structures 1 and 2 (PEtn shows partial substitution).", "The occurrence of two bands for the LPS of RM118orfH when analysed by T-SDS-PAGE (FIG.", "2) is consistent with this conclusion.", "1psA Mutant.", "ESI-MS analysis of LPS-OH from RM118lpsA indicated it to contain glycoforms having an additional Hep residue when compared to RM118orfH (Table 5), the phosphocholine containing Hexl glycoform being the major LPS species (FIG.", "3).", "Linkage analysis was consistent with sequential addition of the heptose to the terminal Hep in structure 2 (Table 6).", "Correspondingly, the 1H NMR spectrum of this O-deacylated LPS sample showed the characteristic pattern in the low-field region (5.0-6.0 ppm) for the LPS tri-heptose inner-core element (HepII, 5.76 ppm; HepI/HepIII, 5.16/5.15 ppm) of H. influenzae (FIG.", "4) (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "This data is consistent with the RM1181psA-derived LPS having the structure 3 (FIG.", "3; Table 1).", "1ic2A Mutant.", "ESI-MS analysis of the O-deacylated LPS samples from strain RM118lic2A revealed the presence of Hex2 glycoforms as the major LPS species (Table 5).", "Compositional analysis of the RM118lic2A LPS indicated the presence of D-glucose as the only neutral hexose, linkage analysis indicating it to be a terminal residue (Table 6).", "A significant proportion of 2-linked heptose residues was also revealed by linkage analysis.", "It is noteworthy, that 2-subsituted heptose residues were not detected in the LPS sample from the lpsA mutant due to substitution of that residue by PEtn groups (cf.", "Structure 3 in FIG.", "3) which are not readily cleaved under the hydrolysis conditions employed in the linkage analysis procedure.", "In accord with these findings, it can be concluded that LPS from the lic2A mutant differs from.", "that of the lpsA mutant in that it carries a glucose residue at the 2-position of HepIII as shown in structure 4 (Table 1).", "The presence of an additional 1H NMR signal at 4.65 ppm indicated the terminal D-Glcp to have the β-configuration, the upfield shifted value of the resonance for HepII (5.58 ppm) compared to that of the unsubstituted analogue, (5.76-ppm) being indicative of the 1,2-linkage to HepIII (structure 4; Table 1) (Masoud et al., Biochem.", "36:2091-2103, 1996, Schweda et al., Carbohydr.", "Res.", "246:319-330, 1993; Schweda et al, Carbohydr.", "Res.", "272:213-224, 1995).", "Lic 3A Mutant.", "Similarly, a mutant was generated which permitted investigation of sialylated glycoforms.", "These studies confirmed the loss of sialic acid in the relevant lic3A mutant.", "The preparation of the mutant is described in Hood et al., Mol.", "Microbio.", "39:341-350, 2001.The sialylated glycoform was found to be absent in strains containing a lic3A gene disruption.", "Lic3A has been demonstrated to have a sialyltransferase activity, which is modified by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety.", "lgtc Mutant.", "In the RM118lgtC mutant, ESI-MS analysis of the O-deacylated LPS sample revealed the presence of Hex3 glycoforms.", "Sugar analysis indicated LPS from the lgtC mutant to contain D-galactose, which by linkage analysis was found to be present as a terminal residue (Table 6).", "Linkage analysis also revealed 4-linked D-Glcp residues consistent with the major Hex3 glycoform (Table 6) being substituted by a lactose moiety at HepIII (structure 5: Table 1).", "The 1H NMR spectrum of the LPS-OH is identical to that previously reported (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999) for the lactose containing Hex3 LPS; glycoform which is present in the parent strain.", "In N. meningitidis, the lgtC gene has been shown to encode a 1,4-α-galactosyltransferase (Wakarchuk et al., Protein Eng.", "11:295-302, 1998).", "A similar function was demonstrated for lgtC from RM118 by examination of the transferase activity of the recombinant enzyme and by analysis of the LPS from the RM118lgtC mutant.", "lgtD Mutant.", "A mixture of Hex3 and Hex4 LPS glycoforms were elaborated by H. influenzae RM118 in which the lgtD gene is inactivated (Table 5).", "In accord, two bands were observed upon T-SDS-PAGE analysis of the LPS, one corresponding in electrophoretic mobility to that from the lgtC mutant and a slower migrating band (FIG.", "2).", "LPS from this mutant strain contained terminal and 4-linked D-Galp residues (Table 6).", "A comparison of the 1D 1H NMR spectra with those of the parent strain and its lgtC mutant, pointed to the presence of an α-D-Galp-(1→4)-β-D-Galp unit in the Hex4 glycoform, a signal at 5.01 ppm being indicative of the terminal α-D-Galp residue (structure 6; Table 1).", "The lgtd gene product was investigated for glycosyltransferase activity with the synthetic acceptor, FCHASE-PK.", "A comparison of the parent RM118 and the lgtD mutant strains in the CE assay showed loss of β-GalpNAc transferase activity in the mutant (FIG.", "5).", "1gtF Mutant.", "Mutation of the lgtF gene in H. influenzae gave a strain from which the LPS neither reacted with MAb TEPC-15 (data not shown) nor showed the characteristic PCho methyl proton signal (3.24 ppm) in their 1H NMR spectrum.", "Linkage analysis indicated that the LPS lacked the terminal β-D-Glcp residue, containing only mono-3-substituted HepI residues (Table 6).", "A similar distribution of glycoforms, as found in the parent strain LPS, differing in the length of the oligosaccharide chain from HepIII was observed for LPS-OH from the lgtF mutant in its ESI-MS (Table 5).", "It is noteworthy that full extension of the globotetraose unit from HepIII can occur in the absence or the presence of the β-D-Glcp residue at HepI (FIG.", "6).", "Applicant has shown that the parent -strain can elaborate mixed populations of LPS glycoforms in which the galabiose unit is elongated by the addition of a terminal β-D-GalpNAc residue giving the globotraose units β-D-GalpNAc-(1→3)-α-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Glcp (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "lic1 Mutant.", "ESI-MS analysis of O-deacylated LPS from the lic1 mutant gave a similar heterogenous mixture of glycoforms (Table 5) as that observed in the parent strain (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999), but lacking the presence of PCho substituents.", "The licd locus has been shown to contain genes responsible for expression of PCho substituents at the 6-position of the β-D-Glcp attached to the 3,4-disubstituted heptose (HepI) in RM118 (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999; Weiser et al., Infect.", "Immun.", "65:943-950, 1997).", "Examination of the 1H-NMR spectrum of RM118lic1 O-deacylated LPS revealed the absence of the characteristic PCho methyl proton signal at 3.24 ppm.", "Additionally the LPS from this licd mutant, as expected (Weiser et al., Infect.", "Immun.", "65:943-950, 1997), did not react with Mab TEPC-15 (data not shown).", "Example 2 Substitutions and Modifications of Inner Core Moiety In NTHi strain 2019, HepI is substituted by lactose (R1=β-D-Galp-(1→4)-β-D-Glcp; R2/R3=H).", "(Phillips et al., Biochemistry 31:4515-4526, 1992).", "In NTHi strain 375, HepIII is substituted by sialylated lactose in a minor glycoform population (R1=β-D-Glcp, R2=H, R3=NeuSAc-β-D-Galp-(1→4)-β-D-Glcp-(1→2)) (Hood et al., Mol.", "Micro.", "33:679-692, 1999).", "This example summarizes the results of structural investigations of LPS from 4 NTHi strains by using methylation analysis, electrospray ionization mass spectrometry (ESI-MS) and NMR.", "H. influenzae non-typeable strains 486, 176, 285 and 1159 were obtained from the strain collection of Prof. Eskola (Hood et al., Mol.", "Microb.", "1999) as part of a Finnish otitis media cohort study and are isolates obtained from the inner ear.", "Bacteria were grown in brain heart infusion (BHI) broth (Difco™ (3.7%, w/v) containing nicotinamide adenine dinucleotide (NAD) (2 μg/mL), hemin (10 μg/mL) and neuraminic acid (NeuAc; 50 μg/mL) at 37° C. LPS was obtained from lyophilized bacteria by using the phenol-chloroform-petroleum ether method, followed by ultracentrifugation (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "Detailed structural studies were made through the use of MS-based methods and high-field NMR techniques on oligosaccharide samples.", "obtained from LPS samples either by O-deacylation with anhydrous hydrazine (37° C., 1 h) or cleavage of the Kdo ketosidic linkage with dilute aqueous acetic acid (100° C., 2 h).", "Compositional sugar analyses of LPS from NTHi strains 486, 176, 285 and 1158 all indicated D-glucose (Glc), 2-amino-2-deoxy-D-glucose (GlcN) and L-glycero-D-manno-heptose (Hep) as major components identified by GLC-MS of their corresponding alditol acetate-and 2-butyl glycoside derivatives.", "In addition, D-galactose (Gal) was identified in strains 486, 176 and 1158.Furthermore, D-glycero-D-manno-heptose was found to be a major component in strain 1158.This sugar was recently identified for the first time as a component in H. influenzae LPS in NTHi strain 9274 (Rahman et al., Glycobiology 9:1371-1380, 1999).", "Treatment of O-deacylated LPS (LPS-OH) with neuraminidase followed by high-performance anion-exchange chromatography and ESI-MS indicated sialic acid (NeuAc).", "to be a major component in NTHI strain 486.Minor amounts.", "were detected in NTHi strains 176, 285 and 1158.ESI-MS of LPS-OH samples indicated heterogeneous mixtures of glycoforms in all strains.", "Part of the ESI-MS spectrum of LPS-OH from NTHi 486 is shown in FIG.", "10.Proposed compositions for the major LPS glycoforms of the NTHi strains are shown in Table 7.All glycoforms contain the conserved PEtn-substituted triheptosyl inner-core moiety attached via a phosphorylated Kdo linker to the putative O-deacylated lipid A (Lipid A-OH).", "PCho is present in all strains as a major component.", "The presence of two PCho in a Hex2-glycoform is indicated for strain 1158.The shortest glycoform (Hexl) is observed for strain 285.Methylation analyses data of the LPS samples is given in Table 8.It is noteworthy that only traces of 2-substituted Hep, an integral component of previously published H. influenzae structures, was detectable in strains 176 and 486.Instead, significant amounts of 3-substituted Hep were detected pointing to a new substitution pattern in the common L-α-D-Hepp-(1→2)-L-α-D-Hepp-(1→3)-[β-D-Glcp-(1→4)-]-L-α-D-Hepp-(1→5)-α-Kdop inner-core element of H. influenzae LPS.", "The complete structures for the major glycoforms in strains 176 and 486 were determined by NMR and the results are summerized in FIG.", "11.Methylation analysis of strain 285 revealed, inter alia, high amounts of terminal L,D-Hep in addition to 3,4-disubstituted Hep which indicated an absence of substitution at HepII and HepIII of the triheptosyl inner-core moiety (i.e.,R2 and R3=H).", "The complete structure of the Hex1 glycoform in this strain (FIG.", "12) was determined by NMR on LPS-OH (data not shown).", "Methylation analysis of strain 1158 showed, inter alia, 6-substituted D-Glc and terminal D,D-Hep which could be attributed to the structural element, D-α-D-Hepp-(1→6)-β-D-Glcp-(1→4)-L-α-D-Hepp which is evidenced by NMR (data not shown).", "The major LPS glycoform was found to be substituted at HepII (R2=α-D-Glcp) and HepIII (R3=PCho→6)-β-D-Glcp).", "A tentative structure of the major glycoform in NTHi 1158 is given in FIG.", "13.Structural Details of NTHi 176.Partial acid hydrolysis of LPS with dilute acetic acid afforded an insoluble lipid A and core oligosaccharide fractions which were separated by GPC (FIG.", "14) giving Fr.1, Fr.2 and Fr.3.The methylation analysis data for these fractions are presented in Table 8.The presence of 3- and 6-substituted Gal and 4-substituted GlcN in Fr.1 indicated the presence of novel structural features for his oligosaccharide.", "CE-ESI-MS on Fr.1 revealed, inter alia, major doubly charged ion at m/z 1214.0 indicating a considerably higher molecular weight glycoform (HMG) in this fraction than observed in Fr.2 and Fr.3.A similar observation was made for NTHi 285 (see below).", "The ESI-MS spectrum of Fr.2 (data not shown) indicated the major glycoforms to have the composition PCho1•Hex2•Hep3•PEtn•AnKdo-ol and PCho1•Hex3•Hep3•PEtn•AnKdo-ol The ESI-MS for Fr.3 (data not shown) indicated major glycoforms with the respective compositions Hex3•Hep3•PEtn•AnKdo-ol and Hex4•Hep3•PEtn•AnKdo-l The structure of the Hex3 glycoform in Fr.2 could be determined by detailed 1H-NMR analysis.", "The 1H NMR spectrum of Fr.2 is shown in FIG.", "11a.", "The characteristic signal for the methyl groups of PCho was observed at δ 3.24.Anomeric resonances of HepI-HepIII were identified at 5.03-5.13, 5.83 and 5.26, respectively.", "Subspectra corresponding to Glc I, Glc II and Gal were identified in the 2D COSY and TOCSY spectra at δ 4.56, 4.62 and 4.64, respectively.", "Chemical shift data point to Glc II and Gal being terminal residues in agreement with methylation analyses.", "The down field shifted value for H-6,6′ of Glc I at δ 4.3 indicated this residue to be substituted with PCho at this position.", "Interresidue NOE connectivities between proton pairs Glc II H-1/Glc I H-4, Glc I H-1/Hep I H-4,6 (FIG.", "15) established the sequence of a disaccharide unit and its attachment point to HepI as β-D-Glcp-(1→4)-[PCho→]-β-D-Glcp-(1→4)-L-α-D-Hepp-(1→.", "Interresidue NOE between H-1 of Gal and H-3/H-2 of HepIII gave evidence for the β-D-Galp-(1→3)-L-α-D-Hepp-(1→unit.", "From the combined data it could thus be concluded that the PCho substituted Hex3 glycoform has the structure numbered 2 in FIG.", "16.Methylation analysis of Fr.2 showed a considerable amount of terminal Hep and it could be concluded that the PCho substituted Hex2 glycoform observed in ESI-MS spectra has the structure numbered 3 in FIG.", "16.The 1H-NMR spectrum of Fr.3 is shown in FIG.", "11b.", "In agreement with ESI-MS data it is evident that PCho is not expressed to the high degree as in Fr.2 since the signal for methyl protons of PCho is of low intensity.", "Signals for anomeric protons of HepI-III resonate at approximately identical chemical shifts as the corresponding ones in Fr.2.At δ 5.26 the anomeric resonance of an α-linked terminal Glc residue (Glc-III) is observed.", "The anomeric region for β-linked hexoses was more heterogeneous than in Fr.2.In 2D spectra spin systems for terminal Glc residues were observed at δ 4.50 and 4.45.A 4-substituted Glc and two terminal Gal residues were identified at δ 4.56, 4.61 and 4.54.NOE data confirmed the presence of the structural element Glcp-(1→4)-β-D-Glcp-(1→4)-L-α-D-Hepp-(1→ .", ".", ".", "as well as Gal linked to HepIII.", "In addition, NOE connectivities confirmed the α-linked Glc to be attached to the 3-position of HepII.", "The combined evidence leads to the structure of the Hex4 glycoform as shown in the structure numbered 4 in FIG.", "16.In agreement with methylation analysis showing 2-substituted Hep and terminal Hep, the Hex3 glycoforms are concluded to have the structures numbered 5 and 6 in FIG.", "16.Structural Details of NTHi 486.The structure of the major LPS of glycoform NTHi 486 was established following extensive use of ESI-MS, NMR and methylation analysis on LPS-OH and oligosaccharide material (OS-1) obtained after mild acid hydrolysis (FIG.", "17).", "As for strain 176, HepIII is substituted at the O-3 position, However in this case, by a glucose residue.", "NTHi 486 LPS is highly sialylated with NeuSAc linked to the terminal β-galactose of a lactose moiety.", "In the 1H NMR spectrum of LPS-OH, five discrete signals of approximately equal area were observed between δ 5.8 and 5.0.Three of these signals corresponded to the H-1 signals of the three heptose residues (HepI-HepIII) in the inner-core region.", "The anomeric signal of an α-linked glucose residue (Glc I) was identified at δ 5.28 (L 3.8 Hz), anomeric signals corresponding to β-linked hexoses were identified between δ 4.52 and 4.42.In the 1H NMR spectrum of OS-1, anomeric resonances of the heptoses as well as one acetylation site were observed at δ 5.83-5.75 (1H, not resolved) and δ 5.14-5.04 (3H, not resolved).", "An intense signal corresponding to the methyl protons of one O-acetyl group was observed at δ 2.17, which correlated to a 13C signal at δ 21.0 in the HSQC-spectrum.", "The sequence of the glycoses within the inner-core region was established from transglycosidic NOE connectivities relating anomeric and aglyconic protons on adjacent residues.", "Signals for the methyl protons of PCho were observed at δ 3.21 (LPS-OH) and δ 3.23 (OS-1) and spin-systems for ethylene protons from this residue and from PEtn were similar to those observed earlier (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999).", "1H-31P NMR correlation studies of LPS-OH and OS-1 demonstrated PCho and PEtn to be linked to Glc I and HepII residues, respectively.", "Characteristic signals from the H-3 methylene protons of sialic acid were.", "observed at δ 1.79 (H-3ax, J3ax,3eq=12.3 Hz) and (δ 2.73 (H3eq, J3eq,4=4.3 Hz) in the 1H NMR spectrum of LPS-OH.", "Interresidue NOE between H-3ax of Neu5Ac and H-3 of a Gal residue confirmed the sialic acid to be 2,3-linked to galactose as indicated by the methylation analyses, α-D-NeuSAc-(2→3)-β-D-Galp-(1→.", "Several chemical shift values for a number of nuclei in HepIII of OS-1 differed considerably from the corresponding chemical shifts in LPS-OH.", "Downfield shifts were obtained for H-2 (+0.99 ppm), H-1 (+0.03 ppm), H-3 (+0.22 ppm) and C-2 (+1.2 ppm) of HepIII in OS-1, while C-1 (−3.3 ppm) and C-3 (−2.5 ppm) were shifted upfield.", "Thus, it was indicated that HepIII was acetylated at the O-2-position.", "The acetylation site was further supported by the HMBC experiment, where a correlation was seen between the carbonyl carbon (δ 174.0) and H-2 of HepIII.", "In addition, a crosspeak was observed.", "between the carbonyl carbon and the methyl protons of the O-acetyl group, thereby establishing the identity of the substituent.", "ESI-MS and methylation analysis of LPS from the lpsA mutant of NTHi 486 indicated an absence of chain extension from HepIII.", "Methylation analysis of O-deacylated material showed terminal Glc, terminal Hep, 3,4-disubstituted Hep, 2,3-disubstituted Hep and 6-substituted GlcN in the relative proportions 34:39:20:3:4.In the ESI-MS spectrum of LPS-OH, two major triply charged ions at m/z 812.9/853.9 could be seen, corresponding to PCho•Hex2•Hep3•PEtn1-2•P1•Kdo1•Lipid A-OH.", "High Molecular Weight Glycoforms in VTHi 176 and 285.After mild acid hydrolysis on LPS from NTIHi 285, gel filtration showed a minor fraction containing a higher molecular weight glycoform together with a main fraction which contained the glycoform shown in FIG.", "12.Sugar analysis of this high molecular (HMW) fraction indicated D-Glc, D-Gal, D-GlcNAc and L,D-Hep and methylation analysis gave terminal Gal, 3-substituted Gal, 6-substituted Gal, 4-substituted GlcNAc, terminal Hep and 3,4-disubstituted Hep.", "CE-ESI-MS showed, inter alia, a major doubly charged ion at m/z 1052.MS/MS on this ion gave a similar fragmentation pattern as m/z 1214 observed for HMW of NTHi 176 (see above).", "In particular a daughter ion was observed at m/z 692 corresponding to a composition PchoHex-Hex-HexNAc.", "Example 3 Preparation of Monoclonal Antibodies (MAb) to Heamophilus influenzae Rd lic1lpsA Double mutant Female BALB/c mice, 6-8 weeks of age, were immunised intraperitoneally with formalin-killed Rd licllpsA whole cells.", "Each mouse received 108 cells in 0.5 ml PBS per injection.", "The mice were boosted on day 14, 35 and trial bled on day 45.The two mice showing the highest antibody titre to the homologous LPS were given two final injections on day 56, an ip injection as given previously and an intravenous injection in 0.1 ml PBS.", "The fusion was performed three days following the last injection.", "Stimulated spleen cells from the two immunised mice were fused with SP2/O-Ag14 myeloma cells in a ratio of 10:1 in 33% PEG1450.Putative hybrids surviving the hypoxanthine/aminopterin/thymidine (HAT) selection, were screened by ELISA against homologous Rd lic1lpsA LPS and heterologous Rd LPS.", "Hybridomas producing antibody of interest were cloned twice using limiting dilution to ensure stability and clonality.", "Ig subclass was determined on spent supernatant using an EIA mouse MAb isotyping kit (Amersham Canada, Oakville, ON).", "Clones were expanded as ascitic fluid by intraperitoneal injection of 106 hybridoma cells in BALB/c mice 10-14 days following ip treatment with 0.5 ml 2,6,10,14-tetramethyl-pentadecane (pristane).", "Ascitic fluid was tapped 7-14 days post-injection.", "Indirect ELISA.", "Culture supernatant and ascitic fluid were assayed against purified LPS in 96-well Nunc Maxisorp EIA plates.", "Wells were coated at 37° C. for 3 h with 1.0 μg LPS in 100 μl 0.05M carbonate buffer (pH 9.8) containing 0.02 M MgCl2 and then blocked with 200 μl 1% BSA-PBS for 1 h at room temperature.", "Following washing with PES-T (0.05% Tween 20), samples of culture supernatant or ascites, serially diluted in 1% BSA-PBS, were added and incubated 1-3 h at room temperature.", "The plates were then washed and alkaline phosphatase-labelled goat anti-mouse IgG (Cedarlane Laboratories, Hornby, ON), diluted 1:3000 in 1% BSA-PBS, was added for 1 h at room temperature..", "The plates were developed with p-NPP Phosphate Substrate System (Kirkegaard & Perry Laboratories, Gaithersberg, Md.).", "After 30-60 minutes, the plates were scanned at 410 nm in a Dynatech EIA plate reader.", "Results.", "Immunisation of BALB/c mice with formalin-killed whole cells of Rd lic1lpsA, fusion, and initial ELISA screening against the homologous LPS and heterologous Rd LPS resulted in the establishment of 12 hybridomas.", "Following testing of the 12 MAbs against a panel of Rd mutant LPS, two MAbs, LLA5, IgG2a and LLA4, IgG2b, were chosen for further testing.", "LLAS was found to cross react with 14 out of 25 non-typable strain LPS whereas LLA4 recognised the homologous LPS.", "Structural analysis revealed that LLA5 was detecting high molecular weight glycoforms while LLA4 bound to an inner core epitope present in the homologous strain.", "Example 4 Preparation of the Hi Rdlic1lpsA LPS-OH-BSA Conjugate LPS from H. influenzae double mutant strain Rdlic1lpsA was isolated and purified by the phenol water extraction protocol and O-deacylated by treatment with anhydrous hydrazine according to the procedures described earlier.", "Sugar analysis indicated the oligosaccharide portion of the LPS-OH to contain L-glycero-D-manno-heptose and glucose as the only detectable aldoses.", "ESI-MS of the LPS-OH revealed a doubly charged ion at m/z 1056.5 corresponding to a molecular mass of 2114.9 Da which is consistent with the expected composition of Glc(HepIII•PEtn•Kdo•P-Lipid A-OH(1).", "1H NMR clearly showed well defined anomeric signals from the heptose, residues at 5.16 (HepI), 5.15 (HepIII) and 5.76 ppm (HepII) for the conserved triheptosyl moiety similar to that of LPS-OH observed for the RdlpsA single mutant.", "No signal due to Pcho methyl resonances (Risberg et al., Eur.", "J. Biochem.", "261:171-180, 1999) was detectable in the 1H NMR.", "Example 5 Immunisation with Carbohydrate(CHO)-BSA Conjugate Followed by, Non-Conjugated CHO The LPS-OH from the previous example can be conjugated to BSA according to the procedure described in Gu et al., Infect.", "Immun.", "64:4047-4053, 1996.Alternatively, it 20 can be conjugated to BSA via the Kdo carboxyl group with M2C2H (4(4-N-maleimidomethyl) cyclohexane 1-carboxyl hydrazide ½ dioxane) according to the procedure developed by Wei Zou of National Research Council of Canada as described in FIG.", "8.In summary, LPS-OH was conjugated to BSA via selective activation of the Kdo carboxyl group with EDC and the use of the linker strategy shown in the above scheme.", "Female BALB/c mice, 6-8 weeks of age, were immunised intraperitoneally with Rd liclipsA odA LPS-BSA conjugate.", "Each mouse received 2 μg of carbohydrate in 0.2 ml Ribis complete adjuvant (Cedarlane Laboratories Ltd, Hornby, ON) per injection.", "The mice were boosted on day 21 and 42 with an equivalent amount of conjugated vaccine.", "On day 137 the mice received a final ip injection containing 10 μg 1003 lic1lpsA odA LPS in Ribi and serum was collected on day 147.Example 6 Preparation of Monoclonal Antibodies LLA4 and LLA5 to Haemophilus influenzae Strains Strains with defined mutations in the LPS biosynthetic machinery can be used to produce monoclonal antibodies (Mabs) to defined LPS oligosaccharide structures.", "Murine Mabs were raised against H. influenzae mutant strains containing the conserved inner-core moiety.", "For example, BALB/c mice were immunized with formalin-killed whole cells of Rd lic1lpsA.", "Initial ELISA screening against the homologous LPS and heterologous Rd LPS resulted in the establishment of 12 hybridomas.", "Following testing of the 12 MAbs against a panel of Rd mutant LPS, two MAbs, LLA5, IgG2a and LLA4, IgG2b, were chosen for further testing.", "LLA4 was found to recognize an inner core epitope present in the homologous strain.", "In ELISA testing, LLA5 showed cross reactivity with purified LPS from a majority strain from a genetically diverse culture collection (14 out of 25 non-typeable strain LPS) (Table 3).", "The epitope recognized by Mab LLA5 was present in the RdlpsA single mutant and in the more truncated RdliclorfH double mutant which lacks the HepIII residue (Table 4).", "The LLA5 epitope is not present in LPS from the single mutant, RdlgtF.", "As mentioned above the lgtF gene is required for addition of the β-D-Glc residue to the 4-position of HepI.", "Surprisingly, a comparison of the LPS from the 25 strains by structural analysis techniques known in the art indicated that Mab LLA5 was detecting a lacto-N-neotetraose (LNnT) -containing oligosaccharide epitope arising from chain extension from the HepI unit of the inner core moiety.", "Strains that were not recognized by Mab LLAS were characterized by the absense of the LNnT-containing chain extensions.", "H. influenzae strains that express the LNnT-containing chain extension only express it to a minor extent under standard laboratory growth conditions.", "It was not previously identified in strain Rd due to low expression levels (Risberg et al., Eur J. Biochem.", "261:717-180, 1999).", "It has now been isolated and characterized by liberating the core oligosaccharide from the LPS of strain Rd (RM 118) by mild acid hydrolysis and separation on gel permeation chromatography on Biogel P-4 in the same fashion as in FIG.", "14, procedures known to those skilled in the art.", "The LNnT-containing core oligosaccharide fraction elutes as a minor high molecular weight component labeled ‘HMW-fraction’.", "This is a minor component compared to the larger peak (centered at 400) which comprised the major glycoforms identified for strain Rd (Risberg et al., Eur J. Biochem.", "261:717-180, 1999).", "The HMW core oligosaccharide from strain Rd was characterized as having LNnT-containg chain extensions from HepI by tandem mass spectrometry (MS/MS) techniques (Thibault et al., Methods in Molecular Biology, Vol 145,: Bacterial toxins: Methods and Protocols (Holst, O., ed.)", "pp 327-344, Humana Press, 1999 and references therein).", "The fragmentation pattern in the ESI-MS/MS was indicative of the presence of the LNnT oligosaccharide chain extension having the sequence Gal-GlcNAc-Gal-Glc and capped by the terminal sugar unit, PEtn-GalNAc (FIG.", "1).", "When strain Rd is grown in medium containing sialic acid, LPS glycoforms containing the sialylated oligosaccharides are expressed.", "Applicant has found sialalyl lactose having the structure α-Neu5Ac-(2→3)-β-D-Galp- (1→4)-β-D-Glc attached as an oligosaccharide extention from HepIII.", "Furthermore this oligosaccharide extention has been identified in several NTHi strains.", "The phase-variable gene, lic3A encodes the sialyltransferase that adds CMP-NeuSAc to the lactose acceptor.", "Sialylated analogues of LNnT chain extentions are detectable in the LPS in strain Rd when the organism is grown under the conditions described.", "Sialylated LNnT oligosaccharide chain extentions from the inner-core LPS having the structure α-Neu5Ac-(2→3)-β-D-Galp-(1→4)-β-D-GlcpNAc-(1→3)-β-D-Galp-(1→4)-β-D-Glcp are readily detectable in the RdlgtClic3A double mutant by ES-MS of O-deacylated LPS.", "Applicant has confirmed the structure of LPS in the conserved inner core has sialylated LNnT-containing chain extensions using MS/MS techniques, high field nuclear magnetic resonance techniques, and methylation analysis, all of which are procedures of structural analysis known to those skilled in the art.", "Applicant has found that H. influenzae utilizes a block mechanism for adding the LNnT-containing oligosaccharide chain extensions which involves genes from the rfb locus.", "There is an absolute correlation between the presence of the rfb gene and LNnT formation in all Hi strains investigated.", "LPS from the NTHi stains that are reactive with Mab LLA5 also show a high molecular weight (HMW) fraction in the gel permeation chromatograms after mild acid hydrolysis.", "For example, LPS from NTHi strain 285 shows the minor HMW fraction for which the presence of LNnT-containing chain extensions, also capped by a Petn-GalNAc unit, are Characterized from the MS/MS fragmentation pattern in the same fashion as shown in FIG.", "1.Further confirmation for this LNnT-containing oligosaccharide chain extension was obtained from an MS/MS/MS experiment.", "No detectable HMW fraction is found in the mild acid hydrolysates of strains that are LLA5-nonreactive, for example NTHi strain 1247.TABLE 1 Structures of the major LPS glycoforms of increasing oligosaccharide chain length from HepIII in mutant stains of H. influenzae RM118.Structure Glycoform Mutant Strain(s) Substitution Pattern (R3) 3 Hex1 LpsA H 4 Hex2 lic2A β-D-Glcp 5 Hex3 lgtC,lgtD β-D-Galp-(1 → 4)-β-D-Glcp 6 Hex4 LgtD α-D-Galp-(1 → 4)-β-D-Gal-(1 → 4)-β-D-Glc TABLE 2 LPS-related genes investigated in strain RM118 in this study.", "HI numbers are the ORF designations given by TIGR for the H. influenzae genome sequence data base.", "The genes given in bold are those selected for detailed comparative analysis of the expressed LPS glycoforms.", "Gene HI number Reference kdtA 0652 1 lgtC 0259 1 lgtD 1578 1 lpsA 0765 1 orfZ 0260.1 1 opsX 0261 1 orfH 0523 1 rfaF 1105 1 lic2A 0550 2 lic1 1537-1540 3 lgtF 0653 4 cld 0866 1 galU 0812 1 kfiC 0868 1 lsg1 0867 1 orfM 0260 1 orfE 0869 1 orfO 0870 1 orfY 0871 1 pgmB 0740 1 pgmC 1337 1 rfe 1716 1 rfbP 0872 1 rfbB 0873 1 1.Hood et al., Mol.", "Microbiol.", "22:951-965, 1996 2.High et al, Mol.", "Microbiol.", "9:1275-1282, 1993 3.Weiser et al., J. Bacteriol.", "172:3304-3309, 1990 4.Applicant's study herein TABLE 3 Rd lic1lpsA odA-BSA plus 1003 lic1lpsA odA LPS Day 147 sera & LLA5 MAb vs Haemophilus mutant & non-typable strain LPS EIA OD410 Strains serum #1 serum #2 LLA5 1003 lic1lpsA 2.329 1.575 — odA Rd lic1lpsA 0.746 0.742 1.414 odA 1268 1.143 0.240 1.371 1247 0.571 0.250 — 1209 0.870 0.162 0.988 1233 0.436 0.135 1.451 1181 0.702 0.244 1.452 1232 0.568 0.270 1.661 981 0.474 0.182 — 486 0.512 0.287 — 1292 0.433 0.184 1.675 1008 0.830 0.172 — 1158 0.763 0.154 — 1124 0.524 0.127 — 1003 2.014 0.914 — 667 0.606 0.252 1.239 285 0.658 1.350 1.334 176 0.360 0.237 1.444 162 0.462 0.251 1.388 1180 0.974 0.254 1.464 1159 0.874 0.128 — 723 >2.500 1.253 — 1231 >2.500 1.008 1.404 1200 1.206 0.186 1.420 1207 0.667 0.137 1.040 432 0.530 0.159 — 375 wt 1.860 0.946 — TABLE 4 LLA5 Mab mutant LPS EIA OD410 Rd lic1lpsA odA 0.889 Rd lpsA 1.630 Rd lic1orfH 1.216 Rd lgtF — TABLE 5 Negative ion ESI-MS data and proposed compositions of O-deacylated LPS from H. influenzae strain Rd and mutants.", "Average mass units were used for calculation of molecular weight based on proposed composition as follows: Lipid A-OH, 953.00; Hex, 162.15; HexNAc, 203.19; Hep, 192.17; Kdo-P, 300.16; PEtn, 123.05.PCho, 165.05 Observed Ions(m/z) Molecular Mass (Da) Relative Strain (M-2H)2− (M-3H)3− Observed Calculated Intensityb Proposed Composition OpsXa 616.0 — 1234.0 1235.1 80.0 Kdo-P, Lipid A-OH(—H2O)c 625.0 — 1252.0 1253.1 20.0 Kdo-P, Lipid A-OH RfaF 721.6 — 1445.1 1445.3 100.0 Hep, Kdo-P, Lipid A-OH OrfH 817.5 1637.5 1637.5 10.6 2Hep, Kdo-P, Lipid A-OH 879.2 — 1760.4 1760.5 42.4 2Hep, PEtn, Kdo-P, Lipid A-OH 940.5 — 1883.4 1883.6 15.2 2Hep, 2PEtn, Kdo-P, Lipid A-OH 1042.3 — 2087.5 2087.7 21.2 PCho, Hex, 2Hep, PEtn, Kdo-P, Lipid A-OH 1104.8 — 2211.6 2210.8 10.6 PCho, Hex, 2Hep, 2PEtn, Kdo-P, Lipid A-OH LpsA 1056.3 — 2114.7 2114.9 10.3 Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1139.0 759.0 2280.1 2279.9 69.0 PCho, Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1200.4 800.0 2403.0 2403.0 20.7 PCho, Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH Lic2A 1137.5 758.3 2277.5 2277.1 18.0 2Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1220.0 813.3 2442.5 2443.1 51.0 PCho, 2Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1281.9 854.3 2565.8 2566.1 31.0 PCho, 2Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH LgtC 1301.1 867.1 2604.3 2604.3 80.0 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1362.7 908.2 2727.4 2727.3 20.0 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH LgtD 1300.6 867.1 2603.9 2604.3 62.0 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1362.0 — 2726.0 2727.3 5.0 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH 1381.6 921.2 2765.5 2766.3 28.0 PCho, 4Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1443.3 962.1 2888.9 2889.4 5.0 PCho, 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH LgtF 1137.4 757.8 2276.6 2277.1 18.0 2Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1218.6 812.2 2439.4 2439.2 18.0 3Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1280.0 853.2 2562.3 2562.3 10.0 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH 1320.3 879.9 2642.7 2642.3 38.0 3Hex, 3Hep, PEtn, P, Kdo-P, Lipid A-OH — 920.8 2765.3 2766.3 16.0 HexNAc, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH Licl 1056.2 — 2114.3 2114.9 12.8 Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1117.4 744.9 2237.2 2237.9 7.2 Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH 1218.3 812.1 2439.2 2439.2 20.0 3Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1279.7 852.6 2561.1 2562.3 12.9 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH 1299.2 865.7 2600.3 2601.3 9.0 4Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1360.6 906.5 2722.8 2724.4 10.0 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH 1401.1 933.7 2804.3 2804.5 12.9 HexNAc, 4Hex, 3Hep, PEtn, Kdo-P, Lipid A-OH 1462.5 974.6 2927.0 2927.5 15.1 HexNAc, 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A-OH aData acquired by CE-ESI-MS on a crystal Model 310 CE instrument interfaced to an API 3000 triple quadrupole mass spectrometer (Perkin-Elmer/Sciex) fitted with a bare fused silica capillary column and using 30 mM morpholine-acetate (pH 9.0) containing 5% methanol as the separation buffer as described (30).", "bMeasured from the respective molecular ions in the reconstructed spectrum.", "cThe major ion observed corresponded to the molecular ion −18 (loss of H2O).", "TABLE 6 Linkage analysis data for LPS derived from H. influenzae RM118 mutated in LPS biosynthesis genes.", "Relative detector response for mutant strain Methylated Sugara Tgmb orfH lpsA lic2A lgtC lgtD lgtF Linkage assigment 2,3,4,6-Me4-Glc 1.00 −trc 6.7 22.5 tr tr D-Glcp-(1→ 2,3,4,6-Me4-Gal 1.05 −tr 34.3 22.9 10.3 D-Galp-(1→ 2,3,6-Me3-Glc 1.18 −tr 6.3 33.0 24.4 18.8 → 4)-D-Glcp-(1→ 2,3,6-Me3-Gal 1.17 12.4 16.6 → 4)-D-Galp-(1→ 2,4,6-Me3-Gald 1.20 5.0 12.4 5.4 tr 4.0 10.3 → 3)-D-Galp-(1→ 2,3,4,6,7-Me5-Hep 1.30 27.4 35.4 L,D-Hepp(1→ 3,4,6,7-Me4-Hep 1.44 53.3 15.8 16.0 16.3 → 2)-L,D-Hepp(1→ 2,4,6,7-Me4-Hep 1.47 37.0 19.7 → 3) -L,D-Hepp(1→ 2,6,7-Me3-Hep 1.52 23.7 45.4 12.5 10.6 17.1 → 3,4)-L,D-Hepp-(1→ 2,3,4,6-Me4-GalN 1.58 8.0 D-GalpNAc-1(1→ a2,3,4,6-Me4-Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol-1-d1, etc bRetention times (Tgm) are reported relative to 2,3,4,6-Me4-Glc and values are not corrected for differences in detector response factors.", "cTrace amount detected.", "dAll samples showed detectable levels of 2,4,6,-Me3-Gal.", "TABLE 7 Negative ion ESI-MS data and proposed compositions for major glycoforms in O-deacylated LPS of NTHi strains 176, 486, 285 and 1158.An asterisk (*) indicates predominantions.", "Observed ions (m/z) Molecular Weight (Da) Strain (M-4H)4− (M-3H)3− Observed Calculateda Proposed Composition 176 608.8 811.9 2439.2 2439.2 Hex3.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 609.5 813.0 2442.0 2442.2 PCho.Hex2.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 639.6 853.0 2562.4 2562.1 Hex3.Hep3.PEtn2.P1.Kdo1.Lipid A-OH 640.4 853.8 2565.6 2565.3 PCho.Hex2.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 650.0 867.0 2604.0 2604.3 PCho.Hex3.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 680.9* 908.3 2727.6 2727.3 PCho.Hex3.Hep3.PEtn2.P1.Kdo1.Lipid A-OH 486 690.4 920.8 2765.5 2766.4 PCho.Hex4.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 721.2* 961.8 2888.6 2889.5 PCho.Hex4.Hep3.PEtn2.P1.Kdo1.Lipid A-OH 763.2 1017.9 3056.8 3057.7 Neu5Ac.PCho.Hex4.Hep3.PEtn1.P1.Kdo1.LipidA-OH 793.9 1059.1 3180.0 3180.7 Neu5Ac.PCho.Hex4.Hep3.PEtn2.P1.Kdo1.LipidA-OH 285 568.9 758.7 2279.5 2280.0 PCho.Hex1.Hep3.PEtn1.P1.Kdo1.Lipid A-OH 599.6* 799.8 2402.3 2403.1 PCho.Hex1.Hep3.PEtn2.P1.Kdo1.Lipid A-OH 1158 657.7 877.4* 2634.8 2634.4 PCho.Hex2.Hep4.PEtn1.P1.Kdo1.LipidA-OH 688.6 918.3 2758.4 2757.4 PCho.Hex2.Hep4.PEtn2.P1.Kdo1.LipidA-OH 698.1 931.0 2796.4 2796.6 PCho.Hex3.Hep4.PEtn1.P1.Kdo1.LipidA-OH 699.0 932.5 2800.0 2799.5 PCho2.Hex2.Hep4.PEtn1.P1.Kdo1.LipidA-OH aAverage mass units were used in calculating molecular weights.", "TABLE 8 Linkage analysis data for LPS from NTHi strains 176, 486, 1158, 285 and oligosaccharides derived from NTHi 176 LPS.", "Relative detector response NTHi strains NTHi 176 derived Methylated Sugara 176 486 285 1158 LPS-Pb Fr.1c Fr.2 Fr.3 Linkage assignment 2,3,4,6-Me4-Glc 22 16 7 13 15 26 32 D-Glcp-(1→ 2,3,4,6-Me4-Gal 17 16 2 8 10 16 22 18 D-Galp-(1→ 2,3,6-Me3-Glc 6 27 15 14 7 2 15 4)-D-Glcp-(1→ 2,3,6-Me3-Gal 2 6 2 4)-D-Galp-(1→ 2,4,6-Me3-Gal 5 7 1 5 1 13 3 2 3)-D-Galp-(1→ 2,3,4-Me3-Glc 1 11 1 1 6)-D-Glcp-(1→ 2,3,4-Me3-Gal 6 6)-D-Galp-(1→ 2,3,4,6,7-Me5-Hep 11 1 D,D-Hepp-(1→ 2,3,4,6,7-Me5-Hep 1 52 1 5 L,D-Hepp-(1→ 3,4,6,7-Me4-Hep 1 4 13 12 2)-L,D-Hepp-(1→ 2,4,6,7-Me4-Hep 22 12 12 12 19 14 3)-L,D-Hepp-(1→ 2,6,7-Me3-Hep 22 14 34 16 26 16 23 16 3,4)-L,D-Hepp-(1→ 4,6,7-Me3-Hep 2 5 8 1 2,3)-L,D-Hepp-(1→ 2,3,6-Me4-GlcN 6 4)-D-GlcpNAc-(1→ 2,3,4,-Me4-GlcN 2 2 4 6)-D-GlcpNAc-(1→ a2,3,4,6-Me4-Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol-1-d1, etc bLPS-P represents dephosphorylated LPS-OH cFr.1 represents high molecular weight sample (HMG) of NTHi 176 obtained after mild acid hydrolysis." ] ]

[ [ "Bacterial Toxin Adsorbing Material, Method of Removing the Toxin by Adsorbing, and an Adsorber Formed by Filling the Adsorbing Material Therein", "The invention aims at providing an adsorbent for bacterial toxins, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent.", "Provided are an adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms, a method for removal of bacterial toxins using said adsorbent, and an adsorber packed with said adsorbent." ], [ "1.An adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms.", "2.The adsorbent according to claim 1, wherein the water-insoluble porous material comprises a polystyrene-based material.", "3.The adsorbent according to claim 1, wherein the water-insoluble porous material comprises an active carbon and/or a substance similar thereto.", "4.The adsorbent according to claim 1, wherein the water-insoluble porous material comprises an acrylic material.", "5.The adsorbent according to any one of claims 1 to 4, which is intended for use in adsorbing at least one bacterial toxin selected from the group consisting of superantigens, toxins having enterotoxin activity, and staphylococcus-derived pathogenic factors.", "6.A method for removal of bacterial toxins by adsorption, which comprises the step of contacting the adsorbent according to any one of claims 1 to 5 with a bacterial toxin-containing fluid.", "7.An adsorber for bacterial toxins, which comprises the adsorbent according to any one of claims 1 to 5 as packed in a container having a fluid inlet and a fluid outlet and equipped with a device for preventing the adsorbent from flowing out of the container." ], [ "<SOH> BACKGROUND ART <EOH>A bacterial toxin may be defined as “a substance which is a bacterial metabolite or constituent and causes, in a trace amount, an unfavorable response in the living body”.", "Bacterial toxins are roughly classified into two classes, endotoxins and exotoxins, according to the site of occurrence.", "The former are also called as protein toxins, and the latter as lipopolysaccharides (hereinafter referred to as “LPSs”).", "There are a large number of bacterial toxins, and the number of such toxins so far known now stands at about 200.Typical examples are cholera toxin, staphylococcal α toxin, botulinum toxin, tetanus toxin, enterotoxin (10 species are already known, namely staphylococcal enterotoxin A, B, C1, C2, C3, D, E, G, H, and I; hereinafter referred to as SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, and SEI, respectively), verotoxins, diphtheria toxin, pertussis toxin, Toxic shock syndrome toxin-1 (hereinafter referred to as TSST-1), and endotoxins.", "Among these, enterotoxins and TSST-1 are also called as superantigens.", "In the case of an ordinary antigen, it is taken up by an antigen-presenting cell and fragmented (into peptides composed of 10 to 15 amino acids) therein, and an antigen fragment, in the form bound to a pocket site of a MHC (major histocompatibility complex) class II molecule, is presented on the antigen-presenting cell surface.", "This is recognized by the α chain and β chain of the TCR (T cell receptor) of a specific T cell clone, and the T cell is activated and the immune response goes on.", "On the other hand, in the case of a superantigen, the antigen is not fragmented but binds directly to a MHC class II molecule on the antigen-presenting cell.", "This is further recognized by the TCR on a T cell, whereby the T cell is activated.", "On that occasion, the antigen is recognized through a specific Vβ region of the TCR.", "Unlike ordinary antigens, that antigen is recognized by almost all members of a T cell population expressing this specific Vβ region, and T cells are thereby activated and causing cytokine production.", "Thus, when an individual is exposed to a superantigen, a huge number of T cells are activated as compared with the ordinary specific immune response and the release of a cytokine, for instance, occurs in a short period of time, supposedly causing an abnormal reaction(s) in the living body.", "Known as the superantigen are TSST-1, enterotoxins and exfoliative toxin A (exfoliative A: ETA) produced by Staphylococcus aureus , which is a gram-positive bacterium, and exotoxins produced by streptococci (streptococcal pyrogenic exotoxin A, B, C: SPE-A, SPE-B, SPE-C), among others.", "An enterotoxin is one of toxins produced by bacteria.", "It has various biological activities, such as emetic, pyrogenic and mitogenic activities, and causes food poisoning symptoms or Toxic shock syndrome (hereinafter referred to as TSS).", "“Enterotoxin” is a term formed from “entero”, which means the intestine, and toxin.", "Although it essentially means a toxin causing diarrhea, it has not yet established that an enterotoxin causes diarrhea; hence what it really means is unclear.", "Toxins having enterotoxin activity include toxins having various biological activities, such as emetic, pyrogenic and mitogenic activities, and causing food poisoning symptoms or TSS.", "Known among them are enterotoxins produced by staphylococci, heat-labile enterotoxin (hereinafter referred to as LT) produced by Campylobacter species, and LT and heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli , among others.", "Staphylococci are widely distributed on or in the skin, nasal cavity, oral cavity, pharynx, urinary organs and intestinal tract of various animals including humans and, further, in air, sewage, river, food, and so forth and include a large number of species.", "Among such a large number of Staphylococcus species, it is coagulase-positive Staphylococcus aureus (hereinafter referred to as S. aureus ) that shows pathogenicity in humans.", "S. aureus causes various infectious diseases, such as TSS and staphylococcal scaled skin syndrome (SSSS) and, as pathogens, producing problems such as hospital infections.", "Furthermore, Staphylococcus epidermidis , for instance, may cause endocarditis, meningitis, septicemia, etc., and Staphylococcus saprophticus may cause urinary tract infection, although they are coagulase-negative.", "Known staphylococcal pathogenic factors include various toxins, enzymes and other biologically active substances, such as Clumping factor, fibrinogen-binding protein, fibrinogen-binding protein A, fibrinogen-binding protein B, collagen-binding protein, coagulase, polysaccaride/adhesin, polysaccaride intracellular adhesin, 220-kDa adhesin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, TSST-1, exfoliative toxin A, exfoliative toxin B, protein A, lipase, V8 protease, fatty acid modifying enzyme, panton-valentine leucocidin, leucocidin R, capsular polysaccaride, staphylokinase, α-toxin, β-hemolysin, γ-hemolysin, δ-hemolysin, phospholipase C, metalloprotease (elastase), and hyaluronidase.", "These bacterial toxins cause a great variety of diseases, from such relatively slight ones as food poisoning and traveler's diarrhea to such severe ones possibly leading to death as lethal diarrhea, tetanus, pertussis, botulism, diphtheria, cholera, TSS, and septicemia.", "In the case of septicemia and TSS, for instance, antibiotics, γ-globulin preparations and so forth are used in the treatment thereof, but the mortality is still high.", "As for food poisoning, it is difficult to perfectly prevent the occurrence of food poisoning how much care is taken from the hygienic point of view and, further, even when a food sufficiently heated is taken, it may cause food poisoning if a heat-stable toxin, such as an enterotoxin, is already contained therein.", "As regards the removal of superantigens by adsorption, Japanese Kokai Publication Hei-8-319431 discloses an adsorbent having a specific side chain.", "However, this cannot be said to be satisfactory from the capacity viewpoint.", "The present inventors have previously found that a material with a compound having a log P value (P being the partition coefficient in the octanol-water system) of not less than 2.50 as immobilized thereon can well adsorb TSST-1 (one of bacterial toxins) (Japanese Kokai Publication Hei-10-290833).", "For preparing such adsorbent material, however, a number of steps are required.", "When, for instance, antibodies specific to various toxins respectively are used, it is indeed possible to remove toxins from body fluids such as blood, plasma and serum, culture supernatants, foodstuffs, and drinks, but these have disadvantages, namely they are expensive and, when sterilized, they are denatured and their absorptive ability is markedly reduced.", "As regards endotoxins, various adsorbents are known for removing them from body fluids.", "For example, Japanese Kokoku Publication Hei-1-16389 discloses an adsorbent comprising polymyxin, which is known as an antidote against endotoxins, immobilized on an appropriate carrier.", "This adsorbent is effective against infections with gram negative bacteria but the removal of endotoxins alone cannot be expected to be highly effective against infections with gram positive bacteria or multiple infections with gram positive and gram negative bacteria.", "Furthermore, in recent years, it has been revealed that TSST-1, as a superantigen, activates the immune system and enhances the endotoxin toxicity to a level thousands of times higher.", "Therefore, the occurrence of an endotoxin at a low concentration, at which septicemia will not be caused clinically, is indicated to cause septicemia.", "Accordingly, the advent of a highly effective adsorbent for bacterial toxins, which can be produced in an inexpensive and simple and easy manner, is earnestly desired." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is an object of the present invention to provide an adsorbent for bacterial toxins which is free of such drawbacks of the prior art, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent.", "As a result of intensive investigations for overcoming the above-mentioned disadvantages, the present inventors could discover that when a water-insoluble porous material, which has a mode of pore radius of 20 angstroms to 1,000 angstroms, is used, bacterial toxins can be adsorbed at very high rates.", "The present invention, therefore, is directed to an adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms." ], [ "TECHNICAL FIELD The present invention relates to an adsorbent for bacterial toxins, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent.", "BACKGROUND ART A bacterial toxin may be defined as “a substance which is a bacterial metabolite or constituent and causes, in a trace amount, an unfavorable response in the living body”.", "Bacterial toxins are roughly classified into two classes, endotoxins and exotoxins, according to the site of occurrence.", "The former are also called as protein toxins, and the latter as lipopolysaccharides (hereinafter referred to as “LPSs”).", "There are a large number of bacterial toxins, and the number of such toxins so far known now stands at about 200.Typical examples are cholera toxin, staphylococcal α toxin, botulinum toxin, tetanus toxin, enterotoxin (10 species are already known, namely staphylococcal enterotoxin A, B, C1, C2, C3, D, E, G, H, and I; hereinafter referred to as SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, and SEI, respectively), verotoxins, diphtheria toxin, pertussis toxin, Toxic shock syndrome toxin-1 (hereinafter referred to as TSST-1), and endotoxins.", "Among these, enterotoxins and TSST-1 are also called as superantigens.", "In the case of an ordinary antigen, it is taken up by an antigen-presenting cell and fragmented (into peptides composed of 10 to 15 amino acids) therein, and an antigen fragment, in the form bound to a pocket site of a MHC (major histocompatibility complex) class II molecule, is presented on the antigen-presenting cell surface.", "This is recognized by the α chain and β chain of the TCR (T cell receptor) of a specific T cell clone, and the T cell is activated and the immune response goes on.", "On the other hand, in the case of a superantigen, the antigen is not fragmented but binds directly to a MHC class II molecule on the antigen-presenting cell.", "This is further recognized by the TCR on a T cell, whereby the T cell is activated.", "On that occasion, the antigen is recognized through a specific Vβ region of the TCR.", "Unlike ordinary antigens, that antigen is recognized by almost all members of a T cell population expressing this specific Vβ region, and T cells are thereby activated and causing cytokine production.", "Thus, when an individual is exposed to a superantigen, a huge number of T cells are activated as compared with the ordinary specific immune response and the release of a cytokine, for instance, occurs in a short period of time, supposedly causing an abnormal reaction(s) in the living body.", "Known as the superantigen are TSST-1, enterotoxins and exfoliative toxin A (exfoliative A: ETA) produced by Staphylococcus aureus, which is a gram-positive bacterium, and exotoxins produced by streptococci (streptococcal pyrogenic exotoxin A, B, C: SPE-A, SPE-B, SPE-C), among others.", "An enterotoxin is one of toxins produced by bacteria.", "It has various biological activities, such as emetic, pyrogenic and mitogenic activities, and causes food poisoning symptoms or Toxic shock syndrome (hereinafter referred to as TSS).", "“Enterotoxin” is a term formed from “entero”, which means the intestine, and toxin.", "Although it essentially means a toxin causing diarrhea, it has not yet established that an enterotoxin causes diarrhea; hence what it really means is unclear.", "Toxins having enterotoxin activity include toxins having various biological activities, such as emetic, pyrogenic and mitogenic activities, and causing food poisoning symptoms or TSS.", "Known among them are enterotoxins produced by staphylococci, heat-labile enterotoxin (hereinafter referred to as LT) produced by Campylobacter species, and LT and heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli, among others.", "Staphylococci are widely distributed on or in the skin, nasal cavity, oral cavity, pharynx, urinary organs and intestinal tract of various animals including humans and, further, in air, sewage, river, food, and so forth and include a large number of species.", "Among such a large number of Staphylococcus species, it is coagulase-positive Staphylococcus aureus (hereinafter referred to as S. aureus) that shows pathogenicity in humans.", "S. aureus causes various infectious diseases, such as TSS and staphylococcal scaled skin syndrome (SSSS) and, as pathogens, producing problems such as hospital infections.", "Furthermore, Staphylococcus epidermidis, for instance, may cause endocarditis, meningitis, septicemia, etc., and Staphylococcus saprophticus may cause urinary tract infection, although they are coagulase-negative.", "Known staphylococcal pathogenic factors include various toxins, enzymes and other biologically active substances, such as Clumping factor, fibrinogen-binding protein, fibrinogen-binding protein A, fibrinogen-binding protein B, collagen-binding protein, coagulase, polysaccaride/adhesin, polysaccaride intracellular adhesin, 220-kDa adhesin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, TSST-1, exfoliative toxin A, exfoliative toxin B, protein A, lipase, V8 protease, fatty acid modifying enzyme, panton-valentine leucocidin, leucocidin R, capsular polysaccaride, staphylokinase, α-toxin, β-hemolysin, γ-hemolysin, δ-hemolysin, phospholipase C, metalloprotease (elastase), and hyaluronidase.", "These bacterial toxins cause a great variety of diseases, from such relatively slight ones as food poisoning and traveler's diarrhea to such severe ones possibly leading to death as lethal diarrhea, tetanus, pertussis, botulism, diphtheria, cholera, TSS, and septicemia.", "In the case of septicemia and TSS, for instance, antibiotics, γ-globulin preparations and so forth are used in the treatment thereof, but the mortality is still high.", "As for food poisoning, it is difficult to perfectly prevent the occurrence of food poisoning how much care is taken from the hygienic point of view and, further, even when a food sufficiently heated is taken, it may cause food poisoning if a heat-stable toxin, such as an enterotoxin, is already contained therein.", "As regards the removal of superantigens by adsorption, Japanese Kokai Publication Hei-8-319431 discloses an adsorbent having a specific side chain.", "However, this cannot be said to be satisfactory from the capacity viewpoint.", "The present inventors have previously found that a material with a compound having a log P value (P being the partition coefficient in the octanol-water system) of not less than 2.50 as immobilized thereon can well adsorb TSST-1 (one of bacterial toxins) (Japanese Kokai Publication Hei-10-290833).", "For preparing such adsorbent material, however, a number of steps are required.", "When, for instance, antibodies specific to various toxins respectively are used, it is indeed possible to remove toxins from body fluids such as blood, plasma and serum, culture supernatants, foodstuffs, and drinks, but these have disadvantages, namely they are expensive and, when sterilized, they are denatured and their absorptive ability is markedly reduced.", "As regards endotoxins, various adsorbents are known for removing them from body fluids.", "For example, Japanese Kokoku Publication Hei-1-16389 discloses an adsorbent comprising polymyxin, which is known as an antidote against endotoxins, immobilized on an appropriate carrier.", "This adsorbent is effective against infections with gram negative bacteria but the removal of endotoxins alone cannot be expected to be highly effective against infections with gram positive bacteria or multiple infections with gram positive and gram negative bacteria.", "Furthermore, in recent years, it has been revealed that TSST-1, as a superantigen, activates the immune system and enhances the endotoxin toxicity to a level thousands of times higher.", "Therefore, the occurrence of an endotoxin at a low concentration, at which septicemia will not be caused clinically, is indicated to cause septicemia.", "Accordingly, the advent of a highly effective adsorbent for bacterial toxins, which can be produced in an inexpensive and simple and easy manner, is earnestly desired.", "SUMMARY OF THE INVENTION It is an object of the present invention to provide an adsorbent for bacterial toxins which is free of such drawbacks of the prior art, a method for removal of such toxins by adsorption, and an adsorber packed with said adsorbent.", "As a result of intensive investigations for overcoming the above-mentioned disadvantages, the present inventors could discover that when a water-insoluble porous material, which has a mode of pore radius of 20 angstroms to 1,000 angstroms, is used, bacterial toxins can be adsorbed at very high rates.", "The present invention, therefore, is directed to an adsorbent for bacterial toxins, which comprises a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms.", "DETAILED DISCLOSURE OF THE INVENTION In search of a compound effective in adsorbing bacterial toxins, the present inventors evaluated various water-insoluble materials or modifications thereof for bacterial toxin adsorbing ability.", "As a result, it was found that water-insoluble porous materials, which have a mode of pore radius of 20 angstroms to 1,000 angstroms, are suited for use as adsorbents for bacterial toxins.", "When an adsorbent having a mode of pore radius of not more than 20 angstroms is used, the capacity for adsorbing bacterial toxins is insufficient; this lowers its utility.", "When an adsorbent having a mode of pore radius not less than 1,000 angstroms is used, proteins (mainly albumin) other than bacterial toxins are adsorbed in large amounts, resulting in a substantial decrease in the amount of bacterial toxins adsorbed; the utility thus lowers from the selectivity viewpoint.", "Therefore, the water-insoluble porous material to be used in the present invention preferably has a mode of pore radius of from 20 angstroms to 1,000 angstroms, more preferably from 40 angstroms to 600 angstroms, most preferably from 50 angstroms to 400 angstroms.", "The water-insoluble porous material according to the invention is a material internally having a porous structure, occurring as a solid at ordinary temperature and ordinary pressure and having a very low solubility in water.", "The porous structure of the water-insoluble porous material can be expressed in terms of pore volume, specific surface area, and pore size distribution, among others.", "These indices can be determined, for example, by the gas adsorption method based on the adsorption isotherm for a gas such as nitrogen, mercury porosimetry using a mercury porosimeter, electron microscopy, and small angle X-ray scattering (“Takoshitsutai no Seishitsu to sono Oyo-gijutsu (Porous Materials, Properties and Application Technology)”, pages 248 to 288, edited by Yasushi Takeuchi, published by Fujitec Corporation).", "It is generally considered that mercury porosimetry is preferred for the so-called macropores, namely pores having a pore size of 100 angstroms and larger, and the gas adsorption method for the so-called micropores, namely pores having a pore size of 100 angstroms or smaller, although strict division is impossible.", "However, pores may be deformed by the pressure applied on the occasion of mercury porosimetry depending on the properties of the material in question.", "Therefore, it is necessary to select an adequate measurement method taking the physical properties of the material into consideration.", "The pore size distribution is generally shown in a graphic form, the pore radius (e.g.", "in angstroms) on the X-axis and the value (e.g.", "in mL/g-angstrom) derived, by differentiation, from the cumulative pore volume (e.g.", "in mL/g) on the Y-axis.", "The mode is the value corresponding to the highest frequency in the distribution.", "Thus, the mode of pore radius according to the invention means the pore radius corresponding to the maximum differentiated cumulative pore volume in the pore size distribution determined by such a measurement method as mentioned above.", "The form of the water-insoluble porous material according to the invention includes, but is not limited to, granules, plates, fibers, and hollow fibers, among others.", "The size is not particularly restricted, either.", "The water-insoluble porous material to be used as the adsorbent in the invention is preferably a hydrophobic inorganic material such as active carbon, an organic material intermediate in polarity such as an acrylic material, a hydrophobic organic material such as a polystyrene-based material, or an organic-organic, organic-inorganic or the like composite material resulting from a combination of the materials mentioned above.", "A polystyrene-based material, which is a hydrophobic organic material, is more preferred.", "The polystyrene-based material so referred to herein includes such polymer materials as polymers mainly made of styrene and/or a derivative thereof.", "Thus, it may be a homopolymer of styrene or a derivative thereof, or a copolymer containing polymer constituent(s) other than styrene as well, including a copolymer with divinylbenzene and/or a derivative thereof.", "The acrylic material so referred to herein includes such polymer compounds as polymers mainly made of acrylic acid, methacrylic acid, and/or a derivative thereof.", "Thus, it may be a homopolymer of an acrylic or a derivative thereof, or a copolymer containing a polymer constituent(s) other than acrylics as well.", "It is an organic material intermediate in polarity.", "Polyacrylamide, which is a hydrophilic organic material, is excluded, however.", "Among acrylic polymers, methacrylate ester resins are particularly preferred.", "Furthermore, active carbon and a substance similar thereto, according to the invention, indicates any of active or activated carbon species in general use or a water-insoluble porous material having a carbonized surface.", "Morphologically, active carbon species are classified into powdery active carbon, granular active carbon, fibrous active carbon, molded active carbon and so forth.", "The raw materials thereof are diverse and include, but are not limited to, vegetable ones such as wood chips and wastes from sugar manufacture, mineral ones such as peat and petroleum pitch, synthetic materials such as acrylic resins and phenol resins, and natural materials such as seaweed and grain, among others.", "Their sizes are not particularly restricted, either.", "For the purpose of improving the blood compatibility of the material, this may be treated or modified, to the extent not detracting from its adsorptive affinity for bacterial toxins, by introducing an appropriate side chain or ligand into the material or coating the same with a hydrophilic material, for instance.", "Examples of the side chain or ligand include, but are not limited to, those having a hydroxyl and/or amino group(s), and the hydrophilic material includes, but is not limited to, polymers of hydroxyethyl methacrylate, and cellulose.", "However, in cases where the mode of pore radius is within the above-specified range, the effects of the invention can be achieved without such treatment.", "The bacterial toxin according to the invention is “a substance which is a bacterial metabolite or constituent and causes, in a trace amount, an unfavorable response in the living body” and includes, but is not limited to, El Tor hemolysin of Vibrio cholerae, streptococcal streptolysin O, streptococcal streptolysin S, staphylococcal a toxin, Pseudomonas aeruginosa leucocidin, botulinum toxin, tetanus toxin, enterotoxigenic Escherichia coli heat-stable enterotoxin, Vibrio parahaemoliticus heat-stable hemolysin, verotoxins, diphtheria toxin, pertussis toxin, coagulase, exfoliatin, tetrodotoxin, TSST-1, endotoxins, etc.", "The superantigen according to the invention indicates a substance capable of activating a huge number of T cells as compared with the ordinary specific immune response to thereby cause an abnormal reaction(s) in the living body.", "It includes, but is not limited to, TSST-1, enterotoxins and exfoliative toxin A (exfoliative A: ETA) produced by S. aureus, and exotoxins produced by streptococci (streptococcal pyrogenic exotoxin A, B, C: SPE-A, SPE-B, SPE-C), among others.", "The toxin having enterotoxin activity according to the invention means a toxin having various biological activities, such as emetic, pyrogenic and mitogenic activities, and causing food poisoning symptoms or TSS.", "It includes, but is not limited to, enterotoxins produced by staphylococci, LT produced by Campylobacter, and LT and heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli, among others.", "The staphylococcal pathogenic factor according to the invention indicates a substance produced by staphylococci and causing an abnormal response(s) in the living body.", "It includes, but is not limited to, various toxins, enzymes and other biologically active substances, such as Clumping factor, fibrinogen-binding protein, fibrinogen-binding protein A, fibrinogen-binding protein B, collagen-binding protein, coagulase, polysaccaride/adhesin, polysaccaride intracellular adhesin, 220-kDa adhesin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH, SEI, TSST-1, exfoliative toxin A, exfoliative toxin B, protein A, lipase, V8 protease, fatty acid modifying enzyme, panton-valentine leucocidin, leucocidine R, capsular polysaccaride, staphylokinase, α-toxin, β-hemolysin, γ-hemolysin, δ-hemolysin, phospholipase C, metalloprotease (elastase), and hyaluronidase.", "The adsorbent according to the invention is excellent in bacterial toxin adsorbing capacity, in particular in adsorbing superantigens, toxins having enterotoxin activity, and staphylococcal pathogenic factors.", "The adsorbent for bacterial toxins according to the invention is suited for use in removing, by adsorption, bacterial toxins from body fluids, culture supernatants, foodstuffs, and drinks, in particular removing, by adsorption, bacterial toxins from body fluids.", "The body fluid according to the invention includes blood, plasma, serum, ascitic fluid, lymph, synovia fluid, fractions obtained from these, and other humoral components derived from the living body.", "The method for removal, by adsorption, of bacterial toxins from a body fluid using the adsorbent of the invention includes various techniques.", "The most expedient one comprises drawing out the body fluid, storing the same in a bag or the like, admixing the same with the adsorbent and, after removal of S. aureus and/or other bacterial toxins, recovering the bacterial toxin-free body fluid by filtering off the adsorbent.", "Another technique comprises filling the adsorbent in a container having a body fluid inlet and a body fluid outlet and equipped, at least at the outlet, with a filter allowing the passage of the body fluid but preventing the passage of the adsorbent, and passing the body fluid through the same.", "While either technique is useful, the latter is more expedient from the procedural viewpoint.", "When the container is incorporated in an extracorporeal circulation circuit, bacterial toxins can be removed efficiently from the body fluid, in particular blood, of a patient in an on-line manner.", "The extracorporeal circulation circuit so referred to herein may comprise the adsorbent of the invention alone or a combination thereof with another extracorporeal circulation system for treatment.", "An example of the system to be used combinedly is an artificial dialysis circuit, and the adsorbent can be used in combination with dialysis therapy.", "Now, the adsorber for bacterial toxins according to the invention, in which the above-mentioned adsorbent for bacterial toxins is used, is described referring to FIG.", "1 schematically showing, in cross section, an example of the adsorber.", "In FIG.", "1, 1 indicates a body fluid inlet, 2 a body fluid outlet, 3 the adsorbent for bacterial toxins according to the invention, 4 and 5 each a filter for preventing the adsorbent from flowing out, 6 a column, and 7 the adsorber for bacterial toxins.", "However, the adsorber for bacterial toxins is not limited to such specific example but may have any constitution provided that it comprises a container having a fluid inlet and a fluid outlet and equipped with a device for preventing the adsorbent for bacterial toxins from flowing out of the container and the above-mentioned adsorbent packed therein.", "The above flowing-out preventing device may be a filter made of a mesh, nonwoven fabric, or cotton plug, for instance.", "The shape, material, and size of the container are not particularly restricted.", "As for the shape, a cylindrical one is preferred, however.", "Preferred as the container material are materials resistant to sterilization treatment, specifically including silicone-coated glass, polypropylene, polyvinyl chloride, polycarbonate, polysulfone, polymethylpentene, and the like.", "The capacity of the container is preferably 50 to 1,500 ml, more preferably 100 to 800 ml, most preferably 150 to 400 ml, and the diameter thereof is preferably 2 to 20 cm, more preferably 3 to 15 cm, most preferably 4 to 10 cm.", "BRIEF DESCRIPTION OF THE DRAWING FIG.", "1 is a schematic representation, in cross section, of an example of the adsorber for bacterial toxins according to the invention.", "EXPLANATION OF THE SYMBOLS 1: body fluid inlet 2: body fluid outlet 3: adsorbent for bacterial toxins 4 and 5: filters for preventing the adsorbent from flowing out 6: column 7: adsorber for bacterial toxins.", "BEST MODES FOR CARRYING OUT THE INVENTION The following examples illustrate the present invention in further detail.", "They are, however, by no means limitative of the scope of the invention.", "EXAMPLE 1 Using a styrene-divinylbenzene copolymer (DIAION HP-20: product of Mitsubishi Chemical Corporation) as the adsorbent for bacterial toxins, the following experiment was carried out.", "This material is an adsorbent having a mode of pore radius within the range of 100 angstroms to 200 angstroms.", "Pore sizes were determined by mercury porosimetry.", "TSST-1 (product of Toxin Technologies, Inc.) was used as a bacterial toxin, and the adsorbent was evaluated for its capacity for adsorbing the toxin in fetal bovine serum (FBS).", "The TSST-1 concentration used was about 5 ng/mL.", "Thus, 0.2 mL of the above adsorbent and 1.2 mL of TSST-1-containing FBS were blended, and the mixture was shaken at 37° C. for 2 hours.", "Thereafter, the supernatant was separated from the adsorbent and assayed for the TSST-1 concentration in the supernatant by ELISA (Enzyme Linked Immuno Sorbent Assay).", "The ELISA of TSST-1 was carried out as follows.", "Rabbit anti-TSST-1 IgG (product of Toxin Technologies), a primary antibody, was 1600-fold diluted with a coating buffer, and the dilution was distributed in 100-μl portions into wells of a microplate.", "After overnight standing at 4° C., the plate was washed.", "A 3% bovine serum albumin solution was distributed in 200-μl portions into the wells of the microplate and, after 2 hours of standing at room temperature, the microplate was washed.", "Then, 100 μl each of a standard TSST-1 solution and the supernatant before or after incubation were added to each well of the microplate.", "After 2 hours of standing at room temperature, the plate was washed.", "Rabbit anti-TSST-1 HRP (product of Toxin Technologies), a secondary antibody, was 400-fold diluted with a 1% bovine serum albumin solution, and the dilution was distributed in 100-μl portions into the wells.", "After 2 hours of standing at room temperature, the plate was washed.", "An orthophenylenediamine solution was distributed in 100-μl portions into the wells, followed by 10 minutes of standing at room temperature.", "4 N sulfuric acid was distributed in 100-μl portions into the wells, followed by absorbance measurement at 492 nm.", "The TSST-1 concentrations in the supernatant before and after incubation were determined by comparison with the absorbance of the standard solution.", "EXAMPLE 2 A styrene-divinylbenzene copolymer (DIAION HP-40: product of Mitsubishi Chemical Corporation) was used as the adsorbent for bacterial toxins and evaluated for its capacity for adsorbing TSST-1 under the same conditions as in Example 1.This material is an adsorbent having a mode of pore radius within the range of 200 angstroms to 300 angstroms.", "Pore sizes were determined by mercury porosimetry.", "EXAMPLE 3 A styrene-divinylbenzene copolymer (DIAION HP-50: product of Mitsubishi Chemical Corporation) was used as the adsorbent for bacterial toxins and evaluated for its capacity for adsorbing TSST-1 under the same conditions as in Example 1.This material is an adsorbent having a mode of pore radius within the range of 900 angstroms to 1,000 angstroms.", "Pore sizes were determined by mercury porosimetry.", "EXAMPLE 4 A styrene-divinylbenzene copolymer (DIAION SP-875: product of Mitsubishi Chemical Corporation) was used as the adsorbent for bacterial toxins and evaluated for its capacity for adsorbing TSST-1 under the same conditions as in Example 1.This material is an adsorbent having a mode of pore radius within the range of 20 angstroms to 30 angstroms.", "Pore sizes were determined by nitrogen gas adsorption method.", "EXAMPLE 5 A methacrylate ester resin (Amberlite XAD-7: product of Organo Corporation) was used as the adsorbent for bacterial toxins and evaluated for its capacity for adsorbing TSST-1 under the same conditions as in Example 1.This material is an adsorbent having a mode of pore radius within the range of 500 angstroms to 600 angstroms.", "Pore sizes were determined by mercury porosimetry.", "EXAMPLE 6 Petroleum pitch-derived spherical active carbon was used as the adsorbent for bacterial toxins and evaluated for its capacity for adsorbing TSST-1 under the same conditions as in Example 1.This material is an adsorbent having a mode of pore radius within the range of 20 angstroms to 30 angstroms.", "Pore sizes were determined by nitrogen gas adsorption method.", "COMPARATIVE EXAMPLE 1 The same volume as the adsorbent of physiological saline and TSST-1-added FBS were mixed up, and the mixture was subjected to the same evaluation as in Example 1.The results of Examples 1 to 6 and Comparative Example 1 are shown in Table 1.The percent adsorption was calculated as follows: Percent adsorption (%)=100×(concentration in Comparative Example 1−concentration in the example)/concentration in Comparative Example 1 TABLE 1 TSST-1 concentration Percent after shaking adsorption (ng/mL) (%) Example 1 0.06 98.8 Example 2 0.45 90.8 Example 3 3.81 22.2 Example 4 3.39 30.8 Example 5 1.38 71.8 Example 6 4.30 12.2 Comparative 4.90 — Example 1 EXAMPLE 7 Using a styrene-divinylbenzene copolymer (DIAION HP-20: product of Mitsubishi Chemical Corporation) as the adsorbent for bacterial toxins, the following experiment was carried out.", "This material is an adsorbent having a mode of pore radius within the range of 100 angstroms to 200 angstroms.", "Pore sizes were determined by mercury porosimetry.", "Among the enterotoxins, SEA, SEB, SEC1, CED, and SEE (products of Toxin Technologies) were used as bacterial toxins, and the adsorbent was evaluated for its capacity of adsorbing them in fetal bovine serum (FBS).", "Thus, 0.2 mL of the above adsorbent and 1.2 mL of FBS containing an appropriate amount of one of the enterotoxins were blended, and the mixture was shaken at 37° C. for 2 hours.", "Thereafter, the supernatant was separated from the adsorbent and assayed for the enterotoxin concentration in the supernatant using an ELISA kit (product of r-Biopharm).", "COMPARATIVE EXAMPLE 2 The same volume as the adsorbent of physiological saline and FBS containing one of the enterotoxins were mixed up, and the mixture was subjected to the same evaluation as in Example 7.The results of Example 7 and Comparative Example 2 are shown in Table 2.The percent adsorption was calculated as follows: Percent adsorption (%)=100×(concentration in Comparative Example 2−concentration in Example 7)/concentration in Comparative Example 2 TABLE 2 Concentration after shaking (ng/ml) Percent Comparative adsorption Example 7 Example 2 (%) SEA 0.23 1.17 80 SEB 0.22 2.80 92 SEC1 0.08 0.50 83 SED 0.10 1.47 93 SEE 0.06 1.80 97 INDUSTRIAL APPLICABILITY According to the invention, it is possible to remove, by adsorption, bacterial toxins with good efficiency by using an adsorbent for bacterial toxins which is characterized in that it is made of a water-insoluble porous material having a mode of pore radius of 20 angstroms to 1,000 angstroms." ] ]

[ [ "Anti-graffiti paint formulations and removal", "A paint formulation which contains specific enzymes which remain inactive in an applied surface coating of the paint formulation until specifically activated by a subsequently-applied applicator solution to facilitate removal of the surface coating.", "When the applicator is applied to the surface coating, the enzymes catalyse the hydrolysis of the binder in the paint or surface coating, e.g.", "by cleaving the ester bonds in the 1 and 3 positions of the triglyceride molecules.", "This allows for easy and rapid removal of the coating without damaging or affecting the underlying surface.", "The invention is useful either for general purpose paint removal, or for the cleaning up or removal of unwanted graffiti." ], [ "1-19.", "(canceled) 20.A paint formulation comprising: (a) a mixture of a surface coating base containing at least one binder, wherein the binder is a member selected from the group consisting of alkyd resins, oils, cellulose, acrylic resins, and mixtures of same; and (b) at least one enzyme specific to the at least one binder that can be activated to facilitate subsequent removal of a coating of the paint formulation from a surface on which the coating has been deposited.", "21.The paint formulation of claim 20, which additionally contains a pigment.", "22.A clear sealer coating for unpainted or previously painted surfaces comprising a formulation according to claim 20, wherein the formulation comprises: (a) a mixture of a surface coating containing at least one binder, wherein the binder is a member selected from the group consisting of alkyd resins, oils, acrylic resins and mixtures of same; (b) at least one enzyme specific to said at least one binder which can be activated to facilitate subsequent removal of the clear sealer coating from a surface on which the coating has been deposited.", "23.The paint formulation of claim 20, wherein the enzyme is a lipase enzyme.", "24.The paint formulation of claim 23, wherein the enzyme is a Lipase enzyme, classified by the Chemical Abstracts Service Registry as: Lipase, tricylglycose, CAS NO 9001-620-1.25.The paint formulation of claim 23, wherein the enzyme is one classified by the International Union of Pure and Applied Chemistry as EC 3.1.1.3.26.The paint formulation of claim 23, wherein the enzyme is derived from Thermomyces lanuginosus produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism with an activity of about 100 KLU/g.", "27.The paint formulation of claim 23, wherein the enzyme is derived from Rhizomucor miehei produced from submerged fermentation.", "28.The paint formulation of claim 26, wherein the lipolytic activity of about 100 KLU/g is determined relative to an analytical standard under a set of conditions wherein the reaction substrate is tributyrin, and the reaction occurs at 30° C., and at pH 7.0.29.The paint formulation of claim 20, wherein said at least one enzyme is mixed with solubilized polyoxyethylene (20) oleyl ether non-ionic surfactant.", "30.The paint formulation of claim 28, wherein said mixture of said at least one enzyme with a surfactant is mixed with a liquid hydrocarbon.", "31.The paint formulation of claim 30, wherein the liquid hydrocarbon is a member selected from the group consisting of aromatic hydrocarbons, aliphatic hydrocarbons, and mixtures thereof.", "32.The paint formulation of claim 20, wherein the enzyme is mixed with a paint preparation of a type selected from the group consisting of alkyd, oil, enamel, and paint preparations comprising mixed glycerol esters of long-chain monocarboxylic acids (triglycerides).", "33.The paint formulation of claim 20, wherein the enzyme mixture is mixed with a coating base comprising an alkyd resin, wherein the resin has been produced by condensation and polymerization of a polyhydric alcohol and a polybasic acid, and with a drying oil modifier.", "34.The paint formulation of claim 20, wherein the enzyme is of a member selected from the group consisting of a protease, amylase, cellulase and hemicellulase enzymes.", "35.The clear paint formulation of claim 22, wherein the enzyme is a member selected from the group consisting of cellulase, hemicellulase, enzymes and combinations thereof.", "36.An activator solution for application to a surface painted with a formulation according to claim 20, comprising a mixture of sodium hypochlorite, sodium hydroxide, alkaline salts and a xanthan gum derived from Xanthomonas campestris which when applied to said painted surface will cause said paint to degrade to a point permitting easy removal.", "37.A kit comprising a formulation according to claim 20, and an activator comprising a mixture of sodium hydroxide, sodium hypochlorite, alkaline salts and a xanthan gum derived from Xanthomonas campestris with said activator being separately maintained until removal of the paint formulation from said paint coated surface is desired.", "38.A method for removal of at least a portion of a surface coating comprising the paint formulation of claim 20, comprising the step of applying a further coating comprising an activator formulation containing an enzyme specific to said at least one binder in said formulation to at least portion of the surface coating, wherein the activator formulation promotes the catalysis and subsequent hydrolysis of 1, 3 esters bonds of triglyceride molecules present in said surface coating, thereby causing the degradation thereof.", "39.The method of claim 38, wherein the activator composition comprises demineralized water; sodium hypochlorite, present in said composition in an amount of between 50 to 60 grams per liter; sodium hydroxide, present in said composition in an amount of about 10 grams per liter; and 0.35 xanthan gum, wherein the xanthan gum is derived from Xanthomonas campestris.", "40.The method of claim 38, comprising the additional step of treating the degraded portion of the surface coating with vacuum equipment apparatus that produces hot water and is effective to remove the degraded portion of the surface coating into a contained vessel.", "41.A method of preparation of a paint formulation comprising an enzyme that facilitates subsequent removal of a surface coating of the paint formulation from a surface onto which the formulation has been deposited, comprising the steps of mixing the enzyme with a non-ionic surfactant pre-heated to about 65° C.; adding a hydrocarbon solution to the mixture and leading to rapid cooling thereof; adding the cooled mixture to a hydrophobic paint composition; and subjecting the formulation to final mixing to provide a homogeneous mixture of the paint formulation" ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Graffiti is usually applied on public and other buildings by way of ‘spray-can’ aerosol paints.", "These paints are usually alkyd and/or oil paints or oil enamels which are designed to dry rapidly.", "Their legal use is most helpful in ‘touch-up’ and small job applications where a rapid drying gloss finish is required.", "In the ‘hit and run’ world of so-called graffiti art, they are the most popular paint used.", "Conventional graffiti paint removal methods usually remove or damage the base surface coating upon which the graffiti is applied.", "This invention is specifically designed to remove the graffiti paint only.", "The estimated cost of graffiti removal throughout the world is variously estimated by municipal authorities to be in the hundreds of millions of dollars each year.", "In many countries the call for the removal from sale of oil enamel spray (aerosol) type paint cans is very strong.", "In Australia to date, the movement to ban the sale of such paints has been overcome by a voluntary paint industry initiative; which also includes the insertion of the following warning label on the paint cans: ‘IMPORTANT: Deliberate misuse of this aerosol to damage private property is a crime.’ To fully appreciate the background to this invention, it is important to understand the chemistry of both paint production and enzyme catalysis.", "An enzyme is a protein with catalytic activity that is restricted to a limited set of reactions defining the specificity of the enzyme.", "This invention places specific water-soluble enzyme preparations into oil or alkyd oil based paints or surface coatings.", "The invention provides for the design of a carrier system for the enzyme that allows its incorporation into oil type paints.", "This is described herein below.", "A simple description for a Paint follows.", "A more complete description can be obtained from the National Paint, Varnish and Lacquer Association, 1500 R.I. Avenue, Washington D.C. 20036 United States of America, or in ‘Paints and Surface Coatings’, Theory and Practice Second Edition.", "Edited by R. Lambourne and T. A. Strivens.", "Published by Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB1 6AH, England 1999.Paint is a unfirmly dispersed mixture with variable viscosity and consisting of (1) a drying oil, synthetic resin, or other film forming component called the BINDER; (2) a SOLVENT or THINNER; and (3) an Organic or Inorganic PIGMENT.", "The BINDER AND SOLVENT are collectively called the VEHICLE.", "Paints are used to protect surfaces and to provide decoration.", "This invention places a specifically prepared enzyme mixture into paints which contain oils and/or alkyds as a binder type.", "It is also helpful to understand more of the characterization of the oils and alkyds used in paint preparation.", "Those skilled in the art will have a fully comprehensive understanding of oils and alkyds used in paint manufacture.", "Vegetable oils and vegetable oil fatty acids continue to play an important role in surface coatings due to their availability as a renewable resource.", "Oils are mixed glycerol esters of long chain monocarboxylic acids called fatty acids.", "The Carbon length is generally C 18 Oils used include, Linseed Oil, Soya Bean Oil, Coconut Oil and ‘Tall Oil’.", "Oils are classified as Drying, Semi Drying, or Non-Drying.", "Table 1.0 illustrates typical composition of major oils used in surface coatings.", "TABLE 1.0 9.12, 15 Saturated 9.12 Linolenic Conjugated Acids Oleic Acid Linoleic Acid Acid Acid TUNG 6 7 4 3 90 LINSEED 10 20-24 14-19 48-54 0 SOYA BEAN 15 22-28 52-55 5-9 0 CASTOR OIL 2-4 90-92 3-6 0 0 DEHYDRATED 2-4 6-8 48-50 0 0 CASTOR OIL TALL 3 30-35 35-40 2-5 10-15 COCONUT 89-94 6-8 0-2 0 0 Linseed oil is a good drying oil containing more than 60% of the polyunsaturated linoleic and linolenic acids.", "Alkyd resins are produced by condensation and polymerization of dihydric or polyhydric alcohols and a polybasic acid such as phthalic anhydride and usually with a drying oil modifier.", "These resins are described as long oil alkyds (60% oil) medium oil alkyds (40% oil) or short oil alkyds (less than 40% oil).", "In practical terms, the reaction of glycerol and oil/s in the production of monoglycerides in the presence of a catalyst, does not go to completion and the equilibrium distribution of species include oil, polyol and mono and diglycerides.", "This is important in this invention as the remaining presence of free oil/s allows, when correctly activated, the enzymatic hydrolysis of the oil to effect a gross weakening of the binder allowing for easy removal of the paint." ], [ "FIELD OF THE INVENTION The present invention relates to paint or surface coating formulations having a built-in removal factor or component.", "More specifically, the present invention relate to enzyme preparations for inclusion in alkyd and oil paints and a rapid removal activator applicator designed specifically for paint removal, and more specifically for graffiti removal, and which can be used in general purpose paints or surface coatings.", "The present invention relates generally to alkyd or oil paint formulations, sometimes referred to as enamel paints, which contain specific enzymes, which remain inactive in an applied surface coating of the paint formulation until specifically activated by a subsequently-applied applicator solution.", "Both the inclusion of specific enzymes and the design of the applicator are novel and form an integral part of the invention.", "When the applicator is applied to finished surface coatings containing lipase enzymes, the enzymes catalyze the hydrolysis of the oil binder in the paint or surface coating by cleaving the ester bonds in the 1 and 3 positions of the triglyceride molecules.", "This allows for easy and rapid removal of the coating without damaging or effecting in any way the underlining surface.", "The present invention also relates to methods of producing the enzyme preparation for inclusion in paint and the preparation of the rapid activator removal applicator.", "BACKGROUND OF THE INVENTION Graffiti is usually applied on public and other buildings by way of ‘spray-can’ aerosol paints.", "These paints are usually alkyd and/or oil paints or oil enamels which are designed to dry rapidly.", "Their legal use is most helpful in ‘touch-up’ and small job applications where a rapid drying gloss finish is required.", "In the ‘hit and run’ world of so-called graffiti art, they are the most popular paint used.", "Conventional graffiti paint removal methods usually remove or damage the base surface coating upon which the graffiti is applied.", "This invention is specifically designed to remove the graffiti paint only.", "The estimated cost of graffiti removal throughout the world is variously estimated by municipal authorities to be in the hundreds of millions of dollars each year.", "In many countries the call for the removal from sale of oil enamel spray (aerosol) type paint cans is very strong.", "In Australia to date, the movement to ban the sale of such paints has been overcome by a voluntary paint industry initiative; which also includes the insertion of the following warning label on the paint cans: ‘IMPORTANT: Deliberate misuse of this aerosol to damage private property is a crime.’ To fully appreciate the background to this invention, it is important to understand the chemistry of both paint production and enzyme catalysis.", "An enzyme is a protein with catalytic activity that is restricted to a limited set of reactions defining the specificity of the enzyme.", "This invention places specific water-soluble enzyme preparations into oil or alkyd oil based paints or surface coatings.", "The invention provides for the design of a carrier system for the enzyme that allows its incorporation into oil type paints.", "This is described herein below.", "A simple description for a Paint follows.", "A more complete description can be obtained from the National Paint, Varnish and Lacquer Association, 1500 R.I. Avenue, Washington D.C. 20036 United States of America, or in ‘Paints and Surface Coatings’, Theory and Practice Second Edition.", "Edited by R. Lambourne and T. A. Strivens.", "Published by Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge CB1 6AH, England 1999.Paint is a unfirmly dispersed mixture with variable viscosity and consisting of (1) a drying oil, synthetic resin, or other film forming component called the BINDER; (2) a SOLVENT or THINNER; and (3) an Organic or Inorganic PIGMENT.", "The BINDER AND SOLVENT are collectively called the VEHICLE.", "Paints are used to protect surfaces and to provide decoration.", "This invention places a specifically prepared enzyme mixture into paints which contain oils and/or alkyds as a binder type.", "It is also helpful to understand more of the characterization of the oils and alkyds used in paint preparation.", "Those skilled in the art will have a fully comprehensive understanding of oils and alkyds used in paint manufacture.", "Vegetable oils and vegetable oil fatty acids continue to play an important role in surface coatings due to their availability as a renewable resource.", "Oils are mixed glycerol esters of long chain monocarboxylic acids called fatty acids.", "The Carbon length is generally C18Oils used include, Linseed Oil, Soya Bean Oil, Coconut Oil and ‘Tall Oil’.", "Oils are classified as Drying, Semi Drying, or Non-Drying.", "Table 1.0 illustrates typical composition of major oils used in surface coatings.", "TABLE 1.0 9.12, 15 Saturated 9.12 Linolenic Conjugated Acids Oleic Acid Linoleic Acid Acid Acid TUNG 6 7 4 3 90 LINSEED 10 20-24 14-19 48-54 0 SOYA BEAN 15 22-28 52-55 5-9 0 CASTOR OIL 2-4 90-92 3-6 0 0 DEHYDRATED 2-4 6-8 48-50 0 0 CASTOR OIL TALL 3 30-35 35-40 2-5 10-15 COCONUT 89-94 6-8 0-2 0 0 Linseed oil is a good drying oil containing more than 60% of the polyunsaturated linoleic and linolenic acids.", "Alkyd resins are produced by condensation and polymerization of dihydric or polyhydric alcohols and a polybasic acid such as phthalic anhydride and usually with a drying oil modifier.", "These resins are described as long oil alkyds (60% oil) medium oil alkyds (40% oil) or short oil alkyds (less than 40% oil).", "In practical terms, the reaction of glycerol and oil/s in the production of monoglycerides in the presence of a catalyst, does not go to completion and the equilibrium distribution of species include oil, polyol and mono and diglycerides.", "This is important in this invention as the remaining presence of free oil/s allows, when correctly activated, the enzymatic hydrolysis of the oil to effect a gross weakening of the binder allowing for easy removal of the paint.", "OBJECTS OF THE INVENTION It is an object of this invention to provide a paint formulation which includes an enzyme preparation which facilitates removal of surface coatings based on such paint formulations.", "It is a further object of this invention to provide an alkyd or oil paint preparation which includes a lipase enzyme preparation which can be activated if and when required to facilitate removal of a surface coating or other paint deposit comprising or based on the paint preparation.", "It is another object of this invention to provide a paint formulation which includes an enzyme preparation and an enzyme activator, suitable for removal of unwanted graffiti or for general paint removal when activated.", "It is yet a further object of this invention to provide a surface coating composition for protecting surface structures which either have no primary surface coating, or have an existing primary surface coating which requires protection by means of the application of an outer clear surface coating.", "It is yet another object of this invention to provide an enzyme preparation for inclusion in a paint formulation which facilitates removal of graffiti, or general paint removal.", "It is yet again another object of this invention to provide a method of producing an enzyme preparation and an enzyme activator for inclusion in a paint formulation, and of producing a paint formulation incorporating same.", "It is yet again a further object again of providing a method of graffiti removal and/or of general paint removal of a surface coating or other surface deposit of a paint formulation based on the activation of an enzyme present in the surface coating or deposit.", "It is yet again another object of this invention to provide an anti-graffiti formulation which goes at least some way towards overcoming or at least minimising the prior art problems or limitations outlined above, or for providing a clear alternative choice for consumers.", "These and other objects of this invention will become more apparent from the following description.", "DISCLOSURE OF THE INVENTION According to one aspect of this invention there is provided an enzyme preparation for inclusion in a paint formulation, particularly an alkyd or oil paint formulation, together with a removal activator, which when activated is suitable for general paint removal or for specific graffiti removal.", "According to another aspect of this invention there is provided a paint formulation, and especially an alkyd or oil paint formulation which includes an enzyme preparation and means for activating said enzyme, to facilitate removal of graffiti, or general paint removal, in respect of surface coatings or deposits comprising or based on the said paint formulation.", "According to a further aspect of the invention, there is provided a clear paint formulation, and especially a clear alkyd or oil paint formulation, adapted to provide a clear sealer coating for both unpainted and previously painted surfaces.", "BEST MODE OF CARRYING OUT THE INVENTION More specifically, the present invention provides a paint formulation comprising a surface coating base with one or more alkyd-, oil-, cellulose- or acrylic-type binders, and an enzyme specific to said one or more binders and adapted to be activated or catalysed when required to facilitate subsequent removal of a deposit or surface coating layer of said paint formulation.", "According to a further aspect of the invention, there is provided a paint formulation of the above type, adapted to provide a clear sealer coating for both unpainted and previously painted surfaces, comprising a clear surface coating base together with one or more alkyd-, oil-, cellulose- or acrylic-type binders, and an enzyme specific to said one or more binders.", "According to another aspect of the invention, there is provided a paint formulation adapted to facilitate removal of graffiti, or paint removal in general, in respect of surface coatings or deposits comprising or based on the above paint formulations.", "According to yet a further aspect of the invention, there is provided a method of preparation of a paint formulation containing an enzyme which facilitates subsequent removal of a surface coating of the composition, said method comprising mixing the enzyme with a non-ionic surfactant pre-heated up to about 65° C., adding a hydrocarbon solution followed by rapid cooling of the mixture, and thereafter adding the cooled mixture to a hydrophobic paint composition and final mixing to provide an homogenous mixture of the paint formulation.", "The invention further provides a method of removal of a deposit or of a surface coating of a paint formulation of the type herein described, said method comprising applying to said deposit or surface coating a further coating containing a specific activator to promote enzymatic catalysis and subsequent hydrolysis of the 1, 3 ester bonds of the triglyceride molecules present in the surface coating, thereby causing the surface coating to break down or disintegrate.", "Preferably, the enzyme preparation for inclusion in alkyd and oil paints is a Lipase enzyme, such as that classified by the Chemical Abstracts Service Registry as: Lipase, triocylglycose, CAS NO 9001-62-1.Another suitable enzyme preparation for inclusion in alkyd and oil paints according to the invention is classified by the International Union of Biochemistry as EC 3.1.1.3.An example of such an enzyme according to the invention is the lipase enzyme derived from Thermomyces lanuginosus produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism, ideally with an activity of 100 KJU/g.", "Such an enzyme in aqueous solution with a lipolytic activity of 100 KLU/g is determined relative to an analytical standard under the following conditions: Substrate, Tributyrin.", "Temperature 30 degrees centigrade and pH 7.0.Enzyme types, other than lipase, with specific catalytic activity are also useful, and are further described in the examples of this invention.", "A preferred enzyme preparation according to the invention is where the aqueous enzyme is mixed with a prescribed volume of solubilized polyoxyethylene (20) oleyl ether non-ionic surfactant material.", "Preferably the premixed aqueous enzyme and polyoxyethylene (20) oleyl ether are further mixed with a hydrocarbon liquid.", "The hydrocarbon liquid may be aromatic, aliphatic or mixtures of both aromatic and aliphatic hydrocarbon liquids.", "According to another embodiment, the preparation is mixed with alkyd, oil, enamel or any paint preparation containing some portion of mixed glycerol esters of long chain monocarboxylic acids (tryglycerides).", "Preferably, the enzyme preparation is mixed with a paint containing an alkyd resin and such alkyd resin having been produced by condensation and polymerisation of dihydric or polyhydric alcohols and a polybasic acid such as phthalic anhydride together with a drying oil modifier.", "New or existing surface structures, which have been finished with paint or other kinds of surface coatings, can also be protected by this invention by way of application of a tough clear sealer coating containing an enzyme preparation and means for activating the enzyme.", "A clear solution with alkyd, oil, cellulose, or acrylic type binders is applied by either spray or brush or roller on such surfaces as painted brick or block, rendered cement surfaces, tiled surfaces, stainless steel surfaces, timber and general high traffic area surfaces which can be at risk of graffiti damage.", "In the event that graffiti paint is applied to such a protected surface the modified activator is capable of both removing the offending graffiti and activating the enzymes present in the clear sealer coating; thus enabling a clean removal of the graffiti and sealer coating without damage to the primary surface coating.", "Upon removal, the area is easily re-protected, by a fresh application of the enzyme-containing clear protective sealer coating.", "The invention also provides a method of producing an activator applicator solution, wherein such solution, when applied to a painted surface containing any of the enzyme preparations described herein, will provide a chemical environment suitable for the enzymatic catalysis and subsequent hydrolysis of the 1,3 ester bonds of the tryglyceride molecules present in the paint, thereby causing the paint film to disintegrate.", "Preferably, the applicator solution contains a mixture of demineralized water, sodium hypochlorite at between 50 and 60 grams per litre and sodium hydroxide at 10 grams per litre and selected alkaline salts at between 0.1 and 0.5 grams per litre and 0.3% xanthan gum derived from Xanthomonas campestris.", "The invention also provides a method of removing the paint, treated as above, by vacuum equipment apparatus which produces in-situ warm to hot water and with either a brush or brushless action vacuums the paint debris into a contained vessel.", "According to one preferred embodiment the enzyme preparation is a lipase enzyme derived from Rhizomucor miehei produced by submerged fermentation.", "The lipase enzyme can be derived from a variety of microorganisms.", "The enzyme preparation according to the invention can be of a non-lipolytic type and may be of any or a mixture of the following types, protease, amylase, cellulase, or hemicellulase.", "The applicant has developed various methods of producing Lipase enzyme and other enzyme solutions, which can be incorporated into any gloss solvent paint preparation.", "The Lipase is classified by the Chemical Abstract Services Registry as ‘Lipase,triocylglycose,CAS No.", "9001-62-1’.", "The corresponding Enzyme Classification Number (International Union Of Biochemistry) is EC 3.1.1.3.Other enzymes and their reactions are more fully described in the examples below.", "The enzyme solution also contains but is not limited to, demineralized water, polyoxyethylene (20) oleyl ether, and liquid hydrocarbon solution/s.", "The activity of the enzyme preparation is expressed in Kilo Lipase Units (KLU).", "The invention also includes the materials and method of manufacture of an applicator suitable for activating the unactivated enzyme solution described above and contained within a dried surface coating.", "The enzyme preparation is designed to operate at a pH of around 11.0.Typical paint preparations described previously have a pH of around 6.8.The enzyme preparation can be activated at a temperature of between 10° C. and 40 C. The activator solution contains warm to hot water—which can be prepared in the special device described below, sodium hydroxide, sodium hypochlorite, alkaline salts, xanthan gum, methylene chloride and sequestering agents and surfactants.", "Upon activation the enzyme preparation hydrolyses the ester bonds in the 1,3 position of the tryglyceride molecules present in the paint binder and weakens the paint film so that cleaning and removal can be affected by the apparatus described below.", "Preferably, the invention also relies upon utilizing a vacuum type water cleaner, similar to those used in carpet cleaning, for one-step removal of the paint film weakened as described above.", "This apparatus produces in-situ warm to hot water and with either a brush or brushes action vacuums the paint debris into a contained vessel and minimizes clean up time.", "DETAILED DESCRIPTION OF THE INVENTION The nature of the present invention may be more clearly understood from the following preferred but non-limiting examples.", "EXAMPLE 1 A black enamel paint containing linseed oil in the binder component of the vehicle was selected.", "In a preferred embodiment of this invention an enzyme solution was prepared for inclusion into this black enamel paint as now described.", "A lipase enzyme solution, containing lipase enzymes derived form Thermomyces lanuginosus produced by submerged fermentation of a genetically modified Aspergillus oryzae microorganism, with an activity of 100 KJL/g was selected.", "This enzyme preparation is a 1,3- specific lipase, in that it cleaves the ester bonds 1 and 3 of a triglyceride.", "This enzyme protein has an IUB Number of 3.1.1.3 and a CAS Number of 9001-62-1 and an EINECS Number of 232-619-9.This enzyme has maximum activity at pH 11.0.The pH of the selected black enamel paint is 6.8.This enzyme has optimum activity at 38° C. The enzymes as so far described are contained in an aqueous solution containing between 1-10% w/w protein enzymes.", "The following description describes how to incorporate this aqueous solution into a hydrophobic enamel paint.", "A quantity sufficient of polyoxyethylene (20) oleyl ether is heated to, between 28° C. and 65° C. To this heated non-ionic surfactant is added a volume of aqueous lipase enzyme preparation as previously described.", "This is well mixed.", "To this mixture is added a 100% hydrocarbon solution with a boiling point in the range 155° C.-165° C. and a relative density of 0.85-0.91.This is well mixed and rapidly cools the mixture.", "This complete solution is now capable of being added to the hydrophobic enamel paint.", "A prescribed aliquot of this solution is added to 1000 mls of the black enamel paint and well mixed in.", "This paint-enzyme preparation is then applied by fine horse hair brush to wooden, steel and concrete surfaces and allowed to dry in the normal way.", "Untreated black enamel paint is applied to adjoining portions of the wood, steel, concrete surfaces and allowed to dry.", "The rapid enzyme activator solution is prepared in the following way.", "To demineralized water is added Sodium Hypochlorite at between 50 and 60 grams per litre and Sodium Hydroxide at 10 grams per litre and selected Alkaline salts between 0.1 and 0.5 grams per litre.", "To this combined and well mixed solution is added up to 0.3% xanthan gum derived from Xanthomonas campestris.", "Sufficient non-ionic surfactant is added to improve surfactant qualities of the mixture.", "The final pH of the mixture is adjusted to pH 11.0.The applicator is applied at ambient temperatures to the prepared painted surfaces described above.", "The applicator is carefully applied to the total painted areas.", "Due to its high viscosity the applicator can remain on vertical surfaces for considerable periods.", "The combination of pH, penetration, time and temperature work to activate the dormant lipase enzyme catalysts.", "At various times frames, commencing at 20 minutes, a carpet cleaner type vacuum cleaner capable of heating water and scrubbing and vacuuming surfaces is employed to wash and scrub all treated and non treated painted surfaces.", "The water temperature is 39° C. The enzyme contained painted surfaces are easily removed.", "The non-enzyme containing painted surfaces remain intact and retain gloss and colour.", "EXAMPLE 2 An enzyme protein described as Lipase, in an aqueous solution and with an IUB number of 3.1.1.3 and a CAS number of 9001-62-1 and an EINECS number 232-619-9 and a prescribed lipolytic activity of 100 KLU/g was selected.", "This activity is determined relative to an analytical standard under the following conditions: Substrate: Tributyrin Temperature: 30° C. pH: 7.0 Tribuyrin is described in the Merck Index, Twelfth Edition, and Published by Merck Research Laboratories, Division of Merck and Co. Inc., Whitehouse Station, N.J., USA 1996, as follows: Tributyrin.", "Butanoic acid 1,2,3-propanetriyl ester; glyceryl Tributyrate.", "C15H26O6 Mol.", "Weight 302.37.C, 59.58% H, 8.67% O, 31.75% (C3H7COO)3C3H5.Prepared by esterification of glycerol with excess butyric acid.", "Oil liquid.", "Bitter taste.", "Insoluble in water.", "The enzyme described above is a 1,3- specific lipase that cleaves the ester bonds in positions 1 and 3 of a triglyceride.", "The enzyme has a lipolytic activity of between 60 and 100% in a temperature range of between 10° C. and 60° C. Optimum activity is between 30° C. and 40° C. Optimum lipolytic activity is found at pH 11.0.The enzyme described is classified as non-toxic.", "The enzyme solution is biodegradable.", "By exhaustive experimentation, the inventors discovered that relatively minor amounts of the described enzyme can be contained in the paint solution to be effective.", "Effectiveness is the ability that when an activator is applied the contained enzymes weaken the binding capability within a paint film so that easy removal is facilitated.", "In this example an amount equivalent to 0.16% of active enzyme in a litre of paint solution is selected for inclusion in a non-ionic hydrocarbon carrier.", "So that no greater than 1% of total additive is added to a finely balanced paint formulation sufficient polyoxyethylene (20) oleyl ether is heat sobulised to absorb the enzyme preparation and mixed well.", "To the cooled and well mixed mixture is added sufficient liquid hydrocarbon solution with a boiling point of between 155° C.-165° C., and a relative density of 0.85-091.The solution is added to black enamel paint so that the total addition on a weight/weight basis is less than 1%.", "EXAMPLE 3 It is most important that the enzyme preparation is delivered to the paint emulsion in as colourless condition as possible.", "Paint formulators are most careful in pigment quality and quantity selection.", "In coloured paints the slightest variation can cause significant colour and tone changes.", "The inventors have discovered a means of first incorporating the enzyme stock solution in a non-ionic surfactant and then combining this solution with a hydrocarbon solvent which enables the total liquid to be successfully incorporated into an oil type paint.", "The solution is colourless to very slight white-yellow.", "The rate of addition of enzyme effectively delivered by this solution, created no colour change in several samples of paint, when measured by spectrophotometer.", "A selected polyoxyethylene (20) oleyl ether is employed as the non-ionic surfactant.", "This material is semi solid at temperatures of up to 25° C. The first difficulty to overcome in this process is to liquefy the surfactant at temperatures which will not activate the lipase enzyme.", "It was discovered that by heating the surfactant and enzyme solution to 25° C., mixing vigorously and adding rapidly the required volume of hydrocarbon liquid, the enzyme mixture could be prepared without loss of activity.", "The mixing must be vigorous.", "The hydrocarbon liquid rapidly cools the mixture.", "The enzyme solution can be either added to a finished paint or incorporated in the build of the paint.", "In this example, as in the previously cited examples, the enzyme solution is added to a finished black enamel paint.", "Addition is at ambient temperature and with moderate, non-foaming agitation.", "The enzyme-included paint is applied to various surface types and tested by application of the activator, described previously.", "EXAMPLE 4 In paint manufacture care is given to the properties known as sedimentation and flocculation.", "The pigments employed usually have a density greater than the resin solution of the paint.", "Therefore under the influence of gravity pigments will tend to settle according to Stokes law.", "This factor is considered carefully in the selection of the hydrocarbon material used to disperse the pigment.", "Aliphatic or aromatic hydrocarbons may be used.", "Either Aliphatic hydrocarbon solvent (white spirit) or Aromatic hydrocarbon solvent (toluene) may be used in this invention for incorporation of the enzyme into paint.", "Toluene (methylbenzene) is slightly soluble in water.", "This is helpful in the context of the invention.", "The paint manufacturer may prefer white spirit to toluene, for greater pigment absorption and the development of a stronger dispersion.", "As in previous examples, the selected Lipase enzymes are incorporated into a non-ionic surfactant and either aromatic or aliphatic hydrocarbons, or mixtures of both aromatic and aliphatic are used for dispersion into the built paint.", "The use of surfactant further ensures that this invention does not alter the pre-existing sedimentation characteristics present in the paint.", "The addition of the co-mixture does not alter the rheological characteristics of the paint.", "EXAMPLE 5 In biological terms the usual tendency for natural decay mechanisms is for a lowering of pH values to acidic.", "This phenomenon is an important consideration in this invention.", "A paint or surface coating containing the enzyme systems, so far described in the above examples, must retain its usual characteristics when used for legitimate purposes.", "The enzyme systems employed are deliberately selected to operate in high alkaline, high pH conditions.", "Unless the paint system is subjected to constant caustic bombardment the enzyme mechanism will not be activated.", "If such an eventuality were to occur naturally it is unlikely that any paint system could maintain gloss, appearance and durability for prolonged periods.", "To further safeguard legitimate paint applications the enzyme system can only be activated to catalysis conditions by complex alkaline systems and slightly elevated temperatures.", "EXAMPLE 6 New or existing surface structures, which have been finished with paint or other kinds of surface coatings, can be protected by this invention by way of application of a tough clear sealer coating containing a specially prepared enzyme solution according to the invention.", "A clear solution with alkyd, oil, cellulose, or acrylic type binders is applied by either spray or brush or roller on such surfaces as painted brick or block, rendered cement surfaces, tiled surfaces, stainless steel surfaces, timber and general high traffic area surfaces which can be at risk of graffiti damage.", "Cellulose nitrate (nitrocellulose) containing lacquers and acrylic lacquers, as well as vinyl preparations containing either organic esters of cellulose or cellulose derivatives such as hydroxy ethyl cellulose, can be prepared which contain enzyme mixtures, which when activated are capable of weakening these types of binders.", "Once weakened removal of the sealer paint film proceeds as previously described.", "Cellulase or hemicellulase enzymes or combinations of each can be utilised in the enzyme preparation included in the clear sealer coating/s.", "Cellulase enzymes derived from either Aspergillus niger or Trichoderma viride capable of decomposing cellulosic polysaccharides into smaller fragments are selected for inclusion in an admixture, which also contains lipase enzymes, non-ionic surfactant and hydrocarbon solutions.", "A novel cellulase enzyme derived from a thermophilic soil fungus, Thielatia terrestris, is selected for inclusion in a clear surface coating, this enzyme is only activated to catalysis by high temperature.", "This feature is particularly suitable for graffiti removal as described previously hereinabove.", "An activator containing sodium hydroxide, sodium hypochlorite, alkaline salts, methylene chloride, n-methyl pyrrolidone, xanthan gum and non-ionic surfactant, is added to very hot water and mixed thoroughly.", "The temperature of the water is greater than 78° C. Apparatus previously described, applies the activator to graffiti paint damaged surfaces.", "Such surfaces having been previously coated with the clear sealer coating already described.", "The activator will remove the graffiti paint, irrespective of its type or origin.", "The enzyme contained clear sealer coating beneath the graffiti damage, will be weakened by the action of the enzyme catalysis triggered by the activator.", "A complete removal of the graffiti and the enzyme containing clear sealer coating can be effected without damage to the primary surface coating.", "Once dry, the primary surface can again be protected by application of the clear enzyme/s containing sealer protective coating.", "Although the invention has been described with reference to preferred embodiments and examples, the present invention has been shown and it will be apparent to those having ordinary skill in the art that a number of changes, modifications or alterations to the invention described herein may be made, none of which depart from the spirit of the present invention.", "All such changes, modifications, and alterations should therefore be seen as being within the scope of the present invention.", "It should be appreciated that the present invention provides a substantial advance in paint formulations and the removal thereof providing all of the herein-described advantages without incurring any relative disadvantages.", "The words “comprise”, “comprises” and “comprising”, as used herein, are used in the inclusive sense of “having” or “including”, and not in the exclusive sense of “consisting only of”." ] ]

[ [ "Process and mould for thermoforming containers", "The present invention relates to a method ol manufacturing water-soluble containers using a horizontal intermittent motion thermolorming machine which comprises the steps of: a) locating a first water-soluble film overa mould, said mould containing a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3 mm and between an edge of the mould and the closest cavity is at least 1.5 mm; b) thermoforming the first film to produce a plurality of pockets; c) at least partially filling the pockets with a composition; and d) sealing the plurality ol the at least partially filled pockets.", "The cavities are positioned in the array such that there are a plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge ol the mould, for receiving support means fitted to the machine for supporting the film." ], [ "1.A process for producing a water-soluble container using a horizontal intermittent motion thermoforming machine which comprises the steps of: a) locating a first water-soluble film over a mould, said mould containing a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3 mm and between an edge of the mould and the closest cavity is at least 1.5 mm; b) thermoforming the first film to produce a plurality of pockets; c) at least partially filling the pockets with a composition; and d) sealing the plurality of the at least partially filled pockets, wherein the cavities are positioned in the array such that there are a plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge of the mould, for receiving support means fitted to the machine for supporting the film.", "2.A process as claimed in claim 1 in which step d) comprises placing a second water-soluble film on top of the at least partially filled pockets and sealing the films together.", "3.A process as claimed in any one of the preceding 4.A process as claimed in any one of the preceding claims in which the depth of the cavities lies in the range of 10 to 80% of the shortest dimension of the mouth cavity.", "5.A process as claimed in any one of the preceding claims in which the depth of the cavities lies in the range of 40 to 60% of the shortest dimension of the mouth cavity.", "6.A process as claimed in any one of the preceding claims in which the cavity bases are planar.", "7.A process as claimed in any one of claims 1 to 4 in which the cavity bases are rounded.", "8.A process as claimed in claim 6 in which the rounded bases have a radius of 20 mm.", "9.A process as claimed in any one of the preceding claims in which corners formed where the cavity side walls meet each other are rounded.", "10.A process as claimed in claim 9 in which the side wall corners have a radius of 10 mm.", "11.A process as claimed in any one of the preceding claims in which edges formed where the cavity side walls meet an upper surface of the mould are rounded.", "12.A process as claimed in claim 11 in which the side wall-mould upper surface edges have a radius of lmm.", "13.A process as claimed in any one of the preceding claims in which bottom corners, formed where the cavity side walls meet the cavity base, are rounded.", "side wall-base bottom corners have a radius of 10 mm.", "15.A process as claimed in claim 13 or claim 14 in which air bores are located in the side wall base bottom corners.", "16.A process as claimed in claim 15 in which the air bores have a diameter of 0.1 mm to 1 mm.", "17.A process as claimed in claim 16 in which the air bores have a diameter of 0.4 mm to 0.5 mm.", "18.A process as claimed in any one of the preceding claims in which the shortest dimension of the planar surface between two adjacent cavities lies in the range of 4 mm to 10 mm and between an edge of the mould and the closest cavity lies in the range of 2 mm to 5 mm.", "19.A process as claimed in any one of the preceding claims in which a continuous strip of uninterrupted planar surface is provided between adjacent rows of cavities.", "20.A process as claimed in any one of the preceding claims in which a continuous strip of uninterrupted planar surface is provided between every other pair of adjacent rows of cavities.", "21.A mould for use in a thermoforming process for manufacturing water-soluble containers from water-soluble films, in which said mould contains a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3mm and between an edge of the mould and the closest cavity is at least 1.5 mm, and uninterrupted planar surface of the mould from a leading to a trailing edge of the mould.", "22.A mould as claimed in claim 21 in which the depth of the cavities lies in the range of 10 to 80% of the shortest dimension of the cavity mouth.", "23.A mould as claimed in claim 21 or 22 in which the depth of the cavities lies in the range of 40 to 60% of the shortest dimension of the cavity mouth.", "24.A mould as claimed in any one of claims 21 to 23 in which the cavity bases are planar.", "25.A mould as claimed in any one of claims 21 to 24 in which the cavity bases are rounded.", "26.A mould as claimed in claim 25 in which the rounded bases have a radius of 20mm.", "27.A mould as claimed in any one of claims 21 to 26 in which corners formed where the cavity side walls meet each other are rounded.", "28.A mould as claimed in claim 27 in which the side wall corners have a radius of 10 mm.", "29.A mould as claimed in any one of claims 21 to 28 in which edges formed where the cavity side walls meet an upper surface of the mould are rounded.", "30.A mould as claimed in claim 29 in which the side wall-mould upper surface edges have a radius of lmm.", "31.A mould as claimed in any one of claims 21 to 30 in which bottom corners, formed where the cavity side walls meet the cavity base, are rounded.", "32.A mould as claimed in claim 31 in which the side wall-base bottom corners have a radius of 10 mm.", "33.A mould as claimed in claim 31 or claim 32 in which air bores are located in the side walls base bottom corners.", "34.A mould as claimed in claim 33 in which the air bores have a diameter of 0.1 mm to 1 mm.", "35.A mould as claimed in claim 34 in which the air bores have a diameter of 0.4 mm to 0.5 mm.", "36.A mould as claimed in any one of claims 21 to 35 in which the shortest dimension of the planar surface between two adjacent cavities lies in the range of 4mm to lOmm and between an edge of the mould and the closest cavity lies in the range of 2 mm to 5 mm.", "37.A mould as claimed in any one of claims 21 to 36 in which a continuous strip of uninterrupted planar surface is provided between adjacent rows of cavities.", "38.A mould as claimed in any one of claims 21 to 37 in which a continuous strip of uninterrupted planar surface is provided between every other pair of adjacent rows of cavities.", "39.A mould as claimed in any one of claims 21 to 38 in which air bores are lacated.", "40.A container formed by the process of any one of the preceding claims." ], [ "The present invention relates to a method of manufacturing water-soluble containers and a mould for use therein.", "It is known to package chemical compositions which may be of a hazardous or irritant nature in water soluble or water dispersible materials such as films.", "The package can simply be added to water in order to dissolve or disperse the contents of the package into the water.", "For example, WO 89/12587 discloses a package which comprises an envelope of a water soluble or water dispersible material which comprises a flexible wall and a water-soluble or water-dispersible heat seal.", "The package may contain an organic liquid comprising, for example, a pesticide, fungicide, insecticide or herbicide.", "It is also known to package detergents in water-soluble or water-dispersible containers.", "For example, WO 94/14941 discloses a water-soluble or water-dispersible capsule containing an aqueous dishwasher detergent.", "The capsule is made of gelatin.", "CA-A-1,112,534 discloses a packet made of a water-soluble material in film form enclosing within it a paste-form, automatic dishwasher-compatible detergent composition.", "The water-soluble material may be, for example, polyvinyl alcohol, polyethylene oxide or methyl cellulose.", "Example 1 illustrates an embodiment wherein a poly(vinyl alcohol) (PVOH) film is made into a 5 cm square packet by heat sealing its edges, and the packet is filled with a composition which contains 8.5 wt.% water.", "In fields such as detergents for domestic use, an attractive appearance for an article is extremely desirable.", "However in the prior art, such as that described above, a bag is simply formed from a single sheet of water-soluble film.", "The film is folded and the edges heat-sealed to form the bag.", "The bag is then filled and heat-sealed.", "This produces a rather flat, limp envelope containing the product.", "Furthermore there may be a lack of uniformity between different bags because of their flexible nature.", "We have discovered that this type of product is not deemed to be attractive by an average consumer.", "It is known to form water-soluble containers by thermoforming a water-soluble material.", "For example, WO 92/17382 discloses a package containing an agrochemical such as a pesticide comprising a first sheet of non-planar water-soluble or water-dispersible material and a second sheet of water-soluble or water-dispersible material superposed on the first sheet and sealed to it by a continuous closed water-soluble or water-dispersible seal along a continuous region of the superposed sheets.", "It is stated to be advantageous to ensure that the package produced is evacuated of air or the contents are under reduced pressure to provide increased resistance to shock.", "Furthermore, when the package contains a liquid, the liquid must be an organic liquid which must be reasonably dry and typically contains less than 2 to 3% of water to ensure that it does not attack the water-soluble package and cause leakage.", "EP-A-654,418 describes self-standing flexible pouches which may contain, for example, liquid detergent compositions for refilling other containers.", "In order to avoid folding of the pouch, which can lead to cracking and leakage, the bag is inflated before it is sealed.", "In order to improve the strength of packages containing liquids, it is also known to provide the package with residual inflatability.", "Thus, for example, EP-A-524,721 describes a water-soluble package which contains a liquid, wherein the package is inflatable to a volume which is greater than the initial volume of the package.", "Thus the package is filled to less than its complete capacity, and the unused capacity may be partially, but not totally, filled with a gas such as air.", "The unused capacity which does not contain gas provides the residual inflatability.", "We have now surprisingly discovered a water-soluble container which contains a liquid composition can be given an attractive three-dimensional appearance by using a thermoforming technique, such as that disclosed in WO 92/17382, on a PVOH film and ensuring that the liquid composition has a reasonably large water content of at least 3 wt% free water, based on the weight of the aqueous composition.", "Immediately after the containers are prepared, they have a limp, unattractive appearance.", "However, after storage for a short while, for example, from a few minutes to a few hours, they develop a more attractive three-dimensional appearance, and also appear to look fuller.", "They can also be said to have a “puffed-up” appearance.", "Although not bound by this theory, it is believed that the water in the aqueous composition shrinks the PVOH film around the liquid composition to provide the attractive appearance.", "In other words the PVOH film attempts to recover its original shape when contacted with the aqueous composition.", "In our co-pending application entitled “Improvements in or Relating to Aqueous Compositions” we describe a process for producing a container as defined above which comprises the steps of: b) filling the pocket with the aqueous composition; c) placing a second PVOH film on top of the filled pocket; and d) sealing the first film and second film together.", "The method of forming the container is similar to the process described in WO 92/17382.A first PVOH film is initially thermoformed into a mould to produce a non-planar sheet containing a pocket, such as a recess, which is able to retain the aqueous composition.", "The pocket is generally bounded by a flange, which is preferably substantially planar.", "The pocket may have internal barrier layers as described in, for example, WO 93/08095.The pocket is then filled with the aqueous composition, and a second PVOH film is placed on the flange and across the pocket.", "The second PVOH film may or may not be thermoformed.", "The pocket may be completely filled, or only partly filled, for example to leave an air space of from 2 to 20%, especially from 5 to 10%, of the volume of the container immediately after it is formed.", "Partial filling may reduce the risk of rupture of the container if it is subjected to shock and reduce the risk of leakage if the container is subjected to high temperatures.", "The films are then sealed together, for example by heat sealing across the flange.", "A suitable heat sealing temperature is, for example, 120° C. to 195° C., for example 140° C. to 150° C. A suitable sealing pressure is, for example, from 250 kPa to 800 kPa.", "Examples of sealing pressures are 276 kPa to 552 kPa (40 p.s.i.", "to 80 p.s.i.", "), especially 345 kPa to 483 kPa (50 p.s.i.", "to 70 p.s.i.)", "or 400 kPa to 800 kPa (4 to 8 bar), especially 500 kPa to 700 kPa (5 to 7 bar) depending on the heat sealing machine used.", "Suitable sealing dwell times are at least 0.4 seconds, for example 0.4 to 2.5 seconds.", "Other methods of sealing the films together may be used, plate, insert bonding, fraction sealing or spin welding.", "An adhesive such as water or an aqueous solution of PVOH may also be used.", "The adhesive can be applied to the films by spraying, transfer coating, roller coating or otherwise coating, or the films can be passed through a mist of the adhesive.", "The seal desirably is also water-soluble.", "It is, however, extremely difficult to manufacture products using PVOH and other materials having similar physical characteristics, partly because of their hygroscopic nature, but mainly due to the fact that the material is very soft and floppy, making it extremely difficult to handle and cut.", "In most thermoforming, vacuum forming or other similar forming processes, the films used have a degree of strength and rigidity.", "Thus friction drives are generally, although not exclusively, used to support the films and to transport them through the machine during the process.", "PVOH and similar films do not have this strength or rigidity and would stretch, thin and tear if subjected to such handling.", "Furthermore, thermo- and other such forming processes impose a significant amount of drawing and stretching of the material.", "As such the known method of thermoforming using PVOH materials utilises a single mould for each moulded product, with each PVOH film placed manually over each mould.", "This means that the amount of material available for deforming is greater, but it is a very labour intensive, slow and therefore costly process to achieve the manufacture of this type of product.", "We have discovered that standard horizontal intermittent motion thermoforming machines, such as those supplied by Multivac, Doyen and Tiromat, can be used to produce thermoformed containers from PVOH and films of a particular to the drive system, in order to run such films at normal production speeds.", "It is therefore an object of the present invention to provide an improvement in the process for manufacturing such containers, to enable a plurality of water-soluble containers to be formed simultaneously.", "A further objective is to provide a tool for use in a process for producing a plurality of water-soluble containers made from PVOH or other films of a similar physical nature or the like, at each stroke of an horizontal intermittent motion thermoforming machine.", "Yet another objective is to provide an improved process for producing multiple containers on a production scale.", "The invention therefore provides a process for producing a water-soluble container using a horizontal intermittent motion thermoforming machine which comprises the steps of: a) locating a first water-soluble film over a mould, said mould containing a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3 mm and between an edge of the mould and the closest cavity is at least 1.5 mm; b) thermoforming the first film to produce a plurality of pockets; c) at least partially filling the pockets with a composition; and d) sealing the plurality of the at least partially plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge of the mould, for receiving support means fitted to the machine for supporting the film.", "The invention further provides a mould for use in a thermoforming process for manufacturing water-soluble containers from water-soluble films, in which said mould contains a plurality of pocket forming cavities, defined by side walls and a base, in a 2-dimensional array, each cavity being surrounded by a planar surface of the mould on all sides in which the shortest dimension of the planar surface between two adjacent cavities is at least 3mm and between an edge of the mould and the closest cavity is at least 1.5 mm, and in which the cavities are positioned in the array such that there are a plurality of continuous strips of uninterrupted planar surface of the mould from a leading to a trailing edge of the mould.", "The invention will now be described, in further detail, by way of example only, with reference to and as shown in the accompanying drawings in which:- FIG.", "1 is an end elevation of a mould used in the present invention; FIG.", "2 is a side sectional elevation of the mould of FIG.", "1 on the line I-I; FIGS.", "3 to 5 are respectively plan views and cross sectional side elevations of a section of the mould of FIG.", "1 showing the dimensions of the cavities; FIG.", "6 is a plan view of the mould of FIG.", "1; and FIG.", "8 shows support rails supporting a web of material on a horizontal intermittent thermoforming machine.", "FIGS.", "1 and 2 show a mould 10 used for thermoforming a plurality of containers from PVOH or films having similar physical characteristics on a horizontal intermittent thermoforming machine comprising a series of stations as shown in FIG.", "7.These are the forming area 30, at which the film 31 is supplied from a reel to the moulds 10 and where the first thermoforming step takes place to form pockets; the filling station 32, at which the pockets are filled; the sealing station 33, to which a further film 34 is supplied to seal the pockets; the cooling station 35; and the cutting station 36 where the sealed containers are separated from each other by shear knives 38.Each mould 10 comprises a 2-dimensional array of pocket forming cavities 11.Although the Figures illustrate a regular array of 6×7 cavities 11 to form 42 containers simultaneously, the number and relative positioning of the cavities 11 may be varied.", "Essentially the surface dimensions of the mould are determined by the width and draw of the machine on which it is to be used.", "The best arrangement of the individual cavities 11 is determined according to the following considerations.", "Each cavity 11 must be surrounded by a planar surface 18 on all sides, to allow for subsequent sealing of the second film to the first films.", "This dimension should be at least 1.5 mm, but is preferably in the range of 2 mm to 5 mm.", "Thus the distance between any cavity and the edge of the mould 10 is at least 1.5 mm and the distance between any two cavities 11 is at least 3 mm.", "The maximum distance is obviously determined by the size As the materials used are very flexible, the web of film tends to sag.", "In order to enable all of the cavities 11 to be filled, support means must be fitted to the machine, from the end of the thermoforming station to the start of the filling station, and also preferably to the cutting station 36, to support the web of film.", "The support means may be provided by rails, bars, filaments, wires, rope, cable or the like.", "Most preferred are wires or rails.", "Where rails 1 are used, as shown in FIG.", "7, the leading ends of the rails may have a smooth cam surface 2 for lifting the web.", "The support means can be intermittent or, more preferably, continuous.", "FIG.", "8 shows how the web is drawn down from the thermoforming station by being held by grippers 3 which are pulled apart to provide some tension in the web.", "Too much tension will displace the thermoformed pockets.", "However, not enough tension is provided so that the web remains flat for filling.", "The support rails 1 maintiain the web as a substantially flat surface.This places an extra constraint on the arrangement of the cavities within the space available i.e.", "there must be clear channels 21 (see arrows Z on FIG.", "6) through the pattern of cavities 11 from the leading edge of the mould 10 to the trailing edge.", "It is preferred that these channels 21 are available between each cavity 11 (across the web i.e.", "on the leading edge), but this is not essential, depending on the number of cavities 11 across the leading edge.", "At least every other cavity should be supported.", "Located in the mould 10 beneath the cavities 11 are air channels 15, which communicate with the cavities 11 via air bores 16.The number and positioning of the air bores 15 has an effect on how the film is drawn into the cavities 11 during the thermoforming process, and configuration of the cavities 11 used.", "In particular they must be designed and arranged to effect the most even deformation of the film into the cavities 11.In a preferred embodiment the air bores 15 are located in the regions where the end and side walls 12, 13 of the cavities 11 join the cavity base 14.The holes are preferably of 0.1 mm to 1 mm diameter and more preferably 0.4 mm to 0.5 mm.", "Vacuum release bores 17 are drilled in the cavity bases 14.The shape of the cavities 11 is dictated partly by the intended use of the containers, but also by the processing constraints.", "A particularly convenient shape for an automatic dishwasher composition is illustrated in FIG.", "3 to 5.The dimensions of the cavities are determined by the required fill volume of the containers and any constraints resulting from the intended use of the containers.", "For example, if the containers are to be used as refill sachets for a trigger spray, the width of the containers, and therefore the cavities is determined by the diameter of the spray bottle neck.", "If the containers are to be used for a dishwasher product, all three dimensions are determined by the dispenser into which the containers will eventually be placed.", "One particularly suitable embodiment which we have found for a dishwasher product has a rectangular cavity mouth, the dimensions of which are 29 mm ×39 mm, with rounded corners, having a radius R1 of, preferably, 10 mm.", "The depth of the cavities depends partly on the area of the cavity mouth, to ensure that the film, can be drawn down without over thinning and tearing.", "This can also be affected by the area of film available between adjacent cavities 11.Referring to FIG.", "6, the upper surface 18 of the mould 10 can clearly be seen.", "The gaps between the cavities 11 are marked as dimensions preferably about 1:1.X and Y are desirably from 5 to 13 mm, preferably 7 to 12 mm, preferably about 10 mm.", "The preferred depth is in the range of 10 to 80% of the shortest dimension of the cavity mouth, and more preferably in the range of 40 to 60 %.", "A preferred depth of the cavities 11 where the mouth of the cavities 11 is 29 mm by 39 mm is 16 mm.", "The corners 19 formed where the end and side walls 12,13 of the cavities 11 join the cavity base 14, are preferably radiussed to avoid over thinning or tearing of the film, as it is drawn down the side walls 13 and the corners 19.The corners 19 preferably have a radius R2 and R3 of between 8 mm and 10 mm.", "The cavity base 14 may be planar or rounded.", "Especially where a greater cavity depth is used, such as 18 mm or 19 mm, it may be preferable to have a rounded base 14 to prevent regions of thicker material from being drawn directly downward to the centre of the base 14.A suitable radius for the base 14, in particular where the cavity depth is 18 mm, is 20 mm.", "The use of a rounded base 14 means that the positioning and direction of the air bores 16 may be different from those used with flat-bottomed cavities 11.This changes the way in which the film is drawn into the cavities 11.The edges 20, where the cavity end and side walls 12,13 join the upper surface 18 of the mould 10, are preferably rounded to allow for a smooth movement of the film over the edges 20 during the thermoforming process, to minimise the risk of the film snagging or tearing.", "The radius R4 is preferably small, e.g.", "1 mm, as it is difficult to fill this area of the cavities 11 without risk of fouling the sealing area.", "Another dimension which must be carefully controlled For cavities of the dimensions given above, it is preferred that the spacing between the cavities lies in the range of 9 mm to 16 mm.", "The draft angle of the side walls 12, 13 is preferably 3° to 5° to assist in the release of the containers.", "However, for certain very soft materials, such as PVOH, draft angles may not be necessary.", "The sizing of the mould 10, incorporating an array of cavities 11 in this manner, enables the film to be supported.", "The width of the web of film is determined by the width of the machine in which the mould is fitted.", "The mould is designed to fit the width of the machine with a suitable “overhang” of film, which can be used for transporting the film.", "It is suggested that small clips or grippers attached to a plurality of driven chains would enable the films to be transported appropriately.", "The grippers preferably toe-out to provide tension as the web of film moves through the machine.", "A first PVOH film is thus positioned over the mould 10 and thermoformed in a known manner to form a plurality of pockets.", "The pockets are then filled with an aqueous or other composition and a second film brought into position over the plurality of pockets.", "The second film may be the same as the first film or another material and is heat, or otherwise sealed, to the parts of the first film remaining on the upper surface 18 of the mould, as described previously.", "The filled containers may then be separated from each other.", "Alternatively, they may be left conjoined and, for example, perforations provided between the individual containers so that they can be separated easily at a later stage, for example by a consumer.", "If the containers are separated, the flanges may be left in dimensional appearance.", "Generally the flanges remaining should be as small as possible for aesthetic purposes while bearing in mind that some flange is required to ensure the two films remain adhered to each other.", "A flange having a width of 1 mm to 10 mm is desirable, preferably 1.5 mm to 6 mm, most preferably about 5 mm.", "For containers of compositions having a high water content, the containers may then be left for a while to attain their attractive appearance, or may be immediately packaged into boxes for retail sale, and left to attain their attractive appearance in the boxes.", "The containers may themselves be packaged in outer containers if desired, for example non-water soluble containers which are removed before the water-soluble containers are used.", "If more than one film is used for the containers, the films may be identical or different.", "The film may be partially or fully alcoholised or hydrolysed, for example, it may be from 40 to 100%, preferably 70 to 92%, more preferably about 88% or about 92%, alcoholised or hydrolysed, polyvinyl acetate film.", "The degree of hydrolysis is known to influence the temperature at which the PVOH starts to dissolve in water.", "88% hydrolysis corresponds to a film soluble in cold (i.e.", "room temperature) water, whereas 92% hydrolysis corresponds to a film soluble in warm water.", "An example of a preferred PVOH is ethoxylated PVOH.", "The film may be cast, blown or extruded.", "It may also be unorientated, mono-axially oriented or bi-axially oriented.", "It is possible for suitable additives such as plasticisers, lubricants and colouring agents to be added to the film.", "Components which modify the properties of the polymer may also be added.", "Plasticisers are generally used in an amount of up to 20 wt%, for example, from 15 to 20 wt%.", "Lubricants are generally used in an on the total number of the composition used to form the film.", "Suitable plasticisers are, for example, pentaerythritols such as depentaerythritol, sorbitol, mannitol, glycerine and glycols such as glycerol, ethylene glycol and polyethylene glycol.", "Solids such as talc, stearic acid, magnesium stearate, silicon dioxide, zince stearate or colloidal silica may also be used.", "It is also possible to include one or more particulate solids in the films in order to accelerate the rate of dissolution of the container.", "This solid may also be present in the contents of the container.", "Dissolution of the solid in water is sufficient to cause an acceleration in the break-up of the container, particularly if a gas is generated, when the physical agitation caused may, for example, result in the virtually immediate release of the contents from the container.", "Examples of such solids are alkali or alkaline earth metal, such as sodium, potassium, magnesium or calcium, bicarbonate or carbonate, in insoluble in cold water at 20° C. and only become soluble example, acidic substances having carboxylic or sulfonic acid groups or salts thereof.", "Examples are cinnamic, tartaric, mandelic, fumaric, maleic, malic, palmoic, citric and naphthalene disulfonic acids.", "The film is generally cold water (20° C.) soluble, but, depending on its degree of hydrolysis, may be insoluble in cold water at 20° C. and only become soluble in warm water or hot water having a temperature of, for soluble in cold water, or water at a temperature of up to say 35° C., steps must be taken to ensure that an aqueous composition contained inside the container does not dissolve the film from the inside.", "Steps may be taken to treat the inside surface of the film, for example by coating it with a semi-permeable or partial water barrier or dissolve or disperse into microscopic particles when described in more detail in EP-A-518,689 and WO 97/27743.taken to adapt the composition to ensure that it does not dissolve the film.", "For example, it has been found that ensuring the composition has a high ionic strength or contains an agent which minimises water loss through the walls of the container will prevent the composition from dissolving the PVOH film from the inside.", "This is described in more detail in EP-A-518,689 and WO 97/27743.It is particularly important to avoid pinholes in the film through which leakage of the contained composition may occur.", "It may therefore be appropriate to use a laminate of two or more layers of a different or the same film, as pinholes are unlikely to coincide in two layers of material.", "When first and second films are used to form the thickness of the second film will generally be from 20 to generally have a thickness before thermoforming of 20 to 500 μm, especially 70 to 400 μm, for example 70 to 300 μm or 90 or 110 to 150 μm.", "The thickness of the second PVOH film may be less than that of the first film as the second film will not generally be thermoformed so localised thinning of the sheet will not occur.", "The thickness of the second film will generally be from 20 to 150 μm or 160 μm, preferably from 40 or 50 to 90 or 100 μm, more preferably from 50 to 80 μm.", "The films may be chosen, if desired, such that they have the same thickness before the first film is thermoformed, or have the same thickness after the first from 25 to 35 g, and a laundry composition may weigh from composition which is encapsulated by a substantially constant thickness of film.", "The containers of the present invention generally use.", "For example, a dishwashing composition may weigh from 15 g to 20 g, a water-softening composition may weigh from 25 to 35 g, and a laundry composition may weigh from 10 to 40 g, especially 20 to 30 g or 30 to 40 g. The containers may have any shape achievable by thermoforming.", "For example they can take the form of a cylinder, cube or cuboid, i.e.", "a rectangular parallelepiped whose faces are not all equal.", "In general, because the containers are not rigid, the sides are not planar, but rather are convex.", "If the container is formed from a thermoformed film and a planar film, the seam between the two films will appear nearer one face of the container rather than the other.", "Apart from the deformation of the container due to the shrinkage of the film discussed above, deformation may also occur at the stage of manufacture if desired.", "For example, if the pocket is filled with a gelled composition having a height greater than that of the pocket, the second film will be deformed when placed on top of the pocket.", "A shaped sealing platen is required to achieve this effect.", "In general the maximum dimension of the filled part of the container (excluding any flanges) is 5 cm.", "For example, a rounded cuboid container may have a length of 1 to 5 cm, especially 3.5 to 4.5 cm, a width of 1.5 to 3.5 cm, especially 2 to 3 cm, and a height of 1 to 2.5 cm, especially 1 to 2 cm, and more especially 1.25 to 1.75 cm.", "The container desirably contains an aqueous composition which is a fabric care, surface care or dishwashing composition.", "Thus, for example, it may be a dishwashing, water-softening, laundry or detergent composition or a rinse aid.", "In this case the container is preferably suitable for use in a domestic washing machine such as a laundry washing machine or a dishwashing machine.", "The composition may also be a concentrated refill composition, for example a trigger-type spray as used in domestic situations.", "Such a composition can simply be added to water already held in the spray container.", "Examples of surface care compositions are those used to clean, treat or polish a surface.", "Suitable surfaces are, for example, household surfaces such as worktops, as well as surfaces of sanitary ware, such as sinks, basins and lavatories.", "The composition preferably contains greater than 3 wt% free water based on the weight of the aqueous composition, in order to ensure that the container has an attractive appearance.", "The actual amount of water present in the composition may be in excess of the amount of free water, since the total water content includes water of solvation and water held within a gelled matrix.", "Free water can be determined by a standard loss-on-drying determination test carried out at 60° C. for 3 hours at 200 mbar (20 kPa).", "Desirably the composition contains more than 10 wt%, 15 wt%, 20 wt%, 25 wt% or 30 wt% total water, but desirably less than 80 wt% total water, more desirably less than 70 wt%, 60 wt%, 50 wt% or 40 wt% total water.", "It may, for example, contain from 30 to 65 wt% total water.", "The remaining ingredients of the composition depend on the use of the composition.", "Thus, for example, the compositions may contain surface active agents such as an anionic, nonionic, cationic, amphoteric or zwitterionic surface active agents or mixtures thereof.", "Examples of anionic surfactants are straight-chained or branched alkyl sulfates and alkyl polyalkoxylated sulfates, also known as alkyl ether sulfates.", "Such surfactants may be produced by the sulfation of higher C8-C20 fatty alcohols.", "ROSO3−M+ wherein R is a linear C8-C20 hydrocarbyl group and M is a water-solubilising cation.", "Preferably R is C10-C16 alkyl, for example C12-C14, and M is alkali metal such as lithium, sodium or potassium.", "Examples of secondary alkyl sulfate surfactants are those which have the sulfate moiety on a “backbone” of the molecule, for example those of formula: CH2(CH2)n(CHOSO3−M+) (CH2)mCH3 wherein m and n are independently 2 or more, the sum of m+n typically being 6 to 20, for example 9 to 15, and M is a water-solubilising cation such as lithium, sodium or potassium.", "Especially preferred secondary alkyl sulfates are the (2,3) alkyl sulfate surfactants of formulae: CH2(CH2)x(CHOSO3−M+) CH3 and CH3 (CH2)x(CHOSO3−M+) CH2CH3 for the 2-sulfate and 3-sulfate, respectively.", "In these formulae x is at least 4, for example 6 to 20, preferably 10 to 16.M is a cation, such as an alkali metal, for example lithium, sodium or potassium.", "Examples of alkoxylated alkyl sulfates are ethoxylated alkyl sulfates of the formula: RO(C2H4O)nSO3−M+ wherein R is a C8-C20 alkyl group, preferably C10-C18 such forming cation such as lithium, sodium, potassium, ammonium, alkylammonium or alkanolammonium.", "These compounds can provide especially desirable fabric cleaning performance benefits when used in combination with alkyl sulfates.", "The alkyl sulfates and alkyl ether sulfates will generally be used in the form of mixtures comprising varying alkyl chain lengths and, if present, varying degrees of alkoxylation.", "Other anionic surfactants which may be employed are salts of fatty acids, for example C8-C18 fatty acids, especially the sodium, potassium or alkanolamine salts, and alkyl, for example C8-C18, benzene sulfonates.", "Examples of nonionic surfactants are fatty acid alkoxylates, such as fatty acid ethoxylates, especially those of formula: R(C2H4O)nOH wherein R is a straight or branched C8-C16 alkyl group, preferably a C9-C15, for example C10-C14 or C12-C14, alkyl group and n is at least 1, for example from 1 to 16, preferably 2 to 12, more preferably 3 to 10.The alkoxylated fatty alcohol nonionic surfactant will frequently have a hydrophilic-lipophilic balance (HLB) which ranges from 3 to 17, more preferably from 6 to 15, most preferably from 10 to 15.Examples of fatty alcohol ethoxylates are those made from alcohols of 12 to 15 carbon atoms and which contain about 7 moles of ethylene oxide.", "Such materials are commercially marketed under the trademarks Neodol 25-7 and Neodol 23-6.5 by Shell Chemical Company.", "Other about 5 moles of ethylene oxide; Neodol 23-9, an ethoxylated primary C12-C13 alcohol having about 9 moles of ethylene oxide; and Neodol 91-10, an ethoxylated C9-C11 primary alcohol having about 10 moles of ethylene oxide.", "Alcohol ethoxylates of this type have also been marketed by Shell Chemical Company under the Dobanol trademark.", "Dobanol 91-5 is an ethoxylated C9-C11 fatty alcohol with an average of 5 moles ethylene oxide and Dobanol 25-7 is an ethoxylated C12-C15 fatty alcohol with an average of 7 moles of ethylene oxide per mole of fatty alcohol.", "Other examples of suitable ethoxylated alcohol nonionic surfactants include Tergitol 15-S-7 and Tergitol 15-S-9, both of which are linear secondary alcohol ethoxylates available from Union Carbide Corporation.", "Tergitol 15-S-7 is a mixed ethoxylated product of a C11-C15 linear secondary alkanol with 7 moles of ethylene oxide and Tergitol 15-S-9 is the same but with 9 moles of ethylene oxide.", "Other suitable alcohol ethoxylated nonionic surfactants are Neodol 45-11, which is a similar ethylene oxide condensation products of a fatty alcohol having 14-15 carbon atoms and the number of ethylene oxide groups per mole being about 11.Such products are also available from Shell Chemical Company.", "Further nonionic surfactants are, for example, C10-C18 alkyl polyglycosides, such as C12-C16 alkyl polyglycosides, especially the polyglucosides.", "These are especially useful when high foaming compositions are desired.", "Further surfactants are polyhydroxy fatty acid amides, such as C10-C18 N-(3-methoxypropyl) glycamides and ethylene oxide-propylene oxide block polymers of the Pluronic type.", "Examples of cationic surfactants are those of the quaternary ammonium type.", "Examples of amphoteric surfactants are C10-C18 amine oxides and the C12-C18 betaines and sulfobetaines.", "The total content of surfactants in the composition is desirably 0.1 to 95 wt%, especially 60 or 75 to 90 wt%.", "The total content of surfactants in the laundry or detergent composition is desirably 60 to 95 wt%, especially 75 to 90 wt%.", "Desirably, especially in a laundry composition, an anionic surfactant is present in an amount of 50 to 75 wt%, a nonionic surfactant is present in an amount of 5 to 20 wt%, and/or a cationic surfactant is present in an amount of from 0 to 10 wt% and/or a amphoteric surfactant may be present in an amount of from 0 to 10 wt%.", "Desirably, in a dishwashing composition, the anionic surfactant is present in an amount of from 0.1 to 50 wt%, a non-ionic surfactant is present in an amount of 0.5 to 20 wt% and/or a cationic surfactant is present in an amount of from 1 to 15 wt%.", "These amounts are based On the solids content of the composition, i.e.", "excluding any water or solvent which may be present.", "The compositions, particularly when used as laundry washing or dishwashing compositions, may also comprise enzymes, such as protease, lipase, amylase, cellulase and peroxidase enzymes.", "Such enzymes are commercially available and sold, for example, under the registered trade marks Esperese, Alcalase, Savinase, Termamyl, Lipolase and Celluzyme by Nova Industries A/S and Maxatasc by International Biosynthetics, Inc. Desirably the enzymes are present in the composition in an amount of from 0.5 to 3 wt%, especially 1 to 2 wt%.", "Dishwasher compositions usually comprise a detergency builder.", "Suitable builders are alkali metal or ammonium phosphates, polyphosphates, phosphonates, polyphosphonates carbonates, bicarbonates borates, polyhydroxysulfonates, polyacetates, carboxylates and polycarboxylates such as citrates.", "The builder is desirably present in an amount of up to 90 wt%, preferably 15 to 90 wt%, more preferably 15 to 75 wt%, relative to the total content of the composition.", "Further details of suitable components are given in, for example, EP-A-694,059, EP-A-518720 and WO 99/06522.The compositions may, if desired, comprise a thickening agent or gelling agent.", "Suitable thickeners are polyacrylate polymers such as those sold under the trade mark CARBOPOL, or the trade mark ACUSOL by Rohm and Haas Company.", "Other suitable thickeners are xanthan gums.", "The thickener, if present, is generally present in an amount of from 0.2 to 4 wt%, especially 0.5 to 2 wt%.", "The compositions can also optionally comprise one or more additional ingredients.", "These include conventional detergent composition components such as further surfactants, bleaches, bleach enhancing agents, builders, suds boosters or suds suppressors, anti-tarnish and anti-corrosion agents, organic solvents, co-solvents, phase stabilisers, emulsifying agents, preservatives, soil suspending agents, soil release agents, germicides, phosphates such as sodium tripolyphosphate or potassium tripolyphosphate, pH adjusting agents or buffers, non-builder alkalinity sources, chelating agents, clays such as smectite clays, enzyme stabilizers, anti-limescale agents, colourants, dyes, hydrotropes, dye transfer inhibiting agents, brighteners and perfumes.", "If used, such optional ingredients will generally constitute no more than 10 wt%, for example from 1 to 6 wt%, of the The builders counteract the effects of calcium, or other ion, water hardness encountered during laundering or bleaching use of the compositions herein.", "Examples of such materials are citrate, succinate, malonate, carboxymethyl succinate, carboxylate, polycarboxylate and polyacetyl carboxylate salts, for example with alkali metal or alkaline earth metal cations, or the corresponding free acids.", "Specific examples are sodium, potassium and lithium salts of oxydisuccinic acid, mellitic acid, benzene polycarboxylic acids, C10-C22 fatty acids and citric acid.", "Other examples are organic phosphonate type sequestering agents such as those sold by Monsanto under the trade mark Dequest and alkyl hydroxy phosphonates.", "Citrate salts and C12-C18 fatty acid soaps are preferred.", "Other suitable builders are polymers and copolymers known to have builder properties.", "For example, such materials include appropriate polyacrylic acid, polymaleic acid, and polyacrylic/polymaleic and copolymers and their salts, such as those sold by BASF under the trade mark Sokalan.", "The builders generally constitute from 0 to 3 wt%, more preferably from 0.1 to 1 wt%, by weight of the compositions.", "Compositions which comprise an enzyme may optionally contain materials which maintain the stability of the enzyme.", "Such enzyme stabilizers include, for example, polyols such as propylene glycol, boric acid and borax.", "Combinations of these enzyme stabilizers may also be employed.", "If utilized, the enzyme stabilizers generally constitute from 0.1 to 1 wt% of the compositions.", "The compositions may optionally comprise materials which serve as phase stabilizers and/or co-solvents.", "alkanolamines such as mono-, di- and triethanolamines and monoisopropanolamine can also be used, by themselves or in combination with the alcohols.", "The phase stabilizers and/or co-solvents can, for example, constitute 0 to 1 wt%, preferably 0.1 to 0.5 wt%, of the composition.", "The compositions may optionally comprise components which adjust or maintain the pH of the compositions at optimum levels.", "Examples of pH adjusting agents are NaOH and citric acid.", "The pH may be from, for example, 1 to 13, such as 8 to 11 depending on the nature of the composition.", "For example, a dishwashing composition desirably has a pH of 8 to 11, a laundry composition has a pH of 7 to 9, and a water-softening composition has a pH of 7 to 9." ] ]

[ [ "Method for determining the earthquake protection of buildings", "According to the invention, in the method for determining the earthquake protection of buildings, the natural frequency of a building is determined.", "Moreover, the natural frequency of the ground surrounding the building is determined.", "Thereafter, a rating value for the building is calculated based on a comparison of the natural frequency of the building to the natural frequency of the ground.", "Based on this rated value, the earthquake protection of a building can be determined.", "Preferably, a failure probability for the building and an average damage to the building are calculated on the basis of the rating value, depending on the magnitude of the earthquake." ], [ "1.A method for determining the earthquake protection of buildings, the method comprising the following steps: determining the natural frequency of a building; determining the natural frequency of the ground, calculating a rating value for the building on the basis of a comparison of the natural frequency of the building to the natural frequency of the ground, and calculating an average degree of damage on the basis of the rating value depending on the magnitude of the earthquake.", "2.The method of claim 1, wherein the natural frequency of the building is determined by rotating an eccentrically supported mass of an exciting device with an increasing number of rotations, whereby the building is caused to oscillate, by picking up the oscillations of the building by means of an acceleration pick-up sensor, and by determining the natural frequency of the building by the spectrum of the oscillations picked up.", "3.The method of claim 2, wherein the exciting device is mounted to the building above 9/10 of the total height thereof.", "4.The method of one of claims 1-3, wherein a basic rating specific to the building is taken from a stored building table for calculating the rating value.", "5.The method of one of claims 1-3, wherein the rating value is calculated as a function of the frequency difference between the natural frequency of the building and the natural frequency of the ground.", "6.The method of claim 4 or 5, wherein the basic rating is reduced by a frequency risk value specific to the building, if  Natural   frequency   of   building - natural frequency   of   ground  Natural    frequency   of    building is greater than a threshold value specific to the building.", "7.The method of one of claims 4-6, wherein the basic rating is reduced by a ground risk value depending on the nature of the ground.", "8.The method of one of claims 4-7, wherein, in addition, one or more building examination calculations are performed and the basic rating is changed by a structure value of the building, depending on the calculation result.", "9.The method of claim 8, wherein each building examination calculation is rated positive or negative, depending on a threshold value, and wherein the basic rating is increased, if all building examination calculations have been rated positive.", "10.The method of claim 9, wherein building criteria are defined and the basic rating is increased only if all building criteria are positive.", "11.The method of one of claims 1-10, wherein a failure probability is calculated on the basis of the rating value.", "12.", "(canceled)" ], [ "The present invention refers to a method for determining the earthquake protection of buildings.", "In particular for buildings in earthquake-prone areas, it is crucial to observe construction regulations specifically issued for these areas, in order to avoid damage to the building upon the occurrence of an earthquake.", "However, such construction regulations are often not met with for economic reasons so that a high economic damage is incurred in case of an earthquake.", "Moreover, structural deficiencies cause the death of a great number of people.", "Presently, the safety of buildings in case of an earthquake is evaluated merely by visual inspection.", "A multitude of deficiencies, such as the quality of the materials used and the like, cannot be assessed in this way.", "Such visual inspections are suitable only to find substantial deficiencies.", "The resulting economic damage to a building cannot be estimated in this manner.", "Another problem of assessing the damages occurring in case of an earthquake is the existence of only large-area maps of earthquake zones.", "These maps are made up from experiences gathered from previous earthquakes and from geologic studies.", "Due to the large areas of the individual earthquake regions, the maps always only give average values for areas of several 1,000 km2.However, the subsoil is not homogeneous in areas of such large sizes, but varies rather often.", "Even minor variations of the subsoil, such as a soft intermediate layer, for example, can alter the behavior of the subsoil fundamentally in case of an earthquake.", "This change in behavior of the subsoil with respect to the average value stated in the map can be so substantial as to make impossible an estimate of the degree of damage to a building in case of an earthquake of a given magnitude.", "It is the object of the invention to provide a method for determining the earthquake protection of buildings with which to better determine the damage to a building to be expected in case of an earthquake.", "The object is solved according to the invention with the features of claim 1.In the method of the invention, the natural frequency of a building is determined.", "The natural frequency of a building is the smallest frequency at which the building moves when oscillating freely.", "The invention further provides for the determining of the natural frequency of the ground.", "Thereafter, a rating number for the building is calculated based on a comparison of the building's natural frequency to the natural frequency of the ground.", "The rating number is directly related to the damage to be expected in case of an earthquake of a given magnitude.", "Based on the rating number, the expected damage to a building can be predicted.", "Determining the natural frequency of a building is advantageous in that, independent of construction regulations and the question to what degree they have been materialized, an actual building-specific measured value is calculated.", "This measured value serves as the basis of the present method.", "The natural frequency of the ground is determined by reading it from a table.", "Such tables are tables corresponding to related maps and containing the natural frequencies of the ground with respect to large earthquake areas.", "Although this natural frequency represents an average value for a large area and is no characteristic value for the ground surrounding the building, comparing these two frequencies already yields a rating number allowing for a substantially more exact prediction on the expected damage to a building than would be possible by a mere visual inspection.", "The natural frequency of the ground is preferably calculated using the Nakamura method allowing for a more exact location-specific natural frequency of the ground to be determined.", "In the Nakamura method, first, the spectrum of the horizontal component and of the vertical component of existing ground oscillations is determined.", "The maximum value of the division of the horizontal spectrum and the vertical spectrum yields the natural frequency of the ground.", "The natural frequency of a building is preferably assessed by rotating an eccentrically supported mass of an exciting device with an increasing number of rotation.", "Thereby, the building is caused to oscillate.", "The oscillations of the building are picked up by an acceleration pick-up sensor or a geophone.", "Thereafter, the natural frequency of the building is determined from the oscillation spectrum picked up.", "Thus, by simply accelerating a mass, it is possible to cause a building to oscillate such that the natural frequency of the building can be calculated.", "For example, this method can be practiced while the building is still inhabited.", "Therefore, the effort for determining the natural frequency is extremely low.", "Preferably, the natural frequency of the building is determined by a Fourier transformation of the time response of the building during the acceleration of the mass.", "Particularly good results can be achieved if the exciting device is fastened to the building above 9/10 of the total height thereof.", "Preferably, the exciting device is arranged on the roof of the building, Preferably, the rating number for a building is calculated on the basis of a building-specific basic rating.", "To this purpose, a computer stores a table with building-specific basic ratings in the form of a data base.", "For a determination of the rating number, the suer simply has to select the type of building.", "Then, the computer which is connected to the acceleration pick-up sensor automatically records the oscillations of the building.", "From these, the computer calculates the natural frequency of the building, preferably using a Fourier transformation, as described above.", "In a further step, the computer uses an acceleration pick-up sensor to record the ground acceleration and transform it into the ground frequency.", "Instead of recording the natural frequency of the ground, the computer may also take the average natural frequency of the ground for a large area from a stored file.", "Then, the computer finds the building-specific basic rating from the stored building table.", "By comparing both natural frequencies and with corresponding consideration to the basic rating, the rating number is determined.", "Taking the basic rating into consideration, which is based on a statistic evaluation of experiences and studies, has the advantage of taking the building type into account.", "When calculating the rating number, particular consideration is given to the difference between the natural frequencies of the building and the ground.", "Most preferably, the building-specific rating is reduced by a frequency risk number also specific to the building, if the difference between the two frequencies is below a fixed threshold.", "This is done with the following equation:  Natural   frequency   of   building - natural frequency   of   ground  Natural    frequency   of    building ( Eq .", " 1 ) If this quotient is smaller than a building-specific threshold number, the building-specific basic rating is reduced by the frequency risk value.", "Thus, a reduced rating is obtained for a small difference between the two natural frequencies.", "As a consequence, the building will be damaged already in case of a minor earthquake, and this can be determined correspondingly using the present method.", "According to the invention, the building-specific basic rating is reduced by a ground risk value depending on the nature of the ground.", "These are values, for example, obtained from a statistic evaluation of scientific experiences and studies and accounting for offsets in the ground, intermediate layers and the like, for example.", "A further change in the basic rating is effected by the computer if, depending on one or a plurality of building examination calculations, weakenings or reinforcements are to be expected.", "Effected building examination calculations performed by the computer will be detailed below in connection with the drawings.", "Preferably, the building examination calculation is taken into account by the computer evaluating each building examination calculation as positive or negative with respect to a threshold value.", "The basic number will be increased if all building examination calculations have been evaluated as positive.", "Simply speaking, this means that a building fulfills important criteria of earthquake standards.", "Preferably, the basic rating is increased only if additional, predefined building criteria are met.", "The rating obtained is preferably used to calculate a mean damage probability as a function of the earthquake magnitude.", "Thus, the calculation yields the extent of the damage as a function of the earthquake magnitude.", "Knowing this extent, it is possible to make a simple estimate what degree of damage to a building is to be expected during an earthquake of a given magnitude.", "In addition to the mean damage rate (MDR), a failure probability, i.e.", "the probability of a complete destruction of the building, is calculated on the basis of the rating.", "Thus, it is possible, for example, to determine the degree of probability at which a collapse or a complete economic destruction of the building has to be expected for a certain magnitude of an earthquake.", "The above method for determining the earthquake protection of buildings is particularly suited for insurance companies.", "Using this method, insurance companies have a tool for appraising the risk of damage and to calculate the insurance fees and the like from this appraisal.", "The following is a detailed description of a preferred embodiment of the invention with reference to the accompanying drawings.", "In the Figures: FIGS.", "1a and 1b show a flow chart of a preferred embodiment of the method according to the present invention, FIG.", "2 illustrates an example of a diagram of the acceleration vs. time and of the amplitude vs. frequency derived therefrom for a building made to oscillate by the exciting devices, FIG.", "3 a schematic representation for calculating the earthquake-equivalent forces, and FIG.", "4 a schematic representation for calculating the overturning moment of the building about the founding.", "First, in step a), building-specific data are inputted, such as the address of the building, the exact position of the building, possibly with GPS data, etc.", "Next, the building type is entered.", "In total, a file in the computer, into which the data are entered, stores 15 different building types in a table.", "For example, these are wooden, steel or steel concrete buildings of different types.", "Each building type is associated to a basic rating specific to the building.", "The basic rating results from empiric values of geologists, seismologists, engineers engaged in statical calculations, and the like.", "It represents a kind of basic stability of the building.", "Based on this basic rating, a computer performs the calculation of the earthquake protection of buildings as described below.", "To enter the basic rating, it is merely necessary to select the building type and the location-specific data since the basic rating required therefor is stored in a file in the computer.", "In step b), ground-specific data in the form of ground classifications are entered or retrieved.", "These are assessments of the structure of the ground layer the building is located on.", "Specifically, consideration is given to the strength and the thickness of ground layers and the like.", "Such data are known, for example, from geologic maps and earlier studies in the relevant area.", "Depending on the nature of the ground, a ground-specific rating is determined.", "Preferably, the computer again contains a file listing individual types of ground so that one merely has to select the type of ground, while the computer automatically assigns a numerical value to the respective type of ground.", "Then, in the preferred embodiment of the method, this numerical value is subtracted from the basic rating.", "For a ground advantageous for the earthquake protection of a building, the basic rating is not altered.", "Possibly, the basic rating may also be increased if the ground strongly attenuates the oscillations occurring during an earthquake, for example.", "In step c), the natural frequency of the building is determined.", "This is done, as described before, by mounting an exciting device on the roof or in a higher floor of the building.", "The exciting device comprises an eccentrically supported mass that is rotated with the rotational speed increasing.", "Thereby, the building is caused to oscillate.", "These oscillations are measured by an oscillation pick-up sensor or a geophone.", "The measured results are automatically transmitted to the computer.", "The measured result is the acceleration versus time.", "The top diagram in FIG.", "2 illustrates an example of such an oscillation curve.", "Using the computer, the acceleration diagram is transformed into an amplitude diagram.", "The bottom diagram in FIG.", "2 illustrates a corresponding amplitude diagram.", "The conversion of the acceleration values into amplitude values is performed using a Fourier transformation.", "This is effected by the following equation: G  ( ω ) = ∫ - ∞ + ∞  g  ( t ) ·  -    ω   t    t ( Eq .", " 2 ) Here, G(ω) represents the frequency range and g(t) represents the function of the acceleration depending on time.", "With the help of this transformation, the diagram of the amplitude vs. the frequency is determined according to the bottom diagram of FIG.", "2.This diagram gives the natural frequency of the building.", "The natural frequency of a building is the frequency with the highest amplitude.", "In the example illustrated, this is true for the frequency f=1,133 Hz.", "The natural frequency is thus determined automatically using an exciting device, an acceleration pick-up sensor and a computer.", "The natural frequency of the ground is determined in step d).", "As described above, the same may be stored as a table in the computer, the table listing area-dependent natural frequencies of the ground.", "Since the position of the building to be examined had been entered in step a), in step d), the computer can automatically read the corresponding value of the natural frequency of the ground from the corresponding table.", "A better result can be achieved by obtaining the natural frequency of the ground, since the exact natural frequency of the ground at the location of the building is then taken into consideration.", "This is not true for known natural frequencies of the ground, since these are available only for large areas, i.e.", "in the form of an average value.", "The natural frequency of the ground may be obtained by means of an acceleration pick-up sensor or, preferably, a geophone, since the ground is constantly caused to oscillate by micro-seismic movements.", "The natural frequency of the ground is obtained using the Nakamura method.", "In the subsequent step e), the natural frequency is evaluated by comparing the natural frequency of the building to the natural frequency of the ground.", "To this end, the following calculation is performed:  Natural   frequency   of   building - natural frequency   of   ground  Natural    frequency   of    building ( Eq .", " 3 ) This ratio is compared to a threshold value specific to the building.", "This threshold value may be stored in a table as a numerical value for each type of building.", "Since this counter value differs only slightly for the various building types, it is possible to store a common, empirically obtained threshold value for all buildings or groups of buildings.", "If the value obtained from equation 3 is smaller than the threshold value, the basic value specific to the building and to the location is automatically decreased by a predetermined value.", "The result obtained is a preliminary rating value for the earthquake protection of the building.", "A definitely more exact estimate of the earthquake protection of the building could already be calculated from this rating value than would be possible with the known methods, such as a visual inspection.", "In step f) of the present method, earthquake equivalent forces are calculated.", "The calculation of earthquake equivalent forces is necessary for the subsequent steps.", "The earthquake equivalent forces are calculated according to the calculation methods formulated in Eurocode 8 (Design of structures for earthquake resistance DIN ENV, 1998; part 1, 2 § 3.3.2.2.)", "or on the basis of the calculation method in NEHRP (National Earthquake Hazard Reduction Program; 1988).", "According to Eurocode 8, an overall earthquake force Fb is calculated first as follows: Fb=Sd(T1)·W (Eq.", "4) Here, Sd(T1) represents the ordinate of the design spectrum (Eurocode 8; part 1.1; § 4.2.4.)", "for a basic oscillation time (T1), (T1) is the basic oscillation time of the building for the translation movement in the direction viewed, and W is the total weight of the building.", "Then, the earthquake equivalent forces F1 are calculated.", "For example, these are the earthquake equivalent forces F1, F2 and F3 (FIG.", "3) acting as horizontal forces on the individual floors.", "The individual earthquake equivalent forces are calculated by: F i = F b · Z i · W i ∑ Z j · W j ( Eq .", " 5 ) Here, Zi, Zj are the heights of the masses mi, mj above the plane on which the earthquake effect (founding) acts, and Wi, Wj are the weight of the respective masses mi, mj.", "The masses mi, mj are calculated according to Eurocode; part 1, 2; § 3.1.Using this calculation, the individual earthquake equivalent forces F1, F2, F3 acting on the floors are determined (FIG.", "3).", "In step g), the tilt resistance of the building is calculated based on the earthquake equivalent forces (FIG.", "4).", "To this end, an equivalent force F of the forces F1, F2, F3 is set at ⅔ of the total height of the building.", "The tilt moment Mkipp is calculated from: M kipp = Fx  2 3  h ( Eq .", " 6 ) Further, a resistance moment Mw of the building is calculated to determine the tilt resistance: M w = G · b 2 ( Eq .", " 7 ) Here, G is the total weight of the building, b is the width of the building, and h is the height of the building.", "When determining the loads, consideration is given to the fact that the building not only has its actual own weight, but also an operation weight, i.e.", "inventory and people.", "The tilt moment Mkipp is compared to the resistance moment Mw.", "Based on this comparison, the calculation of the tilt resistance made in step g) is rated “positive” or “negative”.", "For example, a ratio of both moments could be compared to a threshold value specific to a building.", "The result of the calculation in step g), i.e.", "the rating “positive” or “negative” is stored temporarily.", "In step h), a deformation control is performed.", "Here, the transverse forces occurring in each support of a building are calculated, the transverse force in each support corresponds to the total transverse force of the floors divided by the number of supports in a simplified approximation.", "From this, a relative floor displacement is calculated for each floor by: v c · q d · ( h 12  E ) · ( K b + K c K b · K c ) ( Eq .", " 8 ) Here, Vc is the shear force in a support, qd is the behavior coefficient of the displacement, h is the height of the floor, E is the module of elasticity K b = I L and K c = I h , where I is the moment of inertia and L is the length of the bar.", "The result is compared to an allowable deformation.", "Depending on a predetermined threshold specific to the building that is stored in a corresponding table in the computer, the comparison leads to the rating “positive” or “negative”.", "Again, this rating is stored temporarily.", "In step i), a shearing strain control is performed for the supports.", "From the transverse force in each support, the shearing strain in the respective support is calculated depending on the geometric conditions.", "These values are compared to the average common allowable shearing strain.", "The allowable strains are also stored in a table in the computer, depending on the material used.", "For the allowable strains, one preferably calculates with a high security factor since the quality of the material used, i.e., for example, the quality of the steel or the steel concrete, is not known exactly.", "The comparison again leads to a rating “positive” or “negative”.", "Again, this rating is stored temporarily.", "In the next step j), a shearing strain control of the walls is performed.", "To this ed, the shearing strain in the walls is calculated from the respective transverse force on a floor: Shearing   strain = Transverse   force   on   floor Sum   of   all   cross  -  sectional   areas   of   the   laterally    reinforcing   walls ( Eq .", " 9 ) This value is compared to an allowable shearing strain.", "The same is stored in a corresponding table according to the allowable strain when making the calculation of step i), again calculating with a high security factor since the exact quality of the material is not known.", "The rating result is latched.", "In step k), the last building examination calculation is the control of the diagonal reinforcements.", "The tensile stress and compressive strain in the reinforcements are calculated and subsequently compared to the allowable values: Tensile  /  compr .", " stress =  Transverse   force on   floor  Total   cross  -  sectional area   of   reinforcements · Mean   length   of reinforcements Mean   distance Between   supports ( Eq .", " 10 ) The reinforcements are beams generally extending obliquely between building supports.", "Again, the results of this calculation are compared to allowable values stored in a table.", "The comparison yields the ratings “positive” or negative”.", "Once more, this rating is latched.", "From the building examination calculations in steps g) to k), a respective result “positive” or “negative” is known and stored in the computer.", "In the next step l), the building examination calculations of steps g) to k) are evaluated.", "The evaluation leads to a building structure value being subtracted from or added to the basic rating.", "Preferably, a value is added if all building examination calculations were “positive”.", "As soon as one of the building examination calculations is “negative”, the basic rating that possibly has been changed in step e) because of the natural frequencies occurring, remains unchanged in step l).", "It is also possible to change the basic rating after each of steps g) to k).", "Instead of subtracting or adding fixed values, factors may be introduced by which the basic rating is multiplied.", "Based on the calculation results, a rating value is obtained.", "This is comprised of the basic rating and the corresponding subtracted or added values from the calculations in steps e) and l).", "In step m, the failure probability is calculated.", "This is the probability of the building being destroyed completely by an earthquake depending on the magnitude thereof, a complete destruction meaning at least an economically complete destruction such that rebuilding the edifice is not lucrative.", "The failure probability is calculated by: Rating value of building=−log(failure probability) (Eq.", "11) In parallel to step m), the average damage is calculated in step n) as a function of the earthquake intensity.", "This is a percentage of the damage in case of a weak, medium or strong earthquake, for example.", "The individual numerical values allow for an estimate of the degree of damage to a building in case of a corresponding earthquake.", "The damage is calculated by a mathematic approximation of the logarithmic normal distribution.", "The region below the damage distribution of the average damage probability of 60% to 100% corresponds to a probability of damage of more than 60%.", "The average damage probability q is calculated from: Rating   value   of    the   building = - ln  ( q ) ln  ( 10 )   where   q = ( b 1 · t + b 2 · t 2 + b 3 · t 3 + b 4 · t 4 + b 5 · t 5 ) · Zx ;   Zx = 0.39894228 ( - x 2 · 1 / 2 ) ;   t = 1 ( 1 + p · X )   and   X = ( ln  ( 60 ) - ln  ( x m ) ) S ( Eq .", " 12 ) Here, xm is the average value of the logarithmic-normal damage distribution, s is the standard deviation of the logarithmic-normal damage distribution, b1=0.31938153 b2−0.356563782 b3=1.781477937 b4=−1.821255978 b5=1.330274429 p=0.2316419 According to the invention, it is not necessary to perform all steps a) to m) or n), respectively.", "The essential steps are steps c), d) and e), from which a result regarding the earthquake protection of a building may already be derived.", "Thus, it is possible, for example, to perform step m) or n) immediately after step e).", "Likewise, individual steps in which a building examination calculation is performed, i.e.", "one or more of the steps g) to k), could be omitted.", "Further, a combination or a weighting of these individual steps is possible.", "Since the basic rating is preferably altered by addition or subtraction, the sequence of the individual steps is interchangeable.", "For example, a simple calculation for a steel frame building is performed as follows: A steel frame building has a location with a high earthquake intensity, thus giving a basic rating of 4.5.For soft ground with soft to hard intercalations of clay in a depth of about 10 m, a ground risk value of 0.6 is subtracted so that the basic rating has decreased to 3.9 even after step b).", "After determination of the natural frequency of the ground and of the building, the natural frequencies are evaluated in step e).", "Is this evaluation greater than 15%, a value of 0.8 will be subtracted from the basic rating already reduced.", "If this value is not greater than 15% after comparison of the natural frequencies, the rating value, i.e.", "the already reduced basic rating, will remain unchanged.", "Subsequently, the building examination calculations are performed in steps g) to k).", "If all building examination calculations are “positive”, the value is increased by 2.As soon as a calculation is “negative”, the value remains unchanged.", "If the compared value of the natural frequencies is greater than 15% and at least one building examination calculation is “negative”, a value of 3.7 is obtained.", "From this, a failure probability of 10−3.7 is calculated.", "Thus, the failure probability is 0.02%.", "For a rating value of 3.7, an average damage of 15% is obtained from equation 12." ] ]

[ [ "Solution for preparing stool specimens for diagnostic purposes", "The present invention relates to a new solution for the preparation of stool samples for diagnostic tests.", "The solution allows samples to be prepared simply before use in an immunological detection process and also provides a high level of sensitivity, specificity and reproducibility of the tests.", "The solution must contain at least one buffer substance, one detergent and one blocking reagent.", "The Invention also relates to a process for the analysis of a stool sample for diagnostic purposes using the solution according to the invention." ], [ "1.Process for the analysis of a stool sample for the diagnosis of an H. pylori infection comprising the following steps: a) bringing the sample into contact with a solution that has a pH in a range from 7.0 to 8.0 containing: at least one buffer substance selected from PBS, glycine buffer (0.1 M glycine, 140 mM NaCI), HEPES ([4-(2-hydroxyethyl)-piperazino]-ethane sulfonic acid) and MOPS (3-Morpholino-1-propane sulfonic acid); the zwitterionic detergent Chaps (3-[(3-chloramidopropyl)-dimethylammonium]-1-propane sulfonate) in a concentration of 0.01 to 1.5% (v/v), preferably in a range from 0.05 to 0-3% (v/v), very particularly preferred in a range from 0.1 to 0.15% (v/v); and the blocking reagent mouse serum in a concentration of 0.05 to 5%, preferably in a range from 0.1 to 1%, very particularly preferred in a range from 0.4 to 0.6%; if necessary, gentamicin sulfate and/or Proclin TM 300 as the stabiliser; and if necessary a complexing agent, preferably selected from EDTA and EGTA, particularly preferred EDTA, preferably in a concentration of 1 mM; b) carrying out an immuno assay with the sample treated according to step a); and c) taking of a measurement signal that was obtained within the scope of the immuno assay.", "2.Process according to claim 1, characterized in that the immuno assay is an ELISA.", "3.Process according to claim 1, characterized in that the stool sample treated with the solution is applied to a filter strip." ], [ "The present invention relates to absolution for the preparation of stool samples for diagnostic tests and a process for the analysis of a stool sample for diagnostic purposes.", "Within diagnostics, stool samples are routinely tested for the presence of bacteria, viruses, parasites and other organisms.", "With immunological tests, for example, antigens of the corresponding organisms can be detected.", "Diagnostic detection processes of this type comprise the taking of stool samples, usually a few steps for the preparation of the stool sample and the actual immunological test process in which the presence or absence of the agent to be detected, e.g.", "an antigen is demonstrated on the basis of a reaction, such as a colour reaction.", "In contrast to invasive techniques, such as endoscopy and biopsy, which are generally stressful for the organism and are often also associated with a high equipment requirement and a health risk non-invasive techniques such as stool sampling and subsequent analysis provide a simple opportunity to detect organisms that live in the digestive tract.", "The use of immunological test procedures to analyse stool samples, however, can be difficult for several reasons: Handling the stool samples is unpleasant and the preparation of stool samples is laborious, complex and time-consuming.", "In order to be able to use stool samples in an immunological test process, impurities which could disrupt the test procedure must first be removed from them.", "Normally the preparation of stool samples therefore involves several stages such as dilution, centrifugation or flitration, for example.", "U.S. Pat.", "No.", "5,198,365, for example, describes a method for stool preparation through sample dilution by a factor of 10 to 100.This type of sample dilution, however, has a disadvantageous effect on the sensitivity of the test and the incubation times when carrying out the test.", "Vellacott et al.", "(Lancet 32 (1): 249 (1981)) describe an immunological test for the detection of occult blood in the stool in which a centrifugation phase is required for the sample preparation.", "Hasan et al.", "(J. Clin.", "Micro.", "32: 249 (1994)) describe an immuno-diagnostic test for the detection of Vibric cholera in clinical samples.", "In this process, the sample has to be purified first using a separate filter.", "Accordingly, the state of the art describes procedures for the diagnosis, e.g., of Entamoeba histolytica (Haque (1993), J. Infect.", "Dis.", "167: 247-9), enterohaemorrhagic Escherichia coli (EHEC; Park (1995), J. Clin.", "Microbiol.", "34: 988-990), torovirus-like particles (Koopmans (1993), J. Clin.", "Mlcrobiol.", "31: 2738-2744) or Taenia saginata (Machnicka (1996), Appl.", "Parasitol.", "37: 106-110) from stool.", "The element that these processes have in common is that one or several separate process stages for sample treatment precede the actual test.", "Samples are generally prepared by suspending the stool sample in a suitable suspension buffer.", "The composition of this buffer has a major influence on the sensitivity and specficity of the test.", "Excluding the detection of falsely positive samples often proves to be particularly problematic.", "The detection of falsely positive samples depends essentially on the composition of the sample buffer components.", "In addition, the protein content of the sample affects the viscosity of the sample suspension and thus influences the flow behaviour of the sample suspension.", "Sample preparation should be optimised in terms of the following: high reproducibility, high sensitivity, high specificity and low viscosity.", "The present invention was based on the technical problem of providing a solution that allows the simplest possible sample preparation whilst giving the maximum reproducibility, sensitivity and specificity of the proof.", "A further technical problem was in providing a process as simple as possible for analysing stool samples for diagnostic purposes.", "The process should guarantee the simplest possible sample handling whilst retaining maximum sensitivity, specificity and reproducibility of the results obtained.", "The first problem is solved according to the invention by a solution for the preparation of a stool sample for diagnostic purposes containing at least one buffer substance, at least one detergent and at least one blocking reagent.", "The second problem is solved according to the invention by a process for the analysis of stool samples for diagnostic purposes covering the following steps: (a) bringing the sample into contact with a solution according to the invention; (b) carrying out an immuno assay with the sample treated under step (a); and (c) taking a measurement signal which was obtained within the scope of the immuno essay.", "The solution according to the invention is particularly suitable for the preparation of stool samples in which the diagnosis of a Helicobacter pylori infection is to be carried out.", "Basically, however, preparing the sample using the solution according to the invention allows any pathogens in the digestive tract to be detected.", "In a particularly preferred embodiment, the buffer substance is selected from PBS, TBS, glycine buffer (0.1 M glycine, 140 mM NaCl), HEPES ([4-(2-hydroxyethly)-piperazino]-ethane sulfonic acid), MOPS (3morphollno-1-propane sulfonic acid), whereby PBS is particularly preferred.", "In a further preferred embodiment, the pH of the solution is in a range from 7.0 to 8.0, preferably 7.2 to 7.7, whereby optimum results for the detection of H. pylori are obtained with a pH from 7.3 to 7.5.In a further preferred embodiment, the detergent is a zwitterionic detergent that is preferably selected from Chaps (3-[(3-chloramidopropyl)-dimethylammonium]-1-propane sulfonate) and Zwittergent® (N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate), whereby Chaps is very particularly preferred.", "Detergents are essential for breaking down the sample material.", "In the event of an immunological detection, they release antigen structures (epitopes) in order to allow, in this way, a binding of the antibodies used to the antigen to be detected.", "Normally, non-ionic detergents such as Triton®, Tween® etc.", "are used.", "However, within the framework of the present invention, it has been found, surprisingly, that the use of a zwitterionic detergent has major advantages over the use of non-ionic detergents in that, for example, the detection of falsely positive samples is considerably reduced when the zwitterionic detergent is used.", "The preferred detergent, Chaps, has further advantages over further detergents such as Triton® or Tween®: compared with these detergents, Chaps has fewer denaturing properties (Hjelmeland; U.S. Pat.", "No.", "4,372,888).", "The surface proteins thus, during the breakdown (during sample preparation), remain basically as intact complexes in the membrane.", "The surface proteins are fragmented and denatured to a lesser extent.", "The probability of obtaining antigen epitopes during the breakdown which are sufficient for immunological detection is therefore increased.", "In particular, with immunological detection in sample material that has passed through the digestive tract, the enzymatic degradation of the antigen can already cause a high degree of fragmentation of the sample material.", "The most efficient retention of the residual epitopes left is therefore extremely desirable.", "In a further preferred embodiment, the detergent is in a concentration of 0.01 to 1.5% (v/v), preferably in a concentration of 0.05 to 0.3% (v/v) and, very particularly preferred, in a concentration of 0.1 to 0.15% (v/v).", "The blocking reagent contained in the solution according to the invention may be an animal serum or a protein mixture.", "In a preferred embodiment, the solution according to the invention will contain a blocking reagent that is selected from mouse serum, bovine serum, human serum, rabbit serum, pig serum, foetal calf serum, BSA, skimmed milk powder and peptone, whereby mouse serum is particularly preferred.", "Mouse serum should be particularly preferred if an immuno assay is carried out in which mouse antibodies are used to detect an antigen.", "The use of serum from the same species as the antibodies used for detection (e.g.", "mouse serum in combination with mouse antibodies) has been found to be particularly advantageous for the blocking of non-specific compounds, since the potential non-specific compounds can be almost completely saturated/blocked through serums of the same species.", "The higher saturation of the non-specific compounds in turn increases the sensitivity/specificity of the test.", "In a further preferred embodiment, the blocking reagent is in a concentration of 0.05 to 5%, preferably in a range from 0.1 to 1%, and very particularly preferred in a range from 0.4 to 0.6% in the solution according to the invention.", "The percentage concentration information is given as “% (v/v)” for liquid blocking reagents such as mouse serum, bovine serum, human serum, rabbit serum, pig serum and foetal calf serum and as “% (w/v)” for solid blocking reagents such as BSA, skimmed milk powder and peptone.", "Conventional solutions for sample preparation often show a very high level of serum.", "Often, concentrations of up to 50% serum are used.", "However, the effect of the high serum content is a high viscosity of the prepared sample, thus making handling more difficult.", "This problem does not occur if the solution according to the invention is used: If the solution according to the invention contains serum as the blocking reagent, the serum content in the solution is lower than with conventional solutions for sample preparation.", "This reduces the viscosity of the prepared sample, which is a particular advantage for handling during sample preparation.", "The flow behaviour is improved considerably, which means that the sample buffer is particularly suitable for use in ELISA or immune chromatography tests (test strips).", "Particularly with applications using a test strip, a low viscosity leads to better running behaviour, which allows a higher throughput of samples per unit of time.", "Through using serum of the same species as the detection antibodies, such as mouse serum in combination with mouse antibodies, the level of serum in the conjugate buffer can also be reduced.", "In a further preferred embodiment, the solution contains further additives such as sample stabilising agents.", "The aim of such agents is to ensure that the sample is still suitable even after a long period of storage for immunological test reactions to be carried out and does not provide any distorted results.", "These stabilisers include phenol and antibiotics, such as gentamicin sulfate and Proclin® 300.The solution according to the invention is also particularly advantageous in that it can be used as a basic buffer both for the conjugate (marked detection antibodies) and for the positive and negative control, which means that the test process can be made much simpler.", "In a further preferred embodiment, the solution also contains a complexing agent for metal ions, preferably EDTA or EGTA EDTA is particularly preferable.", "Preferably, the complexing agent will be in a concentration of 1 mM in the solution.", "The solution according to the invention is preferably used within the framework of a test kit, whereby the test kit contains all the reagents to carry out the immuno assay.", "These include, for example, test strips or plates with recesses (e.g.", "micro titre plates), in which the reactions proceed, plus the various detection reagents including the antibodies used and if necessary substrates for detection using ELISA.", "Preferably, all the reagents will be contained in a single pack unit with separate sub-compartments.", "In the process according to the invention for the analysis of a stool sample for diagnostic purposes, the sample is brought into contact with the solution according to the invention.", "Here, the sample is generally suspended in the solution according to the invention.", "After distributing the sample in the solution as homogeneously as possible, the latter is then subjected to an immuno assay and after the completion of the immuno assay, the measurement signal produced is recorded.", "Before the immuno assay, the particular sample elements can be removed by centrifuging or filtration.", "In a preferred embodiment, the immuno assay is carried out as an ELISA.", "Here, the suspended sample is placed in a container which contains the antigen-specific reagents.", "Preferably, within the scope of this ELISA, a sandwich complex is formed from the antigen to be detected and antigen-specific antibodies.", "In an alternative embodiment, the suspended sample is placed on a filter strip which already contains the corresponding detection reagents at previously determined zones.", "Carrying out the immuno assay on a filter has the advantage that undesired particular sample elements of the antigen in question are separated before testing for it.", "A filter may be pre-attached in addition, to give better separation.", "The following examples explain the invention.", "EXAMPLES A Comparison of Various Solutions for Sample Preparation in a One-step ELISA For the test, stool samples from patients from ten different clinics or gastro-enterological practices were available, which had been diagnosed as H. pylori negative (G0) or H. pylori positive (G4) using 13C-urea breath test and/or histological examinations of stomach biopsies.", "H. pylori Stool Sandwich EUSA (One-step Test) The ELISA plate (MaxiSorb Lock well; Nunc) was coated over night at 2-8° C. with 100 μl of a mAK solution (2.0 μg HP25.2 m/2H10 (Connex, Martinsried) per ml carbonate buffer, 0.1 M, pH 9.5).", "The ELISA plates coated in this way were washed 2× with PBS.", "To block the free bonding points, 200 μl blocking buffer (0.3% (w/v) BSA; 20% (w/v) sorbitol in PBS) were added per recess and incubated over night at 2-8° C. The saturated plates were drawn off, dried over night at 28° C. in a circulating air drying cabinet and then stored at 2-8° C. Patient stool was suspended 1:5 (0.1 g stool sample+500 μl sample buffer) in the solution according to the invention (75 mM PBS+0.5% mouse serum+1 mM EDTA+0.05% Proclin® 300+50 μg/ml gentamicin sulfate+10 mM phenol+0.1% (v/v) Chaps) for approx.", "30 sec (Vortex) and then centrifuged for 5 minutes at 5000 g. Per recess, 50 μl of the residue (double to triple determination) were applied to the plate.", "Then 50 μl of the POD-marked antibody HP25.2 m/2H10-POD (Connex, Martinsried) diluted in the sample buffer were added directly to the stool suspension.", "The plates were incubated for 1 hour at room temperature on the shaker (level 4-5).", "After washing four times with washing buffer (75 mM PBS, 0.25% (v/v) Tween®, the perodixase substrate TMB (single-component substrate of neogen) was added (100 μl/recess).", "After 10 minutes, the enzyme reaction was stopped by the addition of 1 N hydrochloric acid (100 μl/recess).", "The colour intensity was then measured at 450 nm against the reference wavelength of 630 nm.", "Example 1 Comparison of the Signal/background Relationship of Various Solutions for Sample Preparation TABLE 1 shows a comparison of various solutions using selected samples which proved particularly difficult for detection purposes.", "HP25.2 m/2H10 (2.0 μg/ml) Capture HP25.2 m/2H10-POD (200 ng/ml) antibodies r-biopharm solution PBS + skimmed Detection sample acc.", "to milk powder (2% antibodies buffer invention (w/v)) Sample buffer Sample OD OD OD CX1002 G0 2.026 0.534 0.678 CX2028 G0 0.059 0.034 0.090 CXT0069-1 G0 0.079 0.021 0.032 CXT0071-1 G0 0.348 0.030 0.051 CXT0072-1 G0 0.079 0.036 0.052 CXT0073-1 G0 0.044 0.036 0.076 CXT0074-1 G0 0.125 0.028 0.058 CXT1038 G4 0.100 0.040 0.074 CXT0053-1 G4 4.458 4.247 4.285 CXT0058-1 G4 0.221 0.146 0.192 CXT0077-1 G4 0.264 0.180 0.198 Total G4 5.607 4.714 4.831 Total G0 2.760 0.719 1.037 Ag-DL in ng/ml 0.33 0.33 0.11 Background 0.102 0.08 0.035 r-biopharm solution available from r-biopharm, Darmstadt OD: optical density DL: detection limit Result: 75 mM PBS+0.5% mouse serum+1 mM EDTA+0.05% (w/v) Proclin® 300+30 μg/ml gentamicin sulfate+0.1% (v/v) Chaps showed the best signal/background ratio of the 3 buffers studied.", "Example 2 Influence of Various Chaps Concentrations in the Solution According to the Invention on Signal/background Ratio and the Sensitivity of the Test Result: The table shows that the addition of Chaps (0.1-0.25% (v/v)) produces a positive G0/G4 signal ratio.", "Capture HP25.2 m/2H10 1.5 μg/ml antibodies HP25.2 m/2H10-POD 0.2 μg/ml) Detection 0.1% (v/v) 0.25% (v/v) 0.5% (v/v) antibodies 0% Chaps Chaps Chaps Chaps Antigen detection 0.3 0.3-1 0.3 0.1-0.3 [ng/ml] Background 0.15 0.067 0.04 0.026 HO delta OD delta OD at 9 0.759 0.89 0.787 1.267 ng/ml <60 kDA Patient samples CXT 73 G0 0.12 0.03 0.04 0.06 CXT 54 G0 0.11 0.03 0.04 0.05 CX 60 G0 0.12 0.065 0.04 0.05 CXT 63 G0 0.07 0.05 0.045 0.05 CXT 62 G0 0.03 0.07 0.03 0.03 CXT 64 G4 0.51 0.94 0.9 0.5 CXT 58-3 04 0.2 0.38 0.27 0.21 Result; The table shows that the addition of Chaps (0.1-0.25% (v/v)) produces a positive G0/G4 signal ratio." ] ]

[ [ "Dispensing Cap", "A dispensing cap (11) for a container, the cap having a closure member (16) movable from a first closed position to an open position in which fluid can pass through the cap, the closure member also being movable between the open position and a second closed position; and a chamber (24) for holding material to be dispensed from the cap, whereby the closure member is initially in the first closed position and when the closure member is moved to the second closed position, the material is released from the chamber." ], [ "1.A dispensing cap for dispensing a material into a container, the cap having a closure member moveable from a first closed position to an open position in which fluid can pass through the cap, the closure member also being moveable between the open position and a second closed position; and a chamber for holding the material to be dispensed from the cap, whereby before use, the closure member is in the first closed position and when the closure member is moved to the second closed position the material is released from the chamber.", "2.The cap of claim 1, wherein the closure member is a spout and when in the open position, fluid can pass through the spout.", "3.The cap of claim 2, including a stem extending through the spout and at least one sealing projection extending from the spout or stem which can seal against a portion of the other of the stem or spout.", "4.The cap of claim 1 which includes first and second sealing projections extending from the stem and when the spout is in the first closed position, the first sealing projection seals against a portion of the spout and when in the second closed position, the second sealing projection seals against a portion of the spout.", "5.The cap of claim 4, which includes an inwardly facing lip mounted on the spout and the first and second sealing projections can seal against the lip.", "6.The cap of claim 1 which includes a sealing member which defines a base section of the chamber and the cap includes actuating means which can be manipulated by a user to actuate the sealing member to release the material from the chamber.", "7.The cap of claim 6, wherein includes an actuating member for moving the sealing member to the material release position.", "8.The cap of claim 7 wherein the actuating member is operatively associated with or integral with the closure member.", "9.The cap of claim 6, wherein the sealing member is substantially U-shaped in cross section and is moveable from a material holding position to an inverted material release position.", "10.The cap of claim 1 having a lower section which includes mounting means for mounting the cap to the container and an upper section which can be removed from the container whilst the lower section remains mounted to the cap and a hinge member connecting the upper and lower sections, the hinge member having a first end attached to the cover and a second end attached to the base and when the cover is in the cover position the first and second ends are angularly offset.", "11.A dispensing cap for a container, the cap having a chamber for holding a material to be dispensed from the cap and into the container, the chamber having a sealing member which is substantially U-shaped in cross section and is moveable from a material holding position to an inverted material release position.", "12.The cap of claim 10, wherein the sealing member is initially biased towards the material holding position and when moved towards the inverted position the bias changes towards the inverted position.", "13.A lid assembly for a container, the assembly including a base mountable about an opening of the container, a cover member moveable between a cover position and a free position and a hinge member connecting the cover and base, the hinge member having a first end attached to the cover and a second end attached to the base and when the cover is in the cover position the first and second ends are angularly offset.", "14.The lid assembly of claim 13 wherein said offset angle is between about 90° and 360°.", "15.The lid assembly of claim 13, wherein the hinge member biases the cover member towards the cover position." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The present invention will be described with particular reference to a cap for use with a beverage or drink container.", "However, it will be appreciated that the dispensing cap may be used with other types of container and no limitation is intended thereby.", "Dispensing caps for dispensing a material contained in the cap into a container upon which the cap is mounted are known.", "Dispensing caps are used where it is desirable to keep components of a mixture separate until prior to use.", "This may be applicable in cases where some ingredients are unstable in solution.", "Dispensing caps typically have a chamber for housing the material to be dispensed.", "In known caps, the chamber is typically separated from the main body of the container by a frangible membrane.", "The cap is typically provided with a means to rupture the membrane and to release the contents of the chamber.", "The membrane is typically ruptured by a piercing member.", "The piercing member may be urged towards the membrane by pushing downwardly on the upper end of the member.", "In some caps, the piercing member must be removed from the cap after use to allow liquid to pass through the cap.", "However, this is often inconvenient to a user and further creates a separate waste item which must be disposed of.", "In order to overcome this difficulty, some earlier caps allow the piercing member to become dislodged and fall into the body of the container after use.", "However, many users would prefer not to have a foreign object floating about in a beverage to be drunk.", "One approach to this problem has been to modify known sipper type caps.", "Sipper caps have a push-pull type mechanism in which a spout is pulled to an open position through which fluid may be drunk and pushed to a closed position.", "Such caps are popular with cyclists and other sports persons as the push pull mechanism requires only a single handed operation when the spout can be gripped by a users teeth or mouth.", "The modified sipper caps have a frangible membrane which can be ruptured by the spout when the spout is pushed towards the closed position.", "However, this requires that the cap is in the open position before first use.", "This is undesirable as it may allow contamination of the material in the cap and/or leakage of fluid from the bottle.", "It is therefore an object of the present invention to provide a dispensing cap which may at least partially overcome the above disadvantages or may provide the public with a useful choice." ], [ "<SOH> BRIEF DESCRIPTION OF THE FIGURES <EOH>FIG.", "1 is a schematic cross sectional view of a preferred dispensing cap of the present invention attached to a beverage container; FIG.", "2 illustrates the cap of FIG.", "1 in a first closed position; FIG.", "3 illustrates the cap of FIG.", "1 in a second closed position; FIG.", "4 illustrates the cap of FIG.", "1 in an open position; FIG.", "5 is a perspective view of a further preferred dispensing cap of the invention in a first closed position; FIG.", "6 is a cross-section of the cap of FIG.", "5 ; FIG.", "7 is a perspective view of the cap of FIG.", "5 in a second closed position; FIG.", "8 is a cross-section of the cap of FIG.", "7 ; FIG.", "9 is a perspective view of the cap of FIG.", "5 in an open position; FIG.", "10 is a cross-section of the cap of FIG.", "9 ; FIG.", "11 is a perspective view of the spout of the cap of FIG.", "5 ; FIG.", "12 is a perspective view of the stem and sealing member of the cap of FIG.", "5 in a pre-release position; FIG.", "13 is a perspective view of the stem and sealing member of FIG.", "12 in the release position; FIG.", "14 is a perspective view of the housing of the cap of FIG.", "5 .", "FIG.", "15 is a cross sectional schematic view of a further preferred dispensing cap of the present invention in a first closed position; FIG.", "16 is a cross sectional schematic view of the cap of FIG.", "15 in a second closed position; FIG.", "17 is a cross sectional schematic view of the cap of FIG.", "15 in an open position; FIG.", "18 is a perspective view of a cap of a further preferred embodiment of the invention; FIG.", "19 is a plan view of the cap of FIG.", "18 and FIG.", "20 is a front view of a preferred lid assembly of a further embodiment of the invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "FIELD OF THE INVENTION The present invention relates to a dispensing cap for use with a container and in particular a dispensing cap for use with a drink or beverage container.", "The present invention also relates to a lid assembly for a container.", "BACKGROUND OF THE INVENTION The present invention will be described with particular reference to a cap for use with a beverage or drink container.", "However, it will be appreciated that the dispensing cap may be used with other types of container and no limitation is intended thereby.", "Dispensing caps for dispensing a material contained in the cap into a container upon which the cap is mounted are known.", "Dispensing caps are used where it is desirable to keep components of a mixture separate until prior to use.", "This may be applicable in cases where some ingredients are unstable in solution.", "Dispensing caps typically have a chamber for housing the material to be dispensed.", "In known caps, the chamber is typically separated from the main body of the container by a frangible membrane.", "The cap is typically provided with a means to rupture the membrane and to release the contents of the chamber.", "The membrane is typically ruptured by a piercing member.", "The piercing member may be urged towards the membrane by pushing downwardly on the upper end of the member.", "In some caps, the piercing member must be removed from the cap after use to allow liquid to pass through the cap.", "However, this is often inconvenient to a user and further creates a separate waste item which must be disposed of.", "In order to overcome this difficulty, some earlier caps allow the piercing member to become dislodged and fall into the body of the container after use.", "However, many users would prefer not to have a foreign object floating about in a beverage to be drunk.", "One approach to this problem has been to modify known sipper type caps.", "Sipper caps have a push-pull type mechanism in which a spout is pulled to an open position through which fluid may be drunk and pushed to a closed position.", "Such caps are popular with cyclists and other sports persons as the push pull mechanism requires only a single handed operation when the spout can be gripped by a users teeth or mouth.", "The modified sipper caps have a frangible membrane which can be ruptured by the spout when the spout is pushed towards the closed position.", "However, this requires that the cap is in the open position before first use.", "This is undesirable as it may allow contamination of the material in the cap and/or leakage of fluid from the bottle.", "It is therefore an object of the present invention to provide a dispensing cap which may at least partially overcome the above disadvantages or may provide the public with a useful choice.", "DESCRIPTION OF THE INVENTION According to a first broad form of the invention, there is provided a dispensing cap for a container, the cap having a closure member movable from a first closed position to an open position in which fluid can pass through the cap, the closure member also being movable between the open position and a second closed position; and a chamber for holding material to be dispensed from the cap, whereby before use, the closure member is in the first closed position and when the closure member is moved to the second closed position, the material is released from the chamber.", "The cap of the present invention is typically used with a beverage or drink container.", "Typically the container is a bottle which may be made from suitable materials such as PET, glass or polyolefin materials.", "The cap may be mounted to the container by any suitable means such as a snap fit, friction fit or be screw threaded.", "The cap is typically a sipper cap of the type in which a drink may be drunk through the cap.", "In this case, the closure member may be in the form of a spout having one or more fluid flow apertures through which liquid may pass.", "The spout is typically slidably mounted within a collar.", "The collar typically has means for attaching the cap to a neck of a bottle.", "The spout is movable from a first closed position to an open position and between the open position and a second closed position.", "Typically when the spout is in the closed position, the fluid flow apertures(s) are blocked by a projection or the like.", "In a preferred form of the invention the cap includes a stem which extends through the center of the spout.", "The spout is moveable with respect to the spout.", "The spout and/or stem may include one or more sealing projections which may be moved in and out of sealing engagement as the spout is moved by a user.", "In one form of the invention, the spout may include a pair of concentric sealing lips.", "The spout may be moved from a first closed position in which the stem seals against a first lip to an open position in which there is no sealing engagement between the spout and stem and a second closed position in which the stem contacts and seals against one or the other of the lips.", "The cap includes a chamber for holding a material to be dispensed into the container.", "The material may be a liquid, powder, granules, tablet or the like.", "Where the container holds a beverage the material may include vitamins, minerals, other nutritional supplements, herbal extracts, medicines, colors, flavors, stabilizers and other additives known in the art.", "When the closure member is moved to the second closed position, material is released from the chamber.", "Typically, the chamber has a sealing member.", "Generally, this sealing member forms a seal which can prevent liquid in the container from coming into contact with the material in the chamber.", "When the closure member is moved to the second closed position, the seal formed by the sealing member is broken so as to release the material.", "The seal may be broken by rupturing, deforming or moving the sealing member from a sealing to a material release position.", "Typically, the seal is broken by a seal breaking member which may be integral with or operatively associated with the closure member.", "Preferably, it is not possible to re seal the chamber after the seal has been broken.", "In a preferred embodiment, the sealing member is cup shaped and is moveable to an inverted position to release the material.", "According to a further broad form of the invention, there is provided a dispensing cap for a container, the cap having a chamber for holding material to be dispensed from the cap and into the container, the chamber having a sealing member wherein the sealing member is substantially U-shaped in cross section and is moveable from a material holding position to an inverted material releasing position.", "The sealing member is typically formed from an elastomeric material which imparts an initial bias to the material holding position.", "However, during inversion, the sealing member is deformed to a substantially flat position, and the bias changes in favor of the inverted position.", "In this way the material can be quickly and efficiently released.", "Typically the sealing member is deformed by means of an annular member which can be actuated by an operator to push against the upper edges of the sealing member.", "The cap of the second broad form may be of the push pull type in which liquid passes through a spout as described above.", "However it will be appreciated that this need not be the case.", "The cap of the second broad form may be of the type which is mounted to the neck of a container in a conventional manner and must be removed from the container to allow the contents of the container to be dispensed therefrom.", "Caps of the push pull type typically include a protective cover or lid which may be associated with a tamper evident seal.", "The protective cover minimizes contamination of the spout and can protect against inadvertent or malicious activation of the cap prior to sale.", "Many users also prefer to replace the protective cover or lid if liquid contents of the container are not drunk at once.", "This is particularly so for cyclists who mount drinking containers to a bicycle frame.", "This mounting position allows dust, road grime and other contaminates to collect on the top of the spout.", "One form of protective cover currently in use is a simple lid which snap fits to the housing of the cap.", "There are a number of disadvantages with such an arrangement.", "First, removal of the lid is a two handed procedure, and when removed the lid is easily lost.", "It can be seen that two handed removal is impractical for many sports people such as cyclists.", "Further, loss of the lid generates a separate waste item.", "To overcome this difficulty it has been proposed to connect the lid to the bottle neck or push pull cap.", "Some caps include a plastics tab connecting the cover to the cap.", "However, the tab is subject to failure after repeated use.", "Premature failure may be avoided by replacing the tab with a flexible plastics strap.", "However, in practice, the straps protrude from the body of the container and may catch on foreign objects.", "More advanced hinge designs have also been proposed.", "However, such designs may significantly add to the overall cost of the container.", "Such difficulties with covers and lids are not limited to caps of the push pull types but are also experienced with conventional types of container closures.", "It is therefore a further object of the present invention to provide a lid assembly which may at least partially overcome the above disadvantages or provide the public with a useful choice.", "According to a further broad form of the invention there is provided a lid assembly for a container, the assembly including a base mountable about an opening of the container, a cover member movable between a cover position and a free position and a hinge member hingedly connecting the cover and base, the hinge member having a first end attached to the cover and a second end attached to the base, and when the cover is in the cover position the first and second ends are angularly offset.", "The lid assembly of the further broad from of the invention may be used with any suitable type of container or cap assembly and is not limited to use with caps of the other forms of the invention or caps of conventional push pull type.", "Typically the first and second ends are angularly offset between about 90 and 360θ, and typically between about 100 to about 220θ.", "It will be appreciated that by being angularly offset the hinge member can extend at least partially about the cover member in a spiral or helical configuration.", "Preferably the hinge member lies substantially flush to the cover and base and does not protrude when the cover is in the cover position.", "The hinge member is typically a strip of a resilient plastics material and can function in the same manner as a coil of a spring to bias the cover towards the closed position.", "This may be advantageous in that should a user fail to return the cover to the cover position, the cover may at least partially return to the covering position, thereby avoiding or minimizing contamination of the underside of the cover and container contents by dirt, dust, airborne microorganisms and other debris.", "The cover and base are typically engagable by known means such as a snap or friction fit.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 is a schematic cross sectional view of a preferred dispensing cap of the present invention attached to a beverage container; FIG.", "2 illustrates the cap of FIG.", "1 in a first closed position; FIG.", "3 illustrates the cap of FIG.", "1 in a second closed position; FIG.", "4 illustrates the cap of FIG.", "1 in an open position; FIG.", "5 is a perspective view of a further preferred dispensing cap of the invention in a first closed position; FIG.", "6 is a cross-section of the cap of FIG.", "5; FIG.", "7 is a perspective view of the cap of FIG.", "5 in a second closed position; FIG.", "8 is a cross-section of the cap of FIG.", "7; FIG.", "9 is a perspective view of the cap of FIG.", "5 in an open position; FIG.", "10 is a cross-section of the cap of FIG.", "9; FIG.", "11 is a perspective view of the spout of the cap of FIG.", "5; FIG.", "12 is a perspective view of the stem and sealing member of the cap of FIG.", "5 in a pre-release position; FIG.", "13 is a perspective view of the stem and sealing member of FIG.", "12 in the release position; FIG.", "14 is a perspective view of the housing of the cap of FIG.", "5.FIG.", "15 is a cross sectional schematic view of a further preferred dispensing cap of the present invention in a first closed position; FIG.", "16 is a cross sectional schematic view of the cap of FIG.", "15 in a second closed position; FIG.", "17 is a cross sectional schematic view of the cap of FIG.", "15 in an open position; FIG.", "18 is a perspective view of a cap of a further preferred embodiment of the invention; FIG.", "19 is a plan view of the cap of FIG.", "18 and FIG.", "20 is a front view of a preferred lid assembly of a further embodiment of the invention.", "BEST METHOD OF PERFORMING THE INVENTION FIG.", "1 illustrates a dispensing cap 11 attached to a beverage container 12.The cap 11 has a housing 13 which has an internal screw thread for attachment to the neck of the container 12.The cap 11 has a top cover 14 which has a snap fit connection to the housing 13.Finger tab 8 facilitates a users grip on the cover 14 during opening.", "The cover 14 is connected to the housing 13 by a flexible connector 15.The cap 11 may be fitted with a tamper evident seal (although this forms no part of the invention).", "The cap 11 has a tubular spout 16 which snugly fits within the housing 13 and is slidable in a vertical direction therein.", "A stem 17 is located within the spout 16.The spout 16 has upper 18 and lower 19 inwardly facing concentric lips.", "The stem 17 also has upper 21 and lower 22 outwardly facing concentric lips.", "FIG.", "2 illustrates the cap 11 in a first closed position after the cover 14 has been lifted away from the housing 13.It can be seen that the lower lip 19 of the spout 16 and the upper lip 21 of the stem 17 contact and provide a seal.", "Lip 22 projects over lip 19.A sealing member 23 is attached to the lower end of the stem 17.The sealing member 23 is in the form of a disc and seals against the inner wall 9 of the housing 13.A chamber 24 is defined by the sealing member 23 and inner wall 9 of the housing and contains a powder 25.FIG.", "3 illustrates the cap 11 in the second closed position.", "The spout 16 has been pushed downwards by a user until upper lip 18 contacts upper lip 21.The lower section 26 of the spout 17 has pushed the outer edges of the sealing member 23 downwards and away from the inner wall 9 of the housing 13.This breaks the seal and allows the powder 25 to be released into the body of the bottle in the direction of arrows A.", "The sealing member 23 is formed from a plastics material.", "The sealing member is designed such that when in the open position, it is biased into that position and cannot be returned to the sealed position.", "It can be seen that during release of the powder, contact between the respective upper portions of the stem 17 and spout 16 provides a seal so as to prevent liquid passing through the spout.", "This allows the bottle to be shaken to disperse and/or dissolve the powder upon its release into the bottle.", "After the liquid has been dispersed, the spout may then be raised to the position shown in FIG.", "4.An outer lip 27 facilitates a user's grip on the spout 16.In this position, fluid can pass through the cap as illustrated by arrows B.", "The cap 11 also has a frangible seal 30 which separates upper and lower sections of the housing 13.When this seal is broken, the upper part of the housing 13, together with the spout 16 and stem 17 may be lifted away from the bottle neck.", "The upper section however remains connected to the lower section through connecting member 31.This procedure allows a user access to the contents of the bottle without activating the spout and releasing the powder.", "This may be desirable if a user does not wish to drink the mixed beverage at that time.", "The upper section of the housing may be pivoted back into place and the powder released at a later stage.", "In this way, a user may also be able to obtain a more concentrated mixture, if desired, by releasing some of the fluid from the bottle before adding the powder.", "The cap 11 may easily be assembled by first providing the housing 13 with the sealing member 23 intact.", "The powder is then added in the desired amount.", "The spout 16 is then slid into the housing.", "Premature activation of the spout to release the powder is inhibited by contact of the lower lip 19 of the spout 16 with the upper lip 21 of the stem 17.", "(The spout is made of a resilient plastics material such that, when required, a user may push the lower lip 19 past the upper lip 21).", "Also, a projection (not illustrated) is provided on the inner wall of the housing at a point just below the lower end of the spout.", "These projections are also resilient which enables a user to be able to push the spout downwards when activating the spout.", "FIGS.", "5 to 14 illustrate a further preferred dispensing cap of the invention.", "The same reference numerals have been used to identify the same or like parts.", "The cap 11 has a housing 13, spout 16 and stem 17.The stem 16 has an upper sealing disc 35 which seals against either the lower lip 19 of the spout 16 as shown in FIG.", "6 or the upper lip 18 of the spout as shown in FIG.", "8.The sealing member 23 is made from an elastomeric plastics material and is cup shaped.", "In the pre-release position as shown in FIG.", "6, the cup shaped sealing member 23 hold the material to be dispensed.", "As the spout 16 is pushed downward by a user, the lower portion of the spout pushes against the outer section of the sealing member.", "The sealing member is initially biased in the position shown in FIG.", "6.As the sealing member approaches a substantially flat position, the bias changes towards the inverted position.", "Thus it is not necessary to push the outer edges of the sealing member 23 all the way to the release position as shown in FIGS.", "8 and 10.The stem 17 has a pair of diametrically opposed arm members 36, 37.These members 36, 37 extend through opposed apertures 38 in the spout 16.The apertures 38 are more clearly seen in FIG.", "11.The ends of the arms 36, 37 are received by vertical slots 39 in the housing 13 (see FIG.", "14).", "This arrangement holds the stem 17 in place relative to the housing 13.As the spout 16 is raised and lowered, the spout 16 is guided by the apertures 38 riding over arms 36, 37.Pushing spout 16 downwards past the second closed position is prevented or inhibited by contact of the upper part of aperture 38 with arms 36, 37.The lower end of the spout 16 has an upturned lip 40.The spout 16 is made from a resilient plastics material which allows the walls of the spout to be resiliently pushed inwardly during assembly to the position illustrated in FIG.", "6 in which the lip 40 is located within the inner wall 9 of the housing 13.As the spout 16 is pushed downwards, to the position shown in FIG.", "8, the resiliency of the spout 16 allows the lip 40 to spring outwardly such that lip 40 now extends below the lower edge of the housing wall 9.As can be seen from FIG.", "10, raising of the spout 16 past the open position of FIG.", "10 back to the first closed position in FIG.", "6 is inhibited by contact of the lip 40 from the housing 13.In practice, this is advantageous as it avoids or prevents a user upon pulling the spout 16 towards the open position from inadvertently moving the spout past the open position to the first closed position.", "The spout 16 is also provided with a number of projecting ribs 41 which facilitate frictional engagement between the inner walls of the housing and the spout 16.This is shown in FIG.", "11.FIGS.", "15 to 17 are schematic cross sectional views of a further preferred dispensing cap 11 of the present invention.", "This cap 11 similar to that of the previous figures and the same reference numerals are used to refer to same or similar features.", "Cap 11 has a housing 13, spout 16, stem 17 and a cup shaped seal 23.However in this form of the invention spout 16 has a single upper sealing lip 50 and stem 17 has upper 51 and lower 52 concentric sealing members.", "The function of this portion will be described below.", "The cap 11 shown in FIGS.", "15 to 17 operates in a similar manner to that described above.", "FIG.", "15 shows the cap 11 before use in a first closed position.", "An operator removes cover 14 to gain access to spout 16.The spout 16 is pushed downwards to a second closed position shown in FIG.", "16 in which lower sealing member 42 of stem 17 seals against lip 50 of spout 16.As the spout 16 is pushed downwards, lower wall 43 of spout 16 pushes against and inverts sealing member 23 thereby releasing the contents of the cap into the bottle.", "FIG.", "17 shows the cap 11 in the open position in which fluid can flow through the spout 16.FIGS.", "18 and 19 are respective perspective and plan views of the cap of FIG.", "17.The housing 13 is separated into upper 64 and lower sections 65 by a frangible section 46 which can be broken such that upper section 64 including spout 16 and stem 17 are removed from the bottle.", "The upper 64 and lower 65 sections are connected by a hinge member 66.The hinge 66 has a first end 67 attached to upper section 64 and a second end 68 attached to lower section 65.The first 67 and second ends 68 are angularly offset 180θ.", "Hinge member 66 sits snugly about the housing 13 and does not protrude as does the connecting member 31 of FIG.", "1.When the upper section 64 is separated from the lower section 65, the hinge member 66 acts like the coil of a helical spring biasing the upper section 64 to the closed position.", "FIG.", "20 shows a lid assembly 70 of a further form of the invention.", "The assembly has a cover 71 and a locking band 72.Frangible tabs 73 connect the cover 71 and locking band 72.These tabs must be broken to open the cover in the first instance and thus function as a tamper evident seal.", "The locking band 72 typically locates in a groove in the neck of a container.", "A hinge member 74 has a first end 75 affixed to the cover 71 and a second end 76 affixed to band 72.Tabs 77, 78 are provided to provide a grip for a user's finger when opening the lid assembly.", "In use the cover 71 is lifted away from the locking band 72.The two parts remain connected by hinge member 74.The hinge member 74 is formed from a resilient plastics material and its resiliency imparts a bias in the cover towards the cover position.", "It may be seen that a dispensing cap of the present invention allows a material to be dispensed into a beverage prior to consumption in a relatively easy and straightforward manner.", "The cap remains sealed during release of the material to allow for mixing of the contents in the bottle by shaking.", "Also, the cap is sealed prior to the initial use.", "Further, the cap is self contained and does not require a separate seal breaking member which may fall into the body of a bottle after use or require separate disposal.", "The lid assembly of the present invention provides a relatively simple and cost effective arrangement for hingedly connecting a cover member about the neck of a bottle.", "It will also be appreciated that various changes and modifications may be made to the invention as described and claimed herein without departing from the spirit and scope of the invention." ] ]

[ [ "Biopanning and rapid analysis of selective interactive ligands (brasil)", "The present invention concerns novel methods of identifying peptide sequences that selectively bind to targets.", "In alternative embodiments, targets may comprise cells or clumps of cells, particles attached to chemicals compounds, molecules or aggregates, or parasites.", "In preferred embodiments, target cells are sorted before exposure to the phage library.", "The general method, Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) provides for rapid and efficient separation of phage that bind to targets, while preserving unbound phage.", "BRASIL may be used in preselection procedure to subract phage that bind non-specifically to a first target before exposing the subtracted library to a second target.", "Certain embodiments concern targeting peptides identified by BRASIL and methods of use of such peptides for targeted delivera of therapeutic agents or imaging agents or diagnosis or treatment of diseases.", "Novel compositions comprising a first phase, second phase, target and a phage library are also disclosed." ], [ "1.A composition comprising: a) a target for phage binding; b) a phage display library; c) a first phase; and d) a second phase; wherein the density of the target is greater than the density of the second phase and the density of the second phase is greater than the density of the first phase.", "2.The composition of claim 1, wherein the target comprises isolated cells or small clumps of cells.", "3.", "(Cancelled) 4.", "(Cancelled) 5.The composition of claim 1, wherein the target is a parasite.", "6.", "(Cancelled) 7.The composition of claim 1, wherein the target comprises a particle attached to a chemical, a compound, a molecule or an aggregate of molecules.", "8.", "(Cancelled) 9.The composition of claim 1, wherein the first phase is an aqueous phase.", "10.", "(Cancelled) 11.The composition of claim 1, wherein the second phase is an organic phase.", "12.The composition of claim 1, wherein the density of the second phase is about 1.02 to 1.04 gm/ml.", "13-21.Cancelled 22.A method comprising: a) exposing a target to a phage display library in an first phase; b) exposing the first phase to a second phase; and c) separating phage bound to the target from unbound phage; wherein bound phage enter the second phase and unbound phage remain in the first phase.", "23.", "(Cancelled) 24.", "(Cancelled) 25.The method of claim 22, further comprising centrifuging the phage bound to the target through an organic phase to form a pellet.", "26.", "(Cancelled) 27.", "(Cancelled) 28.The method of claim 22, wherein the target is a parasite.", "29.", "(Cancelled) 30.The method of claim 22, wherein the target comprises a particle attached to a chemical, a compound, a molecule or an aggregate of molecules.", "31.", "(Cancelled) 32.The method of claim 22, wherein the density of the first phase is about 1.00 gm/ml and the density of the second phase is about 1.02 to 1.04 gm/ml.", "33-35.Cancelled 36.The method of claim 25, further comprising recovering bound phage from the pellet.", "37-40.Cancelled 41.The method of claim 22, further comprising i) prescreening the library against a first target; ii) collecting unbound phage; and iii) screening the unbound phage against a second target.", "42.The method of claim 41, wherein the first target comprises normal cells and the second target comprises diseased cells.", "43.", "(Cancelled) 44.The method of claim 41, wherein the first target is a non-pathogenic organism and the second target is a pathogenic organism.", "45.", "(Cancelled) 46.", "(Cancelled) 47.The method of claim 41, wherein the first target comprises quiescent cells and the second target comprises activated cells.", "48.", "(Cancelled) 49.", "(Cancelled) 50.A targeting peptide prepared by BRASIL (Biopanning and Rapid Analysis of Selective Interactive Ligands).", "51.An expression vector comprising a nucleic acid encoding a targeting peptide according to claim 50.52.The expression vector of claim 51, further comprising a nucleic acid encoding a therapeutic protein or peptide.", "53.The expression vector of claim 52, wherein the therapeutic protein or peptide is a a pro-apoptosis agent, an anti-angiogenic agent, an angiogenic agent, a hormone, a cytokine, a chemokine, a growth factor, a cytotoxic agent, an antibiotic, a survival factor, an anti-apoptotic agent, a hormone antagonist, an antibody or a Fab fragment of an antibody.", "54-58 Cancelled 59.An isolated peptide of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a sequence selected from any of SEQ ID NO:6, [SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of] SEQ ID NO:13 through [SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289] SEQ ID NO:39, SEQ ID NO:114, SEQ ID NO:128 through SEQ ID NO:137, SEQ ID NO:201 through SEQ ID NO:207 or SEQ ID NO:259.60-62.Cancelled 63.The isolated peptide of claim 59, wherein said peptide comprises at least 5 contiguous amino acids of a sequence selected from any of SEQ ID NO:6, [SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of] SEQ ID NO:13 through [SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289] SEQ ID NO:39, SEQ ID NO:114, SEQ ID NO:128 through SEQ ID NO:137, SEQ ID NO:201 through SEQ ID NO:207 or SEQ ID NO:259.64.The isolated peptide of claim 59, wherein said peptide is attached to a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, an antigen, a survival factor, an anti-apoptotic agent, a hormone antagonist, a virus, a cell, a bacterium, a yeast cell or a mammalian cell.", "65.", "(Cancelled) 66.The isolated peptide of claim 59, wherein said peptide is attached to a virus, a bacteriophage, a bacterium, a yeast cell, a liposome, a microparticle, a magnetic bead, a cell or a microdevice.", "67.The isolated peptide of claim 59, wherein said peptide is attached to a eukaryotic expression vector.", "68.A method of targeted delivery comprising: a) selecting a peptide by BRASIL; b) attaching said peptide to a therapeutic agent; and c) providing said peptide and said agent to a subject.", "69.", "(Cancelled) 70.A method of diagnosing a disease state comprising: a) selecting a peptide by BRASIL, wherein said peptide is targeted to cells associated with a disease state; b) administering said peptide to a subject; and c) determining the distribution of said peptide in said subject.", "71.The method of claim 70, wherein said disease state is selected from the group consisting of diabetes, inflammatory disease, arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection, viral infection, cardiovascular disease and degenerative disease.", "72.", "(Cancelled) 73.", "(Cancelled) 74.The method of claim 22, further comprising sorting target cells before they are exposed to the phage library.", "75.The method of claim 74, wherein the cells are sorted by FACS (flourescent activated cell sorting).", "76.The method of claim 75, wherein the cells are obtained from a subject with leukemia patient and leukemia cells are selected for exposure to the phage library.", "77.The method of claim 76, wherein the library is presubtracted against normal cells from the same subject.", "78-87.Cancelled" ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention concerns the fields of molecular medicine and targeted delivery.", "More specifically, the present invention relates to compositions and methods for identification and use of peptides that selectively target organs or tissues.", "In particular, the methods and compositions concern biopanning and rapid analysis of selective interactive ligands (BRASIL).", "2.Description of Related Art Therapeutic treatment of many human disease states is limited by the systemic toxicity of the therapeutic agents used.", "Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells.", "Treatment of cancer and other organ or tissue confined disease states would be greatly facilitated by the development of compositions and methods for targeted delivery to a desired organ or tissue of a therapeutic agent.", "Diagnostic imaging would also be facilitated by the targeted delivery of imaging agents to desired organs, tissues or diseased cells.", "Recently, an in vivo selection system was developed using phage display libraries to identify organ or tissue targeting peptides in a mouse model system.", "Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of immunoglobulins (Smith and Scott, 1985, 1993).", "Such libraries can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in as many as 10 9 permutations.", "(Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al 1998b).", "Intravenous administration of phage display libraries to mice was followed by the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996).", "Phage were recovered that were capable of selective homing to the vascular beds of different mouse organs or tissues, based on the specific targeting peptide sequences expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996).", "A variety of organ and tumor-homing peptides have been identified by this method (Rajotte et al., 1998, 1999; Koivunen et al., 1999; Burg et al., 1999; Pasqualini, 1999).", "Each of those targeting peptides bound to different receptors that were selectively expressed on the vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et al., 2000; Folkman, 1995; Folkman 1997).", "Tumor-homing peptides bound to receptors that were upregulated in the tumor angiogenic vasculature of mice (Brooks et al., 1994b; Pasqualini et al., 2000).", "In addition to identifying individual targeting peptides selective for an organ or tissue (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a; Koivunen et al., 1999), this system has been used to identify endothelial cell surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti, 1999).", "Attachment of therapeutic agents to targeting peptides resulted in the selective delivery of the agent to a desired organ or tissue in the mouse model system.", "Targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature resulted in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b; Ellerby et al., 1999).", "This relative success notwithstanding, cell surface selection of phage libraries has been plagued by technical difficulties.", "A high number of non-binder and non-specific binder clones are recovered when phage libraries are incubated with cell suspensions or monolayers.", "Removal of this background phage binding by repeated washes is both labor-intensive and inefficient.", "Cells and potential ligands are frequently lost during the many washing steps required.", "Thus, there is a need for a rapid and efficient method for in vitro biopanning that retains the selectivity and specificity of in vivo methods, while providing decreased non-specific background.", "Previous studies with phage display libraries have relied on a mouse model system to identify targeting peptides and their receptors, under the assumption that human targeting peptides are homologous.", "However, cell surface receptors may have a very different distribution and function in humans than in mice.", "Further, the mouse model system has been exploited to characterize targeting peptides for only a handful of specific organs.", "A need exists in the art for methods and compositions for identification of targeting sequences selective for human organs, tissues or cell types that can be of clinical use for targeted delivery of therapeutic agents" ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention solves a long-standing need in the art by providing compositions and in vitro methods for identifying targeting peptides that are selective for organs, tissues or cell types.", "In a preferred embodiment, such targeting peptides are identified by collecting samples of one or more organs, tissues, or cell types, separating the samples into isolated cells or small clumps of cells, suspending the cells or clumps in a first phase, exposing the cells or clumps of cells to a phage display library, layering the first phase over a second phase, and centrifuging the two phases so that the cells are pelleted at the bottom of a centrifuge tube.", "In a more preferred embodiment, the first phase is aqueous and the second phase is organic.", "In even more preferred embodiments, the cells are human cells.", "In certain embodiments, phage may be collected from the pellet by exposure to bacteria and phage clones may be plated, isolated and grown up in bulk culture.", "In alternative embodiments, phage inserts may be recovered from the pellet by PCR™ or other amplification techniques and the inserts sequenced to identify the targeting peptides.", "In certain embodiments, the organic phase comprises dibutylphtalate or a mixture of dibutylphthalate and cyclohexane.", "The methods disclosed herein are generally referred to herein as Biopanning and Rapid Analysis of Selective Interactive-Ligands (BRASIL).", "In alternative embodiments, the BRASIL method may be used to identify targeting peptides against virtually any chemical, molecule or complex of molecules.", "The separation of bound and unbound phage is preferably accomplished by partitioning bound phage from an aqueous phase into an organic phase.", "This requires that the target to which the phage bind be either denser than phage, larger than phage or preferably both.", "In preferred embodiments, the target is insoluble in the aqueous phase.", "In order to satisfy this requirement, chemicals, compounds, or molecules may be attached to a large insoluble particle, for example a glass, plastic, ceramic or magnetic bead.", "The skilled article will realize that the invention is not limited to beads and any large and/or dense particle may be used.", "The particle attached target may be exposed to a phage library in an aqueous phasae and phage binding to the target partitioned into an organic phase.", "Although the examples shown herein illustrate the use of centrifugation to partition bound phage into the organic phase, the skilled artisan will realize that other types of partitioning may be used within the scope of the invention.", "For example, for targets attached to magnetic beads, a magnetic field could be imposed to pull the phage bound to beads into an organic phase.", "In embodiments where cells are the targets, the cells may be mammalian cells, human cells, mouse cells or animal cells.", "Alternatively, cells may include any type of prokaryotic or eukaryotic cell, such as bacteria or unicellular microorganisms.", "In preferred embodiments, specific populations of cells may be prepared for use in BRASIL.", "For example, cells from leukemic patients may be sorted using a FACS (fluorescent activated cell sorter, Becton-Dickinson) to sort cancer cells from non-cancer cells.", "A phage library may be screened against cancerous cells only, either with or without a preselection subtraction against normal cells from the same patient.", "The skilled artisan will realize that cell sorting is not limited to leukemic samples, but rather may be practiced with any heterogenous population of cells.", "In certain embodiments, targeting peptides identified by BRASIL are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types in subjects.", "The skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type.", "The skilled artisan will understand that although the targeting peptides disclosed herein are particularly suited for use in human subjects, it is contemplated that they may be of use in other subjects such as mice, dogs, cats, horses, cows, sheep, pigs or any other mammal.", "Certain embodiments concern targeting peptides identified by the BRASIL method.", "One embodiment of the present invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In a preferred embodiment, the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, or even more preferably 5 amino acids or less in size.", "In other preferred embodiments, the isolated peptide of claim 1 comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In certain embodiments, the isolated peptide is attached to a molecule.", "In preferred embodiments, the attachment is a covalent attachment.", "In additional embodiments, the molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, a nucleic acid or an antigen.", "Those molecules are representative only.", "Molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject.", "In preferred embodiments, the pro-apoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO:1), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ ID NO:3) or (KLGKKLG) 3 (SEQ ID NO:4).", "In other preferred embodiments, the anti-angiogenic agent is thrombospondin, angiostatin, endostatin or pigment epithelium-derived factor.", "In further preferred embodiments, the cytokine is interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophage colony stimulating factor).", "Such examples are representative only and are not intended to exclude other pro-apoptosis agents, anti-angiogenic agents or cytokines known in the art.", "In other embodiments, the isolated peptide is attached to a macromolecular complex.", "In preferred embodiments, the attachment is a covalent attachment.", "In other preferred embodiments, the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a cell or a microdevice.", "These are representative examples only.", "Macromolecular complexes within the scope of the present invention include virtually any macromolecular complex that may be attached to a targeting peptide and administered to a subject.", "In other preferred embodiments, the isolated peptide is attached to a eukaryotic expression vector, more preferably a gene therapy vector.", "In another embodiment, the isolated peptide is attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (PPLC) matrix.", "Such attached peptides may be of use, for example, to purify or isolate an antibody, protein, peptide or other ligand that binds to the targeting peptide.", "In certain embodiments, this binding may be used to identify endogenous receptors, ligands or receptor:ligand pairs that are mimicked by the targeting peptide.", "Additional embodiments of the present invention concern fusion proteins comprising at least 3 contiguous amino acids of a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.Certain other embodiments concern compositions comprising the claimed isolated peptides or fusion proteins in a pharmaceutically acceptable carrier.", "Further embodiments concern kits comprising the claimed isolated peptides or fusion proteins in one or more containers.", "Additional embodiments concern kits comprising compositions and apparatus for performing BRASIL.", "Kit components may include, but are not limited to, any composition or apparatus that may be of use in performing BRASIL, such as solutions, buffers, media, organic phase, bacteria, phage libraries, control phage, centrifugation tubes, etc.", "Other embodiments concern methods of targeted delivery comprising selecting a targeting peptide for a desired organ or tissue, attaching said targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing said peptide attached to said molecule, complex or vector to a subject.", "Preferably, the targeting peptide is selected to include at least 3 contiguous amino acids from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In certain preferred embodiments, the molecule attached to the targeting peptide is a chemotherapeutic agent, an antigen or an imaging agent.", "The skilled artisan will realize that within the scope of the present invention any organ, tissue or cell type can be targeted for delivery, using targeting peptides attached to any molecule, macromolecular complex or gene therapy vector.", "Certain embodiments of the present invention concern methods for imaging an organ, tissue, or cell type comprising selecting a peptide targeted to said organ or tissue, attaching an imaging agent to said peptide, administering said peptide to a subject and obtaining an image.", "In preferred embodiments, the targeted cells are associated with a disease or other condition.", "In other preferred embodiments, the targeting peptide comprises at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In other embodiments, the present invention concerns methods of diagnosing a disease state, comprising selecting a peptide targeted to cells associated with such disease state, attaching an imaging agent to said peptide, administering said peptide and imaging agent to a subject suspected of having the disease, and diagnosing the presence or absence of the disease based on the distribution of said peptide and imaging agent within said subject.", "Preferably, the targeting peptide contains at least 3 contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In preferred embodiments, the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection or viral infection.", "In a more preferred embodiment, the disease state is metastatic cancer.", "Additional embodiments concern methods for identifying a receptor for a targeting peptide, comprising contacting said peptide to an organ, tissue or cell containing said receptor, allowing said peptide to bind to said receptor, and identifying said receptor by its binding to said peptide.", "In preferred embodiments, the targeting peptide contains at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.The skilled artisan will realize that the contacting step can utilize samples of organs, tissues or cells, or may alternatively utilize homogenates or detergent extracts of the organs, tissues or cells.", "In certain embodiments, the cells to be contacted may be genetically engineered to express a suspected receptor for the targeting peptide.", "In a preferred embodiment, the targeting peptide is modified with a reactive moiety that allows its covalent attachment to said receptor.", "In a more preferred embodiment, the reactive moiety is a photoreactive group that becomes covalently attached to the receptor when activated by light.", "In another preferred embodiment, the peptide is attached to a solid support and the receptor is purified by affinity chromatography.", "In other preferred embodiments, the solid support comprises magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.", "In certain embodiments, the targeting peptide inhibits the activity of the receptor upon binding to the receptor.", "The skilled artisan will realize that receptor activity can be assayed by a variety of methods known in the art, including but not limited to catalytic activity and binding activity.", "In another preferred embodiment, the receptor is an endostatin receptor, a metalloprotease or an aminopeptidase.", "Other embodiments of the present invention concern isolated nucleic acids of 300 nucleotides or less in size, encoding a targeting peptide.", "In preferred embodiments, the isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less in size.", "In other preferred embodiments, the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector.", "In even more preferred embodiments, the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage.", "In other preferred embodiments, the isolated nucleic acid is operatively linked to a leader sequence that localizes the expressed peptide to the extracellular surface of a host cell.", "Additional embodiments of the present invention concern methods of treating a disease state comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a subject with the disease state.", "Preferably, the targeting peptide includes at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In preferred embodiments the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection and viral infection.", "Another embodiment of the present invention concerns molecular adaptors for targeted gene therapy.", "In a preferred embodiment, the molecular adaptor comprises a Fab fragment of an antibody that is specific for a gene therapy vector, covalently attached to a targeting peptide sequence that provides selective targeting to a desired organ or tissue.", "The skilled artisan will realize that the present invention may include any gene therapy vector that is known in the art.", "The vector binding portion of the molecular adaptor is not limited to Fab fragments of antibodies, but may include any other molecule that can be used to attach a targeting peptide to a gene therapy vector.", "The only requirement is that the gene therapy vector should be selectively targeted to a desired organ or tissue in the presence of the molecular adaptor.", "Another embodiment of the present invention concerns compositions and methods of use of tumor targeting peptides against cancers.", "Tumor targeting peptides identified by the methods disclosed in the instant application may be attached to therapeutic agents, including but not limited to molecules or macromolecular assemblages and administered to a subject with cancer, providing for increased efficacy and decreased systemic toxicity of the therapeutic agent.", "Therapeutic agents within the scope of the present invention include but are not limited to chemotherapeutic agents, radioisotopes, pro-apoptosis agents, cytotoxic agents, cytostatic agents and gene therapy vectors.", "Targeted delivery of such therapeutic agents to tumors provides a significant improvement over the prior art for increasing the delivery of the agent to the tumor, while decreasing the inadvertent delivery of the agent to normal organs and tissues of the subject.", "In a preferred embodiment, the tumor targeting peptide is incorporated into the capsule of a phage gene therapy vector to target delivery of the phage to angiogenic endothelial cells in tumor blood vessels.", "A further embodiment of the present invention concerns methods for identifying new tumor targeting peptides, using phage display libraries that incorporate reporter genes.", "Administration of the reporter gene phage library to a subject with a tumor is followed by recovery of phage from the tumor and identification of tumor targeting peptides by sequencing selected portions of the phage genome that contain the nucleic acid sequence encoding the targeting peptide.", "While these embodiments of the present invention concern tumors, the skilled artisan will realize that within the scope of the present invention other disease states that are localized to specific organs or tissues may also be treated with enhanced therapeutic efficacy and decreased systemic toxicity using the methods and compositions disclosed herein.", "Yet another embodiment of the present invention concerns methods of identifying targeting peptides against antibodies from a subject with a disease state, comprising obtaining a sample of serum from the subject, obtaining antibodies from the sample, adding a phage display library to the antibodies and collecting phage bound to the antibodies.", "In preferred embodiments, the antibodies are attached to a solid support, more preferably attached to protein G attached to beads.", "In another preferred embodiment, a subtraction step is added where the phage display library is first screened against antibodies from a subject who does not have the disease state.", "Only phage that do not bind to these control antibodies are used to obtain phage binding to the diseased subject's antibodies.", "In other preferred embodiments, phage that bind to a target organ or tissue, for example to placenta, may be pre-screened or post-screened against a subject lacking that organ or tissue.", "Phage that bind to the subject lacking the target organ or tissue are removed from the library.", "Other embodiments concern methods of obtaining antibodies against an antigen.", "In preferred embodiments, the antigen comprises one or more targeting peptides.", "The targeting peptides are prepared and immobilized on a solid support, a sample containing antibodies is added and antibodies that bind to the targeting peptides are collected.", "In other preferred embodiments, a phage display library displaying the antigen binding portions of antibodies from a subject is prepared, the library is screened against one or more antigens and phage that bind to the antigens are collected.", "In more preferred embodiments, the antigen is a targeting peptide." ], [ "This application claims the benefit of U.S.", "Provisional Patent Application No.", "60/231,266 filed Sep. 8, 2000, and U.S. patent application Ser.", "No.", "09/765,101, filed Jan. 17, 2001.This invention was made with government support under grants DAMD 17-98-1-8041 and 17-98-1-8581 from the U.S. Army and grants 1R01CA78512-01A1, 1R01CA90810-01 and 1R01CA82976-01 from the National Institutes of Health.", "The government has certain rights in this invention.", "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention concerns the fields of molecular medicine and targeted delivery.", "More specifically, the present invention relates to compositions and methods for identification and use of peptides that selectively target organs or tissues.", "In particular, the methods and compositions concern biopanning and rapid analysis of selective interactive ligands (BRASIL).", "2.Description of Related Art Therapeutic treatment of many human disease states is limited by the systemic toxicity of the therapeutic agents used.", "Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells.", "Treatment of cancer and other organ or tissue confined disease states would be greatly facilitated by the development of compositions and methods for targeted delivery to a desired organ or tissue of a therapeutic agent.", "Diagnostic imaging would also be facilitated by the targeted delivery of imaging agents to desired organs, tissues or diseased cells.", "Recently, an in vivo selection system was developed using phage display libraries to identify organ or tissue targeting peptides in a mouse model system.", "Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of immunoglobulins (Smith and Scott, 1985, 1993).", "Such libraries can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in as many as 109 permutations.", "(Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al 1998b).", "Intravenous administration of phage display libraries to mice was followed by the recovery of phage from individual organs (Pasqualini and Ruoslahti, 1996).", "Phage were recovered that were capable of selective homing to the vascular beds of different mouse organs or tissues, based on the specific targeting peptide sequences expressed on the outer surface of the phage (Pasqualini and Ruoslahti, 1996).", "A variety of organ and tumor-homing peptides have been identified by this method (Rajotte et al., 1998, 1999; Koivunen et al., 1999; Burg et al., 1999; Pasqualini, 1999).", "Each of those targeting peptides bound to different receptors that were selectively expressed on the vasculature of the mouse target tissue (Pasqualini, 1999; Pasqualini et al., 2000; Folkman, 1995; Folkman 1997).", "Tumor-homing peptides bound to receptors that were upregulated in the tumor angiogenic vasculature of mice (Brooks et al., 1994b; Pasqualini et al., 2000).", "In addition to identifying individual targeting peptides selective for an organ or tissue (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a; Koivunen et al., 1999), this system has been used to identify endothelial cell surface markers that are expressed in mice in vivo (Rajotte and Ruoslahti, 1999).", "Attachment of therapeutic agents to targeting peptides resulted in the selective delivery of the agent to a desired organ or tissue in the mouse model system.", "Targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature resulted in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b; Ellerby et al., 1999).", "This relative success notwithstanding, cell surface selection of phage libraries has been plagued by technical difficulties.", "A high number of non-binder and non-specific binder clones are recovered when phage libraries are incubated with cell suspensions or monolayers.", "Removal of this background phage binding by repeated washes is both labor-intensive and inefficient.", "Cells and potential ligands are frequently lost during the many washing steps required.", "Thus, there is a need for a rapid and efficient method for in vitro biopanning that retains the selectivity and specificity of in vivo methods, while providing decreased non-specific background.", "Previous studies with phage display libraries have relied on a mouse model system to identify targeting peptides and their receptors, under the assumption that human targeting peptides are homologous.", "However, cell surface receptors may have a very different distribution and function in humans than in mice.", "Further, the mouse model system has been exploited to characterize targeting peptides for only a handful of specific organs.", "A need exists in the art for methods and compositions for identification of targeting sequences selective for human organs, tissues or cell types that can be of clinical use for targeted delivery of therapeutic agents SUMMARY OF THE INVENTION The present invention solves a long-standing need in the art by providing compositions and in vitro methods for identifying targeting peptides that are selective for organs, tissues or cell types.", "In a preferred embodiment, such targeting peptides are identified by collecting samples of one or more organs, tissues, or cell types, separating the samples into isolated cells or small clumps of cells, suspending the cells or clumps in a first phase, exposing the cells or clumps of cells to a phage display library, layering the first phase over a second phase, and centrifuging the two phases so that the cells are pelleted at the bottom of a centrifuge tube.", "In a more preferred embodiment, the first phase is aqueous and the second phase is organic.", "In even more preferred embodiments, the cells are human cells.", "In certain embodiments, phage may be collected from the pellet by exposure to bacteria and phage clones may be plated, isolated and grown up in bulk culture.", "In alternative embodiments, phage inserts may be recovered from the pellet by PCR™ or other amplification techniques and the inserts sequenced to identify the targeting peptides.", "In certain embodiments, the organic phase comprises dibutylphtalate or a mixture of dibutylphthalate and cyclohexane.", "The methods disclosed herein are generally referred to herein as Biopanning and Rapid Analysis of Selective Interactive-Ligands (BRASIL).", "In alternative embodiments, the BRASIL method may be used to identify targeting peptides against virtually any chemical, molecule or complex of molecules.", "The separation of bound and unbound phage is preferably accomplished by partitioning bound phage from an aqueous phase into an organic phase.", "This requires that the target to which the phage bind be either denser than phage, larger than phage or preferably both.", "In preferred embodiments, the target is insoluble in the aqueous phase.", "In order to satisfy this requirement, chemicals, compounds, or molecules may be attached to a large insoluble particle, for example a glass, plastic, ceramic or magnetic bead.", "The skilled article will realize that the invention is not limited to beads and any large and/or dense particle may be used.", "The particle attached target may be exposed to a phage library in an aqueous phasae and phage binding to the target partitioned into an organic phase.", "Although the examples shown herein illustrate the use of centrifugation to partition bound phage into the organic phase, the skilled artisan will realize that other types of partitioning may be used within the scope of the invention.", "For example, for targets attached to magnetic beads, a magnetic field could be imposed to pull the phage bound to beads into an organic phase.", "In embodiments where cells are the targets, the cells may be mammalian cells, human cells, mouse cells or animal cells.", "Alternatively, cells may include any type of prokaryotic or eukaryotic cell, such as bacteria or unicellular microorganisms.", "In preferred embodiments, specific populations of cells may be prepared for use in BRASIL.", "For example, cells from leukemic patients may be sorted using a FACS (fluorescent activated cell sorter, Becton-Dickinson) to sort cancer cells from non-cancer cells.", "A phage library may be screened against cancerous cells only, either with or without a preselection subtraction against normal cells from the same patient.", "The skilled artisan will realize that cell sorting is not limited to leukemic samples, but rather may be practiced with any heterogenous population of cells.", "In certain embodiments, targeting peptides identified by BRASIL are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types in subjects.", "The skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type.", "The skilled artisan will understand that although the targeting peptides disclosed herein are particularly suited for use in human subjects, it is contemplated that they may be of use in other subjects such as mice, dogs, cats, horses, cows, sheep, pigs or any other mammal.", "Certain embodiments concern targeting peptides identified by the BRASIL method.", "One embodiment of the present invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In a preferred embodiment, the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, or even more preferably 5 amino acids or less in size.", "In other preferred embodiments, the isolated peptide of claim 1 comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a targeting peptide sequence, selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In certain embodiments, the isolated peptide is attached to a molecule.", "In preferred embodiments, the attachment is a covalent attachment.", "In additional embodiments, the molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, a nucleic acid or an antigen.", "Those molecules are representative only.", "Molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject.", "In preferred embodiments, the pro-apoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK)2 (SEQ ID NO:1), (KLAKKLA)2 (SEQ ID NO:2), (KAAKKAA)2 (SEQ ID NO:3) or (KLGKKLG)3 (SEQ ID NO:4).", "In other preferred embodiments, the anti-angiogenic agent is thrombospondin, angiostatin, endostatin or pigment epithelium-derived factor.", "In further preferred embodiments, the cytokine is interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β, tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophage colony stimulating factor).", "Such examples are representative only and are not intended to exclude other pro-apoptosis agents, anti-angiogenic agents or cytokines known in the art.", "In other embodiments, the isolated peptide is attached to a macromolecular complex.", "In preferred embodiments, the attachment is a covalent attachment.", "In other preferred embodiments, the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a cell or a microdevice.", "These are representative examples only.", "Macromolecular complexes within the scope of the present invention include virtually any macromolecular complex that may be attached to a targeting peptide and administered to a subject.", "In other preferred embodiments, the isolated peptide is attached to a eukaryotic expression vector, more preferably a gene therapy vector.", "In another embodiment, the isolated peptide is attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (PPLC) matrix.", "Such attached peptides may be of use, for example, to purify or isolate an antibody, protein, peptide or other ligand that binds to the targeting peptide.", "In certain embodiments, this binding may be used to identify endogenous receptors, ligands or receptor:ligand pairs that are mimicked by the targeting peptide.", "Additional embodiments of the present invention concern fusion proteins comprising at least 3 contiguous amino acids of a sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.Certain other embodiments concern compositions comprising the claimed isolated peptides or fusion proteins in a pharmaceutically acceptable carrier.", "Further embodiments concern kits comprising the claimed isolated peptides or fusion proteins in one or more containers.", "Additional embodiments concern kits comprising compositions and apparatus for performing BRASIL.", "Kit components may include, but are not limited to, any composition or apparatus that may be of use in performing BRASIL, such as solutions, buffers, media, organic phase, bacteria, phage libraries, control phage, centrifugation tubes, etc.", "Other embodiments concern methods of targeted delivery comprising selecting a targeting peptide for a desired organ or tissue, attaching said targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing said peptide attached to said molecule, complex or vector to a subject.", "Preferably, the targeting peptide is selected to include at least 3 contiguous amino acids from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In certain preferred embodiments, the molecule attached to the targeting peptide is a chemotherapeutic agent, an antigen or an imaging agent.", "The skilled artisan will realize that within the scope of the present invention any organ, tissue or cell type can be targeted for delivery, using targeting peptides attached to any molecule, macromolecular complex or gene therapy vector.", "Certain embodiments of the present invention concern methods for imaging an organ, tissue, or cell type comprising selecting a peptide targeted to said organ or tissue, attaching an imaging agent to said peptide, administering said peptide to a subject and obtaining an image.", "In preferred embodiments, the targeted cells are associated with a disease or other condition.", "In other preferred embodiments, the targeting peptide comprises at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In other embodiments, the present invention concerns methods of diagnosing a disease state, comprising selecting a peptide targeted to cells associated with such disease state, attaching an imaging agent to said peptide, administering said peptide and imaging agent to a subject suspected of having the disease, and diagnosing the presence or absence of the disease based on the distribution of said peptide and imaging agent within said subject.", "Preferably, the targeting peptide contains at least 3 contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In preferred embodiments, the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection or viral infection.", "In a more preferred embodiment, the disease state is metastatic cancer.", "Additional embodiments concern methods for identifying a receptor for a targeting peptide, comprising contacting said peptide to an organ, tissue or cell containing said receptor, allowing said peptide to bind to said receptor, and identifying said receptor by its binding to said peptide.", "In preferred embodiments, the targeting peptide contains at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.The skilled artisan will realize that the contacting step can utilize samples of organs, tissues or cells, or may alternatively utilize homogenates or detergent extracts of the organs, tissues or cells.", "In certain embodiments, the cells to be contacted may be genetically engineered to express a suspected receptor for the targeting peptide.", "In a preferred embodiment, the targeting peptide is modified with a reactive moiety that allows its covalent attachment to said receptor.", "In a more preferred embodiment, the reactive moiety is a photoreactive group that becomes covalently attached to the receptor when activated by light.", "In another preferred embodiment, the peptide is attached to a solid support and the receptor is purified by affinity chromatography.", "In other preferred embodiments, the solid support comprises magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.", "In certain embodiments, the targeting peptide inhibits the activity of the receptor upon binding to the receptor.", "The skilled artisan will realize that receptor activity can be assayed by a variety of methods known in the art, including but not limited to catalytic activity and binding activity.", "In another preferred embodiment, the receptor is an endostatin receptor, a metalloprotease or an aminopeptidase.", "Other embodiments of the present invention concern isolated nucleic acids of 300 nucleotides or less in size, encoding a targeting peptide.", "In preferred embodiments, the isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less in size.", "In other preferred embodiments, the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector.", "In even more preferred embodiments, the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage.", "In other preferred embodiments, the isolated nucleic acid is operatively linked to a leader sequence that localizes the expressed peptide to the extracellular surface of a host cell.", "Additional embodiments of the present invention concern methods of treating a disease state comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a subject with the disease state.", "Preferably, the targeting peptide includes at least three contiguous amino acids selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, any of SEQ ID NO:13 through SEQ ID NO:124 or any of SEQ ID NO:128 through SEQ ID NO:289.In preferred embodiments the disease state is diabetes mellitus, inflammatory disease, rheumatoid arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection and viral infection.", "Another embodiment of the present invention concerns molecular adaptors for targeted gene therapy.", "In a preferred embodiment, the molecular adaptor comprises a Fab fragment of an antibody that is specific for a gene therapy vector, covalently attached to a targeting peptide sequence that provides selective targeting to a desired organ or tissue.", "The skilled artisan will realize that the present invention may include any gene therapy vector that is known in the art.", "The vector binding portion of the molecular adaptor is not limited to Fab fragments of antibodies, but may include any other molecule that can be used to attach a targeting peptide to a gene therapy vector.", "The only requirement is that the gene therapy vector should be selectively targeted to a desired organ or tissue in the presence of the molecular adaptor.", "Another embodiment of the present invention concerns compositions and methods of use of tumor targeting peptides against cancers.", "Tumor targeting peptides identified by the methods disclosed in the instant application may be attached to therapeutic agents, including but not limited to molecules or macromolecular assemblages and administered to a subject with cancer, providing for increased efficacy and decreased systemic toxicity of the therapeutic agent.", "Therapeutic agents within the scope of the present invention include but are not limited to chemotherapeutic agents, radioisotopes, pro-apoptosis agents, cytotoxic agents, cytostatic agents and gene therapy vectors.", "Targeted delivery of such therapeutic agents to tumors provides a significant improvement over the prior art for increasing the delivery of the agent to the tumor, while decreasing the inadvertent delivery of the agent to normal organs and tissues of the subject.", "In a preferred embodiment, the tumor targeting peptide is incorporated into the capsule of a phage gene therapy vector to target delivery of the phage to angiogenic endothelial cells in tumor blood vessels.", "A further embodiment of the present invention concerns methods for identifying new tumor targeting peptides, using phage display libraries that incorporate reporter genes.", "Administration of the reporter gene phage library to a subject with a tumor is followed by recovery of phage from the tumor and identification of tumor targeting peptides by sequencing selected portions of the phage genome that contain the nucleic acid sequence encoding the targeting peptide.", "While these embodiments of the present invention concern tumors, the skilled artisan will realize that within the scope of the present invention other disease states that are localized to specific organs or tissues may also be treated with enhanced therapeutic efficacy and decreased systemic toxicity using the methods and compositions disclosed herein.", "Yet another embodiment of the present invention concerns methods of identifying targeting peptides against antibodies from a subject with a disease state, comprising obtaining a sample of serum from the subject, obtaining antibodies from the sample, adding a phage display library to the antibodies and collecting phage bound to the antibodies.", "In preferred embodiments, the antibodies are attached to a solid support, more preferably attached to protein G attached to beads.", "In another preferred embodiment, a subtraction step is added where the phage display library is first screened against antibodies from a subject who does not have the disease state.", "Only phage that do not bind to these control antibodies are used to obtain phage binding to the diseased subject's antibodies.", "In other preferred embodiments, phage that bind to a target organ or tissue, for example to placenta, may be pre-screened or post-screened against a subject lacking that organ or tissue.", "Phage that bind to the subject lacking the target organ or tissue are removed from the library.", "Other embodiments concern methods of obtaining antibodies against an antigen.", "In preferred embodiments, the antigen comprises one or more targeting peptides.", "The targeting peptides are prepared and immobilized on a solid support, a sample containing antibodies is added and antibodies that bind to the targeting peptides are collected.", "In other preferred embodiments, a phage display library displaying the antigen binding portions of antibodies from a subject is prepared, the library is screened against one or more antigens and phage that bind to the antigens are collected.", "In more preferred embodiments, the antigen is a targeting peptide.", "BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention.", "The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.", "FIG.", "1.BRASIL principle.", "A suspension of single cells or small clumps of cells that has been incubated with phage (library or single clones) in an upper first phase is centrifuged over a preferably non-miscible oil lower second phase.", "Because the second phase has an intermediate specific density, upon optimized centrifugation conditions, the cells will enter the lower phase and pellet at the bottom of the tube, carrying with theme only the specifically bound phage.", "The remaining unbound phage stay in the upper phase.", "The cell pellet is then carefully recovered.", "Targeting peptide sequences may be identified by amplification and sequencing of the phage inserts, either with or without recovery of the phage by infection of a host E. coli.", "FIG.", "2A.", "BRASIL method optimization.", "A single-cell suspension of Kaposi sarcoma-derived cells (KS1767) was incubated with increasing titers of a phage displaying an alpha v integrin-binding motif (RGD-4C phage) or a control phage with no peptide insert (Fd phage).", "KS1767 cells were chosen because they express high levels of alpha v integrins.", "Cells and phage were incubated for 4 hours on ice (to prevent phage internalization) and centrifuged for 10 minutes at 10,000 g. The phage bound to the KS1767 cells were recovered by infection of a host E. coli, and plated in LB/tet agar plates at 37° C. overnight.", "Finally, the phage transducing units were counted.", "Extremely low backgrounds were observed.", "Under non-saturated conditions, a conservative mean estimate of the enrichment of RGD-4C phage in relation to Fd phage is 500-1000.This experiment was repeated three times with similar results.", "Mean and standard deviation are shown.", "FIG.", "2B.", "The synthetic RGD-4C peptide—but not the RGE control peptide—in solution inhibited 99.99% of the RGD-4C phage binding to KS1767 cells FIG.", "3.Pre-clearing protocol using BRASIL to selectively remove phage from a phage display library.", "FIG.", "4A.", "Binding of phage clone-19 to immobilized VEGF receptors.", "Clone-19 (black bars) binds to VEGF-R1 and NRP-1 but not VEGF-R2 or BSA.", "No binding of insertless Fd phage could be detected (hash bars).", "FIG.", "4B.", "The binding to the VEGF-R1 (black circle) and NRP-1 (open square) could be completely blocked by 10-20 ng/ml of VEGF165.FIG.", "5.HUVEC cells were cultured in 24-well plates and starved for 24 with basal medium without any sera and supplements.", "Phage clone-19 or RGD.4C (which binds to HUVEC) were added at 1010 T.U.", "per well.", "VEGF165 (20 ng/ml) or basal (starved) medium were added as positive and negative controls.", "Cell proliferation was measured by the MMT method.", "Clone-19 promoted cell proliferation comparably to the positive control (VEGF-165).", "The RGD.4C peptide, which also binds to HUVEC, resulted in a cell proliferation rate only slightly above the negative control.", "FIG.", "6A-6C.", "Binding of selected phage clones to a subconfluent human urothelial cell monolayer.", "Insertless fd-tet phage were included as negative control.", "Results are means of triplicate wells relative to binding of fd-tet phage, that was set as 1.Input of phage was 1×108 T.U.", "per well.", "Bound phage were detected after intensive washing by infection with log phase K91 bacteria and plating of serial dilutions.", "FIG.", "7.Binding of selected phage clones to the human breast cancer cell line MDA-MBA435 (white bars) as well as the urothelial tumor cell lines T24 (light grey bars) and RT4 (dark grey bars).", "Insertless fd-tet phage were included as controls.", "Results are given as mean of triplicate wells relative to binding of fd-tet phage, that was set as 1.Input of phage was 1×108 T.U.", "per well.", "Bound phage were detected after intensive washing by infection with log phase K91 bacteria and plating of serial dilutions.", "FIG.", "8.Inhibition of VHALES (SEQ ID NO:25) phage binding to RT4 tumor cells was inhibited by synthetic VHALE (SEQ ID NO:25) (black squares) but not by the control peptide CARAC (SEQ ID NO:5) (white squares).", "Binding of VHALES (SEQ ID NO:25) phage was 5.4 fold higher than insertless fd-tet phage.", "A subconfluent monolayer of RT4 tumor cells was incubated with 1×108 T.U.", "of VHALES (SEQ ID NO:25) phage per well in presence of increasing amounts of VHALES (SEQ ID NO:25) and control peptide.", "Results are given as mean of triplicate wells.", "Bound phage were detected after intensive washing by infection with log phase K91 bacteria and plating of serial dilutions.", "FIG.", "9.Binding of selected phage clones to porcine urothelium in a dot blot chamber assay.", "Three wells were pooled as one field, and results represent the mean of triplicate fields relative to binding of insertless fd-tet phage, that was set as 1.Input of phage was 1×108 T.U.", "per well.", "Bound phage were detected after intensive washing by infection with log phase K91 bacteria and plating of serial dilutions.", "FIG.", "10.Influence of the GAG-layer on phage binding.", "In the dot blot chamber assay portions of a porcine bladder mucosa were treated with 0.1M HCl for 2 min prior to remove the GAG-layer.", "Binding to treated surface is given relative to untreated surface, that was set as 1.Bound phage were detected after intensive washing by infection with log phase K91 bacteria and plating of serial dilutions.", "FIG.", "11.Binding of selected clones to human bone marrow cells by BRASIL.", "DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As used herein in the specification, “a” or “an” may mean one or more.", "As used herein in the claim(s) in conjunction with the word “comprising” the words “a” or “an” may mean one or more than one.", "As used herein “another” may mean at least a second or more of an item.", "A “targeting peptide” is a peptide comprising a contiguous sequence of amino acids that is characterized by selective localization to a target organ, tissue or cell type, preferably of human origin.", "Selective localization may be determined, for example, by methods disclosed below, wherein the putative targeting peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage.", "In certain embodiments, targeting phage that have been identified by BRASIL are administered to a subject followed by collection of one or more organs, tissues or cell types from the subject and identification of phage found in the target organ, tissue or cell type.", "A phage expressing a targeting peptide sequence is considered to be selectively localized if it exhibits greater binding in the target compared to a control tissue, organ or cell type.", "Another alternative method of determining selective localization is that phage expressing the putative target peptide exhibit at least a two-fold, more preferably at least a three-fold enrichment in the target compared to control phage that express a non-specific peptide or that have not been genetically engineered to express any putative target peptides.", "Another method to determine selective localization is that localization to the target of phage expressing the target peptide is at least partially blocked by the co-administration of a synthetic peptide containing the target peptide sequence.", "“Targeting peptide” and “homing peptide” are used synonymously herein.", "A “phage display library” means a collection of phage that have been genetically engineered to express a set of putative targeting peptides on their outer surface.", "In preferred embodiments, DNA sequences encoding the putative targeting peptides are inserted in frame into a gene encoding a phage capsule protein.", "In other preferred embodiments, the putative targeting peptide sequences are in part random mixtures of all twenty amino acids and in part non-random.", "In certain preferred embodiments the putative targeting peptides of the phage display library exhibit one or more cysteine residues at fixed locations within the targeting peptide sequence.", "A “macromolecular complex” refers to a collection of molecules that may be random, ordered or partially ordered in their arrangement.", "The term encompasses biological organisms such as bacteriophage, viruses, bacteria, unicellular pathogenic organisms, multicellular pathogenic organisms and prokaryotic or eukaryotic cells.", "The term also encompasses non-living assemblages of molecules, such as liposomes, microcapsules, microparticles, microdevices and magnetic beads.", "The only requirement is that the complex contains more than one molecule.", "The molecules may be identical, or may differ from each other.", "A “receptor” for a targeting peptide includes but is not limited to any molecule or complex of molecules that binds to a targeting peptide.", "Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multi-molecular structures, a specific conformation of one or more molecules and a morphoanatomic entity.", "In preferred embodiments, a “receptor” is a naturally occurring molecule or complex of molecules that is present on the lumenal surface of cells forming blood vessels within a target organ or tissue.", "A “subject” refers generally to a mammal.", "In certain preferred embodiments, the subject is a mouse or rabbit.", "In even more preferred embodiments, the subject is a human.", "Phage Display The methods described herein for identification of targeting peptides involve the in vitro administration of phage display libraries.", "Various methods of phage display and methods for producing diverse populations of peptides are well known in the art.", "For example, U.S. Pat.", "Nos.", "5,223,409; 5,622,699 and 6,068,829, each of which is incorporated herein by reference, describe methods for preparing a phage library.", "The phage display technique involves genetically manipulating bacteriophage so that small.", "peptides can be expressed on their surface (Smith and Scott, 1985, 1993).", "The potential range of applications for this technique is quite broad, and the past decade has seen considerable progress in the construction of phage-displayed peptide libraries and in the development of screening methods in which the libraries are used to isolate peptide ligands.", "For example, the use of peptide libraries has made it possible to characterize interacting sites and receptor-ligand binding motifs within many proteins, such as antibodies involved in inflammatory reactions or integrins that mediate cellular adherence.", "This method has also been used to identify novel peptide ligands that serve as leads to the development of peptidomimetic drugs or imaging agents (Arap et al., 1998a).", "In addition to peptides, larger protein domains such as single-chain antibodies can also be displayed on the surface of phage particles (Arap et al., 1998a).", "Previously, amino acid sequences for targeting a given organ or tissue have been isolated by in vivo “biopanning” (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999).", "In brief, a library of phage containing putative targeting peptides is administered to an animal or human subject and samples of organs or tissues containing phage are collected.", "In examples utilizing filamentous phage, the phage may be propagated in vitro between rounds of biopanning in pilus-positive bacteria.", "The bacteria are not lysed by the phage but rather secrete multiple of copies of phage that display a particular insert.", "Phage that bind to a target molecule can be eluted from the target organ or tissue and then amplified by growing them in host bacteria.", "The amplified phage may be administered to a second subject and samples of organs or tissues again collected.", "Multiple rounds of biopanning may be performed until a population of selective binders is obtained.", "The amino acid sequence of the peptides is determined by sequencing the DNA corresponding to the targeting peptide insert in the phage genome.", "The identified targeting peptide can then be produced as a synthetic peptide by standard protein chemistry techniques (Arap et al., 1998a, Smith et al., 1985).", "This approach allows circulating targeting peptides to be detected in an unbiased functional assay, without any preconceived notions about the nature of their target.", "Once a candidate target is identified as the receptor of a targeting peptide, it can be isolated, purified and cloned by using standard biochemical methods (Pasqualini, 1999; Rajotte and Ruoslahti, 1999).", "The in vitro methods disclosed herein also use phage display libraries.", "However, rather than injecting the library into a live host, samples of target organs, tissues or cell types are exposed to the phage display library in vitro.", "Choice of Phage Display System.", "In vivo selection studies performed in mice preferentially employed libraries of random peptides expressed as fusion proteins with the gene III capsule protein in the fUSE5 vector (Pasqualini and Ruoslahti, 1996).", "The number and diversity of individual clones present in a given library is a significant factor for the success of in vivo selection.", "Primary libraries are preferred, which are less likely to have an over-representation of defective phage clones (Koivunen et al., 1999).", "The preparation of a library may be optimized to between 108-109 transducing units (T.U.)/ml.", "A bulk amplification strategy may be applied between rounds of selection.", "Phage libraries displaying linear, cyclic, or double cyclic peptides may be used.", "However, phage libraries displaying 3 to 10 random residues in a cyclic insert (CX3-10C) are preferred, since single cyclic peptides tend to have a higher affinity for the target organ than linear peptides.", "Libraries displaying double-cyclic peptides (such as CX3CX3CX3C; Rojotte et al., 1998) have been successfully used.", "However, the production of the cognate synthetic peptides, although possible, can be complex due to the multiple conformers with different disulfide bridge arrangements.", "Identification of Homing Peptides and Receptors by in vivo Phage Display in Mice.", "In vivo selection of peptides from phage-display peptide libraries administered to mice has been used to identify targeting peptides selective for normal mouse brain, kidney, lung, skin, pancreas, retina, intestine, uterus, prostate, and adrenal gland (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999; Rajotte et al., 1998).", "These results show that the vascular endothelium of normal organs is sufficiently heterogenous to allow differential targeting with peptide probes (Pasqualini and Ruoslahti, 1996; Rajotte et al., 1998).", "A panel of peptide motifs that target the blood vessels of tumor xenografts in nude mice has been assembled (Arap et al., 1998a; reviewed in Pasqualini, 1999).", "These motifs include the RGD-4C, NGR, and GSL peptides.", "The RGD-4C peptide has previously been identified as selectively binding αv integrins and has been shown to home to the vasculature of tumor xenografts in nude mice (Arap et al., 1998a, 1998b; Pasqualini et al., 1997).", "The receptors for the tumor homing RGD4C targeting peptide have been identified as αv integrins (Pasqualini et al., 1997).", "The αv integrins play an important role in angiogenesis.", "The αvβ3 and αvβ5 integrins are absent or expressed at low levels in normal endothelial cells but are induced in angiogenic vasculature of tumors (Brooks et al., 1994; Hammes et al., 1996).", "Aminopeptidase N/CD13 has recently been identified as an angiogenic receptor for the NGR motif (Burg et al., 1999).", "Aminopeptidase N/CD13 is strongly expressed not only in the angiogenic blood vessels of prostate cancer in TRAMP mice but also in the normal epithelial prostate tissue.", "Tumor-homing phage co-localize with their receptors in the angiogenic vasculature of tumors but not in non-angiogenic blood vessels in normal tissues (Arap et al., 1998b).", "Immunohistochemical evidence shows that vascular targeting phage bind to human tumor blood vessels in tissue sections (Pasqualini et al., 2000) but not to normal blood vessels.", "A negative control phage with no insert (fd phage) did not bind to normal or tumor tissue sections.", "The expression of the angiogenic receptors was evaluated in cell lines, in non-proliferating blood vessels and in activated blood vessels of tumors and other angiogenic tissues such as corpus luteum.", "Flow cytometry and immunohistochemistry showed that these receptors are expressed in a number of tumor cells and in activated HUVECs (data not shown).", "The angiogenic receptors were not detected in the vasculature of normal organs of mouse or human tissues.", "The distribution of these receptors was analyzed by immunohistochemistry in tumor cells, tumor vasculature, and normal vasculature.", "Alpha v integrins, CD13, aminopeptidase A, NG2, and MMP-2/MMP-9—the known receptors in tumor blood vessels—are specifically expressed in angiogenic endothelial cells and pericytes of both human and murine origin.", "Angiogenic neovasculature expresses markers that are either expressed at very low levels or not at all in non-proliferating endothelial cells (not shown).", "The markers of angiogenic endothelium include receptors for vascular growth factors, such as specific subtypes of VEGF and basic FGF receptors, and αv integrins, among many others (Mustonen and Alitalo, 1995).", "Thus far, identification and isolation of novel molecules characteristic of angiogenic vasculature has been slow, mainly because endothelial cells undergo dramatic phenotypic changes when grown in culture (Watson et al., 1995).", "Many of these tumor vascular markers are proteases and some of the markers also serve as viral receptors.", "Alpha v integrins are receptors for adenoviruses (Wickham et al., 1997c) and CD13 is a receptor for coronaviruses (Look et al., 1989).", "MMP-2 and MMP-9 are receptors for echoviruses (Koivunen et al., 1999).", "Aminopeptidase A also appears to be a viral receptor.", "Bacteriophage may use the same cellular receptors as eukaryotic viruses.", "These findings suggest that receptors isolated by phage display will have cell internalization capability, a key feature for utilizing the identified peptide motifs as targeted gene therapy carriers.", "Targeted Delivery Peptides that home to tumor vasculature have been coupled to cytotoxic drugs or proapoptotic peptides to yield compounds that were more effective and less toxic than the parental compounds in experimental models of mice bearing tumor xenografts (Arap et al., 1998a; Ellerby et al, 1999).", "The insertion of the RGD-4C peptide into a surface protein of an adenovirus has produced an adenoviral vector that may be used for tumor targeted gene therapy (Arap et al., 1998b).", "BRASIL In certain embodiments, separation of phage bound to the cells of a target organ or tissue from unbound phage is achieved using the BRASIL technique.", "In BRASIL (Biopanning and Rapid Analysis of Selective Interactive Ligands), an organ or tissue is gently separated into cells or small clumps of cells that are suspended in an first phase.", "The first phase is layered over a second phase of appropriate density and centrifuged.", "Cells attached to bound phage are pelleted at the bottom of the centrifuge tube, while unbound phage remain in the first phase.", "This allows a more efficient separation of bound from unbound phage, while maintaining the binding interaction between phage and cell.", "BRASIL may be performed by an ice vitro protocol, where the cells are exposed to the phage library in the aqueous phase before centrifugation.", "In preferred embodiments, the first phase is aqueous and the second phase is organic.", "Specific non-limiting examples of organic phases that may be employed within the scope of the present invention are disclosed below.", "Although the cells shown in the Examples below are primarily human cells, the invention is not limiting for the type of cell that may be used.", "Virtually any type of prokaryotic or eukaryotic cell may be used with BRASIL, including but not limited to human, mouse, mammalian, animal or plant cells, bacteria and unicellular organisms such as amoeba, spores, yeast, molds, algae, Giardia or dinoflagellates.", "In certain embodiments, the cells to be screened by BRASIL may first be sorted, for example using an FACS apparatus (Becton Dickinson) to separate heterogenous populations of cells into homogenous populations of cells.", "In alternative embodiments, the target used to screen the phage library may include non-cellular targets, such as chemicals, compounds, molecules or aggregates of molecules.", "Target molecules of potential use in BRASIL include but are not limited to proteins, proteoglycans, carbohydrates, lipids, glycolipids, sphingolipids and lipoproteins.", "In preferred embodiments, such non-cellular targets may be attached either covalently or non-covalently to a larger particle, such as a glass, plastic, ceramic or magnetic bead.", "Linkers may be used for the attachment to increase the accessibility of the target to the phage targeting peptides.", "In such embodiments, the skilled artisan will realize that other methods of separating bound phage into an organic phase may be used besides centrifugation.", "For example, where magnetic particles are used, the particles may be partitioned into the organic phase by imposition of a magnetic field.", "If the particle is sufficiently large or dense, settling of the particle under the influence of gravity may be used to partition the bound phage into the organic phase.", "The invention is not limiting to the method of partitioning bound phage into an organic phase and any method known in the art for separating phage bound to particles or cells into an organic or other second phase may be used within the scope of the invention.", "The invention is not limiting as to the exact composition of the first and second phases, as long as the cells to be pelleted have a density that is higher than that of the second phase, and the second phase has a density that is higher than the first phase.", "In preferred embodiments, the second phase has a density of about 1.02 to 1.04, while the first phase has a density of about 1.00.The cells or clumps of cells used for BRASIL preferably have a density of greater than 1.04 gm/ml.", "The skilled artisan will realize that specific cell types may vary in density and that optimization of BRASIL by adjustment of phase density may be appropriate.", "In preferred embodiments, in order to prevent mixing and dilution, the first and second phases are immiscible.", "However, step gradient centrifugation using miscible phases is known in the art and may be used in the practice of the present invention, for example using cesium chloride, sucrose, PEG (polyethylene glycol), Ficoll or Percoll solutions of appropriate density.", "A variety of organic solvents of known density are available for use.", "Non-limiting examples of organic solvents with reported densities between 1.02 and 1.04 include diisoamyl phthalate (1.021), phenyl butyrate (1.038), tributyrin.", "(1.035), 9-ethylanthracene (1.041), methyl-diphenylamine (1.048), 1-2-dimethoxy-4-(2-propyl)-benzene (1.039), alpha-phenyl-benzenethanol (1.036), 3-methyl-benzenthiol (1.041), acetaldehyde semicarbazone (1.030), phenylacetaldehyde (1.027) and dibenzylamine (1.026).", "Other organic compounds and their densities may be found, for example, in the Handbook of Chemistry and Physics, 50th Edition, pp.", "C-75 to C-541, the Chemical Rubber Co., Cleveland, Ohio 1969.Of course, it is not necessary that the organic phase be comprised of a single organic solvent and it is contemplated within the scope of the invention that an organic phase of appropriate density may be produced by mixing organic solvents of different densities, as disclosed in the Examples below.", "Additional mixtures may be designed using routine techniques known in the art.", "The skilled artisan will realize that densities often are temperature dependent and that the appropriate densities are obtained at the temperature of the centrifuge used to pellet the cells.", "In various embodiments, that temperature may range from room temperature to about 4° C. For purposes of centrifugation, any organic phase utilized should be a liquid at the temperature used.", "The artisan will further realize that optimization of second phase density may be required for different cell types.", "For example, different densities are observed for rat hepatocytes (1.07-1.10), Kupffer cells (1.05-1.06), human thrombocytes (1.04-1.06), lymphocytes (1.06-1.08), granulocytes (1.08-1.09), erythrocytes (1.09-1.10) and E. coli (1.13).", "All of these cell types would be expected to pellet through a second organic phase of about 1.03 density.", "It is further realized that the osmolarity of the first (aqueous) phase may affect the density of cells, particularly cells that are not bound by a rigid cell wall.", "In preferred embodiments, the osmolarity of the medium is approximately equal to the osmolarity of cells in situ (approximately 150 mM salt concentration).", "A wide variety of media of physiological osmolarity are known in the art, such as phosphate or Tris buffered saline (PBS or TBS).", "The skilled artisan will also realize that organic phases of high toxicity are to be avoided.", "For example, organic solvents such as phenol or formaldehyde that result in denaturation of proteins are undesirable for use as a second phase.", "The toxicity properties of organic solvents are well known in the art.", "In certain embodiments, a subtraction protocol is used with BRASIL to further reduce background phage binding.", "The purpose of subtraction is to remove phage from the library that bind to cells other than the cell of interest, or that bind to inactivated cells.", "In alternative embodiments, the phage library may be screened against a control cell line, tissue or organ sample that is not the targeted cell, tissue or organ.", "After subtraction the library may be screened against the cell, tissue or organ of interest.", "In another alternative embodiment, an unstimulated, quiescent cell line, tissue or organ may be screened against the library and binding phage removed.", "The cell line, tissue or organ is then activated, for example by administration of a hormone, growth factor, cytokine or chemokine and the activated cell line screened against the subtracted phage library.", "Other methods of subtraction protocols are known and may be used in the practice of the present invention, for example as disclosed in U.S. Pat.", "Nos.", "5,840,841, 5,705,610, 5,670,312 and 5,492,807, incorporated herein by reference.", "Proteins and Peptides In certain embodiments, the present invention concerns novel compositions comprising at least one protein or peptide.", "As used herein, a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids.", "For convenience, the terms “protein,” “polypeptide” and “peptide are used interchangeably herein.", "In certain embodiments the size of the at least one protein or peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino acid residues.", "As used herein, an “amino acid residue” refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art.", "In certain embodiments, the residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues.", "In other embodiments, the sequence may comprise one or more non-amino acid moieties.", "In particular embodiments, the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.", "Accordingly, the term “protein or peptide” encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including but not limited to those shown on Table 1 below.", "TABLE 1 Modified and Unusual Amino Acids Abbr.", "Amino Acid Aad 2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine, β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid, piperidine acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib 2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelic acid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn N-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine AIle allo-Isoleucine MeGly N-Methylgflycine, sarcaosine MeIle N-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline Nva Norvaline Nle Norleucine Orn Ornithine Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides.", "The nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.", "One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/).", "The coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art.", "Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.", "Peptide Mimetics Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics.", "Mimetics are peptide-containing molecules that mimic elements of protein secondary structure.", "See, for example, Johnson et al., “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, New York (1993), incorporated herein by reference.", "The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.", "A peptide mimetic is expected to permit molecular interactions similar to the natural molecule.", "These principles may be used to engineer second generation molecules having many of the natural properties of the targeting peptides disclosed herein, but with altered and even improved characteristics.", "Fusion Proteins Other embodiments of the present invention concern fusion proteins.", "These molecules generally have all or a substantial portion of a targeting peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or protein.", "For example, fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.", "Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein.", "Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.", "Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.", "In preferred embodiments, the fusion proteins of the instant invention comprise a targeting peptide linked to a therapeutic protein or peptide.", "Examples of proteins or peptides that may be incorporated into a fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins.", "These examples are not meant to be limiting and it is contemplated that within the scope of the present invention virtually any protein or peptide could be incorporated into a fusion protein comprising a targeting peptide.", "Methods of generating fusion proteins are well known to those of skill in the art.", "Such proteins can be produced, for example, by chemical attachment using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding the targeting peptide to a DNA sequence encoding the second peptide or protein, followed by expression of the intact fusion protein.", "Protein Purification In certain embodiments a protein or peptide may be isolated or purified.", "Protein purification techniques are well known to those of skill in the art.", "These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions.", "The protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).", "Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing.", "An example of receptor protein purification by affinity chromatography is disclosed in U.S. Pat.", "No.", "5,206,347, the entire text of which is incorporated herein by reference.", "A particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even HPLC.", "A purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.", "An isolated or purified protein or peptide, therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.", "Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.", "Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.", "Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure.", "These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.", "A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a “-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.", "Various techniques suitable for use in protein purification are well known to those of skill in the art.", "These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like, or by heat denaturation, followed by: centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques.", "As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.", "There is no general requirement that the protein or peptide always be provided in their most purified state.", "Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments.", "Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme.", "For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system.", "Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.", "Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind to.", "This is a receptor-ligand type of interaction.", "The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix.", "The column material is then able to specifically adsorb the substance from the solution.", "Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.).", "The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.", "The ligand should be coupled in such a way as to not affect its binding properties.", "The ligand should also provide relatively tight binding.", "And it should be possible to elute the substance without destroying the sample or the ligand.", "In various embodiments, affinity chromatography may be performed to purify a targeting peptide, an antibody against a targeting peptide, an antigen that binds to an antibody, an endogenous receptor for a targeting peptide, or a ligand for a targeting peptide.", "Synthetic Peptides Because of their relatively small size, the targeting peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques.", "Various automatic synthesizers are commercially available and can be used in accordance with known protocols.", "See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.", "Short peptide sequences, usually from about 6 up to about 35 to 50 amino acids, can be readily synthesized by such methods.", "Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.", "Antibodies In certain embodiments, it may be desirable to make antibodies against the identified targeting peptides or their receptors.", "The appropriate targeting peptide or receptor, or portions thereof, may be coupled, bonded, bound, conjugated, or chemically-linked to one or more agents via linkers, polylinkers, or derivatized amino acids.", "This may be performed such that a bispecific or multivalent composition or vaccine is produced.", "It is further envisioned that the methods used in the preparation of these compositions are familiar to those of skill in the art and should be suitable for administration to human subjects, i.e., pharmaceutically acceptable.", "Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).", "The term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.", "Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.", "Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).", "Cytokines and Chemokines In certain embodiments, it may be desirable to couple specific bioactive agents to one or more targeting peptides for targeted delivery to an organ or tissue.", "Such agents include, but are not limited to, cytokines, chemokines, pro-apoptosis factors and anti-angiogenic factors.", "The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.", "Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones.", "Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-.alpha.", "and -.beta.", "; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-.beta.", "; platelet-growth factor; transforming growth factors (TGFs) such as TGF-.alpha.", "and TGF-.beta.", "; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -.β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.", "Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression.", "It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment.", "Chemokines include, but are not limited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10.The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines.", "Imaging Agents and Radioisotopes In certain embodiments, the claimed peptides or proteins of the present invention may be attached to imaging agents of use for imaging and diagnosis of various diseased organs or tissues.", "Many appropriate imaging agents are known in the art, as are methods for their attachment to proteins or peptides (see, e.g., U.S. Pat.", "Nos.", "5,021,236 and 4,472,509, both incorporated herein by reference).", "Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the protein or peptide (U.S. Pat.", "No.", "4,472,509).", "Proteins or peptides also may be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.", "Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.", "Non-limiting examples of paramagnetic ions of potential use as imaging agents include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III) samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred.", "Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).", "Radioisotopes of potential use as imaging or therapeutic agents include astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111, 59iron, 32phosphorus, rhenium186, rhenium188, 75selenium, 35sulphur, technicium99m and yttrium90.125I is often being preferred for use in certain embodiments, and technicium99m and indium111 are also often preferred due to their low energy and suitability for long range detection.", "Radioactively labeled proteins or peptides of the present invention may be produced according to well-known methods in the art.", "For instance, they can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.", "Proteins or peptides according to the invention may be labeled with technetium-99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the peptide.", "Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to peptides are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA).", "Also contemplated for use are fluorescent labels, including rhodamine, fluorescein isothiocyanate and renographin.", "In certain embodiments, the claimed proteins or peptides may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.", "Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.", "Preferred secondary binding ligands are biotin and avidin or streptavidin compounds.", "The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Pat.", "Nos.", "3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.", "Cross-Linkers Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.", "Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.", "Heterobifunctional reagents contain two different functional groups.", "By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.", "The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups.", "Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.", "A majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.", "Exemplary methods for cross-linking ligands to liposomes are described in U.S. Pat.", "No.", "5,603,872 and U.S. Pat.", "No.", "5,401,511, each specifically incorporated herein by reference in its entirety).", "Various ligands can be covalently bound to liposomal surfaces through the cross-linking of amine residues.", "Liposomes, in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures.", "The inclusion of PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for cross-linking purposes.", "Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes.", "Ligands are bound covalently to discrete sites on the liposome surfaces.", "The number and surface density of these sites are dictated by the liposome formulation and the liposome type.", "The liposomal surfaces may also have sites for non-covalent association.", "To form covalent conjugates of ligands and liposomes, cross-linking reagents have been studied for effectiveness and biocompatibility.", "Cross-linking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).", "Through the complex chemistry of cross-linking, linkage of the amine residues of the recognizing substance and liposomes is established.", "In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described (U.S. Pat.", "No.", "5,889,155, specifically incorporated herein by reference in its entirety).", "The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimnide residue, allowing coupling in one example, of aldehydes to free thiols.", "The cross-linking reagent can be modified to cross-link various functional groups.", "Cross-linking agents may also be of use to attach chemicals, compounds, molecules or aggregates of molecules to larger particles for use in BRASIL screening.", "Magnetic Beads It is envisioned that particles employed in the instant invention may come in a variety of sizes.", "While large magnetic particles (mean diameter in solution greater than 10 μm) can respond to weak magnetic fields and magnetic field gradients, they tend to settle rapidly, limiting their usefulness for reactions requiring homogeneous conditions.", "Large particles also have a more limited surface area per weight than smaller particles, so that less material can be coupled to them.", "In preferred embodiments, the magnetic beads are less than 10 μm in diameter.", "Various silane couplings applicable to magnetic beads are discussed in U.S. Pat.", "No.", "3,652,761, incorporated herein by reference.", "Procedures for silanization known in the art generally differ from each other in the media chosen for the polymerization of silane and its deposition on reactive surfaces.", "Organic solvents such as toluene (Weetall, 1976), methanol, (U.S. Pat.", "No.", "3,933,997) and chloroform (U.S. Pat.", "No.", "3,652,761) have been used.", "Silane deposition from aqueous alcohol and aqueous solutions with acid have also been used.", "Ferromagnetic materials in general become permanently magnetized in response to magnetic fields.", "Materials termed “superparamagnetic” experience a force in a magnetic field gradient, but do not become permanently magnetized.", "Crystals of magnetic iron oxides may be either ferromagnetic or superparamagnetic, depending on the size of the crystals.", "Superparamagnetic oxides of iron generally result when the crystal is less than about 300 angstroms (Å) in diameter; larger crystals generally have a ferromagnetic character.", "Dispersible magnetic iron oxide particles reportedly having 300 Å diameters and surface amine groups are prepared by base precipitation of ferrous chloride and ferric chloride (Fe2+/Fe3+=1) in the presence of polyethylene imine, according to U.S. Pat.", "No.", "4,267,234.These particles are exposed to a magnetic field three times during preparation and are described as redispersible.", "The magnetic particles are mixed with a glutaraldehyde suspension polymerization system to form magnetic polyglutaraldehyde microspheres with reported diameters of 0.1 μm.", "Polyglutaraldehyde microspheres have conjugated aldehyde groups on the surface which can form bonds to amino containing molecules such as proteins.", "While a variety of particle sizes are envisioned to be applicable in the disclosed method, in a preferred embodiment, particles are between about 0.1 and about 1.5 μm diameter.", "Particles with mean diameters in this range can be produced with a surface area as high as about 100 to 150 m2 /gm, which provides a high capacity for bioaffinity adsorbent coupling.", "Magnetic particles of this size range overcome the rapid settling problems of larger particles, but obviate the need for large magnets to generate the magnetic fields and magnetic field gradients required to separate smaller particles.", "Magnets used to effect separations of the magnetic particles of this invention need only generate magnetic fields between about 100 and about 1000 Oersteds.", "Such fields can be obtained with permanent magnets which are preferably smaller than the container which holds the dispersion of magnetic particles and thus, may be suitable for benchtop use.", "Although ferromagnetic particles may be useful in certain applications of the invention, particles with superparamagnetic behavior are usually preferred since superparamagnetic particles do not exhibit the magnetic aggregation associated with ferromagnetic particles and permit redispersion and reuse.", "The method for preparing the magnetic particles may comprise precipitating metal salts in base to form fine magnetic metal oxide crystals, redispersing and washing the crystals in water and in an electrolyte.", "Magnetic separations may be used to collect the crystals between washes if the crystals are superparamagnetic.", "The crystals may then be coated with a material capable of adsorptively or covalently bonding to the metal oxide and bearing functional groups for coupling with various target molecules.", "Non-Magnetic Beads, Flow Cytometry and FACS In another embodiment, the target of interest may be non-covalently or covalently attached to non-magnetic beads, such as glass, polyacrylamide, polystyrene or latex.", "Targets may be attached to such beads by the same techniques discussed above for magnetic beads.", "After exposure of bead to phage library, those phage bound to beads may be separated from unbound phage by, for example, centrifugation.", "In certain embodiments, cells to be screened by BRASIL may be presorted using some form of flow cytometry.", "Non-limiting examples of flow cytometry methods are disclosed in Betz et al.", "(1984), Wilson et al.", "(1988), Scillian et al.", "(1989), Frengen et al.", "(1994), Griffith et al.", "(1996), Stuart et al.", "(1998) and U.S. Pat.", "Nos.", "5,853,984 and 5,948,627, each incorporated herein by reference in its entirety.", "U.S. Pat.", "Nos.", "4,727,020, 4,704,891 and 4,599,307, incorporated herein by reference, describe the arrangement of the components comprising a flow cytometer and the general principles of its use.", "In the flow cytometer, beads, cells or other particles are passed substantially one at a time through a detector, where each particle is exposed to an energy source.", "The energy source generally provides excitatory light of a single wavelength.", "The detector comprises a light collection unit, such as photomultiplier tubes or a charge coupled device, which may be attached to a data analyzer such as a computer.", "The beads, cells or particles can be characterized by their response to excitatory light, for example by detecting and/or quantifying the amount of fluorescent light emitted in response to the excitatory light.", "Beads or cells exhibiting a particular characteristic can be sorted using an attached cell sorter, such as the FACS Vantage™ cell sorter sold by Becton Dickinson Immunocytometry Systems (San Jose, Calif.).", "Nucleic Acids Nucleic acids according to the present invention may encode a targeting peptide, a receptor protein or a fusion protein.", "The nucleic acid may be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA.", "Where incorporation into an expression vector is desired, the nucleic acid may also comprise a natural intron or an intron derived from another gene.", "Such engineered molecules are sometime referred to as “mini-genes.” A “nucleic acid” as used herein includes single-stranded and double-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogs.", "It is contemplated that a nucleic acid within the scope of the present invention maybe of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater nucleotide residues in length.", "It is contemplated that targeting peptides, fusion proteins and receptors may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence.", "The design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables (see Table 2 below).", "In preferred embodiments, the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest.", "Codon preferences for various species of host cell are well known in the art.", "TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU In addition to nucleic acids encoding the desired targeting peptide, fusion protein or receptor amino acid sequence, the present invention encompasses complementary nucleic acids that hybridize under high stringency conditions with such coding nucleic acid sequences.", "High stringency conditions for nucleic acid hybridization are well known in the art.", "For example, conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleotide content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.", "Vectors for Cloning, Gene Transfer and Expression In certain embodiments expression vectors are employed to express the targeting peptide or fusion protein, which can then be purified and used.", "In other embodiments, the expression vectors are used in gene therapy.", "Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.", "Elements designed to optimize messenger RNA stability and translatability in host cells also are known.", "Regulatory Elements The terms “expression construct” or “expression vector” are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed.", "In preferred embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter.", "A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.", "The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.", "The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.", "Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent and under the control of a promoter that is capable of being expressed in a human cell.", "Generally speaking, such a promoter might include either a human or viral promoter.", "In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase promoter can be used to obtain high-level expression of the coding sequence of interest.", "The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.", "Where a cDNA insert is employed, typically one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript.", "The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals.", "Also contemplated as an element of the expression construct is a terminator.", "These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.", "Selectable Markers In certain embodiments of the invention, the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct.", "Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.", "Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants.", "For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.", "Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.", "Immunologic markers also can be employed.", "The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product.", "Further examples of selectable markers are well known to one of skill in the art.", "Delivery of Expression Vectors There are a number of ways in which expression vectors may introduced into cells.", "In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome.", "The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome, and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).", "Preferred gene therapy vectors are generally viral vectors.", "Although some viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression.", "However, adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression making them ideally suited for rapid, efficient, heterologous gene expression techniques for preparing replication infective viruses are well known in the art.", "Of course in using viral delivery systems, one will desire to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the cell, animal or individual receiving the vector construct.", "A non-limiting method of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.", "DNA viruses used as gene vectors include the papovaviruses (e.g., simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).", "One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector.", "Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.", "“Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein.", "The expression vector comprises a genetically engineered form of adenovirus.", "Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).", "In contrast to retroviral infection, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.", "Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.", "Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.", "So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.", "Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity.", "Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.", "The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.", "The E1 region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.", "The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication.", "These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990).", "The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP).", "The MTP, (located at 16.8 m.u.)", "is particularly efficient during the late phase of infection, and all the mRNAs issued from this promoter possess a 5□-tripartite leader (TPL) sequence which makes them preferred mRNAs for translation.", "In currently used systems, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector.", "Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process.", "Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.", "Generation and propagation of adenovirus vectors which are replication deficient depend on a unique helper cell line, designated 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977).", "Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E1, the D3, or both regions (Graham and Prevec, 1991).", "In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA.", "Combined with the approximately 5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector.", "More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-bome cytotoxicity.", "Also, the replication deficiency of the E1-deleted virus is incomplete.", "For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).", "Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.", "Alternatively, the helper cells, may be derived from the cells of other mammalian species that are permissive for human adenovirus.", "Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.", "As discussed, the preferred helper cell line is 293.Racher et al., (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.", "In one format, natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium.", "Following stirring at 40 rpm, the cell viability is estimated with trypan blue.", "In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) are employed as follows.", "A cell innoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking is initiated.", "For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05.Cultures are left stationary overnight, following which the volume is increased to 100% and shaking is commenced for another 72 hr.", "Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention.", "The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention.", "This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.", "A typical vector applicable to practicing the present invention is replication defective and will not have an adenovirus E1 region.", "Thus, it are most convenient to introduce the polynucleotide encoding the gene at the position from which the E1-coding sequences have been removed.", "However, the position of insertion of the construct within the adenovirus sequences is not critical.", "The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al., (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.", "Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo.", "This group of viruses can be obtained in high titers, e.g., 109-1011 plaque-forming units per ml, and they are highly infective.", "The life cycle of adenovirus does not require integration into the host cell genome.", "The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells.", "No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.", "Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991).", "Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993).", "Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).", "Other gene transfer vectors may be constructed from retroviruses.", "The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).", "The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.", "The integration results in the retention of the viral gene sequences in the recipient cell and its descendants.", "The retroviral genome contains three genes, gag, pot, and env.", "that code for capsid proteins, polymerase enzyme, and envelope components, respectively.", "A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.", "Two long terminal repeat (LTR) sequences are present at the 5□ and 3□ ends of the viral genome.", "These contain strong promoter and enhancer sequences, and also are required for integration in the host cell genome (Coffin, 1990).", "In order to construct a retroviral vector, a nucleic acid encoding protein of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.", "In order to produce virions, a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components, is constructed (Mann et al., 1983).", "When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).", "The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer.", "Retroviral vectors are capable of infecting a broad variety of cell types.", "However, integration and stable expression require the division of host cells (Paskind et al., 1975).", "There are certain limitations to the use of retrovirus vectors.", "For example, retrovirus vectors usually integrate into random sites in the cell genome.", "This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al., 1981).", "Another concern with the use of defective retrovirus vectors, is the potential appearance of wild-type replication-competent virus in the packaging cells.", "This may result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.", "However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).", "Other viral vectors may be employed as expression constructs.", "Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed.", "They offer several attractive features for various mammalian cells (Friedmann, 19.89; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).", "Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention.", "These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).", "Some of these techniques may be successfully adapted for in vivo or ex vivo use.", "In a further embodiment of the invention, the expression construct may be entrapped in a liposome.", "Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium.", "Multilamellar liposomes have multiple lipid layers separated by aqueous medium.", "They form spontaneously when phospholipids are suspended in an excess of aqueous solution.", "The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers.", "Also contemplated are lipofectamine-DNA complexes.", "Liposome-mediated nucleic acid delivery and expression of foreign DNA ill vitro has been very successful.", "Wong et al., (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.", "Nicolau et al., (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.", "A number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells, respectively.", "Also, anti-metabolite resistance can be used as the basis of selection for dhfr: that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hlygro, that confers resistance to hygromycin.", "Pharmaceutical Compositions Where clinical applications are contemplated, it is necessary to prepare pharmaceutical compositions—expression vectors, virus stocks, proteins, antibodies and drugs—in a form appropriate for the intended application.", "Generally, this will entail preparing compositions that are essentially free of impurities that could be harmful to humans or animals.", "One generally will desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells.", "Buffers also are employed when recombinant cells are introduced into a patient.", "Aqueous compositions of the present invention comprise an effective amount of the protein or peptide, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.", "Such compositions also are referred to as inocula.", "The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.", "As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.", "The use of such media and agents for pharmaceutically active substances is well known in the art.", "Except insofar as any conventional media or agent is incompatible with the proteins or peptides of the present invention, its use in therapeutic compositions is contemplated.", "Supplementary active ingredients also can be incorporated into the compositions.", "The active compositions of the present invention may include classic pharmaceutical preparations.", "Administration of these compositions according to the present invention are via any common route so long as the target tissue is available via that route.", "This includes oral, nasal, buccal, rectal, vaginal or topical.", "Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial or intravenous injection.", "Such compositions normally would be administered as pharmaceutically acceptable compositions, described sipra.", "The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.", "In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.", "It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.", "The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.", "The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.", "The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.", "In many cases, it are preferable to include isotonic agents, for example, sugars or sodium chloride.", "Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.", "Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.", "Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.", "In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.", "Therapeutic Agents In certain embodiments, chemotherapeutic agents may be attached to a targeting peptide or fusion protein for selective delivery to a tumor.", "Agents or factors suitable for use include any chemical compound that induces DNA damage when applied to a cell.", "Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing.", "Most chemotherapeutic agents fall into the following categories: alkylating, agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.", "Chemotherapeutic agents and methods of administration, dosages, etc.", "are well known to those of skill in the art (see for example, the “Physicians Desk Reference”, Goodman & Gilman's “The Pharmacological Basis of Therapeutics” and in “Remington's Pharmaceutical Sciences”, incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein.", "Some variation in dosage will necessarily occur depending on the condition of the subject being treated.", "The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.", "Examples of specific chemotherapeutic agents and dose regimes are also described herein.", "Of course, all of these dosages and agents described herein are exemplary rather than limiting, and other doses or agents may be used by a skilled artisan for a specific patient or application.", "Any dosage in-between these points, or range derivable therein is also expected to be of use in the invention.", "Alkylating Agents Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating.", "This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.", "An alkylating agent, may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines.", "They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.", "Antimetabolites Antimetabolites disrupt DNA and RNA synthesis.", "Unlike alkylating agents, they specifically influence the cell cycle during S phase.", "Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds.", "Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.", "Natural Products Natural products generally refer to compounds originally isolated from a natural source, and identified has having a pharmacological activity.", "Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art.", "Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.", "Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis.", "They operate during a specific phase during the cell cycle.", "Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.", "Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia.", "Taxoids include but are not limited to compounds such as docetaxel and paclitaxel.", "Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules.", "Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity.", "They include such compounds as vinblastine (VLB) and vincristine.", "Antitumor Antibiotics Antitumor antibiotics have both antimicrobial and cytotoxic activity.", "These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes.", "These agents are not phase specific so they work in all phases of the cell cycle.", "Examples of antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithrarnycin) and idarubicin.", "Hormones Corticosteroid hormones are considered chemotherapy drugs when they are implemented to kill or slow the growth of cancer cells.", "Corticosteroid hormones can increase the effectiveness of other chemotherapy agents, and consequently, they are frequently used in combination treatments.", "Prednisone and dexamethasone are examples of corticosteroid hormones.", "Progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate have been used in cancers of the endometrium and breast.", "Estrogens such as diethylstilbestrol and ethinyl estradiol have been used in cancers such as breast and prostate.", "Antiestrogens such as tamoxifen have been used in cancers such as breast.", "Androgens such as testosterone propionate and fluoxymesterone have also been used in treating breast cancer.", "Antiandrogens such as flutamide have been used in the treatment of prostate cancer.", "Gonadotropin-releasing hormone analogs such as leuprolide have been used in treating prostate cancer.", "Miscellaneous Agents Some chemotherapy agents do not qualify into the previous categories based on their activities.", "They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin.", "It is contemplated that they are included within the compositions and methods of the present invention.", "Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis-DDP).", "An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer.", "A substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis and malignant melanoma.", "A methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease.", "An adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.", "Regulators of Programmed Cell Death Apoptosis, or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al., 1972).", "The Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems.", "The Bcl-2 protein, discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986).", "The evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.", "Subsequent to its discovery, it was shown that Bcl-2 acts to suppress cell death triggered by a variety of stimuli.", "Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies.", "These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, A1, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).", "Non-limiting examples of pro-apoptosis agents contemplated within the scope of the present invention include gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK)2 (SEQ ID NO:1), (KLAKKLA)2 (SEQ ID NO:2), (KAAKKAA)2 (SEQ ID NO:3) or (KLGKKLG)3 (SEQ ID NO:4).", "Angiogenic Inhibitors In certain embodiments the present invention may concern administration of targeting peptides attached to anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline.", "Dosages The skilled artisan is directed to “Remington's Pharmaceutical Sciences” 15th Edition, chapter 33, and in particular to pages 624-652.Some variation in dosage will necessarily occur depending on the condition of the subject being treated.", "The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.", "Moreover, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA Office of Biologics standards.", "EXAMPLES The following examples are included to demonstrate preferred embodiments of the invention.", "It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice.", "However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.", "Example 1 BRASIL Probing molecular diversity at the cell surface level is important for the identification of targeting peptides and the development of targeted therapies.", "As opposed to purified receptors, membrane-bound proteins are more likely to preserve their functional conformation.", "Many cell surface receptors require homo- or hetero-dimeric interactions that occur only within the cell membrane environment.", "Combinatorial approaches allow the selection of cell membrane ligands in an unbiased functional assay, without any preconceived notions about the nature of the cellular receptors.", "Thus, unknown receptors can be targeted.", "Despite these advantages, it is often difficult to isolate specific ligands due to the high complexity of targets expressed simultaneously on a given cell population.", "To address these problems, a new in vitro approach has been developed to improve the selection of phage at the level of single cells or small clumps of cells.", "This method, Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL), is based on a procedure that allows cell-phage complexes to be separated from the remaining unbound phage in a single differential centrifugation step (FIG.", "1).", "As described herein, BRASIL has been successfully used to isolate phage in various cell systems.", "BRASIL involves the addition of cells to centrifuge tubes containing an first (preferably aqueous) phase layered over a second (preferably organic) phase, as described below.", "Upon centrifugation, the cells and any bound phage end up in a pellet at the bottom of the second phase, while non-bound phage remain in the upper first phase.", "This gentle separation technique helps to preserve the phage-receptor interaction for targeting peptides that are not tightly bound to receptor.", "In preferred embodiments, the first phase is aqueous and the second phase is organic.", "As organic phases are generally immiscible with aqueous phases, this prevents mixing and dilution of the phase components and consequent changes in density.", "Use of an aqueous phase for binding of phage to cells is preferred, as it mimicks the in vivo environment in which protein interactions normally occur.", "An organic second phase is also preferred since it is likely to reduce background by interfering with non-specific hydrophobic interactions, while retaining specifically bound phage by increasing the strength of ionic interactions or hydrogen bonding.", "BRASIL may be used to isolate phage displaying peptide sequences that bind to specific markers of different cell subpopulations from any selected organ, tissue or cell type.", "Cell subpopulations may be purified ex-vivo by Ficoll gradient and/or identified by Fluorescent Activated Cell Analysis (FACS) before the BRASIL method is implemented.", "This improved in vitro panning method may be used to retrieve phage that bind to markers found only in certain cell subpopulations.", "Fine Needle Aspirations (FNAs) of organs are excellent sources of cells to perform biopanning with BRASIL.", "The skilled artisan will realize that the BRASIL technique is applicable for identifying targeting peptides directed against a wide range of organs, tissues and cell types.", "Materials and Methods Reagents and Cells A phage library displaying random cyclic peptides with the structure CX6C (C, cysteine; X, any amino acid residue) was used for the screenings.", "Phage libraries and clones were produced according to Koivunen et al.", "(1999), using known methods (Smith, 1985; Smith and Scott 1993).", "Kaposi's sarcoma cells (KS1767 cell line) were maintained in minimal essential medium (MEM) supplemented with 10% fetal calf serum (Gibco-BRL, Rockville, Md.).", "Dibutyl phthalate, and cyclohexane (Sigma-Aldrich, St. Louis, Mo.)", "were obtained commercially.", "Peptides used were synthesized to greater than 95% purity, cyclized, and analyzed by HPLC and mass spectrometry (AnaSpec, San Jose, Calif.).", "Ex-vivo Procedure In an exemplary in vitro procedure, prostate cells were harvested, washed and re-suspended in medium containing 1% BSA (100-300 μl).", "A phage library (109 phage) was added and left on ice for 4 h. After transfer to a 400 μl eppendorf tube containing 200 μl of dibutylphtalate (Sigma), the mixture was centrifuged for 10 min at 10,000 g. The tube bottom (containing the cell-phage complexes) was snap-frozen in liquid nitrogen to prevent cross-contamination with unbound phage in the upper aqueous phase.", "The frozen tube was carefully cut with a sharp razor blade and the pellet was transferred to a fresh Falcon tube.", "PBS (100-200 μl) was added and the pellet homogenized by repeated pipeting.", "After adding 1 ml of E. coli K91kan bacteria and incubation for 1 hr, LB containing tetracycline was added and the admixture -was grown overnight in a 37° C. shaker.", "Phage bound to pelleted cells were recovered as bacterial plaques.", "Careful steps were taken to prevent any aggregates that could bias the biopanning results.", "The BSA-containing media was filtered through a 0.22 micron sterile mesh and the phage centrifuged in solution for 5 min at 16,000 g immediately prior to addition to the cells.", "This study showed that bound phage could be harvested from prostate cells using the BRASIL method (not shown).", "BRASIL Method Optimization Cells were harvested with phosphate-buffered saline (PBS) and 5 mM EDTA, washed with MEM, and re-suspended in MEM containing 1% BSA at 106 cells/ml and incubated with phage in 1.5 ml Eppendorf tubes.", "After 4 h, 100 μl of the cell-phage suspension was gently transferred to the top of a non-miscible organic lower phase (200 μl in a 400 μl-Eppendorf tube) and centrifuged at 10,000 g for 10 minutes.", "The preferred organic phase combination consisted of a mixture of dibutyl phthalate:cyclohexane (9:1 [v/v]; ρ=1.03 g/ml).", "BRASIL has been attempted with other phthalate admixtures with the appropriate density (for example, dibutyl phhalate:diisooctyl phthalate; 4:6 [v/v]) with similar results.", "The tube was snap frozen in liquid nitrogen, the bottom of the tube sliced off, and the cell-phage pellet transferred to a new tube.", "Bound phage were rescued by infection with 200 μl of E. coli K91kan host bacteria in log phase.", "To evaluate binding specificity, phage and cells were incubated with the cognate or control synthetic peptides for competition assays.", "Binding Assays with Phage Clones KS1767 cells were detached with cold EDTA and re-suspended in MEM containing 1% BSA.", "RGD-4C phage (Pasqualini et al., 1997) were used as a defined ligand that displays a specific oxv integrin-binding motif.", "The cell suspension was incubated with RGD-4C phage or a control phage with no peptide insert (fd-tet phage).", "Increasing amounts of either phage were added to the cells in suspension and the cell-phage admixture was incubated for 4 hr on ice.", "BRASIL was performed on ice to minimize post-binding events such as ligand-receptor internalization by the target cells.", "The cells were separated by centrifugation through the organic phase as described above.", "Bound phage were recovered and phage TU were counted.", "To compare BRASIL to conventional cell panning methods that require an additional washing step, 200 μl of the cell suspension were incubated with phage for 4 hr on ice.", "Unbound phage from 100 μl aliquots were removed either by centrifuging over the organic phase or by washing the cells three times with 1 ml of PBS containing 0.3% BSA.", "Each condition was repeated at least three times.", "Competitive inhibition was tested with the synthetic RGD-4C peptide, containing the targeting sequence CDCRGDCFC (SEQ ID NO:9) were compared to control peptides CARAC (SEQ ID NO:5) or GRGESP (SEQ ID NO:10) used at the same molar ratios.", "Results The BRASIL technique was tested using RGD4C phage that bind to alpha-v integrins (Pasqualini et al., 1997) and the KS1767 cell line, which expresses high levels of alpha-v integrins.", "It was first determined if the oil mixture would interfere with the infection rate.", "Increasing amounts of oil were added to a bacterial culture and phage added to them.", "After 1 hr infection, the cells were plated and the number of tetracycline resistant colonies (infected by phage) counted.", "No significant difference between the control (no oil added) and the oil mixtures could be detected (data not shown) suggesting that the oil mixture does not interfere with the infection rate and recovery of phage.", "It was determined whether phage would pellet at the bottom of the tube if no cells were present.", "109 TU of Fd (insertless) phage were added to the medium and centrifuged.", "No Fd phage could be detected at the bottom of the tube or in the oil phase.", "Next, it was tested if phage could be carried specifically by the cells to the oil phase and then recovered by infection with bacteria.", "For this, increasing amounts of RGD4C phage or Fd phage were added to KS1767 cells in suspension, incubated for 4 hr on ice and then centrifuged over the oil.", "As shown in FIG.", "2A, the number of phage recovered from the cells increased with the number of phage added.", "The ratio between the number of RGD4C to Fd phage recovered (enrichment) varied consistently from 100 to 500-fold.", "The binding of the RGD4C phage to the KS1767 cells was specific and was mediated by the peptide expressed in the pIII protein, since a competition experiment with the corresponding soluble peptide completely inhibited the binding of the RGD4C phage binding to KS1767 cells, bringing the number of phage bound close to the number of Fd phage (background) (FIG.", "2B).", "Negative control peptides (CARAC, SEQ ID NO:5 or GRGESP, SEQ ID NO:10) had no effect on RGD4C phage binding to KS1767 cells (not shown).", "The recovery of phage with or without the snap-freeze step was compared.", "No substantial decrease was noted in the amounts of test phage recovered (data not shown).", "The recovery of phage with BRASIL was compared to standard biopanning methods requiring a washing step.", "The number of RGD4C phage recovered by BRASIL was significantly higher (t test, P<0.01) than the number of the same phage recovered when a conventional phage-cell binding strategy involving washing was used (not shown).", "Conversely, significantly lower background (t test, P<0.01) with the negative control phage was observed (not shown).", "Given the significant increase in recovery of specific phage and the substantial decrease in background, the overall accuracy improved consistently by more than one order of magnitude when BRASIL was used relative to conventional cell panning methods.", "Example 2 VEGF Targeting Peptide Identified by BRASIL Diabetic retinopathy is the formation of new blood vessels (angiogenesis) in the retina and cornea, induced by hyperglycemia.", "Although the neovascularization process of the retina is not fully understood, growth factors, especially vascular endothelial growth factors (VEGFs) play an important role in this process.", "Intraocular neovascularization is a pathological complication of many eye diseases and is the leading cause of blindness in the world.", "Although hyperglycemia per se seems to be the main cause of diabetic retinopathy (Engerman and Kern, 1986), it is the induction of growth factors that start the angiogenic process.", "Among several possible candidates, vascular endothelial growth factor (VEGF) seems to be most important mediator of the ischemia-induce neovascularization since several anti-VEGF therapies prevent ocular angiogenesis in animal models.", "VEGF is produced by several retinal cell types (ganglion cells, RPE, pericytes, endothelial cells, astrocytes and Müller cells).", "Its expression is upregulated by hypoxia and it diffuses freely through the eye.", "Angiogenesis is a complex process, which seems to be balanced by the presence of activators and inhibitors (Hanahan and Folkman, 1996).", "VEGF is a major activator and regulator of both physiological and pathological neovascularization (reviewed by Ferrara and Davis-Smyth, 1997).", "It is a relative specific mitogen for vascular endothelial cells and elicits a pronounced angiogenic response in a variety of in vivo models.", "VEGF belongs to a multigene family with 5 members described so far: VEGF, VEGF-B, VEGF-C, VEGF-D, P1GF and the orf virus VEGF (also called VEGF-E).", "Alternative exon splicing of the genes produces multiple species of mRNA with distinct biological effects (Tischer et al., 1991; Veikkola and Alitalo, 1999).", "VEGFs are also produced as homo- and heterodimers, although very little is known about the function of the VEGF heterodimers.", "Materials and Methods Cells and Reagents Recombinant human VEGF165 (Pharmingen, San Diego, Calif.), recombinant human VEGFR-1 (Oncogene Research Products, Boston, Mass.", "), recombinant rat NRP-1/Fc, rat NRP-2/Fc, human VEGFR-2/Fc (all three receptor/chimeras with the Fc region of human IgG1), PDGF-BB, anti-VEGFR-1 (polyclonal anti-Flt1), and anti-human VEGF polyclonal antibody (R&D Systems Minneapolis, Minn.) were all obtained commercially.", "HUVEC (human umbilical vein endothelial cells) were purchased from Clonetics and cultured according to the manufacturer's instructions.", "Anti-mouse CD13 antibodies R3-63 and 2M-7 were produced and characterized as described (Hansen et al., 1993).", "The anti-M13 polyclonal antibody (Amersham-Pharmacia) was obtained commercially.", "Anti-CD31 antibody was purchased from Pharmingen (CA), anti-smooth muscle actin conjugated to Cy3 or FITC was purchased from Sigma.", "Anti-desmin polyclonal serum was purchased from Daiko.", "Aminopeptidase-N (leucine aminopeptidase) was purchased from Sigma.", "HUVEC were cultured and used between passages 2 and 8, according to the manufacturer's protocol (Clonetics, San Diego, Calif.).", "In order to minimize receptor-mediated internalization, cells and media were kept on ice unless otherwise stated.", "BRASIL Cells were harvested with PBS, 5 mM EDTA (5 minutes), washed with PBS and ressuspended in MEM containing 1% BSA (MEM 1% BSA) at 106 cells/ml.", "Phage was added to the cell suspension and incubated on ice.", "After 4 hr, 100 μl of the cell suspension was transferred to 400 μl eppendorf tubes containing 200 μl of dibutyl phtalate:cyclohexane mixture (9:1) and centrifuged at 10,000 g for 10 minutes.", "Cells with bound phage migrated to the bottom of the tube within the oil phase and the unbound phage remained at the top of the oil in the soluble phase.", "The tubes were snap-frozen in liquid N2, the pellet cut off, transferred to a new eppendorf and phage rescued by infection with 200 μL of E. coli K91kan cells in log-phase, then diluted and plated onto LB plates supplemented with tetracycline.", "HUVEC Biopanning by BRASIL Phage Display A two-step biopanning strategy was designed to isolate phage that bind to angiogenic endothelial cells.", "To decrease non-specific binding, the phage library was pre-cleared on starved HUVEC cells before panning on the same cell line stimulated with VEGF165.After centrifugation through the organic phase, phage bound to the VEGF165-stimulated HUVEC pellet were recovered by bacterial infection, amplified, and subjected to two more rounds of selection.", "Phage peptide libraries were obtained, expanded and manipulated as described (Pasqualini et al., 1999).", "HUVEC at 80% confluence cultured in endothelial basal medium (EBM-2; Clonetics) without supplements for 24 hr were defined as “starved HUVEC.” The medium was then replaced by EBM-2 supplemented with 20 ng/ml VEGF165 and the cells cultured under these conditions for another 18 hr were defined as “VEGF165-stimulated HUVEC.” Both, starved and VEGF165-stimulated HUVEC were harvested with ice-cold PBS and 5 mM EDTA, washed once with EBM-2 plus 1% BSA, and re-suspended in the same medium at 107 cells/ml.", "In the pre-clearing step, starved HUVEC (106 cells) were incubated with 109 TU of unselected CX6C phage library for 2 hr on ice; the mixture was then centrifuged through the organic phase.", "In a screening step, the unbound phage left over in the aqueous upper phase (supernatant) was transferred to a fresh tube and incubated with VEGF165-stimulated HUVEC (106 cells).", "After 4 hr on ice, the cell-phage complexes were separated by centrifugation through the organic lower phase.", "The phage population in the cell pellet was recovered by infection of 200 μl of E. coli K91kan host bacteria growing in log phase.", "This procedure was repeated 3 times using the phage obtained from the previous round.", "After the third round of biopanning, 32 phage were randomly selected and sequenced for analysis.", "Binding Assays on Purified Receptors.", "Human VEGFR-1, human VEGFR-2, rat NRP-1, and rat NRP-2 (1 μg in 50 μl of PBS) were immobilized on microtiter well plates overnight at 4° C. The wells were washed twice with PBS, blocked with PBS containing 3% BSA for 2 h at room temperature, and then incubated with 109 TU of either CPQPRPLC (SEQ ID NO:6) phage, CNIRRQGC (SEQ ID NO:11) phage, or fd-tet phage in 50 μl of PBS/1.5% BSA.", "After 1 hr at room temperature, wells were washed nine times with PBS and phage were recovered by bacterial infection.", "Serial dilutions were plated onto Luria-Bertani (LB) medium supplemented with tetracycline.", "VEGF165, PDGF-BB, or synthetic peptides were used at the indicated concentrations and pre-incubated with the immobilized proteins to evaluate competitive inhibition of phage binding.", "ELISA with either polyclonal anti-VEGFR-1 serum or anti-human IgG (VEGFR-2, NRP-1, and NRP-2) confirmed the presence and concentration of the immobilized receptors on the microtiter plates.", "To show that the VEGF receptors were functionally active, VEGF165 (50 ng/ml) was incubated with the immobilized receptors for 2 hr at room temperature.", "Following three washes, VEGF165 binding was evaluated by ELISA by using anti-VEGF specific antibodies (data not shown).", "Results Biopanning on VEGF Stimulated HUVEC An advantage of BRASIL is that the unbound phage left in the upper aqueous phase can be used for a new round of panning with minimal loss.", "This approach was used to first pre-clear the phage display library with starved HUVEC before biopanning with VEGF165-activated HUVEC (FIG.", "3).", "The VEGF165-activated cells were then collected by BRASIL and the phage bound to them amplified and submitted to another round of selection.", "To test the selection method, 21 phage randomly chosen clones were examined for binding to starved HUVEC and to VEGF-stimulated HUVEC.", "Fourteen out of 21 clones (67%) had a greater than 150% enhancement (range, 1.5 to 8.7-fold; median, 2.2-fold) in the ratio of cell binding upon VEGF stimulation normalized to control insertless phage (data not shown).", "After three rounds of BRASIL selection on VEGF165-activated cells, 34 phage were randomly selected for sequencing.", "Alignment analysis of the 34 insert sequences revealed that 24 clones (70%) of the phage recovered by BRASIL displayed peptide motifs that could be mapped to sequences present in VEGF family members (not shown).", "Peptides with homology to the VEGF family are shown in Table 3 below.", "A phage clone displaying a peptide sequence CPQPRPLC (SEQ ID NO:6, referred to hereafter as “clone 19”) was very similar in sequence to a portion of the VEGF-B isoform 167 protein.", "Three different peptides contained the motif IRRE/Q.", "The motif IRRE/Q did not show substantial homology with known protein sequences and further experiments focussed on CPQPRPLC (SEQ ID NO:6).", "TABLE 3 Targeting peptides with homology to VEGF family members Clone # Sequence Homologies Peptide #1 CEGESASC SEQ ID NO:40 VEGF-D Peptide #3 CVPMRLQC SEQ ID NO:41 VEGF-A, VEGF-B, VEGF-C, PlGF-1, PlGF-2 Peptide #4 CLGKGSVC SEQ ID NO:42 VEGF-A, VEGF-D Peptide #6 CLSPIGEC SEQ ID NO:43 VEGF-A Peptide #7 CNLSVPAC SEQ ID NO:44 VEGF-A, VEGF-D Peptide #9 CIIGSYVC SEQ ID NO:45 PlGF-1, PlGF-2 Peptide #11 CADVLRPC SEQ ID NO:46 VEGF-D Peptide #12 CWRSVEVC SEQ ID NO:47 VEGF-B, VEGF-C Peptide #13 CSIRRESC SEQ ID NO:48 VEGF-C, VEGF-D Peptide #17 CAVVFSQC SEQ ID NO:49 VEGF-B Peptide #18 CLANLQTC SEQ ID NO:50 VEGF-A, VEGF-C Peptide #19 CPQPRPLC SEQ ID NO:6 VEGF-B, PlGF-1, PlGF-2 Peptide #21 CNIRRQGC SEQ ID NO:51 VEGF-C, VEGF-D Peptide #23 CIRREKRC SEQ ID NO:52 VEGF-C, VEGF-D, PlGF-1, PlGF-2 Peptide #24 CAGKSSNC SEQ ID NO:53 VEGF-D Peptide #25 CRECGERC SEQ ID NO:54 PlGF-1 Peptide #26 CMARQARC SEQ ID NO:55 VEGF-A Peptide #28 CLPISSSC SEQ ID NO:56 VEGF-D Peptide #29 CGRAKVRC SEQ ID NO:57 PlGF-1, PlGF-2 Peptide #30 CASGSENC SEQ ID NO:58 VEGF-D Peptide #31 CMRGKGLC SEQ ID NO:59 VEGF-A, PlGF-2 Peptide #32 CAGGGAYC SEQ ID NO:60 VEGF-A, VEGF-B Peptide #33 CAAAPIRC SEQ ID NO:61 VEGF-B, VEGF-C Peptide #36 CGRDSKQC SEQ ID NO:62 VEGF-D Other HUVEC binding peptides that were not homologous to VEGF included CVFAILAC (SEQ ID NO:128), CGVQYVNC (SEQ ID NO:129), CSYKANSC (SEQ ID NO:130), CYQSSSGC (SEQ ID NO:131), CRGGGRLC (SEQ ID NO:132), CGSDRWLC (SEQ ID NO:133), CLVYNPAC (SEQ ID NO:134), CIPGTSLC (SEQ ID NO:135), CATEAVGC (SEQ ID NO:136) and CWGGNQAC (SEQ ID NO:137).", "In vitro phage display was used with different recombinant VEGF receptors to determine if the clone 19 peptide bound to one or more of the VEGF receptors.", "As shown in FIG.", "4, clone-19 bound to human VEGF-R1 as well as to rat Neuropilin-l (NRP-1) but not to the human VEGF-R2.This result is consistent with the binding profile of VEGF-B (Olofsson et al., 1999).", "The lack of binding to VEGF-R2 was not due to absence of activity, since all three immobilized receptors showed similar VEGF165 binding activity (data not shown).", "The clone 19 phage exhibited over a 1,000-fold enrichment of binding to VEGF-R1 over fd-tet phage (not shown).", "Clone 19 phage did not bind to the neutropilin-2 (NRP-2) receptor (not shown).", "The VEGF165 and VEGF-B isoforms are known to compete for binding to VEGF-R1 (Olofsson et al., 1999).", "The interaction of clone-19 with VEGF-R1 and NRP-1 could be blocked by competition with VEGF165 (FIG.", "4A) but not by up to 200 ng/ml of PDGF-BB (data not shown).", "The competition with VEGF165 was concentration dependent and 100% inhibition was obtained with as low as 10 ng/ml of VEGF165 (FIG.", "4B).", "Binding of clone 19 phage could also be blocked by the cognate peptide CPQPRPLC (SEQ ID NO:6), but with differential effects (not shown).", "The CPQPRPLC (SEQ ID NO:6) peptide was approximately 100-fold more efficient in blocking phage binding to VEGF-R1 than to NRP-1 (not shown).", "These results show that CPQPRPLC (SEQ ID NO:6) is a chimeric VEGF-B-family mimeotope that interacts specifically with VEGFR-1 and NRP-1.VEGF-B167 is a possible mitogen for HUVEC cells (Olofsson et al, 1996).", "As shown in FIG.", "5, 1010 T.U.", "of phage clone-19 significantly induced proliferation of HUVEC compared to unstimulated cells or the RGD4C phage, which also binds to HUVEC.", "VEGF-B has two mRNA splice variants generated by the use of different, but overlapping, reading frames of exon 6 (isoforms 167 and 186), which diverge in sequence in their carboxy termini (Olofsson et al., 1999).", "The pentapeptide sequence PRPLC is found in the VEGF-B167 carboxy terminus region encoded by exon 6B, starting at the second residue after the boundary between exons 5 and 6B.", "PRPLC is a neuropilin-1 (NRP-1) binding domain (Makinen et al, 1999).", "On the other hand, the tetrapeptide sequence PQPR, which overlaps with PRPLC and also with the clone 19 peptide, is found in the carboxy terminal of VEGF-B186, and is encoded by exon 6A.", "PQPR is embedded within a 12-residue known NRP-1 binding site (Makinen et al, 1999).", "HUVEC cells were also panned against a CX7C phage library.", "The targeting phage peptide sequences identified are shown in Table 4 below.", "TABLE 4 CX7C Peptides binding to HUVEC cells.", "CTSWWFWSC SEQ ID NO:138 CEWSGIWAC SEQ ID NO:139 CNPLFWWWC SEQ ID NO:140 CGGWLEPPC SEQ ID NO:141 CEWWPEWLC SEQ ID NO:142 CARYLWSWC SEQ ID NO:143 CAWWRFGLC SEQ ID NO:144 CRGEWGMMC SEQ ID NO:145 CFWPFESWC SEQ ID NO:146 CSNAWVHAC SEQ ID NO:147 CSWYWWLGC SEQ ID NO:148 CGGWLFPPC SEQ ID NO:149 CIEWGSRDC SEQ ID NO:150 CVRSSVVAC SEQ ID NO:151 CEDSSRANC SEQ ID NO:152 CGGWLFPPC SEQ ID NO:153 CLLVGQVRC SEQ ID NO:154 CPRYLFWLC SEQ ID NO:155 CYRSAGAGC SEQ ID NO:156 CGGWLFPPC SEQ ID NO:157 CTRVGPKRC SEQ ID NO:158 CKSGQIAVC SEQ ID NO:159 CWWPWGGWC SEQ ID NO:160 CDWGLWWLC SEQ ID NO:161 CRGWADRKC SEQ ID NO:162 CGGWLFPPC SEQ ID NO:163 CTQVRFSGC SEQ ID NO:164 CPWWWFGEC SEQ ID NO:165 CGGWLFPPC SEQ ID NO:166 Discussion A VEGF receptor ligand was identified with the sequence CPQPRPLC (SEQ ID NO:6) that resembles the motif PRPLC (an NRP-1 binding site found in VEGF-B167) and the motif PQPR (embedded within a 12-residue NRP-1-binding epitope of VEGF-B186) (Makinen et al., 1999).", "Thus, the VEGF-B mimetope CPQPRPLC (SEQ ID NO:6) appears to be a chimera between binding sites in different VEGF-B isoforms.", "These results suggest that the carboxy terminal regions of both VEGF-B isoforms may bind to and activate VEGF-R1 and NRP-1.They also suggest that the peptide CPQPRPLC (SEQ ID NO:6) may mimic the effects of both VEGF-B isoforms in its interactions with the VEGF-R1 and NRP-1 receptors.", "The observed differential effects on VEGF-R1 and NRP-1 using the synthetic peptide CPQPRPLC (SEQ ID NO:6) to compete with phage binding suggests that the peptide chimeric motif interacts with VEGF receptors differentially.", "This may be due to the number of binding sites in each receptor or the affinity of the binding sites for the chimeric peptide.", "These results show that BRASIL will be of use to target cell populations derived from patient samples.", "The method can easily be used, for example, in tandem with fine needle aspirates of solid tumors or fluorescence activated cell sorting of white blood cells from patients with leukemia.", "Because unbound phage in the upper aqueous phase may be recovered with minimal losses, pre-clearing strategies are facilitated by BRASIL.", "This allows improved protocols for targeting peptide identification by phage display, for example by subtracting phage binding to cells from normal individuals before isolation of phage binding to diseased cells.", "The BRASIL method allows a decrease in non-specific background of phage binding.", "Multiple samples and several rounds of pre-clearing and selection can be performed in a few hours, allowing method automation and facilitating high-throughput screening.", "Data (shown below) suggest that BRASIL may enable targeting of organs with a significant reticuloendothelial component such as spleen, liver, and bone marrow which has not been feasible with currently available in vivo phage display technology (Pasqualini et al., 2000).", "The method may also be used with phage displaying larger polypeptides or folded proteins such as enzymes or antibodies (not shown), providing a phage display based approach to high throughput screening for novel inhibitors or activators of naturally occurring enzymes, receptors or other proteins.", "The data show that BRASIL is superior to conventional protocols for identifying targeting ligand-receptor pairs and to probing the molecular diversity of cell surfaces.", "Example 3 BRASIL with a Leukemia Cell Line The BRASIL protocol has also been performed with the Molt-4 leukemia cell line and a CX5C library, using the methods described above.", "The library was presubtracted against a normal Molt-4 cell line and then screened against a Molt-4 cell line transformed with a gene encoding the CD-13 protein.", "Molt-4 leukemia targeting peptides are listed in Table 5 below.", "TABLE 5 Targeting peptides against the Molt-4 leukemia cell line CEKRWGC SEQ ID NO:167 CSVWFGC SEQ ID NO:7 CKQRGVC SEQ ID NO:168 CQVRLSC SEQ ID NO:169 CTWDKRC SEQ ID NO:170 CRSPMKC SEQ ID NO:186 CTLFRNC SEQ ID NO:171 CPTMTEC SEQ ID NO:187 CRGSAVC SEQ ID NO:172 CSVWFGC SEQ ID NO:188 CAISVGC SEQ ID NO:173 CSVWYGC SEQ ID NO:189 CTNPQRC SEQ ID NO:174 CSVWYGC SEQ ID NO:190 CDSWPLC SEQ ID NO:175 CWILEQC SEQ ID NO:191 CENGSRC SEQ ID NO:176 CMATLRC SEQ ID NO:192 CGGSSQC SEQ ID NO:177 CRKLGGC SEQ ID NO:193 CGREGPC SEQ ID NO:178 CRAREMC SEQ ID NO:194 CSGRSGC SEQ ID NO:179 CQAWQRC SEQ ID NO:195 CQQGRYC SEQ ID NO:180 CKDRWGC SEQ ID NO:196 CVKQMRC SEQ ID NO:181 CYSDKKC SEQ ID NO:197 CSVWWGC SEQ ID NO:182 CGNHQKC SEQ ID NO:198 CSGPC SEQ ID NO:183 CPNDSLC SEQ ID NO:199 CEGHQSC SEQ ID NO:184 CQGTWIC SEQ ID NO:200 CNVWYGC SEQ ID NO:185 CMVYFGC SEQ ID NO:8 A consensus sequence identified for the leukemic cell line targeting peptides was CXVWXGC (SEQ ID NO:201).", "Example 4 Identification of Targeting Peptides for Urothelial Tissue by BRASIL Targeting peptides for urothelial tissue have not previously been identified by phage display.", "The present example further demonstrates the utility of the BRASIL method for identifying novel targeting peptides and illustrates additional embodiments of the methods and compositions.", "Materials and Methods Materials The human cell lines T24, RT4, MDA-MB435S, and MOLT-4 were obtained from the American Type Culture Collection (Manassas, Va.).", "All tissue culture media were from LifeTechnologies (NY).", "Cells were grown under standard conditions at 37° C. with 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin, and 100 mg/ml streptomycin.", "Human urothelial cells were isolated from fresh ureter specimens and cultured using supplemented Keratinocyte SFM Medium.", "Pig bladders were obtained from Dr. K. Wright (Department of Veterinary Medicine, M.D.", "Anderson Cancer Center, Houston, Tex.).", "Phage display libraries were prepared and amplified with the K91kan E. coli strain as described above.", "Synthetic peptides were from Anaspec (San Jose, Calif.).", "Panning of Phage Urothelial cells were isolated from fresh ureter specimens of patients undergoing nephrectomy for renal cell carcinoma.", "Ureters were freed of connective and fat tissue, slit open and the mucosa gently scraped into PBS under sterile conditions.", "Cells were then pelleted and resuspended in RPMI/10% BSA.", "Approximately 1×107 cells in 200 μl RPMI/10% BSA were then incubated with 1×107 cfu of a cyclic CX7C-phage library, a linear X6-library or amplified phage from a previous round of bipanning for 4 hours on ice.", "In two separate experiments the library or amplified phage from previous rounds were precleared on 1×107 MOLT4 cells for 30 min on ice prior to adding to the normal urothelial cells.", "After incubation, panning was continued using the BRASIL method described above.", "In brief, cells were placed on an oil cushion consisting of 90% dibutylphatalate and 10% cyclohexan (Sigma, St.Louis, Mo.", "), in a 400 μl Eppendorf tube and pelleted for 10 min at 10,000 rpm in an Eppendorf centrifuge.", "The tubes were then frozen in liquid nitrogen and the lower part of the tube containing the cell pellet cut off.", "The pellet was reinfected with 1 ml of log-phase K91 bacteria for 1 hour after removal of excess oil and amplified over night.", "Small aliquots were plated out for single colony picking and sequencing.", "Up to 3 rounds were performed.", "Sequencing and Alignments After each round the peptide inserts of 94 randomly selected phage clones were sequenced by DNA sequencing using the primer 5′-CCCTCATAGTTAGCGTAACGATCT-3′ (SEQ ID NO:12) and the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer, Norwalk, Conn.).", "Peptide sequences were aligned using the ClustalW alignment program (European Bioinformatics Institute homepage, (http://www2.ebi.ac.uk/clustalw/).", "Enriched peptide sequences were aligned to protein databases using the BLAST program of the National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST/).", "Similarity was defined as percentage of positive matches in the area aligned by the program.", "Phage Attachment and Competition Experiments Binding of selected phage was examined with human adherent primary urothelial cells, the breast cancer cell line MA-MB-435 and the transitional carcinoma cell lines RT4 and T24.All cells were grown to subconfluency in 48 well plates and free binding sites were blocked with 800 μl 30% FCS/DMEM (blocking medium) for 1 hour at 37° C. The blocking solution was then replaced by 200 μl 10% FCS/DMEM (washing medium) containing 1×108 cfu of each phage per well.", "After incubation for 2 hours at 4° C. to prevent unspecific endocytosis, unbound phage were removed by washing 7 times with 500 μl washing medium.", "For competition experiments increasing concentrations of the corresponding peptide or a control peptide (CARAC, SEQ ID NO:5) were added during the incubation.", "Bound phage were determined by infection with 500 μl log phase K91 culture and plating of serial dilutions.", "Values represent means of serial dilutions of triplicates wells and are given relative to binding of insertless fd-tet phage.", "To determine binding to intact mucosa a novel dot blot chamber assay was developed, placing the bladder or ureter specimen into a dot blot chamber (Biorad, Hercules, Calif.), with the mucosa facing upwards, thus generating up to 96 equally large fields of mucosa.", "Blocking and washing in the dot blot chamber was performed as above but with 400 μl of the corresponding medium and infection was performed with 400 μl of log-phase K91kan culture per well.", "Three wells were pooled as one well.", "Removal of the glucosaminoglycan-(GAG-) layer on intact mucosa samples was performed as a dot blot chamber assay.", "Half of the wells were incubated with 200 μl 0.1M HCl per well for 2 min, intensely rinsed with blocking solution and both parts blocked as before.", "Results are given relative to fd-tet phage to untreated mucosa, that was set to 1.Results To identify peptide motifs that interact with the human bladder wall, clones were selected from phage display peptide libraries by successive rounds of affinity panning on freshly isolated urothelial cells from surgical ureter specimens.", "In four experiments two different libraries, a cyclic 7 mer and a linear 6 mer library, were panned on human urothelial cells, with or without prior subtraction against MOLT-4 leukemia cells using the BRASIL method.", "Up to four rounds of selection were performed and 94 clones sequenced after every round.", "Five peptide motifs were identified by aligning all obtained sequences with the ClustalW program (Table 6).", "TABLE 6 Selection of Peptides Binding to Human Urothelial Cells Peptide Sequence Shared Motif Found in Round CGQEISGLC* (SEQ ID NO:13) ISGL (SEQ ID NO:35) 2 EVISGL (SEQ ID NO:14) 1 LISGVL (SEQ ID NO:15) 1 ELLSGL (SEQ ID NO:16) 1 GRLSGSL (SEQ ID NO:17) 2 CLRSGGLTC (SEQ ID NO:18) GGLS (SEQ ID NO:36) 2 LGGLSA (SEQ ID NO:19) 2 CWGGLSGLC* (SEQ ID NO:20) 1 CGLSLK (SEQ ID NO:21) 1 GLSARH (SEQ ID NO:22) 1# (2x) CEFGLSEVC (SEQ ID NO:23) 1 SKHALE (SEQ ID NO:24) HALE (SEQ ID NO:37) 1# VHALES* (SEQ ID NO:25) 3 VFALEG (SEQ ID NO:26) 2 CRIRMSAGC* (SEQ ID NO:27) MSAG (SEQ ID NO:38) 1# AMSAGV (SEQ ID NO:28) 2 CMIAGLGRC (SEQ ID NO:29) 1# CRVIVGPRC (SEQ ID NO:30) RVTXG (SEQ ID NO:39) 1# CRVFAGKRC (SEQ ID NO:31) 1 DRVTLG* (SEQ ID NO:32) 3 CRVTRGHGC (SEQ ID NO:33) 1# CIRVEAGSC (SEQ ID NO:34) 1# Phage selected after subtraction to MOLT-4 cells are indicated by a #.", "Phage chosen for binding assays are indicated by *.", "Phage containing the five peptide motifs were amplified, carefully titered and their binding to cultured human urothelial cells tested in a subconfluent monolayer.", "Insertless fd-tet phage were used as a negative control.", "Phage containing the consensus motifs bound up to 12.7 times higher to cultured urothelial cells than insertless fd-tet phage (FIG.", "6).", "Binding was specific for urothelial cells as determined by a lack of binding to the human breast cancer cell line MDA-MB435S, derived from a metastatic, ductal mammary carcinoma (FIG.", "7).", "Binding to the urothelial tumor cell lines T24, derived from a poorly differentiated recurrent transitional cell carcinoma and RT4, derived from a transitional cell papilloma was also examined.", "All phage, except CWGGLSGLC (SEQ ID NO:20) bound to RT4, while only CGQEISGLC (SEQ ID NO:13) phage bound to T24 tumor cells (FIG.", "7).", "VHALES (SEQ ID NO:25) phage apparently bound only to RT4 tumor cells.", "Binding specificity of VHALES (SEQ ID NO:25) was verified by competetive phage binding inhibition (FIG.", "8).", "Binding was 5.4 fold higher than fd-tet phage and binding was reduced by soluble VHALES (SEQ ID NO:25) peptide in a dose-dependent manner, while remaining unchanged by equal amounts of the control peptide CARAC (SEQ ID NO:5) (FIG.", "8).", "Binding of the motifs to intact bladder mucosa was examined using a novel dot blot chamber binding assay that allows the simultaneous testing of phage binding in up to 96 equal parts of bladder or ureter mucosa.", "Binding to intact porcine bladder and human ureter mucosa was determined with this assay.", "Selected phage displaying a consensus motif bound 3.8 to 11.7 times higher to porcine bladder mucosa than fd-tet phage (FIG.", "9).", "Using the same assay the influence of the glucosaminoglycan (GAG) layer on binding of several phage to intact mucosa was examined.", "The GAG-layer was removed as described above.", "Half of the mucosa of human ureter and porcine bladder specimens were treated inside the dot blot chamber with 0.1M HCl for 2 min, extensively washed and the binding assay performed as before.", "Binding of phage after GAG-removal was compared relative to that of untreated mucosa, which was set to 1.The binding of the control phage fd-tet and CRIRMSAGC (SEQ ID NO:27) phage remained unchanged by the treatment, while CWGGLSGLC (SEQ ID NO:20) phage binding increased 4.2 fold.", "VHALES (SEQ ID NO:25) phage binding was reduced by 50% (FIG.", "10).", "When tested on a human ureter sample GAG-removal increased CWGGLSGLC phage binding 3.4 fold (data not shown).", "This suggests a negative influence of the GAG-layer on binding of CWGGLSGLC (SEQ ID NO:20) phage.", "These results show a number of new targeting peptide sequences and conserved motifs targeted to urothelial tissues.", "Further modifications of the BRASIL protocol are demonstrated herein, along with the utility of those novel methods for identification and characterization of targeting peptide sequences.", "The skilled artisan will realize that the disclosed methods and compositions are not limited to urothelial cells or tissues, but rather have broad applicability to a variety of organs, tissues and cell types found in humans.", "Example 5 BRASIL and Stem Cell Screening Another non-limiting example of cell types that may be screened for targeting peptide sequences by BRASIL includes stem cells.", "In the discussion below, the stem cells are obtained from bone marrow.", "However, the skilled artisan will realize that the disclosed methods are applicable for stem cells in general.", "Source of Cells and Culture Mesenchymal cells are primary stem cells derived from bone marrow, obtained by seeding human bone marrow aspirate onto plastic flasks.", "Cells that attach to the flask are the mesenchymal cells.", "Mesenchymal cells were cultured in RPMI 1640 medium supplemented with 20% fetal calf serum at 37° C. (5% CO2) and sub-cultured every 4-5 days.", "KS1767 cells were grown in MEM medium supplemented with 10% fetal calf serum and sub-cultured every 3-5 days.", "Biopanning on Mesenichymal Cells A subtraction strategy was performed in which the phage library was first prescreened against KS1767 cells and phage binding to the KS1767 non-stem cell line were removed.", "The pre-screened library was then screened against mesenchymal cells using the BRAZIL method.", "All procedures were performed at 4° C. All media and solutions used for the biopanning were filtered through a 0.22 μm Millipore filter.", "The mesenchymal and KS1767 cells were washed with PBS and incubated with PBS plus 5 mM EDTA for 10 minutes on ice to promote detachment of the cells from the plastic.", "Cells were collected by aspirating the medium, washed by centrifugation with RPMI 1640 medium and re-suspended at 106 cells/ml in RPMI 1640 medium supplemented with 0.5% BSA (bovine serum albumin).", "A CX7C phage display library (or phage obtained from the previous round of biopanning) was added to the KS cells (109 T.U.", "of phage per 105 cells) and incubated for 1-2h on ice.", "Unbound phage were selected by BRASIL after KS1767 cells were exposed to phage and centrifuged over dibutyl phthalate:cyclohexane (6:1) at 4° C. Under these conditions, cells carrying bound phage pellet at the bottom of the tube.", "Unbound phage remain in the upper (aqueous) phase.", "The upper phase was carefully transferred to a new tube containing 105 mesenchymal cells and further incubated for 4 h on ice.", "Bound phage attached to mesenchymal cells were then selected by BRASIL.", "The mesenchymal cell suspension with phage was centrifuged over dibutyl phthalate:cyclohexane (6:1) at 4° C. Mesenchymal cells carrying the bound phage pelleted at the bottom of the tube.", "The tube was snap frozen at −80° C. for 10 minutes and the bottom of the tube containing the pellet of cells with bound phage was cut off, transferred to a new tube and the pellet carefully ressuspended with 200 μl of a log-phase E.coli K91 culture to recover the phage.", "After 20 minutes of infection, 20 ml of LB medium (Luria-Bertani) was added and the cells cultured for 16-18 h at 37° C. with agitation for phage amplification.", "After the initial selection, phage obtained from a previous round was used for the next round of selection.", "After 3 rounds of biopanning, individual colonies were selected for sequencing.", "The stem cell binding peptides are listed in Table 7 below.", "TABLE 7 Stem cell (mesenchymal) targeting peptides CLGRLTVLC (SEQ ID NO:63) CERSIGFAC (SEQ ID NO:74) CSVPVSSSC (SEQ ID NO:75) CTAWFIESC (SEQ ID NO:64) CYPGYDSYC (SEQ ID NO:76) CPWYWFGTC (SEQ ID NO:77) CSYGRASLC (SEQ ID NO:65) CECRGDCYC (SEQ ID NO:78) CVKKGGFWC (SEQ ID NO:79) CDAGPWTAC (SEQ ID NO:66) CSMTKLGAC (SEQ ID NO:80) CVGVGRSRC (SEQ ID NO:67) CGVLKPYLC (SEQ ID NO:81) CWWPWGWGC (SEQ ID NO:94) CTNPWSPVC (SEQ ID NO:68) CSWWTFGFC (SEQ ID NO:95) CNSRAGSVC (SEQ ID NO:96) CGGSYDEVC (SEQ ID NO:69) CLRLSMSAC (SEQ ID NO:97) CNSRAGSVC (SEQ ID NO:98) CAPMEWSVC (SEQ ID NO:70) CMSGNTERC (SEQ ID NO:99) CGHLGSVYC (SEQ ID NO:100) CTRVHGLAC (SEQ ID NO:71) CVLADPTGC (SEQ ID NO:101) CESLSHVDC (SEQ ID NO:72) CECRGDCYC (SEQ ID NO:102) CWWGWWGTC (SEQ ID NO:103) CLWTQSSGC (SEQ ID NO:73) CWKGFGWWC (SEQ ID NO:104) CKRSATILC (SEQ ID NO:105) CSERIARVC (SEQ ID NO:86) CPWYWLGWC (SEQ ID NO:87) CIEGRRGLC (SEQ ID NO:106) CGRKNEWAC (SEQ ID NO:88) CARDRIIAC (SEQ ID NO:89) CPWYWLGWC (SEQ ID NO:107) CGQMNREVC (SEQ ID NO:90) CDAYPLFFC (SEQ ID NO:91) CVRQGEDAC (SEQ ID NO:108) CWKGFGWWC (SEQ ID NO:92) CSLAVPLAC (SEQ ID NO:109) CLGSGSGSC (SEQ ID NO:93) CGWFSWFGC (SEQ ID NO:113) CMMHGLAAC (SEQ ID NO:110) CRVDFSKGC (SEQ ID NO:114) CSSLATVVC (SEQ ID NO:115) CDWWTTAWC (SEQ ID NO:111) CMYRTSLAC (SEQ ID NO:116) CLAAVYQSC (SEQ ID NO:117) CGWWGLWPC (SEQ ID NO:112) CSRRVIGAC (SEQ ID NO:118) CSWWNWFGC (SEQ ID NO:119) CPWYWFGTC (SEQ ID NO:82) CSRRPEVVC (SEQ ID NO:120) CWVADGYRC (SEQ ID NO:83) CVTGNRGC (SEQ ID NO:121) CVSWWFWGC (SEQ ID NO:122) CECRGDCYC (SEQ ID NO:84) CGWFSWWGC (SEQ ID NO:123) CSWWRFGYC (SEQ ID NO:124) CSHAVMPWC (SEQ ID NO:85) Receptor Identification The phage “D5” containing the peptide sequence CRVDFSKGC (SEQ ID NO:114) showed significant homology with the leptin hormone (Table 8).", "This region of leptin is conserved in several species (Macaca mulatta, Homo sapiens, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Mus musculus, Rattus nervegicus).", "TABLE 8 Homology between phage D5 and leptin sequences Phage (SEQ ID NO:114) Human leptin (SEQ ID NO:125) Mouse leptin (SEQ ID NO:126) The conserved peptide maps to a loop in between amino acids 90-96 in the protein (Zhang et al, 1997).", "This region of the leptin molecule has been been indicated as important for leptin activity.", "A synthetic peptide DLLHLLAFSKSCSLLP (SEQ ID NO:127) has been reported to block leptin activity in vivo (Grasso et al., 1997) (amino acids in bold indicate those with similarity to clone D5 (CRVDFSKGC, SEQ ID NO:114).", "The present example shows that BRASIL can be used to identify targeting peptides against stem cells.", "The homology between one of the identified peptide sequences and an endogenous hormone further validates the identified sequences.", "The skilled artisan will realize that the methods and targeting peptide sequences identified herein are of potential use for identification and purification of stem cells (for example, by affinity chromatography) and for identification of receptor:ligand pairs present in stem cells.", "Example 6 Bone Marrow Screening by BRASIL A non-limiting example of an organ of specific interest for targeting petides is bone marrow.", "Bone is the preferred site of metastasis in the large majority of patients with prostate cancer (Fidler, 1999).", "This striking selectivity has been viewed as an example of site-specific interactions that were essential to cancer progression (Rak, 1995; Zetter, 1998).", "Despite the clinical relevance, little is known about the mechanisms that control prostate cancer spread to bone.", "In addition, there were no effective strategies for targeting therapeutic agents for the treatment of metastatic prostate cancer (Brodt et. al, 1996).", "A subset of peptides capable of selective homing to bone marrow through the circulation is likely to simulate the behavior of prostate cancer cells during bone metastasis formation.", "The vascular markers targeted by using phage display might also be utilized by tumor cells to metastasize.", "This concept has already been proven to be true for lung-homing peptides.", "Peptides that home to lung blood vessels inhibit experimental metastasis.", "These results fit a “modified seed and soil” model, in which the basis for site-specific metastasis is the presence of homing receptors in blood vessels of certain tissues to which metastasis preferentially occurs.", "Such selective vascular markers are exposed to tumor cells during adhesion, the first step of the metastastic cascade.", "Isolation of bone marrow-homing peptides is of utility for identifying those vascular markers that mediate prostate cancer cell homing during the metastatic process, and for potential therapeutic intervention in preventing metastases to bone, or in selectively imaging and/or treating cancer that has already metastasized to bone.", "Screening of Phage Display Libraries on Human Bone Marrow Fresh human ribs removed during surgery for access to underlying tumors were sectioned to expose the bone marrow surface.", "No significant damage to the bone marrow was inflicted to the tissue and the morphology was well preserved during after the procedure.", "The bone samples were washed (gently) 5 times with ice cold DMEM/0.15% BSA (sterile filtered).", "The marrow was removed by gently scraping cells from the bone.", "Cells were washed twice by centrifugation and resuspension in DMEM/BSA to remove debris and fat.", "Cells were resuspended in DMEM/BSA (about 107 cells per ml) and incubated with a phage display library (109 TU) prepared as described above.", "After incubation for 3 hours on ice, the cells were centrifuged through an organic phase consisting of a 9:1 mixture of dibutylphthalate:cyclohexane.", "Centrifugation occured for 10 min at 10,000×g.", "The bottom of the centrifuge tube was snap frozen at −80° C. and phage were recovered by bacterial infection as described above.", "The selection was repeated for 3 more rounds of BRASIL and 90 clones were sequenced.", "The bone marrow targeting sequences are listed in Table 9 below.", "TABLE 9 Bone Marrow Targeting Peptides Identified by BRASIL CPEVMGSSC SEQ ID NO:202 CSSVVRLGC SEQ ID NO:203 CVGAGLHIC SEQ ID NO:204 CHLEPDWVC SEQ ID NO:205 CALGRWDRC SEQ ID NO:206 CFGGVGSWC SEQ ID NO:207 CGRRDTVDC SEQ ID NO:208 CLVLGGYGC SEQ ID NO:209 CWENRGQFC SEQ ID NO:210 CREQASTGC SEQ ID NO:211 CVVKLRNRC SEQ ID NO:212 CVGLRAPLC SEQ ID NO:213 CQKVARPGC SEQ ID NO:214 CQKFARPGC SEQ ID NO:215 CMWGLSYLC SEQ ID NO:216 CREQRHNLC SEQ ID NO:217 CLVLSASAC SEQ ID NO:218 CLLSGLMGC SEQ ID NO:219 CRGDTKALC SEQ ID NO:220 CVSQLGRVC SEQ ID NO:221 CFVFEAMGC SEQ ID NO:222 CSVIKRGAC SEQ ID NO:223 CGGWVDHRC SEQ ID NO:224 CAVVRNQEC SEQ ID NO:225 CDSPRRPVC SEQ ID NO:226 CTFSGHRLC SEQ ID NO:227 CHTWGGRNC SEQ ID NO:228 CEGAGLVAC SEQ ID NO:229 CFPRVWSRC SEQ ID NO:230 CYWLGGALC SEQ ID NO:231 CDTNQRVVC SEQ ID NO:232 CMRVTKTHC SEQ ID NO:233 CDQNWLVHC SEQ ID NO:234 CTFSGHRLC SEQ ID NO:235 CALSAYRVC SEQ ID NO:236 CGGEEGRRC SEQ ID NO:237 CAEAGGPDC SEQ ID NO:238 CIVMLGWRC SEQ ID NO:239 CGHGVTGRC SEQ ID NO:240 CERGRGAAC SEQ ID NO:241 CAAGEGWWC SEQ ID NO:242 CALSAYRVC SEQ ID NO:243 CLWPWAGEC SEQ ID NO:244 CTHATWLVC SEQ ID NO:245 CSGVSTVRC SEQ ID NO:246 CLVSYMNGC SEQ ID NO:247 CVRTSSQWC SEQ ID NO:248 CLGKGLSSC SEQ ID NO:249 CFTAVEQGC SEQ ID NO:250 CGGIGPRFC SEQ ID NO:251 CVATWCEKC SEQ ID NO:252 CSSELRAAC SEQ ID NO:253 CKGSLDEIC SEQ ID NO:254 CSSVVRLGC SEQ ID NO:255 CLKTEFTAC SEQ ID NO:256 CPGRLWRAC SEQ ID NO:257 CSELGGAGC SEQ ID NO:258 CLGWRAAAC SEQ ID NO:259 CGAMWGMGC SEQ ID NO:260 CIGLSGIEC SEQ ID NO:261 CQKLGWRV SEQ ID NO:262 CLEWLQQVC SEQ ID NO:263 CLVLGEKPC SEQ ID NO:264 CAAGKGLLC SEQ ID NO:265 CAAGKDLLC SEQ ID NO:266 CGAQSPRC SEQ ID NO:267 CLSSVRGWC SEQ ID NO:268 CSESQLAWC SEQ ID NO:269 CSRNSVREC SEQ ID NO:270 CGLVITATC SEQ ID NO:271 CPGSVRVQC SEQ ID NO:272 CRGDTKALC SEQ ID NO:273 CACVRSRNC SEQ ID NO:274 CRADSEGVC SEQ ID NO:275 CNVEASVRC SEQ ID NO:276 CVGNAKLMC SEQ ID NO:277 CQKLARAGC SEQ ID NO:278 CGGRAILLC SEQ ID NO:279 CQLGRAHGC SEQ ID NO:280 CGLVITATC SEQ ID NO:281 CVGATYSRC SEQ ID NO:282 CSAFSVAYC SEQ ID NO:283 CLAWEVYLC SEQ ID NO:284 CQWWLGPLC SEQ ID NO:285 CSLGSFMGC SEQ ID NO:286 CVLGEISWC SEQ ID NO:287 CSGGSGARC SEQ ID NO:288 CPWWMMERC SEQ ID NO:289 Statistical Analysis of the Peptide Motifs A system has been designed to analyze the data resulting from peptide library screenings, adapted from the SAS package.", "The system is available upon request from the M.D.", "Anderson Cancer Center.", "Based on a statistical analysis of the phage sequences listed in Table 9, an LG motif (Leu-Gly) was observed in bone marrow targeting phage.", "Selected clones with the motif showed very high binding to human bone marrow cells compared to the negative control (insertless fd-tet phage).", "The positive control was phage containing an RGD-4C insert, which is known to bind to bone marrow.", "The highest affinity peptide (CLGWRAAAC, SEQ ID NO:259) exhibited binding that was over twice as high as the positive control.", "Binding assays were performed using BRASIL as described above, except that a single phage clone was used in place of the phage library.", "The skilled artisan will realize that the bone marrow targeting peptide sequences identified herein will be of use for numerous applications within the scope of the present invention, including but not limited to targeted delivery of therapeutic agents or gene therapy, in vivo imaging of normal or diseased organs or tissues, identification of receptors and receptor ligands in organs or tissues, and therapeutic treatment of a number of human diseases, particularly metastatic prostate cancer.", "All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.", "While the compositions and methods of this invention have been described in terms of preferred embodiments, it are apparent to those of skill in the art that variations may be applied to the COMPOSITIONS, METHODS and APPARATUS and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.", "More specifically, it are apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.", "All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.", "References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.", "Arap, W., Pasqualini R., and Ruoslahti, E. 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[ [ "CAPACITOR HAVING A DIELECTRIC CERAMIC LAYER", "The invention relates to a capacitor with two or more opposing pairs of electrode layers (1) with an intermediate dielectric layer (2), in which the dielectric layers (2) comprise a ceramic material that contains at least two different components existing in separate phases, in which each component exhibits a perovskite structure that contains silver in the A-positions and niobium and tantalum in the B-positions, and in which the composition of a component A and the composition of a component B are each chosen in such a way that TkεA and TkεB exhibit different signs within a temperature range.", "The suitable mixing ratio of the components can, for example, be realized by stacking green compacts of component A on green compacts of component B in a suitable quantity." ], [ "1.A capacitor comprising: at least two pairs of opposing electrode layers; and an intermediate dielectric layer; wherein the intermediate dielectric layer comprises a ceramic material that contains at least two different components existing in separate phases, the at least two different components have a perovskite structure that contains silver in A-positions and niobium and tantalum in B-positions, and a composition of a first component and a composition of a second component are such that temperature coefficients of respective permittivities TkεA and TkεB have different signs within a temperature range.", "2.The capacitor according to claim 1, wherein a volume share of the first component in a total volume of the first and second components deviates less than 25% from a volume share which is calculated using the following formula: V×SA+(1−V)×SB=0, wherein SA and SB represent slopes of straight lines that approximate respective temperature-dependent curves corresponding to a relative change in permittivities of the first and second components in the temperature range.", "3.The capacitor according to claim 1, wherein the first and second components have a composition of Ag(Nb1-xTax)O3, and wherein for the first component 0.50<1-x<0.70 and for the second component 0.30<1-x<0.50.4.The capacitor according to claim 3, wherein for the first component 0.64<1-x<0.66 and for the second component 0.34<1-x<0.36, and wherein a volume-related mixing ratio of first/second components in the ceramic material is between 40/60 and 50/50.5.The capacitor according to claim 4, wherein the first and second components comprise particles with a dimension of between 5 μm and 500 μm.", "6.The capacitor according to claim 1, wherein a mixture of particles for the first component with particles for the second component in the ceramic material is created by sintering.", "7.The capacitor according to claim 1, wherein the first and second components are in different dielectric layers, which are separated by intermediate electrode layers.", "8.The capacitor according to claim 1, wherein the first and second components comprise particles with a dimension of between 100 nm and 5 μm.", "9.The capacitor according to claim 1, wherein the intermediate dielectric layers have a thickness between 1 nm and 50 μm.", "10.The capacitor according to claim 1, wherein the intermediate dielectric layer comprises stacked metalized ceramic green compacts of the first component and of the second component.", "11.The capacitor according to claim 1, wherein the ceramic material contains boric acid." ], [ "The invention relates to a capacitor with two or more opposing pairs of electrode with an intermediate dielectric layer, the dielectric layers of which comprise a ceramic material.", "The publication by M. Valant and D. Suvorov, “Microwave Ceramics with Permittivity over 400,” The 9th International Meeting on Ferroelectricity, Seoul, South Korea, 1997, Abstract Book, P-05-TH-067 discloses multilayer capacitors whose dielectric layers comprise a ceramic material based on a niobium-based perovskite-like solid solution having the general formula A(B1-xNbx)O3.It was found that ceramics of this type are characterized by a high relative permittivity ε>400.Furthermore, at low frequencies between 100 kHz and 1 MHz, these ceramic materials have dielectric properties that make them suitable for use in multilayer capacitors.", "A ceramic material is known from the publication by A. Kania, “Ag(Nb1-xTax)O3 Solid Solutions—Dielectric Properties and Phase Transitions,” Phase Transitions, 1983, Volume 3, pp.", "131-140, produced from silver, niobium and tantalum, referred to here as ANT, which exists in the form of a solid solution of the two materials AgNbO3 and AgTaO3.The ceramic described in this publication comprises the composition Ag(Nb1-xTax)O3, referred to here as ANTx, wherein x can vary from 0 to 0.7.Depending on the composition, at a temperature of approximately 300 K, the ceramic has an ε of between 800 and 400.From the publication by Matjaz Valant and Danilo Suvorov, “New High Permittivity Ag(Nb1-xTax)O3 Microwave Ceramics: Part 2, Dielectric Characteristics,” J.", "Am.", "Ceram.", "Soc.", "82 (1), pp.", "88-93 (1999), it is known that disk-shaped ceramic bodies made of ANTx with an x parameter between 0.46 and 0.54, exhibit a strong relative change in the relative permittivity ε in the temperature range between −20° C. and 120° C. It was demonstrated in particular that the relative change of ε with varying temperature describes a curve that reaches a maximum between 20° C. and 70° C., and assumes values between −0.07 and 0.01.It is further known from publication WO 98/03446 that by adding lithium, wolfram, manganese or bismuth to ANT, in the case of individual temperatures, the temperature coefficient of the relative permittivity TKε can be reduced to very small values as low as ±−70 ppm/K.", "Although the known ANT materials have a high ε, they have the disadvantage of having relatively high TKε values in the temperature range between −20° C. and 80° C. that is of interest for applications.", "At the same time, a high temperature coefficient of relative permittivity ε results in a high temperature coefficient of the capacitor's capacitance.", "Therefore, the object of the present invention is to provide a capacitor whose dielectric has a low temperature coefficient of relative permittivity.", "According to the invention, this object is achieved by a capacitor according to claim 1.Advantageous embodiments of the invention can be found in the other claims.", "The invention is directed to a capacitor with two or more opposing pairs of electrode layers and an intermediate dielectric layer.", "The dielectric layers comprise a ceramic material containing at least two different components that exist in separate phases.", "Each of the components has a perovskite structure that contains silver in the A-positions and niobium and tantalum in the B-positions.", "The composition of one of the components (component A) and the composition of an additional component (component B) are selected in such a way that the temperature coefficients of their respective permittivities TkεA and TKεB have different signs in a temperature range.", "The ANT material has the advantage of having a high ε>300.In addition, the ceramic material according to the invention has the advantage of having low dielectric loss.", "By mixing two components, each of which has a Tkε with a different sign, temperature dependency of the relative permittivity can be largely compensated so that the ceramic material according to the invention has a smaller Tkε than its components.", "Compensation can be achieved not only partially at fixed temperatures, but also throughout the entire temperature range, within which the individual components have different signs.", "That is, compensation is not limited to individual points on the temperature scale.", "Because the components in the ceramic material according to the invention exist as separate phases, in the event that the ceramic material includes only two different components, the Tkε of the ceramic material can be given by the following Lichtenecker rule formula: Tkε=V×TkεA+(1−V)×TkεB In the above formula, V stands for the volume share of component A in the total volume of the components and TkεA and TkεB indicate the temperature coefficients of corresponding components A and B.", "According to the Lichtenecker rule, suitable selection of the volume share of the component A for a given temperature can result in complete compensation of the temperature coefficients of the corresponding permittivity.", "This Lichtenecker rule is now used to determine an optimum volume share of component A in the total volume of components A and B, in such a way that optimum compensation of the temperature coefficients can be achieved in the temperature range within which the individual components have different signs.", "To this end, according to the invention, the volume share of component A in the total volume of components A and B is selected in such a way that it deviates less than 25% from the volume share V, which is calculated using the following formula: V×SA+(1−V)×SB=0.In this formula, SA and SB correspond to the slopes of straight lines that best approximate the respective temperature-dependent curves showing relative changes in the relative permittivities of component A and/or component B in a given temperature range.", "By applying the Lichtenecker rule for individual temperatures to a temperature range, it is possible to achieve an optimum compensation of temperature coefficients Tkε with different signs.", "Using the arithmetic rule specified above, the mean of the temperature coefficients Tkε, weighted with the volume share, is used to calculate suitable volume shares.", "Because the Lichtenecker rule used for calculation adds the Tkε values in a linear fashion, the compensation of temperature coefficients Tkε operates more efficiently the more closely the temperature-dependent curve of the relative change in the relative permittivity of the individual components approximates a straight line.", "It is therefore desirable to come as close to this linear curve as possible using a suitable composition of the components.", "Such an approximation to linear behavior can be achieved in an especially advantageous way by selecting one of the components with a suitable quantitative ratio of niobium to tantalum.", "In order for the compensation of opposite temperature curves to work, the components A and B must exist in separate phases.", "Tests have shown that a first possibility of realizing separate phases comprises mixing the components A and B as not overly small particles with a particle size of >5 μm.", "In case smaller particle sizes are used, an exchange of material occurs between the components due to diffusion.", "This results in a solid solution representing a new material with new properties.", "Here, a simple “linear superposition” of the components A and B, as described by the Lichtenecker rule, is no longer possible.", "The use of particle sizes >5 μm has the effect of the particles mixing only at the periphery due to the slow diffusion processes, resulting in essentially separate phases for component A and component B.", "An additional possibility for maintaining the two components A and B in separate phases comprises arranging components A and B in different dielectric layers separated by electrode layers.", "These electrode layers can, for example, be metallic layers, in particular silver/palladium electrode layers, which effectively block an exchange of material between components A and B during sintering because they represent a diffusion barrier.", "It is therefore necessary to produce dielectric layers that contain only component B of the ceramic material.", "Separating the individual phases using intermediate electrode layers also makes it possible to arrange components A and B of the ceramic material in the form of particles smaller than those described above in the capacitor.", "Due to the blocked diffusion between component A and component B, particle size ceases to be crucial and components A and B can then, for example, exist in the form of grains between 100 nm and 5 μm in dimension, such as are easily produced by grinding in a standard method for manufacturing ceramics.", "The existence of different components in the form of small grains also makes it possible to produce dielectric layers of the capacitor with a very small thickness between 1 and 50 μm.", "As a result, at the same volume, a significantly larger number of dielectric layers can be inserted as well as more sub-capacitors.", "Such thin dielectric layers between 1 and 50 μm are not possible when using components in the form of large particles, since a single layer of particles would exceed the allowed thickness of the dielectric layer.", "Furthermore, a capacitor is especially advantageous in which the superimposed dielectric layers are created by stacking each metalized ceramic green compact of component A and each metalized green compact of component B, followed by sintering the foil stack.", "The metallization of the green compact forms the electrode layers.", "To a great extent, such a capacitor permits use of known methods for manufacturing multilayer capacitors.", "The order in which the green compacts of component A and/or the green compacts of component B are superimposed is also insignificant due to the fact that the capacitor normally comprises a number of individual sub-capacitors connected in series and/or in parallel.", "In any case, compensation of the temperature coefficients of the relative permittivities of the individual dielectric layers is guaranteed.", "It is particularly advantageous for the capacitor to contain a ceramic material produced with boric acid as a sintering aid.", "Boric acid has the advantage of not lowering the insulation resistance of the ANT ceramic in a disadvantageous manner.", "The following describes the invention in greater detail on the basis of exemplary embodiments and accompanying figures.", "FIG.", "1 shows an example of a capacitor according to the invention in a schematic sectional view.", "FIGS.", "2 through 8 show curves of different ANT materials that can be used as component A or component B for the capacitor according to the invention.", "FIGS.", "3, 4, 5, 6, 7 and 8 additionally show curves of mixtures of component A with component B.", "In the figures, the relative change in capacitance AC/C of the layer sample is shown as a function of temperature.", "The change in capacitance is directly linked with the value Δε/ε via C=ε×A/d.", "FIG.", "1 shows a capacitor according to the invention with electrode layers 1, which can be silver/palladium electrodes, for example.", "These electrode layers 1 are separated by dielectric layers 2.Two types of dielectric layers 2, which are electrically connected via a contact layer 3, intersect in a comb-like fashion, thereby creating a parallel circuit of semi-capacitances.", "The contact layers 3 are silver burnt-in electrodes.", "The dielectric layers 2 comprise, as indicated by letters A and B in FIG.", "1, either a ceramic material of component A or a ceramic material of component B.", "According to the exemplary embodiment in FIG.", "1, four dielectric layers 2 from component A and two dielectric layers 2 from component B are arranged in the capacitor.", "Accordingly, the volume ratio of component A to component B in this capacitor is 4:2.By increasing the total number of dielectric layers 2 and by selecting different numbers of dielectric layers for component A or component B, it is possible to produce random mixing ratios between component A and component B.", "In the following, various ceramic materials suitable for component A and component B are presented with their electrical properties.", "Please note that in each of the following examples, a mixing of the components is possible either in the form of mixed particles or in the form of superimposed dielectric layers separated by electrode layers assigned to a component A or a component B.", "In the samples described below, 1 and 1.5 percent by weight H3BO3 was added to the ceramic material at 950° C. before final calcination.", "The ceramic was then sintered for five hours at 1070° C. Thereafter, the dielectric properties of the materials produced in this fashion were tested at frequencies of 1 MHz and approximately 2 GHz.", "The component B known from the compositions described above (ANTx with x=0.65) was used as component B for the composite ceramic.", "The components were mixed as a particle with a medium grain size of 30.9 μm (component A) and 27.7 μm (component B) and then sintered together.", "In a first series of tests, a component B with 1 percent by weight H3BO3 and a number of possible components A with different excesses of niobium and/or tantalum were tested.", "The results are shown in FIG.", "2.Curves 51 through 54 refer to a component A with varying x content and curve 55 refers to the component B specified above, with x=0.65.Curve 51 describes the composition of component B with x=0.35, curve 52 with x=0.38, curve 53 with x=0.40 and curve 54 with x=0.42.FIG.", "2 shows that, in particular, the composition according to curve 51 exhibits good linearity, which is especially well suited for use as component A in the capacitor according to the invention.", "Ceramic materials with different mixture ratios of component A to component B were produced with the different components A shown in FIG.", "2, as shown by Table 1 below.", "Column 1 of Table 1 shows the excess of niobium of component A used as an x value.", "Column 2 contains the weight-specific ratio of component A to component B.", "Columns 3, 4, 5, 6 and 7 show dielectric core values for the shrinkage S of the samples.", "The last column of Table 1 provides the optimum mixture of component A and component B of each maximum change of the relative permittivity in the temperature range from −20° C. and 120° C. for the respective curve.", "TABLE 1 Relative permittivity and dielectric losses of a composite ceramic with 1 percent by weight H3BO3 as a sintering aid, sintered at 1070° C. for five hours (component B = ANTx with x = 0.65) 1 MHz 2 GHz S Δε/ε max X A/B ε tanδ ε′ Qxf[GHz] [%] [%] 0.42 60/40 386 0.0007 371 597 10.1 1.8 62.5/37.5 390 0.0002 398 566 9.7 70/30 408 0.0004 408 492 10.9 0.40 40/60 328 0.0007 326 664 9.3 50/50 370 0.0004 385 577 9.6 60/40 385 0.0004 395 493 9.8 1.5 0.38 35/65 339 0.0010 355 654 10.2 45/55 347 0.0005 357 592 9.8 55/45 375 0.0005 403 516 10.1 1.2 0.35 30/70 317 0.0005 323 644 9.0 40/60 350 0.0009 363 560 10.0 45/55 341 0.0002 362 539 9.1 0.8 50/55 354 0.0005 375 479 9.7 Table 1 shows that at least the composite ceramics produced with the optimum mixing ratio of component A to component B with various x values of component A are suitable for multilayer capacitors.", "FIG.", "3 shows curves for different composite ceramics with a component A where x=0.42 (8% niobium excess) and with different mixing ratios of component A to component B. Curve 56 shows the 60/40 mixing ratio, curve 57 the 70/30 mixing ratio, curve 58 the 62.5/37.5 mixing ratio, curve 59 the curve for pure component A, and curve 60 the curve for pure component B.", "FIG.", "4 shows curves for different composite ceramics with a component A where x=0.40 (10% niobium excess) and with different mixing ratios of component A to component B. Curve 62 shows the 60/40 mixing ratio, curve 64 the 40/60 mixing ratio, curve 63 the 50/50 mixing ratio, curve 61 the curve for pure component A, and curve 65 the curve for pure component B.", "FIG.", "5 shows curves for different composite ceramics with a component A where x=0.38 (12% niobium excess) and with different mixing ratios of component A to component B. Curve 69 shows the 35/65 mixing ratio, curve 68 the 45/55 mixing ratio, curve 67 the 55/45 mixing ratio, curve 70 the curve for pure component A, and curve 66 the curve for pure component B.", "FIG.", "6 shows curves for different composite ceramics with a component A where x=0.35 (15% niobium excess) and with different mixing ratios of component A to component B. Curve 75 shows the 30/70 mixing ratio, curve 73 the 40/60 mixing ratio, curve 72 the 50/50 mixing ratio, curve 71 shows the 45/55 mixing ratio, curve 76 the curve for pure component A, and curve 71 the curve for pure component B.", "Additional tests examined the effects of increasing the boric acid share from 1 percent by weight to 1.5 percent by weight.", "It was found that the increased boric acid share facilitated sintering of the ANT powder.", "It also obtained slightly higher values for the relative permittivity.", "The dielectric losses, measured at 1 MHz, show no significant change with the H3BO3 concentration, while the Qxf values at 2 GHz are a bit less favorable than with the addition of 1 percent by weight H3BO3.FIG.", "7 shows the curves for an ANTx system produced by adding 1.5 percent by weight H3BO3.The other production parameters were the same as in the samples with 1 percent by weight H3BO3.Curve 77 shows the curve for a component A with x=0.42, curve 78 the curve for a mixture of component A and component B with a weight-specific ratio of 70/30, curve 79 a composite ceramic with a 50/50 mixing ratio and, finally, curve 80 the curve for pure component B with x=0.65.FIG.", "8 shows temperature curves for a composite ceramic according to the invention (1.5 percent by weight H3BO3) with a component A with x=0.35 (15% niobium excess) and with various mixing ratios of component A to component B. Curve 81 shows the component A with x=0.35, curve 82 shows a mixture with a 60/40 mixing ratio, curve 83 with a 55/45 mixing ratio, curve 84 with a 45/55 mixing ratio, and curve 85 the component B with x=0.65.Table 2 below shows, as in Table 1, the dielectric properties and the shrinkage for the component B mixtures with a niobium excess of 8% (x=0.42) and with a niobium excess of 15% (x=0.65).", "For each optimum mixing ratio of component A to component B, the maximum relative change in relative permittivity within the temperature range between −20° C. and 120° C. is given in percent.", "TABLE 2 Relative permittivity and dielectric losses of a composite ceramic with 1.5 percent by weight H3BO3 as sintering aid, sintered at 1070° C. for five hours (component B = ANTx with x = 0.65) 1 MHz 2 GHz S Δε/ε max X A/B ε tanδ ε′ Qxf[Ghz] [%] [%] 0.42 60/40 398 0.0003 395 545 11.5 70/30 408 0.0004 408 492 10.9 1.4 0.35 60/40 414 0.0004 436 360 11.7 0.6 55/45 397 0.0004 435 419 11.2 45/55 341 0.0002 362 539 10.7 In the following, capacitors with special ceramic materials are specified, for which the electrical characteristics of the capacitors were measured.", "For composition A, a calcinated precursor was used consisting of 45.4 percent by weight Nb2O5 and 54.6 percent by weight Ta2O5.After this, 58.9 percent by weight calcinate was mixed with 40.1 percent by weight silver oxide and 1 percent by weight H3BO3, and calcinated again.", "H3BO3 was used as a sintering aid.", "Additional processing of this mixture up to type A green compact was performed using known methods.", "A second precursor was produced to create composition B.", "This precursor comprises a mixture of 24.5 percent by weight Nb2O5 and 75.5 percent by weight Ta2O5.The other process steps up to the first calcination correspond to those used to create composition B.", "Subsequently, 61.5 percent by weight of the calcinate was mixed with 37.5 percent by weight Ag2O and 1 percent by weight H3BO3 and calcinated again.", "This mixture was then further processed as indicated for type A green compacts.", "Two capacitors were produced; in both cases green compact was stacked, pressed together and then sintered with the metal coating found on the green compacts.", "A capacitor 1 was produced with ten dielectric layers of type A, each having a thickness of 20 μm and a surface of 10 mm2.In addition, a capacitor 2 according to the invention was produced with five dielectric layers of type A and five dielectric layers of type B, each with the geometric measurements specified for capacitor 1.Table 3 below shows the electrical characteristics of capacitors 1 and 2.TKC refers to a temperature range between −20° C. and 120° C. TABLE 3 Electrical properties of exemplary multilayer capacitors Capacitor C [nF] tanδ TKC 1 2.7 0.4 × 10−3 −3 .", ".", ".", "6.3% 2 2.4 0.43 × 10−3 −3 .", ".", ".", "1% Table 3 shows that the capacitor 2 according to the invention, which comprises both type A and type B dielectric layers, has a significantly smaller TKC than the capacitor 1 with only one type of dielectric layers.", "The invention is not limited to the exemplary embodiments shown; rather, it is defined in its most general form in claim 1." ] ]

[ [ "Ceramic heater and ceramic joined article", "A ceramic heater capable of stably supporting a semiconductor safer and evenly heating the whole of a semiconductor wafer or the like without generating any warp in the semiconductor wafer or the like.", "The ceramic heater includes a disk-like ceramic substrate, a heating element formed on a surface of or inside the ceramic substrate, and through holes for letting lifter pins pass through the ceramic substrate.", "The number of the formed through holes is three or more, and the through holes are formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate." ], [ "1.A ceramic heater comprising: a disk-like ceramic substrate; a heating element formed on a surface of or inside said ceramic substrate; and through holes for letting lifter pins pass through at said ceramic substrate, wherein three or more of said through holes are formed, and said through holes are formed in an area whose distance from the center of said ceramic substrate is ½ of more of the distance from the center of said ceramic substrate to the outer edge of said ceramic substrate.", "2.The ceramic heater according to claim 1, wherein said through holes are formed at substantially regular intervals on a single circle which has a concentric circle relationship with said ceramic substrate.", "3.A ceramic heater comprising: a disk-like ceramic substrate; a heating element formed on a surface of or inside said ceramic substrate; and through holes for letting lifter pins pass through at said ceramic substrate, wherein the diameter of each of said through holes on a heating face side for heating an object to be heated is larger than the diameter of said through hole on the side opposite to said heating face.", "4.The ceramic heater according to claim 3, wherein each of said through holes comprises a columnar portion and a diameter-increasing portion, the diameter of said diameter-increasing portion becomes larger as the portion is closer to the heating face.", "5.The ceramic heater according to claim 3, wherein the diameter on the heating face side of each of said through holes is from 1.2 to 10 times as large as the diameter on the side opposite to said heating face of said through holes.", "6.A ceramic bonded body comprising: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of said ceramic substrate, wherein the center of an area surrounded by the interface between said ceramic body and said ceramic substrate, or the center of an area constituted by the interface between said ceramic body and said ceramic substrate is 3 to 200 μm apart from the center of the bottom face of said ceramic substrate.", "7.A ceramic bonded body comprising: a disk-like ceramic substrate inside which a conductor is provided; and a cylindrical ceramic body having a cylindrical shape bonded to the bottom face of said ceramic substance, wherein the center of the circle surrounded by the interface between said cylindrical ceramic body and said ceramic substrate is 3 to 200 μm apart from the center of the bottom face of said ceramic substrate.", "8.The ceramic bonded body according to claim 6, wherein said conductor is a heating element, and functions as a hot plate.", "9.The ceramic bonded body according to claim 6, wherein said conductor is an electrostatic electrode, and functions as an electrostatic chuck.", "10.The ceramic bonded body according to claim 6, wherein said ceramic substrate has a diameter of 250 mm or more.", "11.The ceramic heater according to claim 4, wherein the diameter on the heating face side of each of said through holes is from 1.2 to 10 times as large as the diameter on the side opposite to said heating face of said through holes.", "12.The ceramic bonded body according to claim 7, wherein said conductor is a heating element, and functions as a hot plate.", "13.The ceramic bonded body according to claim 7, wherein said conductor is an electrostatic electrode, and functions as an electrostatic chuck.", "14.The ceramic bonded body according to claim 7, wherein said ceramic substrate has a diameter of 250 mm or more." ], [ "<SOH> BACKGROUND ART <EOH>Conventionally, a heater, wafer prober or the like wherein a base material made of a metal such as stainless steel or aluminum alloy is used has been used in semiconductor producing/inspecting devices and so on, examples of which include an etching device and a chemical vapor phase growth device and the like.", "However, such a heater made of metal has the following problems.", "First, the thickness of the heater plate must be as thick as about 15 mm since the heater is made of a metal.", "Because in a thin metal plate, a bend, a strain and so on are generated on the basis of thermal expansion resulting from heating so that a silicon wafer put on the metal plate is damaged or inclined.", "However, if the thickness of the heater plate is made thick, a problem that the heater becomes heavy and bulky arises.", "The temperature of a face for heating an object to be heated such as a silicon wafer (referred to as a heating face hereinafter) and the like is controlled by changing the voltage or current quantity applied to the heating elements.", "However, since the metal plate is thick, the temperature of the heater plate does not follow the change in the voltage or current quantity promptly.", "Thus, a problem that the temperature is not easily controlled is caused.", "Thus, JP Kokai Hei 11-40330 and so forth suggest a ceramic substrate wherein a nitride ceramic or a carbide ceramic, with a high thermal conductivity and a high strength, is used as a substrate and heating elements formed by sintering metal particles are provided on a surface of a plate-form body made of such a ceramic.", "As illustrated in FIG.", "15 , usually, in such a ceramic heater, heating elements 62 are formed inside a ceramic substrate 61 and further through holes for passing lifter pins through are formed in the vicinity of the center.", "This is because by letting the lifter pins pass through the through holes 65 and then moving the pins up and down, a semiconductor wafer can be relatively easily received from the previous line or the semiconductor wafer can be carried to the next line.", "Reference numeral 64 represents bottomed holes 64 for embedding temperature measuring elements such as thermocouples, and reference numeral 63 represents external terminals for connecting the heating elements 62 to a power source.", "As described above, the through holes 65 are arranged in the vicinity of the center of the ceramic substrate 61 .", "This is because in order to work the lifter pins by means of one motor, it is preferred that the positions of the lifter pins are closer to each other.", "In the case that this ceramic heater 60 is used to heat an object to be heated, such as a semiconductor wafer and the like, the temperature distribution in the surface of the ceramic heater 60 is reflected on the semiconductor wafer or the like if the object is heated in the state that the object contacts the heating face of the ceramic heater 10 .", "As a result, it is difficult that the semiconductor wafer or the like is evenly heated.", "In order that the surface temperature of the ceramic heater 60 is made to heat the semiconductor wafer or the like evenly, highly complicated control is required.", "Hence, the temperature control is not easy.", "Thus, when the semiconductor wafer is heated, there can be usually used a method of supporting the semiconductor wafer by means of the lifter pins provided for carrying the semiconductor wafer.", "That is, the lifters pins are held in the state that they project slightly from the surface of the ceramic substrate 61 , and the lifter pins are used to support the semiconductor wafer in the state that the semiconductor wafer is a given distance apart from the surface of the ceramic substrate 61 .", "The semiconductor wafer is then heated.", "According to this method of using the lifter pins, the semiconductor wafer is heated by radiation from the ceramic substrate 61 or convection current since the semiconductor wafer is held in the state that the semiconductor wafer is apart from the surface of the ceramic heater 61 by the given distance.", "Accordingly, the temperature distribution in the surface of the ceramic substrate 61 is not usually reflected directly on the semiconductor wafer, so that the semiconductor wafer is more evenly heated and any temperature distribution is not easily generated in the semiconductor wafer.", "However, when the lifter pins are used to intend to carry the semiconductor wafer, the semiconductor wafer may not be stably supported.", "In this case, there arises a problem that the semiconductor wafer is inclined to get out of position.", "At the time when a semiconductor wafer or a liquid crystal substrate is put thereon and heated (as well as the time of the state that a semiconductor wafer or a liquid crystal substrate is put on the heated ceramic substrate and until the temperature thereof returns to the original temperature, that is, a transition state), there is caused a problem that temperature difference is generated in the object to be heated, such as the semiconductor wafer or the liquid crystal substrate.", "Moreover, a problem that free particles adhere to the object to be heated is also encountered.", "Thus, the present inventors analyzed a cause for which an object to be heated, such a semiconductor wafer or a liquid crystal substrate, (which may be referred to as a semiconductor wafer or the like hereinafter), is inclined or temperature unevenness is generated when the semiconductor wafer or the like is heated.", "As a result, it has been found out that in the case that the lifter pins concentrate around the center, the object to be heated, such as the semiconductor wafer cannot be stably supported and the heat capacity per unit area (volume) in the central portion of the ceramic substrate 61 gets smaller than that in the peripheral portion so that the temperature of the central portion of the ceramic substrate 61 rises easily at the time of heating the ceramic substrate.", "It has also been found out that if the lifter pins are present in the vicinity of the center when a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, is lifted up, the object warps and hence the peripheral portion of the object contacts the ceramic substrate 61 so that free particles are generated.", "When the ceramic heater 60 having these through holes 65 is used to heat an object to be heated such as a semiconductor wafer or a liquid crystal substrate, the temperature in the vicinity of the through holes 65 locally becomes low.", "That is, a cooling spot is generated.", "This results in a problem that the temperature of the semiconductor wafer, the liquid crystal substrate or the like falls in this portion so that the semiconductor wafer, the liquid crystal substrate or the like is not easily heated evenly.", "Furthermore, JP Kokai Hei 4-324276 suggests a ceramic heater in which aluminum nitride, which is a non-oxide ceramic having a high thermal conductivity and a large strength, is used as a substrate; heating elements and conductor filled through holes made of tungsten are made in this aluminum nitride substrate; and Nichrome wires as external terminals are brazed thereto.", "Since such a ceramic heater has a ceramic substrate having a large mechanical strength at high temperatures, the thickness of the ceramic substrate can be made small to make the heat capacity small.", "As a result, the temperature of the ceramic substrate can be caused to follow change in the voltage or electric current quantity promptly.", "A ceramic heater as described above adopts a means for bonding a cylindrical ceramic with a disk-like ceramic to protect wires such as external terminals from reactive gas, halogen gas and the like used in a semiconductor producing step, as described in Japanese Patent gazette Nos.", "2525974 and 2783980, and JP Kokai 2000-114355.However, in the case that the ceramic heater described in Japanese Patent gazette No.", "2525974 is used, it is exposed to reactive gas, halogen gas and the like for a long time and thermal stress concentrates on the bonding interface between the cylindrical ceramic and the disk-like ceramic, (which may be referred to as the interface hereinafter).", "Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated, thereby causing a problem that cracks and the like are generated in the interface and air-tightness of the interface deteriorates so that the wires such as the external terminals are corroded.", "In the ceramic heater described in Japanese Patent gazette No.", "2783980, in the interface thereof, the ceramic particles grow to extend to both sides of the interface, whereby the cylindrical ceramic is bonded to the disk-like ceramic.", "Therefore, the bonding strength of the interface is strong but thermal stress concentrates locally.", "Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated so that cracks and the like may be generated in the interface, the cylindrical ceramic, or the disk-like ceramic.", "For recent semiconductor products, it is required to shorten a time necessary for throughput.", "Thus, it is strongly required to shorten a time for rising or dropping the temperature thereof.", "However, in the ceramic heaters described in Japanese Patent gazette No.", "2525974, JP Kokai Hei 2000-114355 and so forth, a flange portion is formed in the cylindrical ceramic, thereby causing a problem that the thermal capacity increases and temperature-rising speed drops.", "In order to shorten the temperature-rising time, it is necessary to increase the temperature-rising speed.", "In order to shorten the temperature-dropping time, it is necessary to rise the temperature-dropping speed.", "However, if the temperature of the ceramic heater is abruptly risen or dropped, larger thermal stress is generated in the interface and the like.", "Thus, cracks and the like as described above become easy to be generated increasingly." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The present invention has been made in view of the above-mentioned problems.", "An object thereof is to provide a ceramic heater capable of stably supporting an object to be heated, such as a semiconductor wafer, a liquid crystal substrate and the like, and evenly heating the semiconductor wafer or the like.", "Another object of the present invention is to provide a ceramic heater which prevents the generation of cooling spots, and causes no drop in the temperature of a semiconductor wafer, a liquid crystal substrate and the like in the vicinity of through holes formed in a ceramic substrate so that the object to be heated, such as the semiconductor wafer, the liquid crystal substrate and the like can be evenly heated.", "Still another object of the present invention is to provide a ceramic bonded body capable of keeping sufficient air-tightness and largely improving the reliability thereof because of the face that no thermal stress concentrates locally in the bonding interface between a ceramic body having a given shape such as a cylindrical shape or a columnar shape and a disk-like ceramic so that no cracks and the like are generated in this portion.", "In order to attain the above-mentioned objects, a ceramic heater according to the first aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein three or more of the above-mentioned through holes are formed, and the above-mentioned through holes are formed in an area whose distance from the center of the above-mentioned ceramic substrate is ½ or more of the distance from the center of the above-mentioned ceramic substrate to the outer edge of the above-mentioned ceramic substrate.", "According to the above-mentioned ceramic heater, the three or more through holes are present in the peripheral portion of the ceramic substrate; therefore, the lifter pins, which are passed through the through holes, are also present in the peripheral portion of the ceramic substrate and do not concentrate in the central portion so that a semiconductor wafer or the like supported by the lifter pins does not become unstable.", "As a result, even if impact and the like are caused when the ceramic heater is used, the semiconductor wafer or the like does not easily get out of position.", "Thus, an object to be heated, such as the semiconductor wafer, can be stably supported by the lifter pins.", "In the case that the semiconductor wafer or the like is heated to rise the temperature thereof, a difference in the thermal capacity per unit area (volume) between the central portion of the ceramic substrate and the peripheral portion thereof turns into a substantially negligible degree.", "As a result, the thermal capacity per unit volume (area) in the central portion of the ceramic substrate becomes almost equivalent to that in the peripheral portion.", "Thus, the semiconductor wafer or the like can be evenly heated even at the temperature-rising time thereof (at the transition time).", "Furthermore, in the case that the through holes are present in the vicinity of the center, a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, warps when the plate-form object is pushed up by the lifter pins.", "Thus, the outer circumference of the plate-form object scrubs the ceramic substrate surface so that free particles are generated.", "However, according to the ceramic heater of the first aspect of the present invention, such a problem is not caused.", "It is desired that the through holes are formed at substantially regular intervals on a single circle having a concentric circle relationship with the above-mentioned ceramic substrate.", "Since the lifter pins passed through the through holes are widely dispersed on the ceramic substrate and are arranged at regular intervals, a semiconductor wafer or the like can be more stably supported.", "Moreover, the semiconductor wafer or the like can be kept more horizontally so that the distance between the ceramic substrate and the semiconductor wafer or the like is made constant.", "As a result, the semiconductor wafer or the like can be more evenly heated.", "A ceramic heater according to a second aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face.", "In case the ceramic heater is provided with the through holes for passing the lifter pins through, the temperature around side walls of the through holes drops since the side walls of the through holes usually contact gas having a lower temperature than the substrate itself.", "As a result, cooling spots are generated in the heating face.", "When a semiconductor wafer, a liquid crystal substrate or the like is put on this ceramic heater, in the cooling spots heat is taken away by the cooling spots; therefore, the temperature in this portion drops so that evenness in the temperature of the semiconductor wafer, the liquid crystal substrate and the like is lost.", "However, according to the ceramic heater of the second aspect of the present invention, the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face; thus, no solid constituting the substrate is present in portions where cooling spots are generated.", "As a result, the occupation ratio of space becomes large so that the heat capacity thereof gets small.", "Accordingly, the temperature of the semiconductor wafer, the liquid crystal substrate and the like of the portion in the vicinity of the formed through holes hardly drops, so that the object to be heated, such as the semiconductor wafer or the liquid crystal substrate and the like, can be more evenly heated.", "Regarding the through holes with a diameter on the heating face side being larger than that on the bottom face side, in case the through holes are constituted so as to have a columnar portion and a diameter-increasing portion having the diameter becoming larger as the portion is closer to the heating face, that is, so as to have a funnel shape, gas having accumulated heat remains in the funnel-shaped portion and cooling spots themselves are not enlarged.", "Thus, the object to be heated, such as the semiconductor wafer or the liquid crystal substrate, can be more evenly heated.", "Since the through holes having the above-mentioned shape can be relatively easily formed with a drill and the like, the through holes can be effectively formed.", "The wafer or the like is heated while the space of the above-mentioned diameter-increasing portion is not filled.", "This is because when the diameter-increasing portion is filled with a filling member, the ceramic and the filling member are rubbed with each other so that free particles are generated.", "Further, a ceramic bonded body according to a third aspect of the present invention includes: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the above-mentioned ceramic substrate, wherein the center of an area surrounded by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate or the center of an area constituted by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the above-mentioned ceramic substrate.", "In the ceramic bonded body according to the third aspect of the present invention, the ceramic body may be a columnar body or a plate-form body, or may be a hollow body such as a cylindrical body, or may be a filled body having a ceramic-filled structure, which has no cavity inside.", "FIG.", "28 is a sectional view which schematically illustrates a ceramic bonded body 700 using a ceramic body 281 made of a filled body.", "In the ceramic body 281 made of the filled body, external terminals 283 having sockets 285 , and conductive wires 235 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded.", "FIG.", "29 is a sectional view which schematically illustrates a ceramic bonded body 800 using a ceramic body 381 made of a plate-form body.", "In the ceramic body 381 made of the filled body, external terminals 383 having sockets 385 , and conductive wires 335 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded.", "In the case of the columnar body, it may be a triangle pole body 150 or a square pole body 160 or may be a polygonal pole body 170 , as illustrated in FIGS.", "30 (a) to 30 (c).", "In the third aspect of the present invention, the center of the area surrounded by the interface between the ceramic body and the ceramic substrate, or the center of the area constituted by the interface between the ceramic body and the ceramic substrate means the centroid of a figure constituted by being surrounded by the interface, or the centroid of a figure constituted by the interface itself.", "The centroid is defined as intersection points of straight lines for dividing a figure into two exact halves.", "In the case of a circle, the center of the circle is the centroid.", "The most preferred example of the present invention is a ceramic bonded body including: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the ceramic substrate, wherein the center of a circle surrounded by the interface between the cylindrical ceramic body and the ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate.", "Thus, this ceramic bonded body will be described hereinafter.", "For example, in the case of heating a ceramic bonded body wherein the center of a circle surrounded by the interface between a cylindrical ceramic body and a ceramic substrate, (which may be referred to the center A hereinafter), is consistent with the center of the bottom face of the ceramic substrate, (which may be referred to as the center B hereinafter), the direction along which the cylindrical ceramic body expands is consistent with the direction along which the ceramic substrate expands in the above-mentioned interface.", "As a result, thermal stress concentrates locally so that thermal fatigue is generated.", "Thus, cracks and the like are generated.", "However, according to the third aspect of the present invention, that is, the ceramic bonded body wherein the distance between the center A and the center B, (which may be referred to as the distance L), is from 3 to 200 μm apart, when it is heated, the direction along which the cylindrical ceramic body expands is different from the direction along which the ceramic substrate expands.", "As a result, thermal stress can be dispersed so that the generation of cracks and the like can be prevented.", "In the ceramic bonded body having a distance L of less than 3 μm, it is difficult to disperse thermal stress sufficiently.", "If the distance L exceeds 200 μm, thermal stress is conversely concentrated so that cracks are easily generated.", "Furthermore, the temperature distribution in the face for heating a semiconductor wafer gets large.", "It is desired that the above-mentioned conductor is a heating element and the ceramic bonded body functions as a ceramic heater.", "As described above, this ceramic bonded body has a structure capable of dispersing thermal stress so that the thermal stress does not concentrate locally.", "Thus, even if temperature-rising and temperature-dropping thereof are repeated, no thermal fatigue is generated.", "In the ceramic bonded body, a flange portion need not be formed in the bonding face between the cylindrical ceramic body and the ceramic substrate.", "Therefore, the thermal capacity does not increase so that the temperature-dropping speed is not lowered.", "For this reason, the ceramic bonded body can be suitably used as a ceramic heater.", "The heating element may be formed into a layer form or into a line form.", "Moreover, it is desired that the above-mentioned conductor is an electrostatic electrode and the above-mentioned ceramic bonded body functions as an electrostatic chuck.", "This is because any electrostatic chuck is used in a corrosive atmosphere in many cases and a constitution wherein the ceramic substrate and the cylindrical ceramic body are bonded to each other as described above is optimal for this chuck.", "Additionally, the ceramic substrate desirably has a diameter of 250 mm or more.", "If the diameter of the ceramic substrate is 250 mm or more, the effects of the third aspect of the present invention, i.e., the effects of dispersing thermal stress and preventing the generation of cracks and the like get larger.", "This fact can easily be understood from FIG.", "32 , which shows results of Examples.", "That is, in the case of the distance L=0, the generation rate of the cracks is higher as the diameter is larger.", "When the diameter exceeds 250 mm, the rate abruptly becomes large.", "However, by setting L to 3 μm or 200 μm, the crack generation rate can be suppressed at a low level." ], [ "TECHNICAL FIELD The present invention mainly relates to a ceramic heater and a ceramic bonded body which are used in the production and inspection of semiconductors, the field of optics, and so on.", "BACKGROUND ART Conventionally, a heater, wafer prober or the like wherein a base material made of a metal such as stainless steel or aluminum alloy is used has been used in semiconductor producing/inspecting devices and so on, examples of which include an etching device and a chemical vapor phase growth device and the like.", "However, such a heater made of metal has the following problems.", "First, the thickness of the heater plate must be as thick as about 15 mm since the heater is made of a metal.", "Because in a thin metal plate, a bend, a strain and so on are generated on the basis of thermal expansion resulting from heating so that a silicon wafer put on the metal plate is damaged or inclined.", "However, if the thickness of the heater plate is made thick, a problem that the heater becomes heavy and bulky arises.", "The temperature of a face for heating an object to be heated such as a silicon wafer (referred to as a heating face hereinafter) and the like is controlled by changing the voltage or current quantity applied to the heating elements.", "However, since the metal plate is thick, the temperature of the heater plate does not follow the change in the voltage or current quantity promptly.", "Thus, a problem that the temperature is not easily controlled is caused.", "Thus, JP Kokai Hei 11-40330 and so forth suggest a ceramic substrate wherein a nitride ceramic or a carbide ceramic, with a high thermal conductivity and a high strength, is used as a substrate and heating elements formed by sintering metal particles are provided on a surface of a plate-form body made of such a ceramic.", "As illustrated in FIG.", "15, usually, in such a ceramic heater, heating elements 62 are formed inside a ceramic substrate 61 and further through holes for passing lifter pins through are formed in the vicinity of the center.", "This is because by letting the lifter pins pass through the through holes 65 and then moving the pins up and down, a semiconductor wafer can be relatively easily received from the previous line or the semiconductor wafer can be carried to the next line.", "Reference numeral 64 represents bottomed holes 64 for embedding temperature measuring elements such as thermocouples, and reference numeral 63 represents external terminals for connecting the heating elements 62 to a power source.", "As described above, the through holes 65 are arranged in the vicinity of the center of the ceramic substrate 61.This is because in order to work the lifter pins by means of one motor, it is preferred that the positions of the lifter pins are closer to each other.", "In the case that this ceramic heater 60 is used to heat an object to be heated, such as a semiconductor wafer and the like, the temperature distribution in the surface of the ceramic heater 60 is reflected on the semiconductor wafer or the like if the object is heated in the state that the object contacts the heating face of the ceramic heater 10.As a result, it is difficult that the semiconductor wafer or the like is evenly heated.", "In order that the surface temperature of the ceramic heater 60 is made to heat the semiconductor wafer or the like evenly, highly complicated control is required.", "Hence, the temperature control is not easy.", "Thus, when the semiconductor wafer is heated, there can be usually used a method of supporting the semiconductor wafer by means of the lifter pins provided for carrying the semiconductor wafer.", "That is, the lifters pins are held in the state that they project slightly from the surface of the ceramic substrate 61, and the lifter pins are used to support the semiconductor wafer in the state that the semiconductor wafer is a given distance apart from the surface of the ceramic substrate 61.The semiconductor wafer is then heated.", "According to this method of using the lifter pins, the semiconductor wafer is heated by radiation from the ceramic substrate 61 or convection current since the semiconductor wafer is held in the state that the semiconductor wafer is apart from the surface of the ceramic heater 61 by the given distance.", "Accordingly, the temperature distribution in the surface of the ceramic substrate 61 is not usually reflected directly on the semiconductor wafer, so that the semiconductor wafer is more evenly heated and any temperature distribution is not easily generated in the semiconductor wafer.", "However, when the lifter pins are used to intend to carry the semiconductor wafer, the semiconductor wafer may not be stably supported.", "In this case, there arises a problem that the semiconductor wafer is inclined to get out of position.", "At the time when a semiconductor wafer or a liquid crystal substrate is put thereon and heated (as well as the time of the state that a semiconductor wafer or a liquid crystal substrate is put on the heated ceramic substrate and until the temperature thereof returns to the original temperature, that is, a transition state), there is caused a problem that temperature difference is generated in the object to be heated, such as the semiconductor wafer or the liquid crystal substrate.", "Moreover, a problem that free particles adhere to the object to be heated is also encountered.", "Thus, the present inventors analyzed a cause for which an object to be heated, such a semiconductor wafer or a liquid crystal substrate, (which may be referred to as a semiconductor wafer or the like hereinafter), is inclined or temperature unevenness is generated when the semiconductor wafer or the like is heated.", "As a result, it has been found out that in the case that the lifter pins concentrate around the center, the object to be heated, such as the semiconductor wafer cannot be stably supported and the heat capacity per unit area (volume) in the central portion of the ceramic substrate 61 gets smaller than that in the peripheral portion so that the temperature of the central portion of the ceramic substrate 61 rises easily at the time of heating the ceramic substrate.", "It has also been found out that if the lifter pins are present in the vicinity of the center when a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, is lifted up, the object warps and hence the peripheral portion of the object contacts the ceramic substrate 61 so that free particles are generated.", "When the ceramic heater 60 having these through holes 65 is used to heat an object to be heated such as a semiconductor wafer or a liquid crystal substrate, the temperature in the vicinity of the through holes 65 locally becomes low.", "That is, a cooling spot is generated.", "This results in a problem that the temperature of the semiconductor wafer, the liquid crystal substrate or the like falls in this portion so that the semiconductor wafer, the liquid crystal substrate or the like is not easily heated evenly.", "Furthermore, JP Kokai Hei 4-324276 suggests a ceramic heater in which aluminum nitride, which is a non-oxide ceramic having a high thermal conductivity and a large strength, is used as a substrate; heating elements and conductor filled through holes made of tungsten are made in this aluminum nitride substrate; and Nichrome wires as external terminals are brazed thereto.", "Since such a ceramic heater has a ceramic substrate having a large mechanical strength at high temperatures, the thickness of the ceramic substrate can be made small to make the heat capacity small.", "As a result, the temperature of the ceramic substrate can be caused to follow change in the voltage or electric current quantity promptly.", "A ceramic heater as described above adopts a means for bonding a cylindrical ceramic with a disk-like ceramic to protect wires such as external terminals from reactive gas, halogen gas and the like used in a semiconductor producing step, as described in Japanese Patent gazette Nos.", "2525974 and 2783980, and JP Kokai 2000-114355.However, in the case that the ceramic heater described in Japanese Patent gazette No.", "2525974 is used, it is exposed to reactive gas, halogen gas and the like for a long time and thermal stress concentrates on the bonding interface between the cylindrical ceramic and the disk-like ceramic, (which may be referred to as the interface hereinafter).", "Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated, thereby causing a problem that cracks and the like are generated in the interface and air-tightness of the interface deteriorates so that the wires such as the external terminals are corroded.", "In the ceramic heater described in Japanese Patent gazette No.", "2783980, in the interface thereof, the ceramic particles grow to extend to both sides of the interface, whereby the cylindrical ceramic is bonded to the disk-like ceramic.", "Therefore, the bonding strength of the interface is strong but thermal stress concentrates locally.", "Thus, by repeating temperature-rising and temperature-dropping thereof, thermal fatigue is generated so that cracks and the like may be generated in the interface, the cylindrical ceramic, or the disk-like ceramic.", "For recent semiconductor products, it is required to shorten a time necessary for throughput.", "Thus, it is strongly required to shorten a time for rising or dropping the temperature thereof.", "However, in the ceramic heaters described in Japanese Patent gazette No.", "2525974, JP Kokai Hei 2000-114355 and so forth, a flange portion is formed in the cylindrical ceramic, thereby causing a problem that the thermal capacity increases and temperature-rising speed drops.", "In order to shorten the temperature-rising time, it is necessary to increase the temperature-rising speed.", "In order to shorten the temperature-dropping time, it is necessary to rise the temperature-dropping speed.", "However, if the temperature of the ceramic heater is abruptly risen or dropped, larger thermal stress is generated in the interface and the like.", "Thus, cracks and the like as described above become easy to be generated increasingly.", "SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems.", "An object thereof is to provide a ceramic heater capable of stably supporting an object to be heated, such as a semiconductor wafer, a liquid crystal substrate and the like, and evenly heating the semiconductor wafer or the like.", "Another object of the present invention is to provide a ceramic heater which prevents the generation of cooling spots, and causes no drop in the temperature of a semiconductor wafer, a liquid crystal substrate and the like in the vicinity of through holes formed in a ceramic substrate so that the object to be heated, such as the semiconductor wafer, the liquid crystal substrate and the like can be evenly heated.", "Still another object of the present invention is to provide a ceramic bonded body capable of keeping sufficient air-tightness and largely improving the reliability thereof because of the face that no thermal stress concentrates locally in the bonding interface between a ceramic body having a given shape such as a cylindrical shape or a columnar shape and a disk-like ceramic so that no cracks and the like are generated in this portion.", "In order to attain the above-mentioned objects, a ceramic heater according to the first aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein three or more of the above-mentioned through holes are formed, and the above-mentioned through holes are formed in an area whose distance from the center of the above-mentioned ceramic substrate is ½ or more of the distance from the center of the above-mentioned ceramic substrate to the outer edge of the above-mentioned ceramic substrate.", "According to the above-mentioned ceramic heater, the three or more through holes are present in the peripheral portion of the ceramic substrate; therefore, the lifter pins, which are passed through the through holes, are also present in the peripheral portion of the ceramic substrate and do not concentrate in the central portion so that a semiconductor wafer or the like supported by the lifter pins does not become unstable.", "As a result, even if impact and the like are caused when the ceramic heater is used, the semiconductor wafer or the like does not easily get out of position.", "Thus, an object to be heated, such as the semiconductor wafer, can be stably supported by the lifter pins.", "In the case that the semiconductor wafer or the like is heated to rise the temperature thereof, a difference in the thermal capacity per unit area (volume) between the central portion of the ceramic substrate and the peripheral portion thereof turns into a substantially negligible degree.", "As a result, the thermal capacity per unit volume (area) in the central portion of the ceramic substrate becomes almost equivalent to that in the peripheral portion.", "Thus, the semiconductor wafer or the like can be evenly heated even at the temperature-rising time thereof (at the transition time).", "Furthermore, in the case that the through holes are present in the vicinity of the center, a plate-form object to be heated, such as a semiconductor wafer or a liquid crystal substrate, warps when the plate-form object is pushed up by the lifter pins.", "Thus, the outer circumference of the plate-form object scrubs the ceramic substrate surface so that free particles are generated.", "However, according to the ceramic heater of the first aspect of the present invention, such a problem is not caused.", "It is desired that the through holes are formed at substantially regular intervals on a single circle having a concentric circle relationship with the above-mentioned ceramic substrate.", "Since the lifter pins passed through the through holes are widely dispersed on the ceramic substrate and are arranged at regular intervals, a semiconductor wafer or the like can be more stably supported.", "Moreover, the semiconductor wafer or the like can be kept more horizontally so that the distance between the ceramic substrate and the semiconductor wafer or the like is made constant.", "As a result, the semiconductor wafer or the like can be more evenly heated.", "A ceramic heater according to a second aspect of the present invention comprises: a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face.", "In case the ceramic heater is provided with the through holes for passing the lifter pins through, the temperature around side walls of the through holes drops since the side walls of the through holes usually contact gas having a lower temperature than the substrate itself.", "As a result, cooling spots are generated in the heating face.", "When a semiconductor wafer, a liquid crystal substrate or the like is put on this ceramic heater, in the cooling spots heat is taken away by the cooling spots; therefore, the temperature in this portion drops so that evenness in the temperature of the semiconductor wafer, the liquid crystal substrate and the like is lost.", "However, according to the ceramic heater of the second aspect of the present invention, the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face; thus, no solid constituting the substrate is present in portions where cooling spots are generated.", "As a result, the occupation ratio of space becomes large so that the heat capacity thereof gets small.", "Accordingly, the temperature of the semiconductor wafer, the liquid crystal substrate and the like of the portion in the vicinity of the formed through holes hardly drops, so that the object to be heated, such as the semiconductor wafer or the liquid crystal substrate and the like, can be more evenly heated.", "Regarding the through holes with a diameter on the heating face side being larger than that on the bottom face side, in case the through holes are constituted so as to have a columnar portion and a diameter-increasing portion having the diameter becoming larger as the portion is closer to the heating face, that is, so as to have a funnel shape, gas having accumulated heat remains in the funnel-shaped portion and cooling spots themselves are not enlarged.", "Thus, the object to be heated, such as the semiconductor wafer or the liquid crystal substrate, can be more evenly heated.", "Since the through holes having the above-mentioned shape can be relatively easily formed with a drill and the like, the through holes can be effectively formed.", "The wafer or the like is heated while the space of the above-mentioned diameter-increasing portion is not filled.", "This is because when the diameter-increasing portion is filled with a filling member, the ceramic and the filling member are rubbed with each other so that free particles are generated.", "Further, a ceramic bonded body according to a third aspect of the present invention includes: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the above-mentioned ceramic substrate, wherein the center of an area surrounded by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate or the center of an area constituted by the interface between the above-mentioned ceramic body and the above-mentioned ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the above-mentioned ceramic substrate.", "In the ceramic bonded body according to the third aspect of the present invention, the ceramic body may be a columnar body or a plate-form body, or may be a hollow body such as a cylindrical body, or may be a filled body having a ceramic-filled structure, which has no cavity inside.", "FIG.", "28 is a sectional view which schematically illustrates a ceramic bonded body 700 using a ceramic body 281 made of a filled body.", "In the ceramic body 281 made of the filled body, external terminals 283 having sockets 285, and conductive wires 235 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded.", "FIG.", "29 is a sectional view which schematically illustrates a ceramic bonded body 800 using a ceramic body 381 made of a plate-form body.", "In the ceramic body 381 made of the filled body, external terminals 383 having sockets 385, and conductive wires 335 are embedded, and further a lead wire 890 of a temperature measuring element 84 is also embedded.", "In the case of the columnar body, it may be a triangle pole body 150 or a square pole body 160 or may be a polygonal pole body 170, as illustrated in FIGS.", "30(a) to 30(c).", "In the third aspect of the present invention, the center of the area surrounded by the interface between the ceramic body and the ceramic substrate, or the center of the area constituted by the interface between the ceramic body and the ceramic substrate means the centroid of a figure constituted by being surrounded by the interface, or the centroid of a figure constituted by the interface itself.", "The centroid is defined as intersection points of straight lines for dividing a figure into two exact halves.", "In the case of a circle, the center of the circle is the centroid.", "The most preferred example of the present invention is a ceramic bonded body including: a disk-like ceramic substrate inside which a conductor is provided; and a ceramic body bonded to the bottom face of the ceramic substrate, wherein the center of a circle surrounded by the interface between the cylindrical ceramic body and the ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate.", "Thus, this ceramic bonded body will be described hereinafter.", "For example, in the case of heating a ceramic bonded body wherein the center of a circle surrounded by the interface between a cylindrical ceramic body and a ceramic substrate, (which may be referred to the center A hereinafter), is consistent with the center of the bottom face of the ceramic substrate, (which may be referred to as the center B hereinafter), the direction along which the cylindrical ceramic body expands is consistent with the direction along which the ceramic substrate expands in the above-mentioned interface.", "As a result, thermal stress concentrates locally so that thermal fatigue is generated.", "Thus, cracks and the like are generated.", "However, according to the third aspect of the present invention, that is, the ceramic bonded body wherein the distance between the center A and the center B, (which may be referred to as the distance L), is from 3 to 200 μm apart, when it is heated, the direction along which the cylindrical ceramic body expands is different from the direction along which the ceramic substrate expands.", "As a result, thermal stress can be dispersed so that the generation of cracks and the like can be prevented.", "In the ceramic bonded body having a distance L of less than 3 μm, it is difficult to disperse thermal stress sufficiently.", "If the distance L exceeds 200 μm, thermal stress is conversely concentrated so that cracks are easily generated.", "Furthermore, the temperature distribution in the face for heating a semiconductor wafer gets large.", "It is desired that the above-mentioned conductor is a heating element and the ceramic bonded body functions as a ceramic heater.", "As described above, this ceramic bonded body has a structure capable of dispersing thermal stress so that the thermal stress does not concentrate locally.", "Thus, even if temperature-rising and temperature-dropping thereof are repeated, no thermal fatigue is generated.", "In the ceramic bonded body, a flange portion need not be formed in the bonding face between the cylindrical ceramic body and the ceramic substrate.", "Therefore, the thermal capacity does not increase so that the temperature-dropping speed is not lowered.", "For this reason, the ceramic bonded body can be suitably used as a ceramic heater.", "The heating element may be formed into a layer form or into a line form.", "Moreover, it is desired that the above-mentioned conductor is an electrostatic electrode and the above-mentioned ceramic bonded body functions as an electrostatic chuck.", "This is because any electrostatic chuck is used in a corrosive atmosphere in many cases and a constitution wherein the ceramic substrate and the cylindrical ceramic body are bonded to each other as described above is optimal for this chuck.", "Additionally, the ceramic substrate desirably has a diameter of 250 mm or more.", "If the diameter of the ceramic substrate is 250 mm or more, the effects of the third aspect of the present invention, i.e., the effects of dispersing thermal stress and preventing the generation of cracks and the like get larger.", "This fact can easily be understood from FIG.", "32, which shows results of Examples.", "That is, in the case of the distance L=0, the generation rate of the cracks is higher as the diameter is larger.", "When the diameter exceeds 250 mm, the rate abruptly becomes large.", "However, by setting L to 3 μm or 200 μm, the crack generation rate can be suppressed at a low level.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a bottom face view which schematically illustrates a ceramic heater of a first aspect of the present invention.", "FIG.", "2 is a partially enlarged sectional view of the ceramic heater illustrated in FIG.", "1.FIG.", "3 is a bottom face view which schematically illustrates another example of the ceramic heater according to the first aspect of the present invention.", "FIG.", "4 is a partially enlarged sectional view of the ceramic heater illustrated in FIG.", "3.FIGS.", "5(a) to 5(d) are sectional views which schematically illustrate some parts of the process for producing the ceramic heater illustrated in FIG.", "1.FIGS.", "6(a) to 6(d) are sectional views which schematically illustrate some parts of the process for producing the ceramic heater illustrated in FIG.", "3.FIG.", "7 is a graph showing a relationship between temperature difference when temperature is risen up and the position of a through hole.", "FIG.", "8 is a graph showing a relationship between the number of free particles and the position of a through hole.", "FIG.", "9 is a bottom face view which schematically illustrates a ceramic heater of a second aspect of the present invention.", "FIG.", "10 is a partially enlarged sectional view of the ceramic heater illustrated in FIG.", "9.FIG.", "11 is a bottom face view which schematically illustrates another example of the ceramic heater of the second aspect of the present invention.", "FIG.", "12 is a partially enlarged sectional view of the ceramic heater illustrated in FIG.", "11.FIGS.", "13(a) to 13(d) are sectional views which schematically illustrate some parts of the process for producing the ceramic heater illustrated in FIG.", "9.FIGS.", "14(a) to 14(d) are sectional views which schematically illustrate some parts of the process for producing the ceramic heater illustrated in FIG.", "11.FIG.", "15 is a bottom face view of a conventional ceramic heater.", "FIG.", "16 is a graph showing a relationship between temperature difference (ΔT) in a silicon wafer when being heated with a ceramic heater according to Example 4 and (the diameter of its diameter-increasing portion in its heating face)/(the diameter of its columnar portion).", "FIG.", "17(a) is a plan view which schematically illustrates a ceramic bonded body according to a third aspect of the present invention, and FIG.", "17(b) is a sectional view of the ceramic bonded body illustrated in FIG.", "17(a).", "FIG.", "18 is a bottom face view which schematically illustrates a ceramic bonded body of the third aspect of the present invention.", "FIG.", "19 is a sectional view of the ceramic bonded body of the third aspect of the present invention.", "FIG.", "20 is a partially enlarged sectional view which schematically illustrates a ceramic substrate constituting the ceramic bonded body of the third aspect of the present invention.", "FIG.", "21 is a vertically sectional view which schematically illustrates a ceramic substrate constituting an electrostatic chuck which is an example of the ceramic bonded body of the third aspect of the present invention.", "FIG.", "22 is a partially enlarged sectional view which schematically illustrates a ceramic substrate constituting the electrostatic chuck illustrated in FIG.", "21.FIG.", "23 is a horizontally sectional view which schematically illustrates an example of an electrostatic electrode embedded in the ceramic substrate.", "FIG.", "24 is a horizontally sectional view which schematically illustrates another example of the electrostatic electrode embedded in the ceramic substrate.", "FIG.", "25 is a horizontally sectional view which schematically illustrates still another example of the electrostatic electrode embedded in the ceramic substrate.", "FIGS.", "26(a) to 26(d) are sectional views which schematically illustrate an example of the process for producing a ceramic heater which is an example of the ceramic bonded body of the third aspect of the present invention.", "FIG.", "27 is a perspective view which schematically illustrates an example of the method for bonding a ceramic substrate and a cylindrical ceramic body.", "FIG.", "28 is a sectional view which schematically illustrates an example of the ceramic bonded body of the third aspect of the present invention.", "FIG.", "29 is a sectional view which schematically illustrates an example of the ceramic bonded body of the third aspect of the present invention.", "FIGS.", "30(a) to 30(c) are perspective views which illustrate examples of a columnar body constituting the ceramic bonded body of the third aspect of the present invention.", "FIG.", "31 is a graph showing results of Test Examples.", "FIG.", "32 is a graph showing results of Comparative Examples 9 and 10 and Examples 15 and 16.EXPLANATION OF SYMBOLS 10, 20, 30, 40, 70 ceramic heater 11, 21, 31, 41, 71, 91 ceramic substrate 11a, 21a, 31a, 41a, 71a heating face 11b, 21b, 31b, 41b, 71b bottom face 12, 22, 32, 42, 72 heating element 120, 320 conductor containing paste layer 130, 330 filled layer 13, 23, 33, 43 external terminal 13a, 33a, 73, 73′ conductor filled through hole 13b, 33b, 79 blind hole 14, 24, 34, 44, 74 bottomed hole 15, 25, 35, 45, 75 through hole 16, 26, 36, 46 lifter pin 220, 420 metal covering layer 39, 59 semiconductor wafer 50, 100, 500 green sheet 77 cylindrical ceramic body DETAILED DISCLOSURE OF THE INVENTION First, a ceramic heater according to a first aspect of the present invention will be described.", "The ceramic heater according to a first aspect of the present invention includes a disk-like ceramic substrate; a heating element formed on a surface of or inside the above-mentioned ceramic substrate; and through holes for letting lifter pins pass through at the above-mentioned ceramic substrate, wherein three or more of the above-mentioned through holes are formed, and the above-mentioned through holes are formed in an area whose distance from the center of the above-mentioned ceramic substrate is ½ or more of the distance from the center of the above-mentioned ceramic substrate to the outer edge of the above-mentioned ceramic substrate.", "FIG.", "1 is a bottom face view which schematically illustrates a ceramic heater according to the first aspect of the present invention.", "FIG.", "2 is a partially enlarged sectional view which schematically illustrates the ceramic heater in FIG.", "1.In this ceramic heater, heating elements are formed inside its ceramic substrate.", "In the ceramic heater 10, the ceramic substrate 11 is formed in a disk-like form.", "In order to heat the ceramic heater in such a manner that the temperature in the whole of a heating face 11a of the ceramic heater 10 is made even, the heating elements 12 which has a concentrically circular pattern are formed inside the ceramic substrate 11.Conductor-filled through holes 13a are formed just under ends of heating elements 12, and further blind holes 13b for making the conductor-filled through holes 13a exposed are formed in the bottom face 11b.", "An external terminal 13 is inserted into the blind hole 13b, and they are bonded to each other with a brazing material and the like (not illustrated).", "For example, a socket (not illustrated) having a conductive wire is fitted to the external terminal 13.This conductive wire is connected to a power source and the like.", "In the bottom face of the ceramic substrate 11, bottomed holes 14 for inserting temperature measuring elements (not illustrated) into are formed.", "Furthermore, in the ceramic substrate 11, three through holes 15, for passing lifter pins 16 through, are formed at regular intervals on a circle whose distance from the center of the ceramic substrate 11 is 55% of the distance from the center to the outer edge thereof.", "By moving the lifter pins 16 up and down, a semiconductor wafer or the like can be relatively easily received from the previous line, and the semiconductor wafer or the like can be carried to the next line.", "Since the semiconductor wafer or the like is not rubbed with the ceramic substrate, no free particles are generated from the ceramic substrate.", "When the semiconductor wafer 39 is heated, the semiconductor wafer 39 can be held and heated in the state that the wafer is a given distance apart, through the lifter pins 16, from the heating face 11a of the ceramic substrate 11 by being held by the lifter pins 16 in the state that the pins project slightly from the heating face 11a of the ceramic substrate 11.By arranging a member having projected structures, such as pins, on the surface of the ceramic substrate, the semiconductor wafer or the like can be heated in the state that the semiconductor wafer or the like is apart from the heating face of the ceramic substrate in the same manner as in the case of holding the lifter pins in the state that the lifter pins project slightly from the heating face of the ceramic substrate.", "In the ceramic heater according to the first aspect of the present invention, the heating elements may be formed inside the ceramic substrate or may be formed outside the ceramic substrate.", "FIG.", "3 is a bottom face view which schematically illustrates another example of the ceramic heater according to the first aspect of the present invention.", "FIG.", "4 is a partially enlarged sectional view of the ceramic heater in FIG.", "3.In this ceramic heater, heating elements are formed on a surface of the ceramic substrate.", "In the ceramic heater 20, its ceramic substrate 21 is formed in a disk-like form, and heating elements 22 having a concentrically circular pattern are formed on the surface of the ceramic substrate 21.External terminals 23, which are terminals for input and output, are connected through a metal covering layer 220 to both ends of the heating element.", "In the bottom face of the ceramic substrate 21, bottomed holes 24, for inserting temperature measuring elements (not illustrated) into, are formed.", "Additionally, in the ceramic substrate 21 are formed three through holes 25 at regular intervals on a circle whose distance from the center of the ceramic substrate 21 is 75% of the distance from the center to the outer edge of the ceramic substrate.", "In the same way as in ceramic heater 10, in the ceramic heater 20, lifter pins 26 are passed through the through holes 25 and moved up and down, whereby a semiconductor wafer or the like can be carried.", "Moreover, the lifter pins 26 are caused to project from a heating face 21a of the ceramic substrate 21, whereby the semiconductor wafer 39 is held apart from the ceramic substrate 21a.", "In the ceramic substrate according to the first aspect of the present invention, the number of the through holes formed in the ceramic substrate is three or more.", "If the number of the through holes is less than 3, that is, 2 or less, it is difficult to support an object to be heated, such as the semiconductor wafer, stably by the lifter pins passed through the through holes.", "The number of the through holes is not particularly limited if the number is 3 or more.", "However, the number of the through holes formed in the ceramic substrate is desirably 11 or less in order to suppress the generation of cooling spots when the ceramic heater is used.", "The position of the through hole is not particularly limited if the through hole is formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate.", "However, in order to keep the semiconductor wafer or the like horizontal, the through hole is desirably formed in an area whose distance from the center of the ceramic substrate is from 50 to 75% of the distance from the center to the outer edge thereof.", "In the case that the through hole is formed in an area whose distance from the center of the ceramic substrate exceeds 75% of the distance from the center to the outer edge thereof, the semiconductor wafer or the like can be stably supported but it is apprehended that the semiconductor wafer or the like warps since the central portion thereof is not supported.", "From the viewpoint that the semiconductor wafer or the like can be more stably supported and can be more evenly heated, the through holes are desirably formed at substantially regular intervals on a single circle having a concentric circle relationship with the ceramic substrate.", "Since the lifter pins passed through the through holes are widely dispersed in the ceramic substrate and arranged at regular intervals, the semiconductor wafer or the like can be more stably supported and kept in more horizontal manner so that the distance between the ceramic substrate and the semiconductor wafer or the like becomes constant.", "As a result, the semiconductor wafer or the like can be more evenly heated.", "In the case that three through holes described above are formed, the arrangement of the through holes is, for example, as follows: as illustrated in FIG.", "1, an arrangement in which three through holes 15 are formed at regular intervals on a single circle having a concentric circle relationship with the ceramic substrate in an area whose distance from the center of the ceramic substrate 11 is ½ or more of the distance from the center to the outer edge thereof.", "In the case that four through holes are formed, an example of the arrangement thereof is an arrangement in which four through holes are formed at regular intervals on a single circle having a concentric circle relationship with the ceramic substrate in the same area.", "In the case that four or more through holes are formed in the ceramic substrate, one of the through holes may be formed at the center of the ceramic substrate.", "When the semiconductor wafer or the like is held apart from the ceramic substrate by the lifter pins and heated, the central portion of the semiconductor wafer or the like can be prevented from being warped.", "As a result, the distance between the semiconductor wafer or the like and the ceramic substrate is formed constant so that the semiconductor wafer or the like can be evenly heated.", "When the semiconductor wafer or the like is held apart by the lifter pins and heated, the height of the lifter pins passed through the through holes, projecting from the heating face of the ceramic substrate, is desirably from 5 to 5000 μm, that is, the semiconductor wafer or the like is desirably held in the state that the semiconductor wafer or the like is from 5 to 5000 μm apart from the heating face of the ceramic substrate.", "If the height is less than 5 μm, the temperature of the semiconductor wafer or the like is affected by the temperature distribution in the ceramic substrate and made uneven.", "If the height exceeds 5000 μm, the temperature of the semiconductor wafer or the like does not rise easily.", "As a result, in particular, the temperature of the peripheral portion of the semiconductor wafer or the like becomes low.", "The object to be heated and the heating face of the ceramic substrate are desirably from 5 to 500 μm, more desirably from 20 to 200 μm apart from each other.", "The shape of the through holes and the lifter pins, as viewed in plan, is usually a circular shape.", "About the through holes, their diameter on the heating face side, which heats the object to be heated, may be larger than that on the bottom face side.", "This is because the heat capacity of the portion where cooling spots are easily generated can be largely lowered so that the semiconductor wafer can be more evenly heated.", "Furthermore, the diameter of the through holes is desirably from 1 to 100 mm, more desirably from 1 to 20 mm.", "If the diameter is less than 1 mm, the semiconductor wafer or the like cannot be stably put on the lifter pins since the lifter pins passed through the through holes are too thin.", "On the other hand, if the diameter exceeds 100 mm, cooling spots are generated in the heating face of the ceramic heater since the through holes are too large.", "As a result, it is apprehended that the semiconductor wafer or the like cannot be evenly heated.", "The diameter of the lifter pins is desirably substantially equal to that of the through holes for letting the lifter pins pass through.", "When the diameter of the lifter pins is largely different from that of the through holes, that is, when the diameter of the lifter pins is far smaller than that of the through holes, gaps are generated between the lifter pins and the side walls of the through holes.", "Accordingly, heat is radiated from the gaps so that cooling spots are generated in the heating face of the ceramic heater.", "Thus, it is apprehended that the semiconductor wafer or the like cannot be evenly heated.", "In the ceramic heater according to the first aspect of the present invention, the diameter of the ceramic substrate is desirably 200 mm or more for the following reason: as the diameter of the ceramic heater is larger, the semiconductor wafer or the like having a larger diameter, which warps more easily, can be placed; therefore, the constitution of the first aspect of the present invention functions effectively.", "The diameter of the ceramic substrate is desirably 12 inches (300 mm) or more.", "This size is a size which becomes the main current of semiconductor wafers in the next generation.", "The thickness of the ceramic substrate is desirably 25 mm or less.", "This is because the temperature-following character deteriorates if the thickness of the ceramic substrate exceeds 25 mm.", "The thickness is desirably 0.5 mm or more.", "If the thickness is less than 0.5 mm, the ceramic substrate is more easily broken since the strength itself of the ceramic substrate drops.", "More desirably, the thickness exceeds 1.5 and 5 mm or less.", "If the thickness is more than 5 mm, heat is not easily conducted so that the heating efficiency trends to deteriorate.", "On the other hand, if the thickness is 1.5 mm or less, a temperature distribution is generated in the heating face since heat conducted in the ceramic substrate does not diffuse sufficiently.", "Additionally, the strength of the ceramic substrate drops so that it may be broken.", "In the ceramic heater according to the first aspect of the present invention, it is desired that a bottomed hole extending from the side opposite to the heating face, on which an object to be heated are put, to the heating face is formed in the ceramic substrate and further the bottom of the bottomed hole is formed relatively closer to the heating face than to the heating elements and a temperature measuring element (not illustrated), such as a thermocouple, is fitted to this bottomed hole.", "The distance between the bottom of the bottomed hole and the heating face is desirably from 0.1 mm to ½ of the thickness of the ceramic substrate.", "In this way, a temperature-measuring position is closer to the heating face than the heating elements, so that the temperature of the semiconductor wafer or the like can be more precisely measured.", "If the distance between the bottom of the bottomed hole and the heating face is less than 0.1 mm, heat is radiated at the point so that a temperature distribution is formed in the heating face.", "If the distance exceeds ½, the ceramic substrate is easily affected by the temperature of the heating elements so that temperature-control cannot be performed.", "Thus, a temperature distribution is formed as well in the heating face.", "The diameter of the bottomed hole desirably ranges from 0.3 to 5 mm.", "If the diameter is too large, the heat-radiating property becomes large.", "If the diameter is too small, the workability deteriorates so that the distance to the heating face cannot be made even.", "A plurality of the bottomed holes are desirably arranged symmetrically with the center of the ceramic substrate and arranged into a cross form.", "This is because temperatures in the entire heating face can be measured.", "Examples of the temperature measuring element include a thermocouple, a platinum temperature measuring resistor, a thermistor and the like.", "Examples of the thermocouple include K, R, B, S, E, J and T type thermocouples, as described in JIS-C-1602 (1980).", "Of these, the K type thermocouple is preferred.", "Desirably, the size of the connecting part of the thermocouple is equal to or more than the strand diameter thereof, and is 0.5 mm or less.", "This is because if the connecting part is large, the heat capacity is large so that the responsibility deteriorates.", "It is difficult that the size is made smaller than the strand diameter.", "The above-mentioned temperature measuring element may be bonded to the bottom of the bottomed hole with brazing gold, brazing silver and the like or, the above-mentioned temperature measuring element may be inserted into the bottomed hole followed by the step of sealing the bottomed holes with a heat-resistant resin.", "The two may be used together.", "Examples of the heat-resistant resin include thermosetting resins, in particular, epoxy resin, polyimide resin, bismaleimide-triazine resin and the like.", "These resins may be used alone or in combination of two or more.", "As the above-mentioned brazing gold, desired is at least one selected from an alloy of 37 to 80.5% by weight of Au and 63 to 19.5% by weight of Cu, and an alloy of 81.5 to 82.5% by weight of Au and 18.5 to 17.5% by weight of Ni.", "These have a melting temperature of 900° C. or more, and are not easily melted in high temperature regions.", "Examples of the brazing silver include Ag-Cu types.", "The material of the ceramic substrate constituting the ceramic heater according to the first aspect of the present invention is not particularly limited.", "For example, a nitride ceramic or a carbide ceramic is desirable.", "Since a nitride ceramic or a carbide ceramic has a smaller thermal expansion coefficient than metals and a far higher mechanical strength than metals, the ceramic substrate thereof does not warp or bend even if the thickness thereof is made small.", "Therefore, the ceramic substrate can be made thin and light.", "Since the thermal conductivity of the ceramic substrate is high and the ceramic substrate itself is thin, the surface temperature of the ceramic substrate follows temperature change in the heating elements promptly.", "That is, the surface temperature of the ceramic substrate can be controlled by changing voltage or electric current quantity to change the temperature of the heating elements.", "Examples of the nitride ceramic include aluminum nitride, silicon nitride, boron nitride, titanium nitride and the like.", "These may be used alone or in combination of two or more.", "Examples of the carbide ceramic include silicon carbide, zirconium carbide, titanium carbide, tantalum carbide, tungsten carbide and the like.", "These may be used alone or in combination of two or more.", "Of these, aluminum nitride is most preferred.", "This is because its thermal conductivity is highest, that is, 180 W/m·K and aluminum nitride has superior temperature-following character.", "When a nitride ceramic, a carbide ceramic and the like are used as the ceramic substrate, an insulating layer maybe formed if necessary.", "About a nitride ceramic, the volume resistance value thereof drops easily at high temperatures by solid-solution of oxygen and the like, and a carbide ceramic has an electric conductivity so far as the ceramic is not made into high purity.", "By making the insulating layer, a short circuit is prevented between the circuits at high temperatures or even if it contains impurities.", "Thus, the temperature controllability can be ensured.", "The above-mentioned insulating layer is desirably an oxide ceramic.", "Specifically, silica, alumina, mullite, cordierite, beryllia, and the like can be used.", "Such an insulating layer may be formed by spin-coating the ceramic substrate with a sol solution wherein an alkoxide is hydrolyzed and polymerized, and then drying and firing the solution, or by sputtering, CVD and the like.", "The surface of the ceramic substrate may be subjected to oxidization-treatment to deposit an oxide layer.", "The insulating layer is desirably from 0.1 to 1000 μm in thickness.", "If the thickness is less than 0.1 μm, insulating property cannot be ensured.", "If the thickness exceeds 1000 μm, heat conductivity from the heating elements to the ceramic substrate is hindered.", "Furthermore, the volume resistivity of the insulating layer is desirably 10 or more times larger than that of the above-mentioned ceramic substrate (at the same measurement temperature).", "In the case of less than 10 times, a short circuit between the circuits cannot be prevented.", "It is desired that the ceramic substrate contains carbon and the carbon content is from 200 to 5000 ppm.", "This is because the electrodes can be concealed and black-body radiation is easily used.", "The brightness of the ceramic substrate is desirably N6 or less as a value based on the rule of JIS Z 8721.This is because the ceramic having such a brightness is superior in radiant heat capacity and the property of the concealing.", "The brightness N is defined as follows: the brightness of ideal black is made to 0; that of ideal white is made to 10; respective colors are divided into 10 parts in the manner that the brightness of the respective colors is recognized stepwise between the brightness of black and that of white; and the resultant parts are indicated by symbols N0 to N10, respectively.", "Actual measurement thereof is performed by comparison with color signals corresponding to N0 to N10.One place of decimals in this case is made to 0 or 5.When the heating elements are disposed, they may be formed on the surface (bottom face) of the ceramic substrate or may be embedded in the ceramic substrate.", "When the heating elements are formed inside the ceramic substrate, it is desired that the heating elements are formed at positions having a distance, from the face opposite to the heating face, of 60% or less of the thickness.", "In the case of more than 60%, heat conducted in the ceramic substrate does not diffuse sufficiently since the heating elements are too close to the heating face.", "Thus, a temperature dispersion in the heating face is generated.", "When the heating elements are formed inside the ceramic substrate, a plurality of heating element forming layers may be formed.", "In this case, the patterns of the respective layers are desirably in the state that any one of the heating elements is formed on some layer so as to be complementary to each other and, when viewed from a position above the heating surface, the patterns are formed in all areas.", "An example of such a structure having a staggered relation.", "The heating elements may be set inside the ceramic substrate and the heating elements may be partially formed exposed.", "When the heating elements are formed on the surface of the ceramic substrate, the heating face is desirably on the side opposite to the face on which the heating elements are formed.", "This is because temperature evenness in the heating face can be improved since the ceramic substrate plays a role for thermal diffusion.", "When the heating elements are formed on the surface of the ceramic substrate, the following method is preferred: a method of applying a conductor containing paste which contains metal particles to the surface of the ceramic substrate to form a conductor containing paste layer having a given pattern, and firing this to sinter the metal particles on the surface of the ceramic substrate.", "If the metal particles are melted and adhered to each other and the metal particles and the ceramic are melted and adhered to each other in the sintering of the metal, the sintering is sufficient.", "When the heating elements are formed inside the ceramic substrate, the thickness thereof is preferably from 1 to 50 μm.", "When the heating elements are formed on the surface of the ceramic substrate, the thickness of the heating elements is preferably from 1 to 30 μm, more preferably from 1 to 10 μm.", "When the heating elements are formed inside the ceramic substrate, the width of the heating elements is preferably from 5 to 20 μm.", "When the heating elements are formed on the surface of the ceramic substrate, the width of the heating elements is preferably from 0.1 to 20 mm, more preferably from 0.1 to 5 mm.", "The resistance value of the heating elements can be changed dependently on their width or thickness.", "The above-mentioned ranges are however most practical.", "The resistance value becomes larger as the heating elements become thinner and narrower.", "The thickness and the width of the heating elements become larger in the case that the heating elements are formed inside the ceramic substrate.", "However, when the heating elements are formed inside, the distance between the heating surface and the heating elements becomes short so that the evenness of the temperature in the surface deteriorates.", "Thus, it is necessary to make the width of the heating elements themselves large.", "Since the heating elements are formed inside, it is unnecessary to consider the adhesiveness to a nitride ceramic and the like ceramic.", "Therefore, it is possible to use a high melting point metal such as tungsten or molybdenum, or a carbide of tungsten, molybdenum and the like.", "Thus, the resistance value can be made high.", "Therefore, the thickness itself may be made large in order to prevent wire-snapping and so on.", "For these reasons, the heating elements are desirably formed to have the above-mentioned thickness and width.", "By setting the position where the heating elements are formed in this way, heat generated from the heating elements diffuses to the whole of the ceramic substrate while the heat is conducted.", "Thus, a temperature distribution in the face for heating an object to be heated (a semiconductor wafer or the like) is made even so that the temperatures of respective portions of the object to be heated are made even.", "As the pattern of the heating elements in the ceramic heater according to the first aspect of the present invention, for example, a spiral pattern, an eccentrically circular pattern, a pattern of repeated bending lines and the like can be used, as well as the concentrically circular pattern illustrated in FIG.", "1.These may be used together.", "By making the heating element pattern formed in the outermost circumference into a pattern divided in the circumferential direction, minute temperature control can be attained in the outermost circumference of the ceramic heater, the temperature of which is readily lowered.", "Thus, any distribution in the temperature of the ceramic heater can be suppressed.", "Furthermore, the heating element pattern divided in the circumferential direction may be formed not only in the outermost circumference of the ceramic substrate but also inside it.", "The heating elements may have a rectangular section or an elliptical section.", "They desirably have a flat section.", "From the flat section, heat is more easily radiated toward the heating face.", "Thus, a temperature distribution in the heating face is not easily generated.", "The aspect ratio (the width of the heating element/the thickness of the heating element) of the section is desirably from 10 to 5000.Adjusting the aspect ratio into this range makes it possible to increase the resistance value of the heating elements and keep the evenness of the temperature in the heating face.", "In the case that the thickness of the heating elements is made constant, the amount of heat conducted toward the heating face of the ceramic substrate becomes small if the aspect ratio is smaller than the above-mentioned range.", "Thus, a heat distribution similar to the pattern of the heating elements is generated in the heating face.", "On the other hand, if the aspect ratio is too large, the temperature of the portions just above the centers of the heating elements becomes high so that a heat distribution similar to the pattern of the heating elements is generated in the heating face.", "Accordingly, if temperature distribution is considered, the aspect ratio of the section is preferably from 10 to 5000.When the heating elements are formed on the surface of the ceramic substrate, the aspect ratio is desirably from 10 to 200.When the heating elements are formed inside the ceramic substrate, the aspect ratio is desirably from 200 to 5000.The aspect ratio becomes larger in the case that the heating elements are formed inside the ceramic substrate.", "This is based on the following reason.", "If the heating elements are formed inside, the distance between the heating face and the heating elements becomes short so that temperature evenness in the surface deteriorates.", "It is therefore necessary to make the heating elements themselves flat.", "A conductor containing paste used when the heating elements are formed is not particularly limited.", "Preferably, the paste contains a resin, a solvent, a thickener and the like as well as metal particles or a conductive ceramic for ensuring electric conductivity.", "The above-mentioned metal particles are preferably made of, for example, a noble metal (gold, silver, platinum or palladium), lead, tungsten, molybdenum, nickel and the like.", "Of these, the noble metal (gold, silver, platinum or palladium) is more preferred.", "These may be used alone, but are desirably used in combination of two or more.", "These metals are not relatively easily oxidized, and have a resistance value sufficient for generating heat.", "Examples of the above-mentioned conductive ceramic include carbides of tungsten and molybdenum.", "These may be used alone or in combination of two or more.", "The particle diameter of these metal particles or the conductive ceramic particles is preferably from 0.1 to 100 μm.", "If the particle diameter is too fine, that is, less than0.1 μm, they are easily oxidized.", "On the other hand, if the particle diameter exceeds 100 μm, they are not easily sintered so that the resistance value becomes large.", "The shape of the metal particles may be spherical or scaly.", "When these metal particles are used, they maybe a mixture of spherical particles and scaly particles.", "In the case that the metal particles are scaly or a mixture of spherical particles and scaly particles, metal oxides between the metal particles are easily held and adhesion between the heating elements and the nitride ceramic and the like is made sure.", "Moreover, the resistance value can be made large.", "Thus, this case is profitable.", "Examples of the resin used in the conductor containing paste include epoxy resin, phenol resin and the like.", "Examples of the solvent are isopropyl alcohol and the like.", "Examples of the thickener is cellulose and the like.", "It is desired to add a metal oxide to the metal particles in the conductor-containing paste and form the heating elements up to a sintered body of the metal particles and the metal oxide, as described above.", "By sintering the metal oxide together with the metal particles in this way, the nitride ceramic or the carbide ceramic constituting the ceramic substrate can be closely adhered to the metal particles.", "The reason why the adhesion to the nitride ceramic or the carbide ceramic by mixing the metal oxide is improved is unclear, but would be based on the following.", "The surface of the metal particles, or the surface of the nitride ceramic or the carbide ceramic is slightly oxidized so that an oxidized film is formed.", "Pieces of this oxidized film are sintered and integrated with each other through the metal oxide so that the metal particles and the nitride ceramic or the carbide ceramic are closely adhered to each other.", "A preferred example of the above-mentioned metal oxide is at least one selected from the group consisting of lead oxide, zinc oxide, silica, boron oxide (B2O3), alumina, yttria and titania.", "These oxides make it possible to improve adhesion between the metal particles and the nitride ceramic or the carbide ceramic without increasing the resistance value of the heating elements.", "When the total amount of the metal oxides is set to 100 parts by weight, the weight ratio of lead oxide, zinc oxide, silica, boron oxide (B2O3), alumina, yttria and titania is as follows: lead oxide: 1 to 10, silica: 1 to 30, boron oxide: 5 to 50, zinc oxide: 20 to 70, alumina: 1 to 10, yttria: 1 to 50 and titania: 1 to 50.The weight ratio is desirably adjusted within the scope that the total thereof does not exceed 100 parts by weight.", "By adjusting the amounts of these oxides within these ranges, in particular, adhesion to the nitride ceramic can be improved.", "The amount of the metal oxide added in the metal particles is preferably 0.1% or more by weight and less than 10% by weight.", "As the heating elements, a metal foil or a metal wire may be used.", "As the metal foil, a nickel foil or stainless steel foil formed into heating elements by patterning based on etching and the like is preferred.", "The patterned metal foil may be stuck with a resin film and the like.", "Examples of the metal wire include a tungsten wire and a molybdenum wire.", "The area resistivity when the heating elements are formed is preferably from 1 mΩ/□ to 10 Ω/□.", "If the area resistivity is less than 0.1 Ω/□, the resistivity is too small so that the heat quantity is small.", "Thus, the heating elements do not exhibit its original function easily.", "On the other hand, if the area resistivity exceeds 10 Ω/□, the heat quantity becomes too large for applied voltage quantity.", "Thus, in the ceramic substrate wherein the heating elements are formed on the surface of the ceramic substrate, the heat quantity thereof is not easily controlled.", "From the viewpoint of the control of the heat quantity, the area resistivity of the heating elements is more preferably from 1 to 50 mΩ/□.", "However, if the area resistivity is made large, the pattern width (sectional area) can be made large so that a problem of wire-disconnection is not easily caused.", "Therefore, the area resistivity is preferably set to 50 mΩ/□ as the case may be.", "In the case that the heating elements are formed on the surface of the ceramic substrate 21, a metal covering layer 220 (see FIG.", "4) is preferably formed on the surface of the heating elements.", "The metal covering layer prevents a change in the resistance value based on oxidization of the inner metal sintered body.", "The thickness of the formed metal covering layer 220 is preferably from 0.1 to 10 μm.", "The metal used when the metal covering layer 220 is formed is not particularly limited if the metal is a non-oxidizable metal.", "Specific examples thereof include gold, silver, palladium, platinum, and nickel.", "These may be used alone or in combination of two or more.", "Of these metals, nickel is preferred.", "In the heating element 12, a terminal for connection to a power source is necessary.", "This terminal is fixed to the heating element 12 through solder.", "Nickel prevents thermal diffusion from the solder.", "An example of the connecting terminal is an external terminal 13 made of kovar.", "In the case that the heating elements are formed inside the ceramic substrate, no covering is necessary since the surface of the heating elements is not oxidized.", "In the case that the heating elements are formed inside the ceramic substrate 11, a part of the heating elements may be exposed in the surface.", "It is allowable that conductor filled through holes for connecting the heating elements are formed in the terminal portions and terminals are connected and fixed to the conductor filled through holes.", "In the case that the connecting terminals are connected, an alloy such as silver-lead, lead-tin or bismuth-tin can be used as the solder.", "The thickness of the solder layer is desirably from 0.1 to 50 μm.", "This is because this range is a range sufficient for maintaining connection based on the solder.", "The ceramic heater according to the first aspect of the present invention can be used within the temperature range of 100 to 800° C. A ceramic body having a cylindrical shape or the like may be bonded to the bottom face of the ceramic substrate constituting the ceramic heater according to the first aspect of the present invention so as to protect wires such as external terminals.", "In this way, the wires such as external terminals can be prevented from being corroded by reactive gas, halogen gas and the like.", "Furthermore, the ceramic heater according to the first aspect of the present invention can be used when a liquid crystal substrate is heated.", "The following will describe a process for producing the ceramic heater according to the first aspect of the present invention.", "Firstly, a process for producing a ceramic heater wherein heating elements 12 are formed inside a ceramic substrate 11 (see FIGS.", "1 and 2) will be described on the basis of FIG.", "5.", "(1) Step of Forming the Ceramic Substrate First, powder of a nitride ceramic and the like is mixed with a binder, a solvent and so on to prepare a paste.", "This is used to form green sheets 50.As the above-mentioned ceramic powder, aluminum nitride, and the like can be used.", "If necessary, a sintering aid such as yttria, a compound containing Na or Ca, and the like may be added thereto.", "As the binder, desirable is at least one selected from an acrylic resin binder, ethylcellulose, butylcellosolve, and polyvinyl alcohol.", "As the solvent, desirable is at least one selected from α-terpineol and glycol.", "A paste obtained by mixing these is formed into a sheet form by a doctor blade process, to manufacture green sheets.", "The thickness of the green sheets is preferably from 0.1 to 5 mm.", "(2) Step of Printing a Conductor Containing Paste on the Green Sheet A metal paste or a conductor containing paste containing a conductive ceramic, for forming heating elements 12, is printed on the green sheet 50, so as to form a conductor containing paste layer 120.A conductor containing paste filled layer 130 for conductor filled through holes 13a is formed in through holes.", "The conductor containing paste contains metal particles or conductive ceramic particles.", "The average particle diameter of tungsten particles or molybdenum particles is preferably from 0.1 to 5 μm.", "If the average particle is less than 0.1 μm or exceeds 5 μm, the conductor containing paste is not easily printed.", "Such a conductor containing paste may be a composition (paste) obtained by mixing, for example, 85 to 87 parts by weight of the metal particles or the conductive ceramic particles; 1.5 to 10 to parts by weight of at least one binder selected from acrylic resin binders, ethylcellulose, butylcellosolve and polyvinyl alcohol; and 1.5 to 10 parts by weight of at least one solvent selected from α-terpineol and glycol.", "(3) Step of Laminating the Green Sheets Green sheets 50 on which no conductor containing paste is printed are laminated on the upper and lower sides of the green sheet 50 on which the conductor containing paste is printed (see FIG.", "5(a)).", "At this time, the laminating is performed in such a manner that the green sheet 50 on which the conductor containing paste is printed is arranged at a position which has a distance, from the bottom face, of 60% or less of the thickness of the laminated green sheets.", "Specifically, the number of the green sheets laminated on the upper side is preferably from 20 to 50, and that of the green sheets laminated on the lower side is preferably from 5 to 20.", "(4) Step of Firing the Green Sheet Lamination The green sheet lamination is heated and pressed to sinter the green sheets and the inner conductor containing paste layer.", "The heating temperature is preferably from 1000 to 2000° C., and the pressing pressure is preferably from 10 to 20 MPa.", "The heating is performed in the atmosphere of an inert gas.", "As the inert gas, argon, nitrogen and the like can be used.", "Next, three or more through holes 15, for letting lifter pins 16 for supporting a semiconductor wafer 39 pass through, are formed in the resultant sintered body.", "The holes 15 are formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate.", "The through holes 15 are desirably formed at substantially regular intervals on a single circle having a concentric circle relationship with the ceramic substrate 11 for the following reason: the lifter pins, which are passed through the through holes 15, are widely dispersed on the ceramic substrate 11 and arranged at regular interval; therefore, the semiconductor wafer 39 can be kept more horizontal and the distance between the ceramic substrate 11 and the semiconductor wafer 39 is made constant, so that the semiconductor wafer 39 can be more evenly heated.", "Furthermore, bottomed holes 14, for embedding temperature measuring elements such as thermocouples, are formed in the ceramic substrate (see FIG.", "5(b)).", "Thereafter, blind holes 13 are provided to make the conductor filled through holes 13a, for connecting the heating elements 12 to external terminals 13, exposed (see FIG.", "5(c)).", "The above-mentioned steps of making the bottomed holes and the through holes may be applied to the above-mentioned sheet lamination, but is preferably applied to the above-mentioned sintered body.", "This is because the lamination may be deformed in the sintering step.", "The through holes and the bottomed holes can be formed by grinding the surface and then subjecting the surface to blast treatment such as sandblast.", "The external terminals 13 are connected to the conductor filled through holes 13a for connecting the inner heating elements 12, and heated for re-flow.", "The heating temperature is preferably from 200 to 500° C. Furthermore, thermocouples (not illustrated) as temperature measuring elements are fitted into the bottomed holes 14 with brazing silver, brazing gold and the like, and then holes are sealed up with a heat-resistant resin such as polyimide, so as to finish the production of a ceramic heater 10 (see FIG.", "5(d)).", "The following will describe a process for producing a ceramic heater 20 wherein heating elements 22 are formed on the bottom face of a ceramic substrate 21 (see FIGS.", "3 and 4) on the basis of FIG.", "6.", "(1) Step of Forming the Ceramic Substrate A sintering aid such as yttria (Y2O3) or B4C, a compound containing Na or Ca, a binder and so on are blended as appropriate with powder made of a ceramic such as a nitride ceramic, for example, the above-mentioned aluminum nitride or a carbide ceramic, so as to prepare a slurry.", "Thereafter, this slurry is made into a granular form by spray drying and the like.", "The granules are put into a mold and pressed to be formed into a plate form or some other form.", "Thus, a raw formed body (green) is produced.", "Next, this raw formed body is heated and fired to be sintered.", "Thus, a plate made of the ceramic is produced.", "Thereafter, the plate is made into a given shape to produce the ceramic substrate 21.The shape of the raw formed body may be such a shape that the sintered body can be used as it is after the firing (FIG.", "6(a)).", "By heating and firing the raw formed body under pressure, the ceramic substrate 21 having no pores can be produced.", "It is sufficient that the heating and the firing are performed at the sintering temperature or higher.", "The firing temperature is from 1000 to 2500° C. for nitride ceramics or carbide ceramics.", "The firing temperature is from 1500 to 2000° C. for oxide ceramics.", "Furthermore, the ceramic substrate is drilled so as to form three or more through holes 25 for letting lifter pins 26 for supporting a semiconductor wafer 39 pass through, in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate.", "In the same manner as in the ceramic substrate 10, the through holes 25 are desirably formed at substantially regular intervals on a single circle having a concentric circle relationship with the ceramic substrate 21.Additionally, bottomed holes 24, for embedding temperature measuring elements such as thermocouples, are formed in the ceramic substrate (see FIG.", "6(a)).", "(2) Step of Printing a Conductor Containing Paste on the Ceramic Substrate A conductor containing paste is generally a fluid comprising metal particles, a resin and a solvent, and having a high viscosity.", "This conductor containing paste is printed on portions where heating elements 22 are to be formed by screen printing and the like.", "In this way, a conductor containing paste layer is formed.", "The conductor containing paste is desirably formed in such a manner that sections of the heating elements 22 subjected to the firing are rectangular and flat.", "(3) Firing of the Conductor Containing Paste The conductor containing paste layer printed on the bottom face 21b of the ceramic substrate is heated and fired to remove the resin and the solvent and sinter the metal particles.", "Thus, the metal particles are baked onto the bottom face of the ceramic substrate 21 to form the heating elements 22 (FIG.", "6(b)).", "The heating and firing temperature is preferably from 500 to 1000° C. If the above-mentioned oxides are added to the conductor containing paste, the metal particles, the ceramic substrate and the oxides are sintered to be integrated with each other.", "Thus, the adhesiveness between the heating elements 22 and the ceramic substrate 21 is improved.", "(4) Step of Forming a Metal Covering Layer Next, a metal covering layer 220 is deposited on the surface of the heating elements 22 (see FIG.", "6(c)).", "The metal covering layer 220 can be formed by electroplating, electroless plating, sputtering and the like.", "From the viewpoint of mass-productivity, electroless plating is optimal.", "(5) Fitting of Terminals and so on A terminal (external terminal 23) for connection to a power source is fitted up to an end of each of the pattern pieces of the heating elements 22 with solder.", "Thermocouples (not illustrated) are fixed to the bottomed holes 24 with brazing silver, brazing gold and the like.", "The bottomed holes are sealed with a heat-resistant resin such as polyimide and the like, so as to finish the production of a ceramic heater 20 (see FIG.", "6(d)).", "About the ceramic heater according to the first aspect of the present invention, electrostatic electrodes are set inside the ceramic substrate, whereby the ceramic substrate can be used as an electrostatic chuck.", "By forming a chuck top conductive layer on the surface, the ceramic heater can be used as a ceramic substrate for a wafer prober.", "As described above, the ceramic heater according to the first aspect of the present invention can be used as a ceramic heater for a semiconductor producing/inspecting device or for heating a liquid crystal substrate.", "Next, a ceramic heater according to a second aspect of the present invention will be described.", "The ceramic heater according to the second aspect of the present invention includes: a disk-like ceramic substrate; a heating element formed on a surface of or inside the ceramic substrate; and through holes for letting lifter pins into at the ceramic substrate, wherein the diameter of each of the above-mentioned through holes on a heating face side for heating an object to be heated is larger than the diameter of the above-mentioned through hole on the side opposite to the above-mentioned heating face.", "In the ceramic heater according to the second aspect of the present invention, the diameter on the heating face side of the through holes is desirably from 1.2 to 10 times the diameter on the side opposite to the heating face.", "If the diameter on the heating side is less than 1.2 times the diameter on the side opposite to the heating face or exceeds 10 times, the effect of accumulating heat cannot be obtained.", "FIG.", "9 is a plan view which schematically illustrates a ceramic heater according to the second aspect of the present invention.", "FIG.", "10 is a partially enlarged sectional view which schematically illustrates the ceramic heater in FIG.", "9.In this ceramic heater, heating elements are formed inside its ceramic substrate.", "In this ceramic heater 30, the ceramic substrate 31 is formed in a disk-like form.", "In order to heat the ceramic heater in such a manner that the temperature in the whole of a heating face 31a of the ceramic heater 30 is made even, the heating elements 32 which has a concentrically circular pattern are formed inside the ceramic substrate 31.Conductor filled through holes 33a are formed just under ends of heating elements 32, and further blind holes 33b for making the conductor filled through holes 33a exposed are formed in the bottom face 31b.", "An external terminal 33 is inserted into the blind hole 33b, and they are bonded to each other with a brazing material (not illustrated).", "For example, a socket (not illustrated) having a conductive wire is fitted to the external terminal 33.This conductive wire is connected to a power source and the like.", "In the bottom face of the ceramic substrate 31, bottomed holes 34, for inserting temperature measuring elements (not illustrated) into, are formed.", "Furthermore, three through holes 35, for letting lifter pins 36 pass through, are formed in the vicinity of the center of the ceramic substrate 31.As illustrated in FIG.", "10, the through hole 35 is composed of a columnar portion 35a and a diameter-increasing portion 35b the diameter of which becomes larger as the portion is closer to the heating face, and the whole thereof has a funnel shape.", "In portions where cooling spots are easily generated, the solid constituting the substrate is hardly present.", "Instead of it, gas such as air is present.", "Accordingly, the heat capacity of the portion where cooling spots are easily generated gets small.", "Because of the presence of this portion, portions with low temperature are not easily generated in a semiconductor wafer 59.Since gas such as air accumulates heat and remains in the funnel-shaped portion, the semiconductor wafer 59 can be evenly heated.", "In FIG.", "10, the through hole 35 has a shape the diameter of which increases abruptly at a spot from the vicinity of the heating face (specifically a position whose distance from the heating face of the ceramic substrate is ⅔ or less of the thickness of the ceramic substrate) toward the heating face.", "However, the through hole may be a through hole having such a shape that the diameter thereof increases gradually from the vicinity of the bottom face side in order to cause the gas (such as the air) having accumulated heat to remain in the vicinity of the heating face.", "When this ceramic heater 30 is used to heat the semiconductor wafer 59, it is allowable to receive the semiconductor wafer 59 by means of the lifter pins 36, put the semiconductor wafer 59 on the heating face, and send electric current to the ceramic heater so as to be heated.", "It is also allowable to support the semiconductor wafer 59 in the state that the wafer is a given distance apart from the heating face 31a of the ceramic substrate through the lifer pins 36 by holding the lifter pins 36 in the state that they project slightly from the heating face 31a of the ceramic substrate 31, and then heat the semiconductor wafer 59.In particular, when the semiconductor wafer or the like is placed so as to contact the heating face, in general it is easily affected by cooling spots present in the vicinity of the through holes.", "In the second aspect of the present invention, however, the diameter on the heating face side of the through holes is larger as described above.", "Thus, cooling spots are not easily generated, and thus the semiconductor wafer can be more evenly heated than that in the prior art.", "Before and after the step of using the ceramic substrate of the second aspect of the present invention to conduct heating treatment and so forth, the semiconductor wafer or the like can be relatively easily received from the previous line or carried to the next line by letting the lifter pins 36 pass through the through holes 35 and subsequently moving the lifter pins up and down.", "When the semiconductor wafer or the like is supported by the lifter pins 36, the semiconductor wafer or the like is not rubbed with the ceramic substrate.", "Therefore, no free particles are generated from the ceramic substrate.", "By arranging a member having projected structures, such as pins and the like, on the surface of the ceramic substrate, the semiconductor wafer or the like can be heated in the state that the semiconductor wafer or the like is apart from the heating face of the ceramic substrate in the same manner as in the case of holding the lifter pins in the state that the lifter pins project slightly from the heating face of the ceramic substrate.", "In the ceramic heater according to the second aspect of the present invention, the heating elements may be formed inside the ceramic substrate or may be formed outside the ceramic substrate.", "FIG.", "11 is a bottom face view which schematically illustrates another example of the ceramic heater according to the second aspect of the present invention.", "FIG.", "12 is a partially enlarged sectional view which schematically illustrates the ceramic heater in FIG.", "11.In this ceramic heater, heating elements are formed on a surface of the ceramic substrate.", "In the ceramic heater 40, its ceramic substrate 41 is formed in a disk-like form, and heating elements 42 having a concentrically circular pattern are formed on the surface of the ceramic substrate 41.External terminals 43, which are terminals for input and output, are connected through a metal covering layer 420 to both ends of the heating element.", "In the bottom face of the ceramic substrate 41, bottomed holes 44, for inserting temperature measuring elements (not illustrated) into, are formed.", "Additionally, three through holes 45, for letting lifter pins 46 pass through, are formed in the vicinity of the center of the ceramic substrate 41.In the same manner as in the case of the ceramic heater 30, the through hole 45 is composed of a columnar portion 45a and a diameter-increasing portion 45b.", "In portions where cooling spots are easily generated, the solid constituting the substrate is hardly present.", "Accordingly, the heat capacity of the portion where cooling spots are easily generated can be largely lowered.", "Thus, a semiconductor wafer 59 can be evenly heated.", "By letting the lifter pins 46 pass through the through holes 45 and then moving the pins up and down, the semiconductor wafer or the like can be carried and moved.", "By projecting the lifter pins 46 from the heating face 41a of the ceramic substrate 41, the semiconductor wafer 59 can be held in the state that the wafer is apart from the ceramic substrate 41a.", "In the ceramic heater according to the second aspect of the present invention, the shape of the through holes is not particularly limited if the diameter thereof on the heating face side is larger than that on the bottom face.", "As described, the through holes are preferably composed of a columnar portion and a diameter-increasing portion as illustrated in FIG.", "10.The number of the through holes formed in the ceramic substrate is preferably three or more.", "If the number of the through holes is two or less, it is difficult to support an object to be heated, such as a semiconductor wafer, stably by the lifter pins passed through the through holes.", "The number of the through holes is not particularly limited if the number is three or more.", "Since it is also true for the second aspect of the present invention that cooling spots are easily generated in the through holes portions, it is preferred that the number thereof is not very large.", "For example, the number is desirably 11 or less.", "The position where the through holes are formed is not particularly limited.", "Desirably, the through holes are formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge of the ceramic substrate.", "This is because this case makes it possible to support a semiconductor wafer or the like more stably.", "The volume of the peripheral portion is larger than that of the central portion; therefore, if the through holes are formed in the vicinity of the center, the heat capacity of the central portion gets small by making the through holes.", "Thus, the temperature in the central portion is readily made high.", "However, if the through holes are formed in the peripheral portion, difference in the heat capacity between the central portion and the peripheral portion is hardly generated so that the temperature in the heating face can be made even.", "The through holes are desirably formed at substantially regular intervals on a single circle having a concentric circle relationship with the ceramic substrate since a semiconductor wafer or the like can be more stably supported and can be still more evenly heated.", "In the case that four or more through holes are formed in the ceramic substrate, one of the through holes may be formed in the center of the ceramic substrate.", "By the lifter pins, the semiconductor wafer or the like can be held apart from the ceramic substrate and further the central portion of the semiconductor wafer or the like can be prevented form warping when it is heated.", "As a result, the distance between the semiconductor wafer or the like and the ceramic substrate is made constant so that the semiconductor wafer or the like can be evenly heated.", "When a semiconductor wafer or the like is held apart by the lifter pins, it is desired that the height of the lifter pins projecting from the heating face of the ceramic substrate, the pins being passed through the through holes, is from 5 to 5000 μm, that is, that the semiconductor wafer or the like is held from 5 to 5000 μm apart from the heating face of the ceramic substrate.", "If the distance is less than 5 μm, the temperature of the semiconductor or the like is affected by the temperature distribution in the ceramic substrate and is made uneven.", "If the distance exceeds 5000 μm, the temperature of the semiconductor wafer or the like does not rise easily.", "In particular, the temperature in the peripheral portion of the semiconductor wafer or the like drops.", "An object to be heated and the heating face of the ceramic substrate are separated from each other more desirably by a distance of 5 to 500 μm, still more desirably 20 to 200 μm.", "The diameter of the through holes is desirably from 1 to 100 mm, more desirably from 1 to 20 mm.", "In case the through holes have a trapezoidal sectional shape, the diameter of the through holes means the diameter thereof at the position in the middle between the bottom face and the heating face.", "When the through holes are composed of a columnar portion and a diameter-increasing portion, the diameter thereof means the diameter of the columnar portion.", "If the diameter of the through holes is less than 1 mm, the lifter pins passed through the through holes become too thin so that a semiconductor wafer or the like can not be stably put on the lifter pins with ease.", "On the other hand, if the diameter of the through holes exceeds 100 mm, the through holes are too large so that cooling spots are readily generated in the heating face by the effect of gas present inside the through holes.", "Additionally, the heat capacities in a given area of the ceramic substrate are different between the portion where the through holes are present and the portion where the through holes are not present; therefore, the temperature in the heating face readily gets uneven, whereby it is apprehended that the semiconductor wafer or the like cannot be evenly heated.", "The constitutions of parts other than the through holes in the ceramic substrate, which are parts of the ceramic heater of the second aspect of the present invention, such as temperature measuring elements, heating elements, and connecting terminals, are the same as in the first aspect of the present invention.", "Thus, description thereof will not be repeated.", "A ceramic body having a cylindrical shape or the like may be bonded to the bottom face of the ceramic substrate which constitutes the ceramic heater according to the second aspect of the present invention, so as to protect wires such as external terminals.", "In this way, the wires such as the external terminals can be protected from reactive gas, halogen gas and the like.", "Furthermore, the ceramic heater according to the second aspect of the present invention can be used when a liquid crystal substrate is heated.", "The following will describe a process for producing a ceramic heater according to the second aspect of the present invention.", "Firstly, a ceramic heater wherein heating elements 32 are formed inside a ceramic substrate 31 (see FIGS.", "9 and 10) will be described on the basis of FIG.", "13.", "(1) Step of Forming the Ceramic Substrate First, ceramic powder made of a nitride ceramic and the like are mixed with a binder, a solvent and so on to prepare a paste.", "This is used to form green sheets 100.Aluminum nitride and the like can be used as the above-mentioned ceramic powder made of a nitride ceramic and the like.", "If necessary, a sintering aid such as yttria, a compound containing Na or Ca, and the like may be added thereto.", "As the binder, desirable is at least one selected from an acrylic resin binder, ethylcellulose, butylcellosolve, and polyvinyl alcohol.", "As the solvent, desirable is at least one selected from α-terpineol and glycol.", "A paste obtained by mixing these is formed into a sheet form by a doctor blade process, to manufacture green sheets.", "The thickness of the green sheets is preferably from 0.1 to 5 mm.", "(2) Step of Printing a Conductor Containing Paste on the Green Sheet A metal paste or a conductor containing paste which contains paste containing a conductive ceramic, for forming heating elements 32, is printed on the green sheet 100, so as to form a conductor containing paste layer 320.A conductor containing paste filled layer 330 for conductor filled through holes 33a is formed in through holes.", "The conductor containing paste contains metal particles or conductive ceramic particles.", "The average particle diameter of tungsten particles or molybdenum particles is preferably from 0.1 to 5 μm.", "If the average particle is less than 0.1 μm or exceeds 5 μm, the conductor containing paste is not easily printed.", "Such a conductor containing paste may be a composition (paste) obtained by mixing, for example, 85 to 87 parts by weight of the metal particles or the conductive ceramic particles; 1.5 to 10 to parts by weight of at least one binder selected from acrylic resin binders, ethylcellulose, butylcellosolve and polyvinyl alcohol; and 1.5 to 10 parts by weight of at least one solvent selected from α-terpineol and glycol.", "(3) Step of Laminating the Green Sheets Green sheets 100 on which no conductor containing paste is printed are laminated on the upper and lower sides of the green sheet 100 on which the conductor containing paste is printed (see FIG.", "13(a)) At this time, the laminating is performed in such a manner that the green sheet 100 on which the conductor containing paste is printed is arranged at a position which has a distance, from the bottom face, of 60% or less of the thickness of the laminated green sheets.", "Specifically, the number of the green sheets laminated on the upper side is preferably from 20 to 50, and that of the green sheets laminated on the lower side is preferably from 5 to 20.", "(4) Step of Firing the Green Sheet Lamination The green sheet lamination is heated and pressed to sinter the green sheets and the inner conductor containing paste.", "The heating temperature is preferably from 1000 to2000° C., and the pressing pressure is preferably from 10 to 20 MPa.", "The heating is performed in the atmosphere of an inert gas.", "As the inert gas, argon, nitrogen and the like can be used.", "Next, through holes 35, for letting lifter pins 36 for supporting a semiconductor wafer 59 pass through, are formed in the resultant sintered body.", "At this time, the through holes 35 having a columnar portion 35a and a diameter-increasing portion 35b can be formed by: firstly, forming columnar through holes with a drill having an ordinary cutting edge; and subsequently processing the through hole portions from the heating face side, by using a drill having a cutting edge capable of making a conical shaped concave portions.", "Also, by sandblast treatment, through holes having a trapezoidal vertical section, as well as the above-mentioned shaped through holes, can be formed.", "The through holes 35 are desirably formed at substantially regular intervals on a single circle having a concentric circle relationship with the ceramic substrate 31 for the following reason: the lifter pins 36, which are passed through the through holes 35, are widely dispersed on the ceramic substrate 31 and arranged at regular interval; therefore, the semiconductor wafer 59 can be kept more horizontal.", "Furthermore, bottomed holes 34, for embedding temperature measuring elements such as thermocouples, are formed in the ceramic substrate (see FIG.", "13(b)).", "Thereafter, blind holes 33 are provided in order to make the conductor filled through holes 33a, which is for connecting the heating elements 32 to external terminals 33, exposed (see FIG.", "13(c)).", "The above-mentioned steps of making the bottomed holes and the through holes may be applied to the above-mentioned sheet lamination, but is preferably applied to the above-mentioned sintered body.", "This is because the lamination may be deformed in the sintering step.", "The formation of the through holes and the bottomed holes is usually performed after grinding the surface.", "Thereafter, the external terminals 33 are connected to the conductor filled through holes 33a for connecting the inner heating elements 32, and heated for re-flow.", "The heating temperature is preferably from 200 to 500° C. Furthermore, thermocouples (not illustrated) as temperature measuring elements are fitted into the bottomed holes 14 with brazing silver, brazing gold and the like, and then holes are sealed up with a heat-resistant resin such as polyimide, so as to finish the production of a ceramic heater 30 (see FIG.", "13(d)).", "The following will describe a process for producing a ceramic heater 40 wherein heating elements 42 are formed on the bottom face of a ceramic substrate 41 (see FIGS.", "11 and 12) on the basis of FIG.", "14.", "(1) Step of Forming the Ceramic Substrate A sintering aid such as yttria (Y2O3) or B4C, a compound containing Na or Ca, a binder and so on are blended as appropriate with powder made of a ceramic such as a nitride ceramic, for example, the above-mentioned aluminum nitride or a carbide ceramic, so as to prepare a slurry.", "Thereafter, this slurry is made into a granular form by spray drying and the like.", "The granules are put into a mold and pressed to be formed into a plate form or some other form.", "Thus, a raw formed body (green) is produced.", "Next, this raw formed body is heated and fired to be sintered.", "Thus, a plate made of the ceramic is produced.", "Thereafter, the plate is made into a given shape to produce the ceramic substrate 41.The shape of the raw formed body may be such a shape that the sintered body can be used as it is after the firing.", "By heating and firing the raw formed body under pressure, the ceramic substrate 41 having no pores can be produced.", "It is sufficient that the heating and the firing are performed at the sintering temperature or higher.", "The firing temperature is from 1000 to 2500° C. for nitride ceramics or carbide ceramics.", "The firing temperature is from 1500 to 2000° C. for oxide ceramics.", "Furthermore, the ceramic substrate is drilled so as to form through holes 45, for letting lifter pins 46 for supporting a semiconductor wafer 59 pass through.", "The method of forming the through holes is the same as in the case of the above-mentioned ceramic heater having the heating elements inside.", "Additionally, bottomed holes 44, for embedding temperature measuring elements such as thermocouples, are formed in the ceramic substrate (see FIG.", "14(a)).", "(2) Step of Printing a Conductor Containing Paste on the Ceramic Substrate A conductor containing paste is generally a fluid comprising metal particles, a resin and a solvent, and having a high viscosity.", "This conductor containing paste is printed on portions where heating elements 4 are to be formed by screen printing and the like.", "In this way, a conductor containing paste layer is formed.", "The conductor containing paste is desirably formed in such a manner that sections of the heating elements 42 subjected to the firing are rectangular and flat.", "(3) Firing of the Conductor Containing Paste The conductor containing paste layer printed on the bottom face 41b of the ceramic substrate is heated and fired to remove the resin and the solvent and sinter the metal particles.", "Thus, the metal particles are baked onto the bottom face of the ceramic substrate 41 to form the heating elements 42 (FIG.", "14(b)).", "The heating and firing temperature is preferably from 500 to 1000° C. If the above-mentioned oxides are added to the conductor containing paste, the metal particles, the ceramic substrate and the oxides are sintered to be integrated with each other.", "Thus, the adhesiveness between the heating elements 42 and the ceramic substrate 41 is improved.", "(4) Step of Forming a Metal Covering Layer Next, a metal covering layer 420 is deposited on the surface of the heating elements (FIG.", "14(c)).", "The metal covering layer 420 can be formed by electroplating, electroless plating, sputtering and the like.", "From the viewpoint of mass-productivity, electroless plating is optimal.", "(5) Fitting of Terminals and so on A terminal (external terminal 43 for connection to a power source is fitted up to an end of each of the pattern pieces of the heating elements 42 with solder.", "Thermocouples (not illustrated) are fixed to the bottomed holes 44 with brazing silver, brazing gold and the like.", "The bottomed holes are sealed with a heat-resistant resin such as polyimide, so as to finish the production of a ceramic heater 40 (FIG.", "14(d)).", "About the ceramic heater of the second aspect of the present invention, electrostatic electrodes are set inside the ceramic substrate, whereby the ceramic heater can be used as an electrostatic chuck.", "By forming a chuck top conductive layer on the surface, the ceramic heater can be used as a ceramic substrate for a wafer prober.", "The following will describe a ceramic bonded body according to a third aspect of the present invention according to embodiments.", "The third aspect of the present invention is not limited to this description.", "In the following, the ceramic body will be described as a cylindrical ceramic body.", "However, the ceramic body may be a columnar filled body, or may be a triangle polar or square polar hollow body or filled body.", "The ceramic bonded body of the third aspect of the present invention according to an embodiment includes: a disk-like ceramic substrate inside which a conductor is provided; and a cylindrical ceramic body having a cylindrical shape bonded to the bottom face of the ceramic substrate, wherein the center of the circle surrounded by the interface between the cylindrical ceramic body and the ceramic substrate is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate.", "FIG.", "17(a) is a plan view which schematically illustrates a ceramic bonded body according to the third aspect of the present invention, and FIG.", "17(b) is a partially enlarged sectional view which schematically illustrates this ceramic bonded body.", "FIG.", "17 illustrates only a ceramic substrate and the ceramic body in a cylindrical form and does not illustrate a conductor formed inside the ceramic substrate, or other members.", "The ceramic bonded body 1 is formed by bonding the cylindrical ceramic body 7 to the bottom face of the ceramic substrate 2 having a disk-like shape.", "At this time, the face on which the ceramic substrate 2 and the cylindrical ceramic body 7 are bonded is an interface 6.In the ceramic bonded body 1, the distance L between: the center A of the circle surrounded by the interface 6; and the center B of the bottom face of the ceramic substrate 2 is from 3 to 200 μm.", "The method for bonding the ceramic substrate and the cylindrical ceramic body will be described in detail later.", "In the case that the ceramic bonded body according to the third aspect of the present invention is applied to a semiconductor producing/inspecting device, it is desired that a ceramic substrate having therein conductors is fixed to the upper portion of a supporting case having a bottom plate and further wires from the conductors are stored in the cylindrical ceramic body bonded to the bottom face of the ceramic substrate.", "This is for preventing the wires from being exposed to corrosive gas and the like and corroded.", "In the case that the conductors formed in the ceramic substrate constituting a ceramic bonded body of the third aspect of the present invention are heating elements and conductor circuits, the ceramic bonded body functions as a ceramic heater.", "FIG.", "18 is a plan view which schematically illustrates a ceramic heater which is an example of the ceramic bonded body of the third aspect of the present invention, and FIG.", "19 is a sectional view thereof.", "FIG.", "20 is a partially enlarged sectional view of the vicinity of the cylindrical ceramic body illustrated in FIG.", "19.As illustrated in FIG.", "19, in this ceramic heater 70, cylindrical ceramic body 77 is bonded directly to the vicinity of the center of the bottom face 71b of a ceramic substrate 71 having a disk-like shape.", "At this time, the center of the circle surrounded by the interface between the cylindrical ceramic body 77 and the ceramic substrate 71 is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate 71, as described above.", "Since the cylindrical ceramic body 77 is formed to adhere closely to the bottom plate (not illustrated) of the supporting case, the inside and the outside of the cylindrical ceramic body 77 are completely separated.", "As illustrated in FIG.", "18, heating elements 72 formed of circuits having a concentrically circular shape are formed in the ceramic substrate 71.About these heating elements 72, two concentric circles adjacent to each other are connected to each other so as to be a single line as one circuit.", "As illustrated in FIG.", "19, conductor circuits 78 extending toward the center of the ceramic substrate 71 are formed between the heating elements 72 and the bottom face 71b, and ends 72a of the heating element are connected to one end of the conductor circuit 78 through via holes 86.The conductor circuit 78 is formed to extend the ends 72a of the heating element to the central portion.", "In the ceramic substrate 71, a conductor filled through hole 73′ and a blind hole 79 for making the conductor filled through hole 73′ exposed are formed just under the other end of the conductor circuit 78 extending in the vicinity of the inside of the cylindrical ceramic body 77.This conductor filled through hole 73′ is connected to an external terminal 83 whose tip has a T-shape through a solder layer (not illustrated).", "In the case that the ends 72a of the heating element are inside the cylindrical ceramic body 77, no via holes or conductor circuits are necessary.", "Accordingly, the conductor filled through holes 73 are directly fitted to the ends of the heating element and they are connected to the external terminals 83 through a solder layer.", "Sockets 85 having a conductive wire 830 are fitted to these external terminals 83, and this conductive wire 830 is led out from the through holes formed in the bottom plate (not illustrated) and then connected to a power source (not illustrated) and the like.", "On the other hand, temperature measuring elements 84, such as thermocouples, having a lead wire 890 are inserted into the bottomed holes 74 formed in the bottom face 71b of the ceramic substrate 71, and the holes are sealed with a heat-resistant resin, a ceramic (such as silica gel and the like.", "This lead wire 890 is passed through an insulator (not illustrated), and is led out through a through hole (not illustrated) formed in the bottom plate of the supporting case.", "The inside of the insulator is also insulated from the outside thereof.", "Furthermore, through holes 75, for letting lifter pins (not illustrated) pass through, are formed in the vicinity of the center of the ceramic substrate 71.The lifter pins can be formed so as to be moved up and down in the state that an object to be heated, such as a silicon wafer, is put thereon.", "This makes it possible to deliver the silicon wafer to a non-illustrated carrying machine or receive the silicon wafer from the carrying machine and further to heat the silicon wafer put on the heating face 71a of the ceramic substrate 71, or support the silicon wafer 50 to 2000 μm apart from the heating face 71a and heat it.", "The silicon wafer may be heated 50 to 2000 μm apart from the heating face 71a by making: through holes or concave portions in the ceramic substrate 71; inserting supporting pins whose tips are in a spire form or a hemispherical form into the through holes or concave portions; fixing the supporting pins in the state that they project slightly from the ceramic substrate 71; and then supporting the silicon wafer by the supporting piping system.", "A coolant introducing pipe and the like may be fitted to the bottom plate of the supporting case.", "In this case, the temperature of the ceramic substrate 71, the cooling rate and so forth can be controlled by introducing a coolant into this coolant introducing pipe through a pipe.", "As described above, in this ceramic heater 70, the cylindrical ceramic body 77 is bonded to the bottom face 71b of the ceramic substrate 71 and the cylindrical ceramic body 77 is formed to extend to the bottom plate (case wall) of the non-illustrated supporting case; therefore, the inside of the cylindrical ceramic body 77 is completely insulated from the outside thereof.", "Accordingly, by protecting the conductive wires 830 led out from the through holes in the bottom plate by tubular members, even if the ceramic heater 70 is surrounded by an atmosphere containing reactive gas, halogen gas and the like, and even in the state that the reactive gas and the like enter easily the inside of the supporting case, the wires inside the cylindrical ceramic body 77 do not corrode.", "The wires 890 from the temperature measuring elements 84 do not corrode since they are protected from the insulator and so on.", "Furthermore, inert gas and the like may be caused to flow slowly in the cylindrical ceramic body 77 so that reactive gas, halogen gas and the like do not flow in the cylindrical ceramic body 77.In this way, the corrosion of the conductive wires 830 can be still more surely prevented.", "Since the cylindrical ceramic body 77 has a function for supporting the ceramic substrate 71 fixedly, the ceramic substrate 71 can be prevented from being warped by its self weight even when the ceramic substrate 71 is heated to a high temperature.", "As a result, an object to be heated, such as a silicon wafer, can be prevented from being damaged, and further the object to be heated can be heated to have an even temperature.", "The constitutions of the ceramic substrate and temperature measuring elements which are parts of the ceramic heater of the third aspect of the present invention are the same as disclosed in the ceramic heater of the first aspect of the present invention.", "Thus, description thereon is omitted.", "The number of the through holes formed to pass the lifter pins through the ceramic substrate, and the position where they are formed are not limited to the above.", "The shape of the cylindrical ceramic body in the ceramic bonded body of the third aspect of the present invention is a cylindrical shape as illustrated in FIG.", "19, and the inner diameter thereof is desirably 30 mm or more.", "If the diameter is less than 30 mm, the ceramic substrate is not fixedly supported with ease.", "When the ceramic substrate is heated to a high temperature, it is apprehended that the ceramic substrate is warped by its self weight.", "The thickness of the cylindrical ceramic body is desirably from 3 to 20 mm.", "If the thickness is less than 3 mm, the thickness of the cylindrical ceramic body is too small so that the mechanical strength is poor.", "Thus, it is apprehended that the cylindrical ceramic body is damaged by repeating temperature-rising and temperature-dropping thereof.", "If the thickness exceeds 20 mm, the thickness of the cylindrical ceramic body is too large so that the heat capacity gets large.", "Thus, it is apprehended that the temperature-rising rate drops.", "As the ceramic which forms the cylindrical ceramic body, the same material for the above-mentioned ceramic substrate can be used.", "The method for bonding the cylindrical ceramic body to the ceramic substrate will be described in detail later.", "The constitutions of heating elements, external terminals, conductive wires and so on formed inside the ceramic substrate are the same as in the ceramic heater of the first aspect of the present invention.", "Thus, description thereon is omitted.", "In the ceramic heater 70 illustrated in FIGS.", "18, 19 and 20, the ceramic substrate 71 is usually fitted to the upper part of the supporting case (not illustrated).", "In other embodiments, however, the substrate may be put on the upper face of a supporting face having a substrate-receiving portion at its upper end, and fixed with fixing members such as bolts.", "The above-mentioned ceramic heater 70 is desirably used at 100° C. or more, more desirably 200° C. or more.", "The ceramic substrate which constitutes the ceramic bonded body of the third aspect of the present invention is used to product a semiconductor or inspect a semiconductor.", "Specifically, examples thereof include an electrostatic chuck, susceptor, a ceramic heater (hot plate) and the like.", "The above-mentioned ceramic heater is a device wherein only heating elements are formed inside the ceramic substrate.", "This makes it possible to hold an object to be heated, such as a silicon wafer, on the surface of the ceramic substrate or apart from the surface, and heat the object to a given temperature or wash the object.", "Furthermore, the ceramic heater which is an example of the ceramic bonded body of the third aspect of the present invention can be used when a liquid crystal substrate is heated.", "In the case that conductors formed inside the ceramic substrate which constitutes the ceramic bonded body of the third aspect of the present invention are electrostatic electrodes or conductor circuits, the ceramic bonded body functions as an electrostatic chuck.", "FIG.", "21 is a vertical sectional view which schematically illustrates such an electrostatic chuck, and FIG.", "22 is a partially enlarged sectional view thereof.", "FIG.", "23 is a horizontal sectional view which schematically illustrates the vicinity of electrostatic electrodes formed on a substrate which constitutes the electrostatic chuck.", "Inside the ceramic substrate 91 which constitutes this electrostatic chuck 90, chuck positive and negative electrostatic layers 92a and 92b in a semicircular form are arranged oppositely to each other.", "A ceramic dielectric film 94 is formed on these electrostatic electrodes.", "Inside the ceramic substrate 91, heating elements 920 are formed so that an object to be heated, such as a silicon wafer, can be heated.", "If necessary, RF electrodes are embedded in the ceramic substrate 91.The electrostatic electrodes are preferably made of a metal such as a noble metal (gold, silver, platinum or palladium), lead, tungsten, molybdenum, nickel and the like, or a conductive ceramic such as a carbide of tungsten or molybdenum.", "These may be used alone or in combination of two or more thereof.", "As illustrated in FIGS.", "21, 22, in this electrostatic chuck 90, electrostatic electrodes 92a, 92b are formed in the ceramic substrate 91, and a conductor filled through hole 93 is formed just under an end of each of the electrostatic electrodes 92a, 92b.", "A ceramic dielectric film 94 is formed on the electrostatic electrodes 92.Except these matters, the electrostatic chuck has the same constitution as the above-mentioned ceramic heater 70.That is, a cylindrical ceramic body 97 is bonded to the vicinity of the center of the bottom face of the ceramic substrate 91.As described above, at this time the center of the circle surrounded by the interface between the cylindrical ceramic body 97 and the ceramic substrate 91 is 3 to 200 μm apart from the center of the bottom face of the ceramic substrate 91.The conductor filled through holes 93 and conductor filled through holes 930 are formed above the area inside of the cylindrical ceramic body 97.These conductor filled through holes 93, 930 are connected to the electrostatic electrodes 92a, 92b and the heating elements 920, and further connected to external terminals 960 inserted into blind holes 990.A socket 950 having a conductive wire 931 is connected to one end of the external terminal 960.This conductive wire 931 is led out from a through hole (not illustrated).", "In the case of heating elements 920 having an end outside the cylindrical ceramic body 97, via holes 99, conductor circuits 980 and conductor filled through holes 930′ are formed in the same manner as in the case of the ceramic heater 70 illustrated in FIGS.", "18 to 20, whereby ends of the heating elements 920 extend to the inside the cylindrical ceramic body 97 (see FIG.", "22).", "Accordingly, by inserting the external terminals 960 into the blind holes 990 for making the conductor filled through holes 930′ exposed and connecting them, the external terminals 960 can be stored inside the cylindrical ceramic body 97.When this electrostatic chuck 90 is worked, voltages are applied to the heating elements 920 and the electrostatic electrodes 92, respectively.", "In this way, a silicon wafer put on the electrostatic chuck 90 is heated to a given temperature and further electrostatically absorbed on the ceramic substrate 91.This electrostatic chuck may not necessarily have the heating elements 920.FIG.", "24 is a horizontal sectional view which schematically illustrates another electrostatic electrode formed on a substrate for electrostatic chuck.", "A chuck positive electrostatic layer 172, which is composed of a semicircular part 172a and a combteeth-shaped part 172b, and a chuck negative electrostatic layer 173, which is also composed of a semicircular part 173a and a combteeth-shaped part 173b, are arranged face-to-face in such a manner that the teeth of one comb teeth shaped part 172b extend in staggered relation with the teeth of the other comb teeth shaped part 173b.", "FIG.", "25 is a horizontal sectional view which schematically illustrates further another electrostatic electrode formed on a substrate for an electrostatic chuck.", "In this electrostatic chuck, chuck positive electrostatic layers 182a, 182b and chuck negative electrostatic layers 183a, 183b, each of which has a shape obtained by dividing a circle into 4 parts, are formed inside the ceramic substrate 181.The two chuck positive electrostatic layers 182a, 182b and the two chuck negative electrostatic layers 183a, 183b are formed to cross each other.", "In the case that electrodes having a form obtained by dividing an electrode having a circular shape or some other shape are formed, the number of divided pieces is not particularly limited and may be 5 or more.", "Its shape is not limited to a fan-shape.", "The following will describe a process for producing a ceramic heater, as an example of a process for producing the ceramic bonded body of the third aspect of the present invention, referring to FIG.", "26.FIGS.", "26(a) to (d) are sectional views which schematically illustrate some parts of the process for producing a ceramic heater, which is an example of the ceramic bonded body of the third aspect of the present invention.", "(1) Step of Forming Green Sheets First, a green sheet 500 is formed in the same way as in the process for producing the ceramic heater of the first aspect of the present invention.", "Next, the following are produced: a green sheet wherein portions 860 which will be via holes for connecting an end of a heating element to a conductor circuit are formed; and a green sheet wherein portions 730, 730′ which will be conductor filled through hole for connecting the conductor circuit to an external terminal are formed.", "If necessary, the following are formed: portions which will be through holes for letting lifter pins for carrying a silicon wafer pass through; portions which will be through holes for inserting supporting pins for supporting a silicon wafer into; portions which will be bottomed holes for embedding temperature measuring elements such as thermocouples; and the like.", "About the through holes and the bottomed holes, the above-mentioned working may be performed after a green sheet lamination which will be described later is formed, or after the lamination is formed and fired.", "The above-mentioned paste to which carbon is added may be filled into the portions 860, which will be the via holes, and the portions 730, 730′, which will be the conductor filled through holes.", "This is because the carbon in the green sheet reacts with tungsten or molybdenum filled into the conductor filled through holes to form carbides thereof.", "(2) Step of Printing a Conductor Containing Paste on the Green Sheet A metal paste or a conductor containing paste which contains a conductive ceramic is printed on the green sheet wherein the portions 860, which will be the via holes, are formed, so as to form a conductor containing paste layer 720.The conductor containing paste contains metal particles or conductive ceramic particles.", "The average particle diameter of tungsten particles or molybdenum particles, which will be the metal particles, is preferably from 0.1 to 5 μm.", "If the average particle is less than 0.1 μm or exceeds 5 μm, the conductor containing paste is not easily printed.", "Such a conductor containing paste may be a composition (paste) obtained by mixing, for example, 85 to 87 parts by weight of the metal particles or the conductive ceramic particles; 1.5 to 10 to parts by weight of at least one binder selected from acrylic resin binders, ethylcellulose, butylcellosolve and polyvinyl alcohol; and 1.5 to 10 parts by weight of at least one solvent selected from α-terpineol and glycol.", "A conductor containing paste which is usually used when electrostatic electrodes and the like are formed is printed on the green sheet wherein the portions 730, 730′, which will be the conductor filled through holes, are formed, so as to form a conductor containing paste layer 780.", "(3) Step of Laminating the Green Sheets Green sheets 500 on which no conductor containing paste is printed are laminated on the green sheet on which the conductor containing paste is printed, and then the green sheet wherein the conductor containing paste layer 780 is formed is put beneath the resultant.", "Furthermore, green sheets 500 wherein no conductor containing paste layer is printed is printed are laminated beneath this green sheet (see FIG.", "26(a)).", "At this time, the number of the green sheets 500 laminated on the upper side of the green sheet wherein the conductor containing paste layer 720 is printed is made larger than that of the green sheets 500 laminated on the lower side, so that the positions where heating elements to be produced are formed are biased toward the bottom side.", "Specifically, the number of the green sheets 500 laminated on the upper side is preferably from 20 to 50, and that of the green sheets 500 laminated on the lower side is preferably from 5 to 20.", "(4) Step of Firing the Green Sheet Lamination The green sheet lamination is heated and pressed to sinter the green sheets 500 and the inner conductor containing paste layers 720, 780, and so forth, thereby producing a ceramic substrate 71, heating elements 72, conductor circuits 78, and so forth (see FIG.", "26(b)).", "The heating temperature is preferably from 1000 to 2000° C., and the pressing pressure is preferably from 10 to 20 MPa.", "The heating is performed in the atmosphere of an inert gas.", "As the inert gas, argon, nitrogen and the like can be used.", "Next, bottomed holes, for embedding temperature measuring elements, are formed in the bottom face 71b of the ceramic substrate 71 (not illustrated).", "The bottomed holes can be formed by grinding the surface and then performing drilling or blast treatment such as sandblast.", "The above-mentioned bottomed holes or concave portions may be formed after the ceramic substrate 71 and a cylindrical ceramic body 70, which will be described later, are bonded to each other, or may be formed at the same time of laminating and firing the green sheets 500 after portions which will be the bottomed holes are beforehand formed in the green sheets 500.Blind holes 79 are also provided to make the conductor filled through holes 73, 73′, for connecting the inner heating elements 72, exposed.", "The blind holes 79 may also be formed after the ceramic substrate 71 is bonded to the cylindrical ceramic body 77.", "(5) Production of the Cylindrical Ceramic Body Nitride aluminum power and the like are put into a cylindrical mold, and is formed.", "If necessary, the formed body is cut.", "This is sintered at a heating temperature of 1000 to 2000° C. under a normal pressure to produce the cylindrical ceramic body 77.The sintering is performed in an inert gas atmosphere.", "Examples of the inert gas which can be used include argon and nitrogen.", "The size of the cylindrical ceramic body 77 is adjusted in such a manner that the conductor filled through holes 73, 73′ formed inside the ceramic substrate are put in the body 77.Next, end faces of the cylindrical ceramic body 77 are ground to be made flat.", "(6) Bonding of the Ceramic Substrate to the Cylindrical Ceramic Body In the state that the central vicinity of the bottom face 71b of the ceramic substrate 71 contacts the end face of the cylindrical ceramic body 77, the ceramic substrate 71 and the cylindrical ceramic body 77 are heated and bonded to each other.", "At this time, the conductor filled through holes 73, 73′ inside the ceramic substrate 71 are allowed to be above an area inside the inner diameter of the cylindrical ceramic body 77, and further the center of the circle surrounded by the interface between the cylindrical ceramic body 77 and the ceramic substrate 71 is positioned 3 to 200 μm apart from the center of the bottom face of the ceramic substrate 71 to bond the cylindrical ceramic body 77 to the bottom face 71b of the ceramic substrate 71 (see FIG.", "26(c)).", "Specifically, a mask 190 in which an opening 191 is formed as illustrated in FIG.", "27 is put on the bottom face of the ceramic substrate 71 and subsequently the cylindrical ceramic body 77 is fitted into the opening 191 and heated, thereby bonding the ceramic substrate 71 and the cylindrical ceramic body 77 to each other.", "Since the opening diameter of the opening 191 is equal to the outer diameter of the cylindrical ceramic body 77, the distance between the center C of the opening 191 and the center B of the bottom face of the ceramic substrate 71 is equal to the distance L between: the center of the circle surrounded by the interface between the ceramic substrate 71 and the cylindrical ceramic body 77; and the center of the bottom face of the ceramic substrate 71.As the method of bonding the ceramic substrate 71 to the cylindrical ceramic body 77, a method of using brazing gold, brazing silver and the like to perform brazing, a method of using an adhesive made of oxide-based glass and the like to bond them, or some other method can be used.", "The ceramic substrate 71 and the cylindrical ceramic body 77 can also be bonded to each other by a method of applying a ceramic paste comprising the same main component as the ceramic which constitutes the ceramic substrate 71 and the cylindrical ceramic body 77, and then sintering this, or a method of applying a solution containing a sintering aid to bonding faces of the ceramic substrate and the cylindrical ceramic body.", "In the third aspect of the present invention, thermal stress in the bonding faces can be dispersed even if any one of the bonding methods is used.", "Therefore, the air-tightness of the bonded portions of the ceramic substrate 71 and the cylindrical ceramic body 77 can be ensured.", "(7) Fitting of Terminals and so on External terminals 83 are inserted into the blind holes 79 formed inside the inner diameter of the cylindrical ceramic body 77 through a solder or a brazing material, and the solder and the like are heated for re-flow, thereby connecting the external terminals 83 to the conductor filled through holes 73, 73′ (FIG.", "26(d)).", "The heating temperature is preferably from 90 to 450° C. in the case of the solder treatment, and is preferably from 900 to 1100° C. in the case of the treatment with the brazing material.", "Next, the external terminals 83 are connected through sockets 85 to conductive wires 830 connected to a power source (see FIG.", "19).", "Furthermore, thermocouples as temperature measuring elements are inserted into the formed bottomed holes, and the holes are sealed with a heat-resistant resin and the like, whereby a ceramic heater having the cylindrical ceramic body on the bottom face thereof can be produced.", "According to this ceramic heater, after a semiconductor wafer such as a silicon wafer is put on the ceramic heater or the silicon wafer or the like is held by lifter pins, supporting pins or some other member, an operation such as washing can be performed while the silicon wafer or the like is heated or cooled.", "An electrostatic chuck can be produced by forming electrostatic electrodes inside the ceramic substrate when the above-mentioned ceramic heater is produced.", "In this case, however, it is necessary to form conductor filled through holes for connecting the electrostatic electrodes and external terminals, but it is unnecessary to form through holes for inserting supporting pins.", "In the case that electrodes are formed inside the ceramic substrate, it is advisable that a conductor containing paste layer which will be electrostatic electrodes is formed on the surface of a green sheet in the same manner as in the case of forming heating elements.", "BEST MODES FOR CARRYING OUT THE INVENTION The present invention will be described in more detail by way of working examples hereinafter.", "EXAMPLE 1 Production of Ceramic Heater (See FIGS.", "1, 2 and 5) (1) The following paste was used to perform formation by a doctor blade method, so as to form a green sheet 50 having a thickness of 0.47 μm: a paste obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 0.6 μm), 4 parts by weight of alumina, 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "(2) Next, this green sheet 50 was dried at 80° C. for 5 hours, and subsequently portions which would be conductor filled through holes 13a were formed by punching.", "(3) The following were mixed to prepare a conductor containing paste A: 100 parts by weight of tungsten carbide particles having an average particle diameter of 1 μm, 3.0 parts by weight of an acrylic resin binder, 3.5 parts by weight of α-terpineol solvent, and 0.3 part by weight of a dispersant.", "The following were mixed to prepare a conductor containing paste B: 100 parts by weight of tungsten particles having an average particle diameter of 3 μm, 1.9 parts by weight of an acrylic resin binder, 3.7 parts by weight of α-terpineol solvent, and 0.2 part by weight of a dispersant.", "This conductor containing paste A was printed on the green sheet by screen printing, so as to form a conductor containing paste layer 120 for heating elements.", "The printed pattern was made into a concentrically circular pattern as described in FIG.", "1.Moreover, the conductor containing paste B was filled into the portions which would be conductor filled through holes 13a for connection to external terminals 13, so as to form a filled layer 130.Thirty seven green sheets 50 on which no conductor containing paste was printed were laminated on the upper side (heating surface) of the green sheet 50 that had been subjected to the above-mentioned processing, and the same thirteen green sheets were laminated on the lower side of the green sheet 50.The resultant was pressed at 130° C. and a pressure of 8 MPa to form a lamination (see FIG.", "5(a)).", "(4) Next, the resultant lamination was degreased at 600° C. in the atmosphere of nitrogen gas for 5 hours and hot-pressed at 1890° C. and a pressure of 15 MPa for 10 hours to yield a ceramic plate 3 mm in thickness.", "This was cut off into a disk 210 mm in diameter to prepare a ceramic plate having therein heating elements 12 having a thickness of 6 μm and a width of 10 mm.", "(5) Next, the ceramic plate obtained in the (4) was ground with a diamond grindstone.", "Subsequently, bottomed holes 14, for inserting thermocouples into, were formed in the bottom face.", "Moreover, three through holes 15 (diameter: 5.6 mm), for letting lifter pins (diameter: 5 mm) for carrying a semiconductor wafer or the like pass through, were formed (see FIG.", "5(b)).", "The through holes 15 were formed at regular intervals on a circle having a diameter of 116 mm and having a concentric circle relationship with the ceramic substrate 11.The through holes 15 were present in an area whose distance from the center of the ceramic substrate 11 was 55%, that is, ½ or more of the distance from the center to the outer edge thereof.", "(6) Next, the portions above the formed conductor filled through holes 13a were hollowed out to form blind holes 3b (see FIG.", "5(c)).", "Brazing gold made of Ni and Au was used and heated for re-flow to connect external terminals 13 made of kovar to the blind holes 13b (see FIG.", "5(d)).", "(7) Thermocouples (not illustrated) for temperature-control were embedded in the bottomed holes 14 to finish the production of a ceramic heater 10 of the first aspect of the present invention.", "EXAMPLE 2 Production of Ceramic Heater (See FIGS.", "3, 4 and 6) (1) A composition made of 100 parts by weight of aluminum nitride powder (average particle diameter: 0.6 μm), 4 parts by weight of yttria (average particle diameter: 0.4 μm), 12 parts by weight of an acrylic resin binder and an alcohol was subjected to spray-drying to form granular powder.", "(2) Next, this granular powder was put into a mold and formed into a flat plate form to obtain a raw formed body (green).", "(3) next, this raw formed body was hot-pressed at 1800° C. and a pressure of 20 MPa to yield a nitride aluminum plate having a thickness of 3 mm.", "Next, this plate was cut out into a disk having a diameter of 210 mm, so as to prepare a plate made of the ceramic (ceramic substrate 21).", "This ceramic substrate 21 was drilled to form three through holes 25 (diameter: 3.5 mm), for passing lifter pins 26 (diameter: 3 mm) through, and bottomed holes 24 for embedding thermocouples (see FIG.", "6(a)).", "The through holes 25 were formed at regular intervals on a circle having a concentric circle relationship with the ceramic substrate 21 and having a diameter of 158 mm.", "The positions where the through holes 25 were formed were positions whose distance from the center of the ceramic substrate 21 was 75%, that is, ½ or more of the distance from the center to the outer edge thereof.", "(4) A conductor containing paste layer was formed on the ceramic substrate 21 obtained in the above-mentioned step (3) by screen printing.", "The printed pattern was a concentrically circular pattern illustrated in FIG.", "3.The used conductor containing paste was a paste having a composition of 48% by weight of Ag, 21% by weight of Pt, 1.0% by weight of SiO2, 1.2% by weight of B2O3, 4.1% by weight of ZnO, 3.4% by weight of PbO, 3.4% by weight of ethyl acetate and 17.9% by weight of butyl carbitol.", "The conductor containing paste was a Ag-Pt paste, and the silver particles had an average particle diameter of 4.5 μm, and were scaly.", "The Pt particles had an average particle size of 0.5 μm, and were spherical.", "(5) Furthermore, the ceramic substrate 21 was heated and fired at 780° C. after the formation of the conductor containing paste layer, so as to sinter Ag and Pt in the conductor containing paste and bake them onto the ceramic substrate 21.Thus, heating elements 22 were formed (see FIG.", "6(b)).", "The heating elements 22 had a thickness of 5 μm, a width of 2.4 mm and an area resistivity of 7.7 mΩ/□.", "(6) The ceramic substrate 21 manufactured in the above-mentioned (5) was immersed into an electroless nickel plating bath consisting of an aqueous solution containing 80 g/L of nickel sulfate, 24 g/L of sodium hypophosphite, 12 g/L of sodium acetate, 8 g/L of boric acid, and 6 g/L of ammonium chloride to precipitate a metal covering layer (nickel layer) 220 having a thickness of 1 μm on the surface of the silver-lead heating elements 22 (see FIG.", "6(c)).", "(7) Next, by screen printing, a silver-lead solder paste (manufactured by Tanaka Kikinzoku Kogyo K.K.)", "was printed on portions onto which terminal portions 23 for attaining connection to a power source would be set up, to form a solder layer (not illustrated).", "Next, the external terminals 23 made of kovar were put on the solder layer, and heated for re-flow at 420° C. to attach the external terminals 23 onto the surface of the heating elements 22 (see FIG.", "6(d)).", "(8) Thermocouples (not illustrated) for temperature-control were sealed in the bottomed holes 24 with a polyimide to finish the production of a ceramic heater 20 of the first aspect of the present invention.", "EXAMPLE 3 (1) The following paste was used to perform formation by a doctor blade method, so as to form a green sheet 50 having a thickness of 0.47 μm: a paste obtained by mixing 100 parts by weight of SiC powder (manufactured by Yakushima Denko, average particle diameter: 1.1 μm), 4 parts by weight of B4C, 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "Furthermore, 80 parts by weight of borosilicate glass having an average particle size of 1.0 μm, 5 parts by weight of polyethylene glycol and 15 parts by weight of alcohol were mixed to yield a glass paste.", "This glass paste was applied to the formed green sheet.", "(2) Next, this green sheet was dried at 80° C. for 5 hours, and subsequently portions which would be conductor filled through holes were formed by punching.", "(3) The following were mixed to prepare a conductor containing paste A: 100 parts by weight of tungsten carbide particles having an average particle diameter of 1 μm, 3.0 parts by weight of an acrylic type binder, 3.5 parts by weight of α-terpineol solvent, and 0.3 part by weight of a dispersant.", "The following were mixed to prepare a conductor containing paste B: 100 parts by weight of tungsten particles having an average particle diameter of 3 μm, 1.9 parts by weight of an acrylic resin binder, 3.7 parts by weight of α-terpineol solvent, and 0.2 part by weight of a dispersant.", "This conductor containing paste A was printed on the green sheet by screen printing, so as to form a conductor containing paste layer for heating elements.", "The printed pattern was formed into a concentrically circular pattern as described in FIG.", "3.Moreover, the conductor containing paste B was filled into the portions which would be the conductor filled through holes for connection to external terminals, so as to form a filled layer.", "The glass paste was applied to the green sheet which had been subjected to the above-mentioned processing, and further thirty seven green sheets on which no conductor containing paste was printed were laminated on the upper side (heating surface) of the green sheet, and the same thirteen green sheets were laminated on the lower side of the green sheet.", "The resultant was pressed at 130° C. and a pressure of 8 MPa to form a lamination.", "(4) Next, the resultant lamination was degreased at 600° C. in the atmosphere of nitrogen gas for 5 hours and hot-pressed at 1890° C. and a pressure of 15 MPa for 10 hours to yield a ceramic plate 3 mm in thickness.", "This was cut off into a disk 230 mm in diameter to prepare a ceramic plate having therein heating elements having a thickness of 6 μm and a width of 10 mm.", "Furthermore, three through holes for lifter pins, which had a diameter of 5 mm, were formed at regular intervals on a circle having a diameter of 207 mm and having a concentric circle relationship with the ceramic substrate.", "The positions where the through holes were formed were present in an area whose distance from the center of the ceramic substrate was 90%, that is, ½ or more of the distance from the center to the outer edge thereof.", "(5) Next, the ceramic plate obtained in the (4) was ground with a diamond grindstone.", "Furthermore, a sputtering machine (ASP-34, manufactured by Showa Sinku) was used to form a magnesium fluoride film having a thickness of 2 mm on the surface.", "(6) Next, the portions above the formed conductor filled through holes 13a were hollowed out to form blind holes.", "Brazing gold made of Ni and Au was used and heated for re-flow to connect external terminals made of kovar to the blind holes.", "(7) Thermocouples (not illustrated) for temperature-control were embedded in the bottomed holes to yield a ceramic heater.", "TEST EXAMPLE 1 This example was basically the same as in Example 1, but the diameter was set to 330 mm, and the positions of the through holes for lifter pins were at positions whose distance from the center of the ceramic substrate was 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the distance from the center to the outer edge thereof.", "As will be described later, the temperature evenness in the heating face when the temperature of the ceramic heater was risen and the number of free particles were measured.", "COMPARATIVE EXAMPLE 1 Production of Ceramic Heater A ceramic heater was produced in the same way as in Example 1 except that through holes (diameter: 5.6 mm) , for letting lifter pins (diameter: 5.0 mm) pass through, were formed at positions described below.", "That is, three through holes were formed at regular intervals on a circle having a diameter of 63 mm and having a concentric circle relationship with the ceramic substrate.", "The positions where the through holes were formed were positions whose distance from the center of the ceramic substrate was 30% of the distance from the center to the outer edge thereof, and were not in an area whose distance from the center was ½ or more thereof.", "A silicon wafer was placed by means of the lifter pins at each of ceramic heaters according to Examples 1 to 3 and Comparative Example 1, and electric current was sent thereto so as to rise the temperature thereof to 300° C. They were evaluated by the following methods.", "The results are shown in Table 1.The nine ceramic heaters according to Test Example were also evaluated by the following methods.", "The results are shown in FIGS.", "7 and 8.The lifter pins projected by 50 μm from the heating face of the ceramic heater.", "The portions of the silicon wafer supported by the lifter pins were 50 μm apart from the heating face.", "Evaluation Method (1) Temperature Evenness of the In-face Temperature of the Heating Face at the Time of Rising the Temperature The temperature of the ceramic heater was risen to 300° C. in 45 seconds while a silicon wafer having thermocouples was placed.", "The difference in the temperature of the silicone wafer between the highest temperature and the lowest temperature in the step of rising the temperature was examined.", "(2) The Number of Free Particles A semiconductor wafer having a diameter of 200 mm or 300 mm was placed, and a test wherein this semiconductor wafer was pushed up by the lifter pins was carried out 100 times.", "The number of free particles adhering to the wafer was then measured.", "In the free particle-number measurement, arbitrary 10 points were observed with an electron microscope to measure the number of free particles, and the number was converted to the number per cm2.TABLE 1 In-face temperature distribution in the heating face at the time of Distance of the rising the The number of through holes temperature free particles from the center (%) *1 (° C.) *2 (pcs/cm2) Example 1 55 2 68 Example 2 75 1.5 53 Example 3 90 1.8 96 Comparative 30 5 265 Example 1 *1) Distance of the through holes from the center: Percentage of the distance from the center of the ceramic substrate to the through holes to the distance from the center to the outer edge of the ceramic substrate *2) In face temperature distribution (° C.) in the heating face: Maximum of the temperature difference between the highest temperature and the lowest temperature in the heating face at stationary time As is evident from Table 1, the temperature of the ceramic heaters according to the Examples was even at the time of rising the temperature.", "On the other hand, the temperature of the ceramic heater according to the comparative example was largely dispersed at the time of rising the temperature.", "This is because: in the ceramic heater according to the comparative example, the three through holes concentrated to the central portion thereof and the heat capacity per unit area (volume) of the central portion became smaller; therefore, the temperature of the central portion easily got high when the temperature was risen.", "On the other hand, in the ceramic heaters according to the Examples, this is also presumed to be because, the heat capacities per unit area (volume) between the central portion and the peripheral portion were hardly different since the through holes were formed in the peripheral portion, which had a large area (large volume).", "It is understood from FIG.", "7 that for the above-mentioned reason the temperature was even at the time of rising the temperature in the case that the through holes were formed at positions whose distance from the center of the ceramic substrate was 50% or more of the distance from the center to the outer edge thereof.", "It can be understood from FIG.", "8 that the number of free particles got fewer in the case that the through holes were formed at positions whose distance from the center of the ceramic substrate was 50% or more of the distance from the center to the outer edge thereof.", "As described above, it is presumed that: when the wafer warped upwards into a convex form, the outer circumference contacted the ceramic substrate linearly and scratched the ceramic substrate surface, whereby free particles were generated; however, in the first aspect of the present invention, the positions of the through holes for the lifter pins were arranged in an area whose distance from the center was ½ or more of the distance between the outer edge and the center of the substrate, whereby free particles were prevented from being generated.", "EXAMPLE 4 Production of Ceramic Heater (See FIGS.", "9, 10 and 13) (1) The following paste was used to perform formation by a doctor blade method, so as to form a green sheet 100 having a thickness of 0.47 μm: a paste obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 0.6 μm), 4 parts by weight of alumina, 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "(2) Next, this green sheet 100 was dried at 80° C. for 5 hours, and subsequently portions which would be conductor filled through holes 33a were formed by punching.", "(3) The following were mixed to prepare a conductor containing paste A: 100 parts by weight of tungsten carbide particles having an average particle diameter of 1 μm, 3.0 parts by weight of an acrylic resin binder, 3.5 parts by weight of α-terpineol solvent, and 0.3 part by weight of a dispersant.", "The following were mixed to prepare a conductor containing paste B: 100 parts by weight of tungsten particles having an average particle diameter of 3 μm, 1.9 parts by weight of an acrylic resin binder, 3.7 parts by weight of α-terpineol solvent, and 0.2 part by weight of a dispersant.", "This conductor containing paste A was printed on the green sheet by screen printing, so as to form a conductor containing paste layer 320 for heating elements.", "The printed pattern was formed into a concentrically circular pattern as described in FIG.", "9.Moreover, the conductor containing paste B was filled into the portions which would be conductor filled through holes 33a for connection to external terminals 33, so as to form a filled layer 330.Thirty seven green sheets 100 on which no conductor containing paste was printed were laminated on the upper side (heating surface) of the green sheet 100 that had been subjected to the above-mentioned processing, and the same thirteen green sheets were laminated on the lower side thereof.", "The resultant was pressed at 130° C. and a pressure of 8 MPa to form a lamination (see FIG.", "13(a)).", "(4) Next, the resultant lamination was degreased at 600° C. in the atmosphere of nitrogen gas for 5 hours and hot-pressed at 1890° C. and a pressure of 15 MPa for 10 hours to yield a ceramic plate 4 mm in thickness.", "This was cut off into a disk of 310 mm in diameter to prepare a ceramic plate having therein heating elements 32 having a thickness of 6 μm and a width of 10 mm.", "(5) Next, the ceramic plate obtained in the (4) was ground with a diamond grindstone.", "Subsequently bottomed holes 34, for inserting thermocouples into, were formed in the bottom face.", "Moreover, three through holes 35, for letting lifter pins 36 (diameter: 3 mm) for carrying a semiconductor wafer or the like pass through, were formed (see FIG.", "13(b)).", "About the through holes 35, the diameter of their columnar portion 35a was 3.5 mm and the length thereof was 2 mm, and depth (length) of their diameter-increasing portion 35b was 2 mm and the diameter, at the heating face, of the diameter-increasing portion 35b was 7 mm (see FIG.", "10).", "The through holes 35 were formed at regular intervals on a circle having a diameter of 200 mm and having a concentric circle relationship with the ceramic substrate 31.", "(6) Next, the portions above the formed conductor filled through holes 33a were hollowed out to form blind holes 33b (see FIG.", "13(c)).", "Brazing gold made of Ni and Au was used and heated for re-flow to connect external terminals 33 made of kovar to the blind holes 33b (see FIG.", "13(d)).", "(7) Thermocouples (not illustrated) for temperature-control were embedded in the bottomed holes 34 to finish the production of a ceramic heater 30 of the second aspect of the present invention.", "EXAMPLE 5 Production of Ceramic Heater (See FIGS.", "11, 12 and 14) (1) A composition made of 100 parts by weight of aluminum nitride powder (average particle diameter: 0.6μm), 4parts by weight of yttria (average particle diameter: 0.4 μm), 12 parts by weight of an acrylic resin binder and an alcohol was subjected to spray-drying to form granular powder.", "(2) Next, this granular powder was put into a mold and formed into a flat plate form to obtain a raw formed body (green).", "(3) Next, this raw formed body was hot-pressed at 1800° C. and a pressure of 20 MPa to yield a nitride aluminum plate having a thickness of 5 mm.", "Next, this plate was cut out into a disk having a diameter of 310 mm, so as to prepare a plate made of the ceramic (ceramic substrate 41).", "This ceramic substrate 41 was drilled to form three through holes 45 (diameter: 3.5 mm), for letting lifter pins 46 (diameter: 3 mm) pass through, and bottomed holes 44 for embedding thermocouples (see FIG.", "14(a)).", "About the through holes 45, the diameter of their columnar portion 45a was 3.5 mm and the length thereof was 3 mm, and the depth (length) of their diameter-increasing portion 45b was 2 mm and the diameter, in the heating face, of the diameter-increasing portion 45b was 10 mm (see FIG.", "12).", "The through holes 45 were formed at regular intervals on a circle having a concentric circle relationship with the ceramic substrate 41 and having a diameter of 40 mm.", "(4) A conductor containing paste layer was formed on the ceramic substrate 41 obtained in the above-mentioned (3) by screen printing.", "The printed pattern was a concentrically circular pattern illustrated in FIG.", "3.The used conductor containing paste was a paste having a composition of 48% by weight of Ag, 21% by weight of Pt, 1.0% by weight of SiO2, 1.2% by weight of B2O3, 4.1% by weight of ZnO, 3.4% by weight of PbO, 3.4% by weight of ethyl acetate and 17.9% by weight of butyl carbitol.", "This conductor containing paste was a Ag-Pt paste, and the silver particles had an average particle diameter of 4.5 μm, and were scaly.", "The Pt particles had an average particle size of 0.5 μm, and were spherical.", "(5) Furthermore, the ceramic substrate 41 was heated and fired at 780° C. after the formation of the conductor containing paste layer, so as to sinter Ag and Pt in the conductor containing paste and bake them onto the ceramic substrate 41.Thus, heating elements 42 were formed (see FIG.", "14(b)).", "The heating elements 42 had a thickness of 5 μm, a width of 2.4 mm and an area resistivity of 7.7 mΩ/□.", "(6) The ceramic substrate 41 formed in the above-mentioned (5) was immersed into an electroless nickel plating bath consisting of an aqueous solution containing 80 g/L of nickel sulfate, 24 g/L of sodium hypophosphite, 12 g/L of sodium acetate, 8 g/L of boric acid, and 6 g/L of ammonium chloride to precipitate a metal covering layer (nickel layer) 420 having a thickness of 1 μm on the surface of the silver-lead heating elements 42 (see FIG.", "14(c)).", "(7) By screen printing, a silver-lead solder paste (manufactured by Tanaka Kikinzoku Kogyo K. K.) was printed on portions onto which terminal portions 43 for attaining connection to a power source would be set up, to form a solder layer (not illustrated).", "Next, the external terminals 43 made of kovar were put on the solder layer, and heated for re-flow at 420° C. to attach the external terminals 43 onto the surface of the heating elements 42 (see FIG.", "14(d)).", "(8) Thermocouples (not illustrated) for temperature-control were sealed in the bottomed holes 44 with a polyimide to finish the production of a ceramic heater 40 of the second aspect of the present invention.", "COMPARATIVE EXAMPLE 2 Production of Ceramic Heater In the same way as in the prior art, a ceramic heater was produced in the same way as in Example 4 except that columnar through holes (diameter: 3.5 mm), for letting lifter pins (diameter: 3.0 mm) pass through, were formed in the ceramic substrate.", "COMPARATIVE EXAMPLE 3 A ceramic heater was produced in the same way as in Example 4 except that plugs, which were capable of being fitted into the through holes and were made of AlN, were fitted to the through holes.", "A silicon wafer was placed by means of the lifter pins at each of ceramic heaters according to Examples 4 and 5 and Comparative Examples 2 and 3, and electric current was sent thereto so as to rise the temperature thereof to 300° C. They were evaluated by the following methods.", "The lifter pins projected by 50 μm from the heating face of the ceramic heater.", "The portions of the silicon wafer which is supported by the lifter pins were 50 μm apart from the heating face.", "Evaluation Method (1)Temperature Evenness of the In-Face Temperature of the Heating Face at the Time of Rising the Temperature The temperature of the ceramic heater was risen to 300° C. in 45 seconds while a silicon wafer having thermocouples was placed.", "The difference in the temperature of the silicone wafer between the highest temperature and the lowest temperature in the step of rising the temperature was examined.", "The results are shown in Table 2.", "(2) The Number of Free Particles on the Silicon Wafer The temperature of the ceramic heater was risen to 300° C. Thereafter, the number of free particles generated on the silicon wafer was measured.", "The results are shown in Table 2.TABLE 2 In face-temperature distribution in the heating face The number of at the time of rising free particles the temperature (° C.) (pcs/cm2) Example 4 3 30 Example 5 2 30 Comparative Example 2 5 30 Comparative Example 3 2 500 Note) In face-temperature distribution: difference between the highest temperature and the lowest temperature in the silicon wafer As is evident from Table 2, according to the ceramic heaters according to the Examples, the temperature of the silicon wafer was even at the time of rising the temperature.", "On the other hand, according to the ceramic heater according to Comparative Example 2, the temperature of the silicon wafer was largely dispersed at the time of rising the temperature.", "In particular, at portions corresponding to the portions where the through holes were formed, the temperature thereof dropped.", "It appears that the reason for this is as follows: in the ceramic heater according to Comparative Example 2, the heat capacity of cooling spot portions was large since the through holes had a columnar shape and the diameter thereof was not increased in the vicinity of the heating face, and this resulted in a drop in the temperature of silicon wafer at portions corresponding to the portions where the through holes were formed.", "On the other hand, in the ceramic heaters of the Examples, the through holes were composed of the columnar portion and the diameter-increasing portion and the diameter thereof was increased in the vicinity of the heating face; therefore, the heat capacity of portions where cooling spots were easily generated was small.", "For this reason, a dispersion in the temperature of the silicon wafer is considered not be easily generated.", "In the ceramic heater according to Comparative Example 3, the plugs made of AlN were fitted to the through holes.", "Thus, the ceramic heater had no portions where cooling spots were easily generated.", "For this reason, the temperature of the silicon wafer was even at the time of rising the temperature.", "In the ceramic heaters according to Examples 4, 5 and Comparative Example 2, the silicon wafer was placed by way of the lifter pins.", "Thus, free particles were hardly generated.", "However, in the ceramic heater according to Comparative Example 3, the ceramic heater was scrubbed with the plugs made of AlN at the time of the heating, so that a large amount of free particles were generated in the silicon wafer.", "EXAMPLE 6 In the present Example 6, a ceramic heater was produced in the same way as in Example 4 except that the ratio of the diameter of the diameter-increasing portion in the heating face and the columnar portion was changed.", "A silicon wafer having thermocouples was placed on the produced ceramic heater through the lifter pins, and then the wafer was heated to 300° C. The difference between the highest temperature and the lowest temperature in the silicon wafer was examined.", "The result is shown in FIG.", "16.In FIG.", "16, the vertical axis ΔT represents the temperature difference (° C.) between the highest temperature and the lowest temperature in the silicon wafer, and the ratio as the horizontal axis represents the ratio of the diameter of the diameter-increasing portion at the heating face of the ceramic heater to the diameter of the columnar portion (the diameter of the diameter-increasing portion/the diameter of the columnar portion).", "As is clear from FIG.", "16, if the ratio of the diameter of the diameter-increasing portion in the heating face to the diameter of the columnar portion is less than 1.2 or exceeds 10, the temperature difference ΔT in the silicon wafer gets large.", "EXAMPLE 7 Production of Electrostatic Chuck (See FIGS.", "21 to 22) (1) The following paste was used to perform formation by a doctor blade method, so as to form a green sheet having a thickness of 0.47 μm: a paste obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 1.1 μm), 4 parts by weight of yttrium (average particle diameter: 0.4 μm), 12 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "(2) Next, this green sheet 100 was dried at 80° C. for 5 hours, and subsequently the following were produced: a green sheet to which no processing was applied; a green sheet in which through holes for via holes, for connecting heating elements to conductor circuits, were formed by punching; a green sheet in which through holes for via holes, for connecting the conductor circuits to external terminals, were formed by punching; and a green sheet in which through holes for conductor filled through holes, for connecting electrostatic electrodes to the external terminals, were formed by punching.", "(3) The following were mixed to prepare a conductor containing paste A: 100 parts by weight of tungsten carbide particles having an average particle diameter of 1 μm, 3.0 parts by weight of an acrylic resin binder, 3.5 parts by weight of α-terpineol solvent, and 0.3 part by weight of a dispersant.", "The following were mixed to prepare a conductor containing paste B: 100 parts by weight of tungsten particles having an average particle diameter of 3 μm, 1.9 parts by weight of an acrylic resin binder, 3.7 parts by weight of α-terpineol solvent, and 0.2 part by weight of a dispersant.", "(4) The conductor containing paste A was printed on the surface of the green sheets in which the through holes for the via holes were formed by screen printing, so as to print a conductor containing paste layer which would be the heating elements.", "The conductor containing paste A was printed on the surface of the green sheets in which the through holes for the conductor filled through holes, for connecting the conductor circuits to the external terminals, were formed by screen printing, so as to print a conductor containing paste layer which would be the conductor circuits.", "A conductor containing paste layer having an electrostatic electrode pattern having the shape illustrated in FIG.", "23 was formed on the green sheets to which no processing was applied.", "Moreover, the conductor containing paste B was filled into the through holes for the via holes, for connecting the heating elements to the conductor circuits, and the through holes for the conductor filled through holes, for connecting the external terminals.", "Next, the respective green sheets which had been subjected to the above-mentioned processing were laminated.", "First, thirty four green sheets in which only the portions which would be the conductor filled through holes 93 were formed were laminated on the upper side (heating face side) of the green sheet on which the conductor containing paste layer which would be the heating elements was printed, and then the green sheet on which the conductor containing paste layer which would be the conductor circuits was printed was laminated on the just lower side thereof (bottom face side).", "Furthermore, twelve green sheets in which the portions which would be the conductor filled through holes 93, 930 and 930′ were formed were laminated on the lower side thereof.", "The green sheet on which the conductor containing paste layer having the electrostatic electrode pattern was printed was laminated on the topmost portion of the thus-laminated green sheets, and further two green sheets to which no processing was applied were laminated thereon.", "The resultant was pressed at 130° C. and a pressure of 8 MPa to form a lamination.", "(5) Next, the resultant lamination was degreased at 600° C. in the atmosphere of nitrogen gas for 5 hours and then hot-pressed at 1890° C. and a pressure of 15 MPa for 3 hours to yield a nitride aluminum plate 3 mm in thickness.", "This was cut off into a disk of 230 mm in diameter to prepare a ceramic substrate 91 having therein the heating elements 920 having a thickness of 5 μm and a width of 2.4 mm, the conductor circuits 980 having a thickness of 20 μm and a width of 10 mm, and the chuck positive electrostatic layer 92a and the chuck negative electrostatic layer 92b having a thickness of 6 μm.", "(6) Next, the ceramic substrate 91 obtained in the (5) was ground with a diamond grindstone.", "Subsequently, a mask was put thereon, and blast treatment with glass beads was conducted to form bottomed holes 900 for thermocouples in the surface.", "The portions above the formed conductor filled through holes 93 and 930 were hollowed out in the bottom face 91b of the ceramic substrate 91 so as to form blind holes 990.", "(7) A composition obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 1.1 μm), 4 parts by weight of yttria (average particle diameter: 0.4 μm), 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol was used to produce granules by spray-drying.", "The granules were put into a pipe-form mold and sintered at 1890° C. under a normal pressure to produce a cylindrical ceramic body having a length of 200 mm, an outer diameter of 45 mm and an inner diameter of 35 mm.", "(8) To bonding faces of the ceramic substrate 91 and the cylindrical ceramic body 97 was applied a liquid obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 1.1 μm), 4 parts by weight of yttria (average particle diameter: 0.4 μm), 11.5 parts by weight of the acrylic resin binder, 0.5 part by weight of the dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "Thereafter, an end face of the cylindrical ceramic body 97 was brought into contact with the bottom face 91b of the ceramic substrate 91 at a position, in a manner that the blind holes 990 would be inside the inner diameter, of the end face.", "The resultant was heated at 1890° C. to bond the ceramic substrate 91 and the cylindrical ceramic body 97.Specifically, a mask 190 in which an opening 191 as illustrated in FIG.", "27 was formed was put on the bottom face of the ceramic substrate 91.Thereafter, the cylindrical ceramic body 97 was fitted into the opening 191, and the resultant was heated to bond the ceramic substrate 91 and the cylindrical ceramic body 97.The distance L between: the center of the circle surrounded by the interface between the ceramic substrate 91 and the cylindrical ceramic body 97; and the center of the bottom face of the ceramic substrate 91 was set to 5 μm.", "(9) Next, brazing silver (Ag: 40% by weight, Cu: 30% by weight, Zn: 28% by weight, Ni: 1.8% by weight, and the balance: other elements, re-flow temperature: 800° C.) was used to attach external terminals 960 to the blind holes 990 formed in the cylindrical ceramic body 97.Conductive wires 931 were connected to the external terminals 960 through sockets 950.", "(10) Thermocouples for temperature-control were inserted into the bottomed holes 900, and silica sol was filled into the holes.", "The silica sol was hardened and gelation occured at 190° C. for 2 hours to bond the cylindrical ceramic body to the bottom face of the ceramic substrate inside which the electrostatic electrodes, the heating elements, the conductor circuits, the via holes and the conductor filled through holes were formed.", "In this way, a ceramic bonded body wherein the ceramic substrate functioned as an electrostatic chuck was produced.", "EXAMPLE 8 Production of Ceramic Heater (See FIGS.", "18 to 19, and 26) (1) The following paste was used to perform formation by a doctor blade method, so as to form a green sheet having a thickness of 0.47 μm: a paste obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 1.1 μm), 4 parts by weight of yttrium oxide (Y2O3: yttria, average particle diameter: 0.4 μm), 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol.", "(2) Next, this green sheet was dried at 80° C. for 5 hours, and subsequently the following were formed by punching: portions which would be through holes 75, for letting lifter pins for carrying a silicon wafer pass through; portions which would be via holes 860; and portions 730, 730′ which would be conductor filled through holes, as illustrated in FIG.", "18.", "(3) The following were mixed to prepare a conductor containing paste A: 100 parts by weight of tungsten carbide particles having an average particle diameter of 1 μm, 3.0 parts by weight of an acrylic resin binder, 3.5 parts by weight of α-terpineol solvent, and 0.3 part by weight of a dispersant.", "The following were mixed to prepare a conductor containing paste B: 100 parts by weight of tungsten particles having an average particle diameter of 3 μm, 1.9 parts by weight of an acrylic resin binder, 3.7 parts by weight of α-terpineol solvent, and 0.2 part by weight of a dispersant.", "This conductor containing paste A was printed on the green sheets in which the portions 860, which would be the via holes, were formed by screen printing, so as to print a conductor containing paste layer 720 for heating elements.", "The printed pattern was formed into a concentrically circular pattern as illustrated in FIG.", "18.The width of the conductor containing paste layer 720 was set to 10 mm, and the thickness thereof was set to 12 μm.", "Subsequently, the conductor containing paste A was printed on the green sheet in which the portions 730′, which would be the conductor filled through holes, were formed by screen printing.", "In this way, a conductor containing paste layer 780 for conductor circuits was formed.", "The printed shape was a band shape.", "Moreover, the conductor containing paste B was filled into the portions 860, which would be the via holes, and the portions 730 and 730′, which would be the conductor filled through holes.", "Thirty seven green sheets in which no conductor containing paste was printed were laminated on the green sheet which had been subjected to the above-mentioned processing, the sheet having the printed conductor containing paste layer 720.The green sheet on which the conductor containing paste layer 780 was printed was then laminated beneath it.", "Thereafter, twelve green sheets on which no conductor containing paste was printed were laminated beneath it.", "The resultant was pressed at 130° C. and 8 MPa, so as to form a lamination.", "(4) Next, the resultant lamination was degreased at 600° C. in the atmosphere of nitrogen gas for 5 hours and then hot-pressed at 1890° C. and a pressure of 15 MPa for 10 hours to yield a nitride aluminum plate 3 mm in thickness.", "This was cut off into a disk of 230 mm in diameter to prepare a ceramic substrate 71 having therein the heating elements 72 having a thickness of 6 μm and a width of 10 mm, the conductor circuits 78 having a thickness of 20 μm and a width of 10 mm, the via holes 860 and the conductor filled through holes 73, 73′.", "(5) Next, the ceramic substrate 71 obtained in the (4) was ground with a diamond grindstone.", "Subsequently, a mask was put thereon, and blast treatment with glass beads was conducted to form bottomed holes 74 for thermocouples in the surface.", "The portions above the formed conductor filled through holes 73, 73′ were hollowed out in the bottom face 71b of the ceramic substrate 71, so as to form blind holes 79.", "(6) A composition obtained by mixing 100 parts by weight of aluminum nitride powder (manufactured by Tokuyama Corp., average particle diameter: 1.1 μm), 4 parts by weight of Y2O3 (average particle diameter: 0.4 μm), 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols of 1-butanol and ethanol was used to produce granules by spray-drying.", "The granules were put into a cylindrical mold and sintered at 1890° C. under a normal pressure to produce a cylindrical ceramic body 77.", "(7) To bonding faces of the ceramic substrate 71 and the cylindrical ceramic body 77 was applied an aqueous yttrium nitrate (2.61×10−1 mol/L) solution.", "Thereafter, an end face of the cylindrical ceramic body 77 was brought into contact with the bottom face 71b of the ceramic substrate 71 at a position, in a manner that the blind holes 79 would be inside the inner diameter, of the end face.", "The resultant was heated at 1890° C. to bond the ceramic substrate 71 and the cylindrical ceramic body 77.Specifically, a mask 190 in which an opening 191 as illustrated in FIG.", "27 was formed was put on the bottom face of the ceramic substrate 71.Thereafter, the cylindrical ceramic body 77 was fitted into the opening 191, and the resultant was heated to bond the ceramic substrate 71 and the cylindrical ceramic body 77.The distance L: between the center of the circle surrounded by the interface between the ceramic substrate 71 and the cylindrical ceramic body 77; and the center of the bottom face of the ceramic substrate 71 was set to 190 μm.", "(8) Next, brazing silver (Ag: 40% by weight, Cu: 30% by weight, Zn: 28% by weight, Ni: 1.8% by weight, and the balance: other elements, re-flow temperature: 800° C.) was used to attach external terminals 83 to the blind holes 79 formed in the cylindrical ceramic body 77.Conductive wires 830 were connected to the external terminals 83 through sockets 85.", "(9) Thermocouples for temperature-control were inserted into the bottomed holes 74, and silica sol was filled into the holes.", "The silica sol was hardened and gelation was occurred at 190° C. for 2 hours to bond the cylindrical ceramic body to the bottom face of the ceramic substrate inside which the heating elements, the conductor circuits, the via holes and the conductor filled through holes were formed.", "In this way, a ceramic bonded body wherein the ceramic substrate functioned as a ceramic heater was produced.", "EXAMPLE 9 A ceramic bonded body was produced in the same way as in Example 7 except that the following steps were carried out.", "First, the diameter of the ceramic substrate was set to 300 mm, and in the step (7) 100 parts by weight of aluminum nitride powder, 4 parts by weight of yttria, 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols were mixed to produce granules by spray-drying.", "Moreover, conductive wires were bonded to external terminals through sockets to form power source supplying lines.", "The power source supplying lines were put into a mold, and the granules were filled into the mold, and then pressed.", "Furthermore, the granules were subjected to cold isostatic press at a pressure of 1000 kg/cm2, and then sintered at 1890° C. under a normal pressure.", "The resultant was then shaped into a ceramic body formed of a columnar sold body having a length of 200 mm and an outer diameter of 45 mm.", "The distance L between: the center of the bottom face of the ceramic substrate; and the center of the interface between the ceramic body and the ceramic substrate was set to 3 μm.", "EXAMPLE 10 A ceramic bonded body was produced in the same way as in Example 8 except that the following steps were carried out.", "The diameter of the ceramic substrate was set to 320 mm, and in the (6) 100 parts by weight of aluminum nitride powder, 4 parts by weight of yttria, 11.5 parts by weight of an acrylic resin binder, 0.5 part by weight of a dispersant and 53 parts by weight of alcohols were mixed to produce granules by spray-drying.", "Moreover, conductive wires were bonded to external terminals through sockets to form power source supplying lines.", "The power source supplying lines were put into a mold, and the granules were filled into the mold, and then pressed.", "Furthermore, the granules were subjected to cold isostatic press at a pressure of 1000 kg/cm2, and then sintered at 1890° C. under a normal pressure.", "The resultant was then shaped into a ceramic body formed of a columnar sold body having a length of 200 mm and an outer diameter of 45 mm.", "The distance L between: the center of the bottom face of the ceramic substrate; and the center of the interface (circle) between the ceramic body and the ceramic substrate was set to 200 μm.", "EXAMPLE 11 A ceramic bonded body was produced in the same way as in Example 7 except that L was set to 10 μm.", "EXAMPLE 12 A ceramic bonded body was produced in the same way as in Example 8 except that L was set to 50 μm.", "EXAMPLE 13 A ceramic bonded body was produced in the same way as in Example 9 except that L was set to 100 μm.", "EXAMPLE 14 A ceramic bonded body was produced in the same way as in Example 10 except that L was set to 150 μm.", "TEST EXAMPLE 2 Ceramic bonded bodies wherein L was changed from 0 to 240 μm were produced.", "The temperatures of the ceramic bonded bodies were risen to 450° C. At this time, the temperature difference ΔT between the highest temperature and the lowest temperature in the heating face was measured.", "The results are shown in FIG.", "31.It is understood that when L was more than 200 μm, ΔT got large.", "The ceramic bonded bodies had the same constitution as illustrated in FIGS.", "18 to 19.COMPARATIVE EXAMPLE 4 A ceramic bonded body was produced in the same way as in Example 7 except that the ceramic substrate 91 was bonded to the cylindrical ceramic body 97 in such a manner that the center of the circle surrounded by the interface between the ceramic substrate 91 and the cylindrical ceramic body 97 and the center of the bottom face of the ceramic substrate 91 would be at the same position.", "COMPARATIVE EXAMPLE 5 A ceramic bonded body was produced in the same way as in Example 7 except that the distance L between: the center of the circle surrounded by the interface between the ceramic substrate 91 and the cylindrical ceramic body 97; and the center of the bottom face of the ceramic substrate 91 was set to 2 μm.", "COMPARATIVE EXAMPLE 6 A ceramic bonded body was produced in the same way as in Example 8 except that the distance L between: the center of the circle surrounded by the interface between the ceramic substrate 71 and the cylindrical ceramic body 77; and the center of the bottom face of the ceramic substrate 71 was set to 2 μm.", "COMPARATIVE EXAMPLE 7 A ceramic bonded body was produced in the same way as in Example 7 except that the distance L between: the center of the circle surrounded by the interface between the ceramic substrate 91 and the cylindrical ceramic body 97; and the center of the bottom face of the ceramic substrate 91 was set to 205 μm.", "COMPARATIVE EXAMPLE 8 A ceramic bonded body was produced in the same way as in Example 8 except that the distance L between: the center of the circle surrounded by the interface between the ceramic substrate 71 and the cylindrical ceramic body 77; and the center of the bottom face of the ceramic substrate 91 was set to 205 μm.", "The ceramic bonded bodies according to Examples 7 to 14 and Comparative Examples 4 to 8 were subjected to the following evaluation tests.", "The results are shown in Table 3 described below.", "(1) Measurement of the Breaking Strength A bending strength test was performed to measure the breaking strength of the bonding face.", "(2) Heat Cycle Test A heat cycle test wherein the step of keeping each of the ceramic bonded bodies at 25° C. and then heating it to 450° C. was repeated was made 500 times.", "It was checked whether or not a crack was generated in the bonding portion between the cylindrical ceramic body and the ceramic substrate.", "A case in which the generation rate was less than 50% was judged as no generation of any crack, and a case in which the generation rate was 50% or more was judged as crack generation.", "(3) Existence of Corrosion in the Wires and so on Each of the ceramic bonded bodies according to the Examples and the Comparative Examples was fitted to a supporting case, and the temperature thereof was risen to 200° C. in the atmosphere of CF4.Thereafter, the state of corrosion in the wires and so on of the ceramic bonded body was observed with the naked eye.", "Nitrogen gas was introduced, as an inert gas, into the cylindrical ceramic body.", "TABLE 3 Breaking strength (MPa) Heat cycle test Corrosion Example 7 400 No generation of Not generated crack Example 8 410 No generation of Not generated crack Example 9 440 No generation of Not generated crack Example 10 435 No generation of Not generated crack Example 11 411 No generation of Not generated crack Example 12 405 No generation of Not generated crack Example 13 438 No generation of Not generated crack Example 14 430 No generation of Not generated crack Comparative 320 Crack generation Generated Example 4 Comparative 300 Crack generation Generated Example 5 Comparative 285 Crack generation Generated Example 6 Comparative 320 Crack generation Generated Example 7 Comparative 300 Crack generation Generated Example 8 As is evident from the results shown in Table 3, the ceramic bonded bodies according to Examples 7 to 14 had sufficiently large bonding strength in both of the breaking strength test and the heat cycle test, and the wires and so on arranged inside the cylindrical ceramic bodies of these ceramic bonded body were not corroded by CF4 gas.", "On the other hand, in the ceramic bonded bodies according to Comparative Examples 4 to 8, the bonding strength between the cylindrical ceramic body and the ceramic substrate was low, and further the wires and so on arranged inside the cylindrical ceramic body were corroded by CF4 gas.", "It appears that this is because thermal stress concentrated locally in the bonding interface between the cylindrical ceramic and the disk-like ceramic, whereby thermal fatigue was generated so that cracks and the like were generated.", "EXAMPLES 15 AND 16, AND COMPARATIVE EXAMPLES 9 AND 10 L was set to 3 (Example 15), 200μm (Example 16), 0 μm (Comparative Example 9) and 205 μm (Comparative Example 10), respectively, and the diameter of a ceramic substrate was changed from 150 mm to 350 mm.", "The crack generation rates of the thus-produced ceramic bonded bodies were examined.", "The results are shown in FIG.", "32.As is evident from Comparative Examples 9 and 10, the crack generation rate was close to 80% when the diameter was more than 250 mm and practical endurance was not obtained.", "On the other hand, in Examples 15 and 16, the value of the crack generation rate was kept low even if the diameter was more than 250 mm.", "As described above, the present invention can overcome endurance drop generated in ceramic heaters having a diameter of 250 mm or more.", "Industrial Applicability According to the ceramic heater of the first aspect of the present invention, three or more through holes are formed in a ceramic substrate and formed in an area whose distance from the center of the ceramic substrate is ½ or more of the distance from the center to the outer edge thereof; therefore, lifter pins passed through the through holes are also present in the peripheral portion of the ceramic substrate and do not concentrate in the central portion.", "Thus, a semiconductor wafer supported by the lifter pins and the like does not become unstable.", "A difference in the heat capacity in the ceramic substrate is small so that a dispersion in the temperature thereof is small when the temperature is risen.", "Thus, a semiconductor wafer or the like can be evenly heated.", "According to the ceramic heater of the second aspect of the present invention, in through holes formed in a ceramic substrate, the diameter thereof on the side of its heating face for heating an object to be heated is larger than that on the side opposite to the heating face; therefore, the occupation rate of gas in portions where cooling spots are generated gets large so that the heat capacity thereof gets small.", "Accordingly, the temperature of a semiconductor wafer, a liquid crystal substrate and the like of the portion in the vicinity of the formed through holes hardly drops so that the object to be heated, such as the semiconductor wafer or the liquid crystal substrate, can be more evenly heated.", "Furthermore, according to the ceramic bonded body of the third aspect of the present invention, thermal stress does not concentrate locally in the bonding interface between a ceramic body having a given shape such as a cylindrical or columnar shape and a disk-like ceramic so that no crack and the like are generated in this portion; therefore, sufficient air-tightness can be kept.", "Thus, the reliability of the ceramic bonded body can be largely improved." ] ]

[ [ "Diagnosis of illnesses or predisposition to certain illnesses", "The present invention describes a set of oligomer probes (oligonucleotides and/or PNA oligomers), which serve for the detection of the cytosine methylation state in nucleic acids.", "These probes are particularly suitable for the diagnosis of existing diseases by analysis of a set of genetic and/or epigenetic parameters." ], [ "1.Nucleic acids comprising a sequence segment at least 18 bases long of a chemically pretreated DNA according to one of the sequences Seq.", "ID 1 to Seq.", "ID 40712.2.An oligomer (oligonucleotide or peptide nucleic acid (PNA) oligomer) for the detection of the cytosine methylation state in chemically pretreated DNA, containing at least one base sequence with a length of at least 9 nucleotides, which hybridizes to a chemically pretreated DNA (Seq.", "ID 1 to Seq.", "ID 40712).", "3.The oligomer according to claim 2, whereby the base sequence comprises at least one CpG dinucleotide.", "4.The oligomer according to claim 3, further characterized in that the cytosine of the CpG dinucleotide is found in approximately the middle third of the oligomer.", "5.A set of oligomers according to claim 3, comprising at least one oligomer for at least one of the CpG dinucleotides of one of the sequences of Seq.", "ID 1 to Seq.", "ID 40712.6.A set of oligomers according to claim 5 containing at least one oligomer for each of the CpG dinucleotides of one of the sequences of Seq.", "ID 1 to Seq.", "ID 40712.7.A set of at least two nucleic acids according to claim 2, which are utilized as primer oligonucleotides for the amplification of DNA sequences according to at least one of the sequences Seq.", "ID 1 to Seq.", "ID 40712 or segments thereof.", "8.A set of oligonucleotides according to claim 7, further characterized in that at least one oligonucleotide is bound to a solid phase.", "9.A set of oligomer probes for the detection of the cytosine methylation state and/or of single nucleotide polymorphisms (SNPs) in chemically pretreated genomic DNA according to one of the sequences Seq.", "ID 1 to Seq.", "ID 40712, comprising at least ten of the oligomers according to one of claims 2 to 4.10.A method for the production of an arrangement of different oligomers (an array) fixed on a support material for the analysis of disorders related to the methylation state of the CpG dinucleotides of one of the sequences Seq.", "ID 1 to Seq.", "ID 40712, in which at least one oligomer according to one of claims 2 to 4 is coupled to a solid phase.", "11.An arrangement of different oligomers (an array) according to one of claims 2 to 4, which is bound to a solid phase.", "12.The array of different oligonucleotide and/or PNA oligomer sequences according to claim 11, further characterized in that these are arranged on a planar solid phase in the form of a rectangular or hexagonal grid.", "13.The array according to claim 11, further characterized in that the solid phase surface is comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.", "14.A DNA and/or PNA array for the analysis of disorders related to the methylation state of genes, which contains at least one nucleic acid according to one of claims 1 or 2.15.A method for determining genetic and/or epigenetic parameters for the diagnosis of existing diseases or of the predisposition for specific diseases by analysis of cytosine methylations, is hereby characterized in that the following steps are conducted: a) in a genomic DNA sample, cytosine bases that are unmethylated at the 5′-position are converted by chemical treatment to uracil or another base unlike cytosine in its base-pairing behavior; b) from this chemically pretreated genomic DNA, fragments are amplified with the use of sets of primer oligonucleotides according to claim 7 or 8 and a polymerase, whereby the amplified products bear a detectable label; c) the amplified products are hybridized to a set of oligonucleotides and/or PNA probes containing at least one base sequence with a length of at least 9 nucleotides which hybridizes to a chemically pretreated DNA (Seq.", "ID 1 to Seq.", "ID 40712) or, however, to an array of different such oligonucleotides and/or PNA probes bound to a solid phase; d) the hybridized amplified products are then detected.", "16.The method according to claim 15, further characterized in that the chemical treatment is conducted by means of a solution of a bisulfite, hydrogen sulfite or disulfite.", "17.The method according to claim 15, further characterized in that more than ten different fragments are amplified, which are 100-2000 base pairs in length.", "18.The method according to claim 15, further characterized in that the amplification of several DNA segments is conducted in one reaction vessel.", "19.The method according to claim 15, further characterized in that the polymerase is a heat-stable DNA polymerase.", "20.The method according to claim 18, further characterized in that the amplification is conducted by means of the polymerase chain reaction (PCR).", "21.The method according to claim 15, further characterized in that the labels of the amplified products are fluorescent labels.", "22.The method according to claim 15, further characterized in that the labels of the amplified products are radionuclides.", "23.The method according to claim 15, further characterized in that the labels of the amplified products are removable molecular fragments with typical mass, which are detected in a mass spectrometer.", "24.The method according to claim 15, further characterized in that the amplified products or fragments of the amplified products are detected in the mass spectrometer.", "25.The method according to claim 23, further characterized in that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer.", "26.The method according to 23, further characterized in that the detection is carried out and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI).", "27.The method according to claim 15, further characterized in that the genomic DNA was obtained from cells or cell components that contain DNA, whereby sources for DNA comprise e.g., cell lines, biopsies, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and all possible combinations thereof.", "28.A kit, comprising a bisulfite (=bisulfite (disulfite), hydrogen sulfite) reagent as well as oligonucleotides and/or PNA oligomers according to one of claims 2 to 4.29.Use of a nucleic acid comprising a sequence segment at least 18 bases long of a chemically pretreated DNA according to one of the sequences Seq.", "ID 1 to Seq.", "ID 40712, an oligonucleotide or PNA oligomer containing at least one base sequence with a length of at least 9 nucleotides which hybridizes to a chemically pretreated DNA (Seq.", "ID 1 to Seq.", "ID 40712), a kit comprising a bisulfite (=bisulfite (disulfite), hydrogen sulfite) reagent as well as oligonucleotides and/or PNA oligomers according to one of claims 2 to 4, or an array of such oligonucleotides and/or PNA oligomers fixed on a support material for the diagnosis and/or therapy of undesired drug interactions; cancer diseases; CNS malfunctions; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain lesions; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease; malfunction, damage or disorder of the gastrointestinal tract; malfunction, damage or disorder of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunctions, headaches; sexual malfunctions, by analysis of methylation patterns.", "30.The method according to claim 24, further characterized in that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer.", "31.The method according to claim 24, further characterized in that the detection is carried out and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI)." ], [ "<SOH> FIELD OF THE INVENTION <EOH>The levels of observation that have been well studied in molecular biology according to developments in methods in recent years include the genes themselves, the transcription of these genes into RNA and the translation to proteins therefrom.", "During the course of development of an individual, which gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome.", "In this regard, pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome.", "The present invention describes nucleic acids, oligonucleotides, PNA oligomers and a method for the diagnosis of existing diseases or of predisposition for specific diseases." ], [ "FIELD OF THE INVENTION The levels of observation that have been well studied in molecular biology according to developments in methods in recent years include the genes themselves, the transcription of these genes into RNA and the translation to proteins therefrom.", "During the course of development of an individual, which gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome.", "In this regard, pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome.", "The present invention describes nucleic acids, oligonucleotides, PNA oligomers and a method for the diagnosis of existing diseases or of predisposition for specific diseases.", "PRIOR ART The methylation of CpG islands is often equated with transcription inactivity.", "Although there is clear evidence that CpG islands are to be found in promoters of genes, not all CpG islands and methylation sites are localized in known promoters.", "In different tissue-specific and imprinting genes, the CpG islands are localized at considerable distances downstream of the start of transcription, and also many genes possess multiple promoters.", "For a number of diseases, methylation of CpG dinucleotides has been detected as a causal factor.", "In contrast to classical mutations, DNA methylation involves a mechanism that describes a base substitution without modifying the coding function of a gene.", "This interplay between epigenetic modification and classical mutations plays an important role in tumorigenesis.", "For example, focal hypermethylation and generalized genomic demethylation are features of many different tumor types.", "It is assumed that tumorigenesis and tumor progression are caused, first of all, by hypermethylation of induced mutation events, and secondly, by the turning off of genes which control cellular proliferation and/or by the induced reactivation of genes, which are [normally] used only for embryological development, via demethylation.", "In hereditable non-polyposis colorectal cancer, e.g., the majority of mutation-negative cases of colon cancer are based rather on the hypermethylation of the hMLH1 promoter and the associated non-expression of hMLH1, a repair gene for erroneous base pairings (Bevilacqua R A, Simpson A J, Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability.", "Int J Cancer.", "Jul.", "15, 2000 ;87(2):200-3.).", "In the pathogenesis of lung cancer, the loss of expression is correlated with the methylation of CpG islands in the promoter sequence of an RAS effector homolog.", "(Dammann R, Li C, Yoon J H, Chin P L, Bates S, Pfeifer G P, Nucleotide.", "Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3.Nat.", "Genet.", "July 2000 ;25(3):315-9).", "An epigenetic inactivation of the LKB1 tumor supressor gene, including the hypermethylation of the promoter, is associated with the Peutz-Jeghers syndrome (Esteller M, Avizienyte E, Corn P G, Lothe R A, Baylin S B, Aaltonen L A, Herman J G, Epigenetic inactivation of LKB1 in primary tumors associated with the Peutz-Jeghers syndrome.", "Oncogene.", "Jan. 6, 2000;19(1):164-8).", "A plurality of diseases, which are associated with methylation, have in their etiology a close connection with the tumor suppressor genes p16 or p15.Thus a relationship between Mycosis fungoides and hypermethylation of the p16(INK4a) gene is assumed (Navas I C, Ortiz-Romero P L, Villuendas R, Martinez P, Garcia C, Gomez E, Rodriguez J L, Garcia D, Vanaclocha F, Iglesias L, Piris M A, Algara P, p16(INK4a) gene alterations are frequent in lesions of mycosis fungoides.", "Am J Pathol.", "May, 2000; 156(5):1565-72).", "Also, there is a strong correlation between the turning off of the transcription of the p16 gene in gastric carcinoma and the de novo methylation of a few specific CpG sites (Song S H, Jong H S, Choi H H, Kang S H, Ryu M H, Kim N K, Kim W H, Bang Y J, Methylation of specific CpG sites in the promoter region could significantly down-regulate p16(INK4a) expression in gastric adenocarcinoma.", "Int J Cancer.", "Jul.", "15, 2000;87(2):236-40).", "The pathogenesis of cholangiocarcinoma, which is associated with primary sclerosing cholangitis, has been related to the inactivation of the p16 tumor suppressor gene, which is again dependent on the methylation of the p16 promoter (Ahrendt S A, Eisenberger C F, Yip L, Rashid A, Chow J T, Pitt H A, Sidransky D, Chromosome 9p21 loss and p16 inactivation in primary sclerosing cholangitis-associated cholangiocarcinoma.", "J Surg Res.", "Jun.", "1, 2000;84(1):88-93).", "The inactivation of the p16 gene by hypermethylation plays a role in the genesis of leukemia and in the progression of acute lymphoblastic leukemia (Nakamura M, Sugita K, Inukai T, Goi K, Iijima K, Tezuka T, Kojika S, Shiraishi K, Miyamoto N, Karakida N, Kagami K, O-Koyama T, Mori T, Nakazawa S, p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.", "Leukemia.", "June 2000;13(6):884-90).", "In addition, it is postulated that the hypermethylation of the p16 and p15 genes plays a decisive role in the tumorigenesis of multiple myeloma (Ng M H, Wong I H, Lo K W, DNA methylation changes and multiple myeloma.", "Leuk Lymphoma.", "August 1999;34(5-6):463-72).", "The VHL gene, which is inactivated by methylation, appears to participate in predisposition to renal carcinoma (Glavac D, Ravnik-Glavac M, Ovcak Z, Masera A, Genetic changes in the origin and development of renal cell carcinoma (RCC).", "Pflugers Arch.", "1996;431(6 Suppl 2):R193-4).", "A divergent methylation of the 5′-CpG island may participate in nasopharyngeal carcinoma, possibly by the inactivation of transcription of the p16 gene (Lo K W, Cheung S T, Leung S F, van Hasselt A, Tsang Y S, Mak K F, Chung Y F, Woo J K, Lee J C, Huang D P, Hypermethylation of the p16 gene in nasopharyngeal carcinoma.", "Cancer Res.", "Jun.", "15, 1996;56(12):2721-5).", "An inactivation of the p16 protein was detected in liver cell carcinoma.", "Promoter hypermethylation and homozygous deletions are the most frequent mechanisms here (Jin M, Piao Z, Kim N G, Park C, Shin E C, Park J H, Jung H J, Kim C G, Kim H, p16 is a major inactivation target in hepatocellular carcinoma.", "Cancer.", "Jul.", "1, 2000;89(1):60-8).", "DNA methylation as a control of gene expression was detected for the BRCA1 gene for breast cancer (Magdinier F, Billard L M, Wittmann G, Frappart L, Benchaib M, Lenoir G M, Guerin J F, Dante, R Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells FASEB J. August 2000;14(11):1585-94).", "A correlation between methylation and non-Hodgkin's lymphoma is also assumed (Martinez-Delgado B, Richart A, Garcia M J, Robledo M, Osorio A, Cebrian A, Rivas C, Benitez J, Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow-up of non-Hodgkin's lymphomas.", "Br J Haematol.", "April 2000;109(1):97-103).", "CpG methylation also brings about the progression of T-cell leukemia, which is related to a decreased expression of the CDKN2A gene (Nosaka K, Maeda M, Tamiya S, Sakai T, Mitsuya H, Matsuoka M, Increasing methylation of the CDKN2A gene is associated with the progression of adult T-cell leukemia.", "Cancer Res.", "Feb. 15, 2000;60(4):1043-8).", "An increased methylation of the CpG islands was established in bladder cancer (Salem C, Liang G, Tsai Y C, Coulter J, Knowles M A, Feng A C, Groshen S, Nichols P W, Jones P A, Progressive increases in de novo methylation of CpG islands in bladder cancer.", "Cancer Res.", "May 1, 2000;60(9):2473-6).", "Transcription inactivation in esophageal squamous cell carcinomas has been related to the methylation of the FHIT gene, which is associated with the progression of the disease (Shimada Y, Sato F, Watanabe G, Yamasaki S, Kato M, Maeda M, Imamura M, Loss of fragile histidine triad gene expression is associated with progression of esophageal squamous cell carcinoma, but not with the patient's prognosis and smoking history.", "Cancer.", "Jul.", "1, 2000;89(1):5-11).", "Neutral endopeptidase 24.11 (NEP) inactivates the increase of neuropeptides, which participate in the growth of androgen-independent prostate cancer.", "A loss of NEP expression by hypermethylation of the NEP promotors may contribute to the development of neuropeptide-stimulated, androgen-independent prostate cancer (Usmani B A, Shen R, Janeczko M, Papandreou C N, Lee W H, Nelson W G, Nelson J B, Nanus D M, Methylation of the neutral endopeptidase gene promoter in human prostate cancers.", "Clin Cancer Res.", "May 2000;6(5):1664-70).", "Adrenocortical tumors in adults display structural abnormalities in the tumor DNA.", "Among other things, these abnormalities contain an overexpression of the IGF2 gene in correlation with a demethylation of the DNA at this locus (Wilkin F, Gagne N, Paquette J, Oligny L L, Deal C, Pediatric adrenocortical tumors: molecular events leading to insulin-like growth factor 11 gene overexpression.", "J Clin Endocrinol Metab.", "May 2000;85(5):2048-56.Review).", "It is assumed that DNA methylations in several exons in the retinoblastoma gene contribute to the disease (Mancini D, Singh S, Ainsworth P, Rodenhiser D, Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1).", "Am J Hum Genet.", "July 2000;61(1):80-7).", "In chronic myeloid leukemia, a relationship is suspected between the deregulation of the p53 gene and a change in the methylation pattern with progression of the disease (Guinn B A, Mills K I, p53 mutations, methylation and genomic instability in the progression of chronic myeloid leukaemia.", "Leuk Lymphoma.", "July 1997;26(3-4):211-26).", "A relationship with methylation has also been detected for acute myeloid leukemia (Melki J R, Vincent P C, Clark S J. Concurrent DNA hypermethytation of multiple genes in acute myeloid leukemia.", "Cancer Res.", "Aug. 1, 1999;59(15):3730-40).", "A tumor-specific methylation site in the Wilms tumor suppressor gene has been identified (Kleymenova E V, Yuan X, LaBate M E, Walker C L, Identification of a tumor-specific methylation site in the Wilms tumor suppressor gene.", "Oncogene.", "Feb. 12, 1998;16(6):713-20).", "In Burkitt's lymphoma, several promotors have a complete CpG methylation (Tao Q, Robertson K D, Manns A, Hildesheim A, Ambinder R F, Epstein-Barr virus (EBV) in endemic Burkitt's lymphoma: molecular analysis of primary tumor tissue.", "Blood.", "Feb. 15, 1998;91(4):1373-81).", "It is assumed that DNA methylation plays a role in thyroid carcinoma (Venkataraman G M, Yatin M, Marcinek R, Ain K B, Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I-symporter gene methylation status.", "J Clin Endocrinol Metab.", "July 1999;84(7):2449 57).", "Not only are many cancer diseases associated with methylation, but there are also many other diseases that are related to methylation.", "Investigations of inflammatory arthritis have indicated that this disease is associated with a hypomethylation of genomic DNA (Kim Y I, Logan J W, Mason J B, Roubenoff R, DNA hypomethylation in inflammatory arthritis: reversal with methotrexate.", "J Lab Clin Med.", "August 1996;128(2):165-72).", "A methylation-regulated expression has been detected for the ICF syndrome (Kondo T, Bobek M P, Kuick R, Lamb B, Zhu X, Narayan A, Bourc'his D, Viegas-Pequignot E, Ehrlich M, Hanash S M, Whole-genome methylation scan in ICF syndrome: hypomethylation of nonsatellite DNA repeats D4Z4 and NBL2).", "The participation of methylation is suspected in systemic lupus erythematosus (Vallin H, Perers A, Alm G V, Ronnblom L, Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus.", "J Immunol.", "December 1999;163(11):6306-13); and there may also be a relationship between the Duchenne muscular dystrophy gene and a CpG-rich island (Banerjee S, Singh P B, Rasberry C, Cattanach B M, Embryonic inheritance of the chromatin organisation of the imprinted H19 domain in mouse spermatozoa.", "Mech Dev.", "February 2000;90(2):217-26; Burmeister M, Lehrach H, Long-range restriction map around the Duchenne muscular dystrophy gene.", "Nature.", "Dec. 11-17, 1986;324(6097):582-5).", "An epigenetic effect, in which the hypomethylation of the amyloid precursor protein [gene], which is related to the development of the disease, participates, is suspected in Alzheimer's disease (West R L, Lee J M, Maroun L E, Hypomethylation of the amyloid precursor protein gene in the brain of an Alzheimer's disease patient.", "1995;6(2):141-6).", "The methylation state also plays an important role at the chromosomal level.", "For example, in mental retardation syndromes that are associated with the fragility of the X chromosome, the degree of chromosomal fragility is determined by the methylation (de Muniain A L, Cobo A M, Poza J J, Saenz A, [Diseases due to instability of DNA].", "Neurologia.", "December 1995;10 Suppl 1:12-9).", "5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells.", "For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis.", "The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest.", "5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine.", "In addition, in the case of a PCR amplification, the epigenetic information which is borne by the 5-methylcytosines is completely lost.", "A relatively new method that in the meantime has become the most widely used method for investigating DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which, after subsequent alkaline hydrolysis, is then converted to uracil, which corresponds in its base-pairing behavior to thymidine.", "In contrast, 5-methylcytosine is not modified under these conditions.", "Thus, the original DNA is converted so that methylcytosine, which originally cannot be distinguished from cytosine by its hybridization behavior, can now be detected by “standard” molecular biology techniques as the only remaining cytosine, for example, by amplification and hybridization or sequencing.", "All of these techniques are based on base pairing, which is now fully utilized.", "The prior art, which concerns sensitivity, is defined by a method that incorporates the DNA to be investigated in an agarose matrix, so that the diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek, A. et al., Nucl.", "Acids Res.", "1996, 24, 5064-5066).", "Individual cells can be investigated by this method, which illustrates the potential of the method.", "Of course, up until now, only individual regions of up to approximately 3000 base pairs long have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible.", "Of course, this method also cannot reliably analyze very small fragments of small quantities of sample.", "These are lost despite the protection from diffusion through the matrix.", "An overview of other known possibilities for detecting 5-methylcytosines can be derived from the following review article: Rein, T., DePamphilis, M. L., Zorbas, H., Nucleic Acids Res.", "1998, 26, 2255.The bisulfite technique has been previously applied only in research, with a few exceptions (e.g., Zechnigk, M. et al., Eur.", "J. Hum.", "Gen. 1997, 5, 94-98).", "However, short, specific segments of a known gene have always been amplified after a bisulfite treatment and either completely sequenced (Olek, A. and Walter, J., Nat.", "Genet 1997, 17, 275-276) or individual cytosine positions have been detected by a primer extension reaction (Gonzalgo, M. L. and Jones, P. A., Nucl.", "Acids Res.", "1997, 25, 2529-2531, WO-Patent 95-00669) or an enzyme step (Xiong, Z. and Laird, P. W., Nucl.", "Acids Res.", "1997, 25, 2532-2534).", "Detection by hybridization has also been described (Olek et al., WO-A 99-28,498).", "Other publications which are concerned with the application of the bisulfite technique for the detection of methylation in the case of individual genes are: Xiong, Z. and Laird, P. W. (1 7)*, Nucl.", "Acids Res.", "25, 2532; (Gonzalgo, M. L. and Jones, P. A., (1997), Nucl.", "Acids Res.", "25, 2529; Grigg, S. and Clark, S. (1994), Bioassays 16, 431; Zeschnik, M. et al.", "(1997), Human Molecular Genetics 6, 387; Teil, R. et al.", "(1994), Nucl.", "Acids Res.", "22, 695; Martin, V. et al.", "(1995), Gene 157, 261; WO-A 97-46,705, WO-95-15,373 and WO-45,560.(1987)?—Trans.", "Note.", "Matrix-assisted laser desorptions/ionization mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karas, M. und Hillenkamp, F. (1988).", "Laser desorption Ionization of proteins with molecular masses exeeding 10000 daltons.", "Anal.", "Chem.", "60: 2299-2301).", "An analyte is embedded in a light-absorbing matrix.", "The matrix is vaporized by a short laser pulse and the analyte molecule is transported unfragmented into the gaseous phase.", "The analyte is ionized by collisions with matrix molecules.", "An applied voltage accelerates the ions in a field-free flight tube.", "Ions are accelerated to varying degrees based on their different masses.", "Smaller ions reach the detector sooner than large ions.", "MALDI-TOF spectroscopy is excellently suitable for the analysis of peptides and proteins.", "The analysis of nucleic acids is somewhat more difficult (Gut, I. G. and Beck, S. (1995)), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry.", "Molecular Biology: Current Innovations and Future Trends 1: 147-157).", "For nucleic acids, the sensitivity is approximately 100 times poorer than for peptides and decreases overproportionally with increasing fragment size.", "For nucleic acids, which have a backbone with a multiple negative charge, the ionization process via the matrix is basically less efficient.", "In MALDI-TOF spectroscopy, the choice of matrix plays an imminently important role.", "Several very powerful matrices, which produce a very fine crystallization, have been found for the desorption of peptides.", "In the meantime, several effective matrices have been developed for DNA, but the difference in sensitivity has not been reduced thereby.", "The difference in sensitivity can be reduced by modifying the DNA chemically in such a way that it resembles a peptide.", "Phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be converted by simple alkylation chemistry to a charge-neutral DNA (Gut, I. G. and Beck, S. (1995), A procedure for selective DNA alkylation and detection by mass spectrometry.", "Nucleic Acids Res.", "23: 1367-1373).", "The coupling of a charge tag to this modified DNA results in an increase in sensitivity to the same order of magnitude as is found for peptides.", "Another advantage of charge tagging is the increased stability of the analysis in the presence of impurities, which make the detection of unmodified substrates very difficult.", "Genomic DNA is obtained from DNA of cells, tissue or other test samples by standard methods.", "This standard methodology is found in references such as Fritsch and Maniatis, eds., Molecular Cloning: A Laboratory Manual, 1989.Presentation of the Problem The present invention will present oligonucleotides and/or PNA oligomers for the detection of cytosine methylation and a method, which is particularly suitable for the diagnosis of existing diseases or of predisposition for specific diseases by analysis of a set of genetic and/or epigenetic parameters.", "DESCRIPTION The present invention describes a set of at least 10 oligomer probes (oligonucleotides and/or PNA oligomers), which serve for the detection of the cytosine methylation state in chemically pretreated genomic DNA (Seq.", "ID 1 to Seq.", "ID 40712).", "The analysis of a set of genetic and/or epigenetic parameters for the diagnosis of existing diseases or for the diagnosis of predisposition to specific diseases is possible with these probes.", "Genetic parameters in the sense of this invention are mutations and polymorphisms of the claimed nucleic acids (Seq.", "ID 1 to Seq.", "ID 40712) and additional sequences necessary for their regulation.", "Particularly designated as mutations are insertions, deletions, point mutations, inversions and polymorphisms and particularly preferred are SNPs (single nucleotide polymorphisms).", "Polymorphisms, however, can also be insertions, deletions or inversions.", "Epigenetic parameters in the sense of this invention are particularly cytosine methylations and other chemical modifications of DNA bases of the claimed nucleic acids (Seq.", "ID 1 to Seq.", "ID 40712) and additional sequences necessary for their regulation.", "Other epigenetic parameters, for example, are the acetylation of histones, although this cannot be directly analyzed with the described method; however, it is correlated in turn with DNA methylation.", "From said chemically pretreated DNA, segments that are at least 18 base pairs in length from Seq.", "ID 1 to Seq.", "ID 40712 are utilized for the diagnosis.", "Oligomers with a length of at least 9 nucleotides are used as detectors of these segments.", "The oligomers contain at least one CpG dinucleotide.", "The cytosine of the corresponding CpG dinucleotide is found in approximately the middle third of the oligomer.", "It is a deciding factor that at least one oligonucleotide from Seq.", "ID 1 to Seq.", "ID 40712 is present in the respective set of oligomers for at least each of the CpG dinucleotides.", "The oligomers are preferably produced on a support material in a fixed arrangement, whereby at least one oligomer is coupled to a solid phase.", "It is also important in this connection that it is not individual CpG dinucleotides, but the plurality of CpG dinucleotides present in the sequences, which must be analyzed for the diagnosis of genetic and/or epigenetic parameters of the claimed nucleic acids (Seq.", "ID 1 to Seq.", "ID 40712).", "In a particularly preferred variant of the method, all of the CpG dinucleotides present in the sequences are to be investigated.", "In a preferred variant of the method, at least one oligomer is bound to a solid phase.", "In another preferred variant of the method, at least ten of the oligomers are used for the detection of the cytosine methylation state and/or of single nucleotide polymorphisms (SNPs) in chemically pretreated genomic DNA.", "The oligomers are preferably used for the diagnosis of undesired drug interactions; cancer diseases; CNS malfunctions, damage or disorders; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain lesions; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease; malfunction, damage or disorder of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disorder of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction, damage or disease; headaches and sexual malfunctions, by analysis of methylation patterns.", "Also, of the nucleic acids listed in the Appendix (Seq.", "ID 1 to Seq.", "ID 40712), preferably at least one will be used for the analysis of a set of genetic and/or epigenetic parameters for the diagnosis of existing diseases or for the diagnosis of predisposition for specific diseases.", "In addition, a method is described for determining important genetic and/or epigenetic parameters for the diagnosis of existing diseases or for the diagnosis of predisposition for specific diseases, by analysis of cytosine methylations and of single nucleotide polymorphisms (SNPs) in chemically pretreated genomic DNA samples.", "The procedure for this comprises the following steps: In the first step of the method, a genomic DNA sample is chemically treated in such a way that cytosine bases that are unmethylated at the 5′-position are converted to uracil, thymine or another base unlike cytosine in its hybridization behavior.", "This is understood in the following as chemical pretreatment.", "The person of average skill in the art understands that the oligomers fulfill the same objective when thymine is exchanged for uracil in the sequences used.", "The genomic DNA to be analyzed is obtained preferably from the usual sources for DNA, such as, e.g., cell lines, blood, sputum, stool, urine, cerebrospinal fluid, tissue embedded in paraffin, for example, tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological slides and all other possible combinations thereof.", "Preferably, the above-described treatment of genomic DNA with bisulfite (hydrogen sulfite, disulfite) and subsequent alkaline hydrolysis, which converts unmethylated cytosine nuleobases to uracil, is used for this purpose.", "In the second step of the method, fragments from the chemically pretreated genomic DNA are amplified with the use of primer oligonucleotides.", "Preferably, more than 10 different fragments are amplified, which are 100-2000 base pairs in length.", "In a preferred variant of the method, the amplification is preferably conducted with the polymerase chain reaction (PCR), wherein a heat-stable DNA polymerase is preferably used.", "It is preferred according to the invention that the amplification of several DNA segments is conducted in one reaction vessel.", "In a preferred variant of the method, the set of primer oligonucleotides comprises at least two oligonucleotides, whose sequences are inversely complementary or identical to a segment that is at least 18 base pairs long of the base sequences listed in the Appendix (Seq.", "ID 1 to Seq.", "ID 40712).", "The primer oligonucleotides are preferably characterized in that they do not contain a CpG dinucleotide.", "According to the invention, it is also preferred that different oligomers are arranged on a planar solid phase in the form of a rectangular or hexagonal grid.", "In a preferred variant of the method, the amplification occurs by elongation of primer oligonucleotides that are bound to a solid phase.", "This solid-phase surface is preferably comprised of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.", "The amplified products obtained in the second step are then hybridized to a set of oligonucleotides and/or PNA probes or to an array.", "The set used in the hybridization is most preferably comprised of at least 10 oligomer probes.", "The amplified products thus serve as probes, which hybridize to the oligonucleotides previously bound to a solid phase.", "The unhybridized fragments are then removed.", "Said oligomers comprise at least one base sequence with a length of 9 nucleotides, which contains at least one CpG dinucleotide.", "The cytosine of the corresponding CpG dinucleotide is found in approximately the middle third of the oligomer.", "One oligonucleotide is present for each CpG dinucleotide.", "In the fourth step of the method, the unhybridized amplified products are removed.", "In the last step of the method, the hybridized amplified products are detected.", "It is preferred according to the invention that labels, which are introduced on the amplified products at any position of the solid phase at which an oligonucleotide sequence is found, can be identified.", "It is preferred according to the invention that the labels of the amplified products are fluorescent labels.", "It is preferred according to the invention that the labels of the amplified products are radionuclides.", "It is preferred according to the invention that the labels of the amplified products are removable molecular fragments with typical mass, which are detected in a mass spectrometer.", "It is preferred according to the invention that the amplified products, fragments of the amplified products or probes complementary to the amplified products are detected in the mass spectrometer.", "It is preferred according to the invention that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer.", "It is preferred according to the invention that the detection is carried out and visualized by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) or by means of electrospray mass spectrometry (ESI).", "A method is preferred for the diagnosis and/or prognosis of adverse events for patients or individuals, whereby these adverse events are related to genetic and/or epigenetic parameters.", "The use of a method according to the invention is preferred for the diagnosis of existing diseases or of the predisposition for specific diseases by analysis of a set of genetic and/or epigenetic parameters.", "The subject of the present invention is also a kit comprising a reagent containing bisulfite, a set of primer oligonucleotides comprising at least two oligonucleotides, each of whose sequences is a segment that is at least 18 base pairs long and corresponds to the base sequences listed in the Appendix (Seq.", "ID 1 to Seq.", "ID 40712) or are complementary to them for the production of amplified products, oligonucleotides and/or PNA oligomers as well as instructions for conducting and evaluating the described method.", "The following example relates to a fragment of the hMLH1 gene associated with hereditable non-polyposis colorectal cancer, in which a specific CG position is investigated for methylation.", "In the first step, a genomic sequence is treated with the use of bisulfite (hydrogen sulfite, disulfite) in such a way that all of the unmethylated cytosines at the 5-position of the base are modified such that a base that is different in its base pairing behavior is formed, while the cytosines that are methylated in the 5-position remain unchanged.", "If bisulfite in the concentration range between 0.1 M and 6 M is used for the reaction, then an addition occurs at the unmethylated cytosine bases.", "Also a denaturing reagent or solvent as well as a radical trap must be present.", "A subsequent alkaline hydrolysis then leads to the conversion of unmethylated cytosine nucleobases to uracil.", "This converted DNA serves for the detection of methylated cytosines.", "In the second step of the method, the treated DNA sample is diluted with water or an aqueous solution.", "A desulfonation of the DNA (10-30 min, 90-100° C.) at alkaline pH is then preferably conducted.", "In the third step of the method, the DNA sample is amplified in a polymerase chain reaction, preferably with a heat-stable DNA polymerase.", "In the present example, cytosines of the hMLH1 gene, here from a 1551 bp-long 5′-flanking region, are investigated.", "A defined fragment of 719-bp length is amplified for this purpose with the specific primer oligonucleotides AGCMCACCTCCATGCACTG and TTGATTGGACAGCTTGAATGC.", "This amplified product serves as a sample, which hybridizes to an oligonucleotide that has been previously bound to a solid phase, with the formation of a duplex structure, for example, GAAGAGCGGACAG, whereby the cytosine to be detected is found at position 588 of the amplified product.", "The detection of the hybridization product is based on primer oligonucleotides fluorescently labeled with Cy3 and Cy5, which were used for the amplification.", "A hybridization reaction of the amplified DNA with the oligonucleotide occurs only if a methylated cytosine was present at this site in the bisulfite-treated DNA.", "Thus the methylation state of the respective cytosine to be investigated decides the hybridization product." ] ]

[ [ "Self-propelled screening apparatus", "A self-propelled screening apparatus (1) having a chassis (11); a pair of endless tracks (12) supporting the chassis (11); a superstructure (13) rotatably mounted on the chassis (11); a prime mover provided in or on the superstructure (13) and arranged to provide motive power to operate the endless tracks (12) and where the prime mover is rotatable with the superstructure (13); and a screen box (16) mounted on the superstructure (13), also for rotation therewith.", "The screen box (16) has an input (17) for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material.", "The prime mover and the screen box (16) are arranged on opposite sides of the rotational axis of the superstructure (13), to provide a stable weight distribution." ], [ "1.A self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a prime mover provided in or on the superstructure and arranged to provide motive power to operate the endless tracks, said prime mover being rotatable with the superstructure; and a screen box mounted on this superstructure, also for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material; in which the prime mover and the screen box are arranged on opposite sides of the rotational axis of the superstructure, to provide a stable weight distribution.", "2.A self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a superstructure rotatably mounted on the chassis via a slewing ring; and a screen box mounted on the superstructure for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material.", "3.Apparatus according to claim 1, in which the superstructure comprises a housing or canopy in which an engine is mounted, and which serves as a source of power to operate the endless tracks.", "4.Apparatus according to claim 3, in which the engine is arranged to drive driven components of the screen box.", "5.Apparatus according to claim 1, in which the screen box has more than one screen deck, and has a loading chute at an inboard end, and an opposite discharge end over which oversized material can be discharged.", "6.Apparatus according to claim 1, in which the screen box is adjustable inwardly or outwardly of the rotational axis of the superstructure, to move the discharge point relative to the apparatus.", "7.Apparatus according to claim 6, in which the screen box is mounted on, or incorporates a telescopically adjustable frame.", "8.Apparatus according to claim 1, and including a driver's cab from which an operator can control the movement of the apparatus and/or screening operation.", "9.Apparatus according to claim 1, in which the apparatus is arranged to be capable of being operated by remote control.", "10.Apparatus according to claim 1, in which the screenbox is independently adjustably relative to the superstructure.", "11.Apparatus according to claim 1, in which the screenbox is pivotable through up to 90 degrees in either direction from a datum position relative to the superstructure.", "12.Apparatus according to claim 2, in which the superstructure comprises a housing or canopy in which an engine is mounted, and which serves as a source of power to operate the endless tracks.", "13.Apparatus according to claim 12, in which the engine is arranged to drive driven components of the screen box.", "14.Apparatus according to claim 2, in which the screen box has more than one screen deck, and has a loading chute at an inboard end, and an opposite discharge end over which oversized material can be discharged.", "15.Apparatus according to claim 2, in which the screen box is adjustable inwardly or outwardly of the rotational axis of the superstructure, to move the discharge point relative to the apparatus.", "16.Apparatus according to claim 15, in which the screen box is mounted on, or incorporates a telescopically adjustable frame.", "17.Apparatus according to claim 2, and including a driver's cab from which an operator can control the movement of the apparatus and/or screening operation.", "18.Apparatus according to claim 2, in which the apparatus is arranged to be capable of being operated by remote control.", "19.Apparatus according to claim 2, in which the screenbox is independently adjustably relative to the superstructure.", "20.Apparatus according to claim 2, in which the screenbox is pivotable through up to 90 degrees in either direction from a datum position relative to the superstructure." ], [ "This invention relates to a self-propelled screening apparatus which is operative to screen a supply of bulk material into at least one size range of screened material, and to discharge the screened material to a required deposition zone.", "Screening apparatus or plants are used to separate out a supply of bulk material into one or more size ranges of screened material, and typically may be used in quarry locations in order to separate crushed stone into different size ranges of e.g.", "ballast, sand, gravel and the like.", "They may also be used in land or site clearance work, when the bulk material may be rubble, broken concrete, tree roots and soil.", "Some screening apparatus are very large static installations, when intended for long term use at a particular site.", "Other apparatus are designed to be transportable from site to site, either via a low loader, or as a towed vehicle, and such apparatus therefore has to be designed to be convertible between a transport mode and an operating (screening) mode.", "Still further screening apparatus are self-propelled, and which are therefore required to be easily manoeuvrable, for the purposes of loading the apparatus with bulk material at any particular location, and for discharging screened material at a required deposition zone.", "The present invention is concerned with a self-propelled screening apparatus, and seeks to provide, by simple means, an apparatus which is very easily manoeuvrable, and yet also is stable, despite the fact that bulk material is being handled, and the necessary screen or screens to handle the bulk material provide substantial mass, and also generate substantial inertia.", "According to one aspect of the invention there is provided a self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a superstructure rotatably mounted on the chassis; a prime mover provided in or on the superstructure and arranged to provide motive power to operate the endless tracks, said prime mover being rotatable with the superstructure; and a screen box mounted on the superstructure, also for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material; in which the prime mover and the screen box are arranged on opposite sides of the rotational axis of the superstructure, to provide a stable weight distribution.", "According to a second aspect of the invention there is provided a self-propelled screening apparatus which comprises: a chassis; a pair of endless tracks supporting the chassis; a superstructure rotatably mounted on the chassis via a slewing ring; and a screen box mounted on the superstructure for rotation therewith, said screen box having an input for receiving a supply of bulk material, at least one screen deck for screening the bulk material, and an output for discharging screened material.", "The invention therefore provides an easily manoeuvrable apparatus via the endless tracks, and also by means of the rotational adjustment of the screen box relative to the chassis to suit loading and/or screening requirements.", "The superstructure may comprise a housing or canopy in which an engine may be mounted, and which serves as a source of power to operate the endless tracks, and preferably also any driven components of the screen box e.g.", "a vibrating mechanism.", "The engine may be an internal combustion engine, or any suitable power source e.g.", "a hydraulic pump driven by any suitable means.", "The screen box may have more than one screen deck, and may have a loading chute at an inboard end (with respect to the rotational axis) and oversized material i.e.", "material which is too large to pass downwardly through the screening apertures, may pass directly over the top of the upper screen deck to be discharged via an outboard end of the screen box.", "If it is required to provide a facility whereby the discharge from the screen box may be adjustable inwardly or outwardly of the rotational axis of the superstructure on which the screen box is mounted i.e.", "to move the discharge point nearer to, or further from the apparatus, the screen box may be mounted on, or incorporate a telescopically adjustable frame.", "The weight distribution of the apparatus is such that the weight of the screen box (and any material being handled) is balanced by other components of the apparatus on the superstructure e.g.", "the prime mover.", "The screen box may carry out screening operations while the apparatus is static, or on the move if required.", "Also, via the slewing ring, the screen box can take up any required rotational position through 360° about the rotational axis as may be required, and therefore may include discharge of screened material forwardly, or rearwardly of the direction of travel, or transversely thereof e.g.", "at 90°.", "To further provide for flexibility in operation, by enabling additional positional adjustment of the discharge of screened material, the screen box may be adjustably mounted on the superstructure.", "The apparatus may have a driver's cab, and from which an operator can control the movement of the apparatus and/or screening operations.", "Alternatively, the apparatus may be arranged to be capable of being operated by remote control, e.g.", "an infra-red control or a radio controlled system, and which might be provided, for example, in the operating cab of a loading vehicle which is being used to load the apparatus with bulk material.", "A preferred embodiment of self-propelled screening apparatus according to the invention will now be described in detail, by way of example only, with reference to the accompanying drawing, in which: FIG.", "1 is a side view of a self-propelled screening apparatus according to the invention, and showing a screen box mounted on one end thereof, and with respect to the longitudinal axis of the apparatus; and FIG.", "2 is an end view, showing the screen box rotated through 90° to a lateral discharge position.", "Referring now to the drawing, a self-propelled screening apparatus according to the invention is designated generally by reference 10 and which comprises a chassis 11, and a pair of endless tracks 12 which support the chassis 11 and which are driven by any suitable prime mover (not shown) in order to propel the apparatus over the ground.", "Evidently, by providing endless tracks, selective operation of one or more of the tracks provides easy manoeuvrability of the apparatus, and especially in view of the relatively short length of the tracks, and also the relatively short length of the superstructure mounted on the chassis 11, and as described below.", "A superstructure 13 is rotatably mounted on the chassis 11, via a slewing ring 14, whereby the superstructure 13 can be rotated through 360° about a substantially vertical axis passing through the centre of the slewing ring.", "The superstructure 13 includes an engine housing or canopy 15 in which a prime mover can be mounted, and which may be an internal combustion engine, or any other suitable power source e.g.", "a driven hydraulic pump.", "The prime mover provides drive power to operate the endless tracks 12, and also serves to provide drive input (if required) to a screen box 16 which is mounted on the superstructure 13.The screen box 16 is therefore rotatable with the superstructure 13, and can take up an “in-line” position as shown in FIG.", "1, or a transversely extending discharge position shown in FIG.", "2.Other angular adjustments about the rotational axis can be taken up, according to a required deposition zone for screened material.", "If required, additional positional adjustment of the discharge of screened material may be obtained by providing an adjustable mounting of the screen box 16 on the superstructure 13.Although not shown in detail, the screenbox 16 may be pivotally adjusted about pivot axis 16a to alter the deposition point for screened material relative to the superstructure 13, i.e.", "the screenbox 16 may be independently adjustable relative to the superstructure 13, and which also is adjustable relative to chassis 11 and endless tracks 12.In a preferred arrangement, the screenbox 16 may be adjustable in either direction through up to 90° from a datum position relative to the superstructure 13.The screen box 16 has an input in the form of loading chute 17, which receives a supply of bulk material, and at least one screen deck for screening the bulk material, and an output for discharging the screened material.", "Oversized material i.e.", "material which is too large to pass downwardly through the screening apertures, may pass directly over the upper screen deck, to be discharged from the outboard end of the screen box 16.As can be seen in FIG.", "1, the screen box 16 is located at one end of the apparatus i.e.", "is spaced from the rotational axis provided by the stewing ring 14, and therefore to counterbalance this weight, the prime mover is mounted within the housing 15 at the opposite side of the rotational axis.", "This provides a stable arrangement, during operation.", "The prime mover, which is mounted in the housing 15, serves as a source of power to operate the endless track 12, and also drive any components of the screen box e.g.", "a vibrating mechanism, if required.", "The screen box 16 may have more than one screen deck, if it is required to screen the bulk material into more than one screened size range.", "In order to provide a facility whereby discharge from the screen box may be adjusted inwardly or outwardly of the rotational axis of the superstructure (and therefore also of the screen box), the screen box may be mounted on, or incorporate a telescopically adjustable frame, as shown by reference 18.The apparatus 10 may include a driver's cab (not shown), from which an operator can control the movement of the apparatus and operation of the screen.", "Alternatively, the apparatus 10 may be arranged to be operated by remote control e.g.", "an infra-red or radio control system, or via an umbilical.", "The remote control conveniently can be exercised by the driver of another vehicle working in conjunction with the screening apparatus e.g.", "the operator of a loading vehicle delivering supplies of bulk material to the screening apparatus.", "The preferred embodiment of the invention therefore provides a simple, but easily manoeuvrable apparatus which can carry out screening operations, and whose position can be adjusted to suit loading requirements and/or discharge requirements of the screened material.", "There is no need to provide an under-conveyor to work with the screen box, to discharge the screened material.", "Furthermore, the apparatus can work in any angular position of adjustment about the axis of the slewing ring.", "The weight distribution of the components mounted on the superstructure is such as to provide a stable operating system." ] ]

[ [ "Porphyrins and related compounds", "New porphyrins and chlorins including bacteriochlorins, for photodynamic therapy." ], [ "1.N-confused tetrahydroxyphenyl and tetra-alkoxyphenyl porphyrins represented as: and the corresponding chlorins (dihydroporphyrins) and bacteriochlorins (tetrahydroporphyrins), including tautomers in all cases, and their metallates and salts, including internal salts: —OR1 standing for a hydroxy group (R1=hydrogen) or an alkoxy group where R1 is a branched or unbranched alkyl group containing from 1 to 4 carbon atoms, and where OR1 may be at any position on the phenyl ring but particularly meta and there may be zero, one, two or three groups per phenyl ring provided at least one of the rings is substituted and —R standing for hydrogen, when IIB and IIA represent the same molecule, or R═X, a branched or unbranched alkyl group of 1 to 6 carbon atoms, or an aralkyl group the alkyl chain of which contains 1 to 3 carbon atoms, or alternatively R═XY where X is as last and Y consists of 1 to 3 substituents which are attached to any of the carbons in X and may be linked, and which are selected from: ═—SO3H, CO2H and their C1 to C12 esters and amides.", "═—OH or OR2 where R2=a polyhydroxylated alkyl chain from 1 to 20 carbon atoms, e.g.", "a carbohydrate or a folic acid residue.", "═—NR3R4 where R3, R4 are the same or different and may be hydrogen or an alkyl group of 1 to 12 carbon atoms or a polyhydroxylated alkyl chain or folic acid residue as last.", "=Z—P where P is an end-capped polyalkylene preferably polyethylene glycol group of molecular weight of 2,000 to 100,000, preferably 5,000 to 40,000 and very preferably 10,000 to 20,000 Da and Z is a linker group to attach P e.g.", "the —O(C═O)NH(CH2)6NH(C═O)O— group.", "R5=hydrogen, a halogen or a nitro group.", "2.The compounds 2-Aza-21-carba-5,10,15,20-tetra(3-methoxyphenyl)porphyrin (NC-m-TMPP), 2-Aza-21-carba-5,10,15,20-tetra(3-hydroxyphenyl)porphyrin (NC-m-THPP), 2-Aza-21-carba-2-methyl-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N-Me-NC-m-THPP), 2-Aza-21-carba-2-methyl-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (N-Me-NC-m-TMPP), 2-Aza-21-carba-2-methyl-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N-Me-NC-m-THPP), 2-Aza-21-carba-2-propylsulfonate-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (N—PS—NC-m-TMPP), 2-Aza-21-carba-2-propylsulfonate-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N—PS—NC-m-THPP), 2-Aza-21-carba-21-nitro-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (21-NO2—NC-m-TMPP), 2-Aza-21-bromo-21-carba-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (21-Br—NC-m-TMPP), 2-Aza-21-carba-5,10,15,20-tetra-(3-methoxyphenyl)dihydroporphyrin (NC-m-TMPC).", "3.Compounds as described in claim 1 where chain OR1 is attached at the meta position of the phenyl ring provided.", "4.The use of a compound according to claim 1, 2 or 3 in the preparation of a medicament for photodynamic therapy in treatment of cancerous or other diseased tissue, and such medicament and therapy itself.", "5.The use of compounds according to claim 4 where conditions include Barrett's Oesophagus, ARMD, keratinosis, diabetic neuropathy, peripheral vascular disease, coronary artery disease, and disorders arising from bacterial or viral infections." ], [ "<SOH> BACKGROUND <EOH>Certain N-confused porphyrins have been known since 1994 (E. D. Steinberg et al, Tetrahedron 1998, 54, 4151-4202, see 4192 and 4193 specifically).", "They are of a structure that may be represented, when substituted with phenyl rings at the meso positions as herein proposed, as shown in Figure I.", "Recently, Lindsey et al have published a ready synthesis of such N-confused porphyrins (G. R. Geier, D. M. Haynes, J. S. Lindsey, Organic Letters 1999, 1(9), 1455-1458).", "Also known, in our European Patent Specifications Nos.", "0186962 and 0337601 are tetrahydroxyphenyl derivatives of porphyrins, chlorins and bacteriochlorins, for photodynamic therapy (PDT) of cancerous tissue.", "These compounds have good properties and, among them, tetra(3-hydroxyphenyl)dihydroporphyrin or m-THPC is well into the stages of product development for PDT.", "Nevertheless, improvements in PDT compounds in respect of absorption wavelength and hence depths of PDT necrosis, tissue selectivity, water-compatibility, and ease of synthesis are sought.", "PDT compounds also have application as light activated agents with the power to kill viruses and bacteria that are now proving resistant to conventional antibiotics." ], [ "FIELD The invention relates to porphyrins and chlorins for photodynamic therapy, of cancerous and other diseased tissues.", "BACKGROUND Certain N-confused porphyrins have been known since 1994 (E. D. Steinberg et al, Tetrahedron 1998, 54, 4151-4202, see 4192 and 4193 specifically).", "They are of a structure that may be represented, when substituted with phenyl rings at the meso positions as herein proposed, as shown in Figure I.", "Recently, Lindsey et al have published a ready synthesis of such N-confused porphyrins (G. R. Geier, D. M. Haynes, J. S. Lindsey, Organic Letters 1999, 1(9), 1455-1458).", "Also known, in our European Patent Specifications Nos.", "0186962 and 0337601 are tetrahydroxyphenyl derivatives of porphyrins, chlorins and bacteriochlorins, for photodynamic therapy (PDT) of cancerous tissue.", "These compounds have good properties and, among them, tetra(3-hydroxyphenyl)dihydroporphyrin or m-THPC is well into the stages of product development for PDT.", "Nevertheless, improvements in PDT compounds in respect of absorption wavelength and hence depths of PDT necrosis, tissue selectivity, water-compatibility, and ease of synthesis are sought.", "PDT compounds also have application as light activated agents with the power to kill viruses and bacteria that are now proving resistant to conventional antibiotics.", "THE INVENTION We propose the following N-confused tetrahydroxyphenyl and tetra-alkoxyphenyl porphyrins represented as Figures IIA and IIB, and the corresponding chlorins (dihydroporphyrins) and bacteriochlorins (tetrahydroporphyrins), including tautomers in all cases, and their metallates and salts, including internal salts, as new compounds valuable in PDT.", "In formulae IIA and IIB, OR1 stands for a hydroxy group (R1=hydrogen) or an alkoxy group where R1 is a branched or unbranched alkyl group containing from 1 to 4 carbon atoms; OR1 may be at any position on the phenyl ring and there may be zero, one, two or three groups per phenyl ring provided at least one of the rings is substituted.", "A single m-hydroxy group on each phenyl ring is specifically suitable.", "In formula IIB, examples of R (besides hydrogen where IIB and IIA represent tautomers of the same molecule) are: R═X, a branched or unbranched alkyl group of 1 to 6 carbon atoms, or an aralkyl group the alkyl chain of which contains 1 to 3 carbon atoms.", "R═XY where Y consists of 1 to 3 substituents which may be linked and/or attached to any of the carbons in X.", "Examples of Y are the following: ═—SO3H, CO2H and their C1 to C12 esters and amides.", "═—OH or OR2 where R2=a polyhydroxylated alkyl moiety from 1 to 20 carbon atoms, e.g.", "a carbohydrate, or derivatised amino acid such as a folic acid residue.", "═—NR3R4 where R3, R4 are the same or different and may be hydrogen or an alkyl group of 1 to 12 carbon atoms or a polyhydroxylated alkyl chain or derivatised amino acid residue as last.", "=Z—P where P is an end-capped polyalkylene preferably polyethylene glycol group of molecular weight of 2,000 to 100,000, preferably 5,000 to 40,000 and very preferably 10,000 to 20,000 Da and Z is a linker group to attach P e.g.", "the —(C═O)NH(CH2)6NH(C═O)O— group.", "R5=hydrogen, a halogen or a nitro group.", "The invention also extends to the use of compounds as above in the preparation of a medicament for photodynamic therapy, in treatment of tumours or other disease, and such medicament and therapy itself.", "The disclosed compounds are considered to be of use, for example, in the treatment of cancerous conditions such as, but not limited to, head and neck, liver, pancreatic and prostate cancers and other disease conditions such as, but not limited to, Barrett's Oesophagus, Age-related Macular Degeneration (ARMD), keratinoses, diabetic neuropathy, peripheral vascular disease, coronary artery disease, and other disorders arising from bacterial or viral infections.", "STRUCTURES In the above porphyrin formulae it will be recognised that when R═H, IIA and IIB are tautomeric forms and also that formally each represents a number of individual tautomers such as are shown below (all represented without the meso-phenyl groups and with R5═H).", "Corresponding chlorins to formula IIA may be represented by IIIA-D: Corresponding chlorins to formula IIB may be represented by IVA-IVD: Corresponding bacteriochlorins to formula IIA may be represented by VA-F: Corresponding bacteriochlorins to formula IIB may be represented by VIA-F: The invention covers all tautomers of the above compounds, and is not limited to those shown in the diagrams.", "There are no optical isomers.", "DISCUSSION As noted above, N-confused porphyrins (NCPs), or 2-aza-21-carbaporphyrins to give them a more correct IUPAC nomenclature, are a relatively new class of tetrapyrrolic macrocycles recently discovered as by-products from the routine preparation of the structurally isomeric meso-substituted porphyrins.", "They are unusual in that one of the macrocycle's pyrrole rings has been inverted so that what was once a central nitrogen atom has now become an external, quaternisable nitrogen, hence the term ‘N-confused’.", "This nitrogen now has the potential for ready introduction of functionality into the molecule.", "Unusually, the now-central C—H bond is quite labile, allowing metallation of the macrocycle almost as easily as in the porphyrinic isomers (See Figure VII, P. J. Chmielewski et al, J. Chem.", "Soc.", "Perkin Trans.", "2, 1995, 503-509).", "Until very recently, the N-confused porphyrins have remained a laboratory curiosity mainly due to the low yields of these compounds.", "However, authors have remarked on the potential of N-confused porphyrins in the PDT field because of their significantly different UV/Visible spectra when compared to porphyrins.", "Thus, the reduction in symmetry, and hence perturbation of the pi-electronic structure, caused by N-confusion, leads to significant red-shifting and increased intensity of the lowest energy Q band compared to the corresponding porphyrins.", "The intensity of this low energy band can be further increased by quaternisation of the external nitrogen.", "It is also noted that the UV/Visible spectrum is pH dependent.", "With the publication by Lindsey of a synthetic route producing relatively high yields of meso substituted N-confused porphyrins, these unusual compounds were opened up for research.", "For example, though the compounds have been known since 1994 and some studies on coordination properties and reactivity have been reported, photophysical studies had not been performed to ascertain potential as PDT agents.", "In initial experiments, 2-aza-21-carba-5,10,15,20-tetraphenylporphyrin, was synthesised by the method of Lindsey and subsequent reaction with methyl iodide at room temperature led to conversion of the N-confused porphyrin to an N-methyl derivative with a lowest energy Q-band to B-band intensity ratio similar to m-THPC.", "No reduction to the N-confused chlorin was necessary to achieve these comparable intensity ratios.", "This reaction was duly performed on the tetrahydroxyphenylporphyrin derivative, with similar results (See Scheme 1).", "EXAMPLES The following patent examples are given and are summarised in the intervening schemes shown below.", "The synthesis of the N-methyl substituted N-confused porphyrin depicted in Scheme 1 is outlined in examples 1-5.Example 1 2-Aza-21-carba-5,10,15,20tetra(3-methoxyphenyl)porphyrin (NC-m-TMPP): Pyrrole (1.04 ml, 15 mmol) and 3-methoxybenzaldehyde (1.82 ml, 15 mmol) were mixed with methanesulfonic acid (0.68 ml, 10.5 mmol) in a large volume of dichloromethane (1500 ml) at room temperature (RT).", "The reactants were allowed to stir for 30 min and then the reaction was terminated by the addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (3 g, 13.2 mmol).", "After stirring for 1 min, triethylamine (5.8 ml) was added to neutralise the solution and the resultant mixture left to stir for a further 10 min.", "Initial purification was achieved by passing the whole crude mixture through a column (60 mm) of basic alumina (300 g, activity III).", "Everything was collected as a single fraction, evaporated in vacuo to near dryness before being absorbed onto 15-20 g of basic alumina (activity III).", "This was added to the top of a column (50 mm) of basic alumina (300 g, activity III) eluting with 3:1 hexane/dichloromethane.", "The polarity of the eluant was increased from 3:1 to 1:1, to 1:3, and finally 100% dichloromethane.", "The porphyrin eluted first, with the desired N-confused porphyrin (NC-m-TMPP) eluting largely with 100% dichloromethane, and evaporated in vacuo to dryness.", "The product obtained was then refluxed with methanol (21 ml) for 10 min and then allowed to cool in the fridge for 2 h. After filtration the solid was washed with methanol (5 ml) and dried in a vacuum oven (60° C.) for 2 h to give the title compound as a micro-crystalline solid (780 mg, 28%).", "The porphyrin (m-TMPP) was obtained as a minor product (105 mg, 3.8%).", "Increasing the pyrrole concentration to 11.5 mmol or 12.4 mmol and the other reactants accordingly led to a decreased yield of 24%.", "A modified procedure at lower dilution (equivalent to 20 mM) also resulted in a poorer yield of crystallised product (see below).", "To a mixture of pyrrole (2.8 ml, 40 mmol) and 3-methoxybenzaldehyde (4.9 ml, 40 mmol) in dichloromethane (2000 ml) was added at RT with vigorous stirring methanesulfonic acid (2.6 ml, 40 mmol).", "The reactants were allowed to stir for 8 min after which the reaction was terminated by the addition of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (8 g, 35.2 mmol).", "After stirring for 1 min, triethylamine (16 ml) was added to neutralise the solution and the resultant mixture left to stir for a further 10 min.", "Initial purification was achieved by passing the whole crude mixture through a column (60 mm) of basic alumina (400 g, activity III).", "Everything was collected as a single fraction, evaporated in vacuo to 100 ml before being absorbed onto 50-60 g of basic alumina (activity III).", "This was added to the top of a column (60 mm) of basic alumina (600 g, activity III) eluting with 3:1 hexane/dichloromethane.", "The polarity of the eluant was increased from 3:1 to 1:1, to 1:3, and finally 100% dichloromethane.", "The porphyrin eluted first, with the desired N-confused porphyrin (NC-m-TMPP) eluting largely with 100% dichloromethane, and evaporated in vacuo to dryness.", "The product obtained was then refluxed with methanol (30 ml) for 10 min and then allowed to cool in the fridge for 2 h. After filtration the solid collected was washed with methanol (10 ml) and then dried in a vacuum oven (60° C.) for 2 h to give the title compound as a micro-crystalline solid (1.6 g, 22%).", "NC-m-TMPP Physical Data: δH (360 MHz, CDCl3) −5.01 (1H, s, H-21), −2.43 (2H, bs, NH), 3.99 (6H, s, OCH3), 4.04 (3H, s, OCH3), 4.19 (3H, s, OCH3), 7.30-7.36 (4H, m, CHar), 7.61-7.78 (8H, m, CHar), 7.88-7.97 (4H, m, CHar), 8.59-8.66 (4H, m, H-8, 12, 13, 17), 8.81 (1H, s, H-3), 8.95 (1H, d, J18, 17 4.8 Hz, H-18), 9.02 (1H, d, J7, 8 4.9 Hz, H-7); λmax (CH2C12): 280 (ε 23 009), 441 (ε 19 8126), 540 (ε 10 858), 582 (ε 12 724), 725 (ε 11 930); m/z (+ve FAB, NOBA) 735 (MH+, 9.9%), (Found: MH+ 735.29711.C48H39N4O4 requires 735.29713).", "Example 2 2-Aza-21-carba-5,10,15,20-tetra(3-hydroxyphenyl)porphyrin (NC-m-THPP): NC-m-TMPP (200 mg, 0.27 mmol) was dissolved in dry dichloromethane (15 ml) and the solution was cooled to an external temperature of −78° C. A 1.0M solution of boron tribromide in dichloromethane (2.5 ml, 2.5 mmol) was added dropwise and the resultant reaction mixture stirred at −78° C. for 2 h. This was then allowed to warm up slowly to RT with continued stirring overnight.", "Next day, the mixture was cooled to an external temperature of −10° C. and excess methanol (3 ml) carefully added to quench the reaction.", "Excess triethylamine (4 ml) was then added slowly to neutralise the HBr given off and the resultant solution stirred for 30 min after which the mixture was then evaporated in vacuo to dryness.", "Water (30 ml) was added to the residue and the product extracted using ethyl acetate (4×50 ml).", "The combined organic extracts were then dried (Na2SO4) and concentrated in vacuo to dryness to give the crude product.", "This crude product was dissolved in methanol (10 ml) and acetic acid (0.2 ml) and then heated to reflux.", "Water (15 ml) was added gradually ensuring the resultant mixture remained at reflux, after which the mixture was allowed to cool to RT and then placed in the fridge overnight.", "The solid formed was filtered, washed with 40% methanol in water (10 ml) and dichloromethane (3×10 ml) and then dried (vacuum oven, 60° C., 4 h) to yield the title compound (0.17 g, 89%).", "NC-m-THPP Physical Data: δH (360 MHz, CD3OD) −4.16 (1H, bs, H-21), 7.22-7.72 (16H, m, CHar), 8.21-8.25 (4H, m, H-8, 12, 13, 17), 8.37 (1H, s, H-3), 8.48 (1H, d, J18, 17 4.0 Hz, H-18), 8.62 (1H, bs, H-7); λmax (CH3OH): 281 (ε 20 861), 439 (ε 139 386), 538 (ε 6 225), 582 (ε 6 883), 727 (ε 7 673); m/z (+ve FAB, NOBA) 679 (MH+, 15.3%), (Found: MH+ 679.23450.C44H31N4O4 requires 679.23453).", "Example 3 2-Aza-21-carba-2-methyl-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N-Me-NC-m-THPP): To a solution of NC-m-THPP (40 mg, 0.059 mmol) in dimethylformamide (10 ml) was added methyl iodide (2 ml).", "The mixture was allowed to stand at RT for 24 h. After removal of the solvent the residue was dissolved in ethyl acetate (20 ml) and extracted with saturated aqueous sodium bicarbonate solution (2×20 ml).", "The organic layer was dried (Na2SO4) and evaporated in vacuo.", "The residue was recrystallised from methanol/water to give N-Me-NC-m-THPP (40 mg, 98%).", "N-Me-NC-m-THPP Physical Data: λmax (CH3OH) 358, 464, 576, 782.Alternatively, the steps performed in examples 2 & 3 may be reversed as follows.", "Example 4 2-Aza-21-carba-2-methyl-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (N-Me-NC-m-TMPP): To a stirred solution of NC-m-TMPP (500 mg, 0.68 mmol) in dichloromethane (50 ml) was added methyl iodide (4.25 ml, 68.1 mmol) and the resultant mixture was stirred at RT overnight.", "The reaction mixture was then heated under reflux for 1 h after which thin layer chromatography (TLC) analysis (100% dichloromethane) indicated that all the starting material had been consumed.", "Once cool, the mixture was washed with saturated aqueous sodium bicarbonate solution (2×25 ml) and water (2×25 ml), dried (Na2SO4) and the solvent removed in vacuo to yield the crude material.", "This crude product was dissolved in methanol (40 ml) and triethylamine (1 ml) and then heated to reflux for 10 min.", "The solution was allowed to cool slowly to RT and placed in the fridge for 4 h. The solid formed was filtered, washed with methanol (20 ml) and then dried (vacuum oven, 60° C., 2 h) to yield the title compound (0.42 g, 83%).", "N-Me-NC-m-TMPP Physical Data: δH (360 MHz, CDCl3) 1.26 (1H, s, H-21), 3.52 (3H, s, NCH3), 3.96 (6H, s, OCH3), 4.00 (3H, s, OCH3), 4.01 (3H, s, OCH3), 7.15-7.25 (4H, m, CHar), 7.33 (1H, d, JAB 1.4 Hz, H-3), 7.44-7.57 (12H, m, CHar), 7.57-7.61 (2H, m, H-7, 18), 7.84 (2H, m, H-12, 13), 7.93 (1H, d, J17, 18 4.6 Hz, H-17), 8.02 (1H, d, J8,7 4.7 Hz, H-8); λmax (CH2Cl2): 274 (ε 24 744), 365 (ε 38 788), 447 (ε 96 105), 582 (ε 8 003), 656 (ε 11 341), 710 (ε 12 856); m/z (+ve FAB, NOBA) 749 (MH+, 100% matrix subtracted), (Found: MH+ 749.31277.C49H41N4O4 requires 749.31278).", "Example 5 2-Aza-21-arba-2-methyl-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N-Me-NC-m-THPP): N-Me-NC-m-TMPP (200 mg, 0.27 mmol) was dissolved in dry dichloromethane (20 ml) and the solution was cooled to an external temperature of −78° C. A 1.0M solution of boron tribromide in dichloromethane (2.6 ml, 2.6 mmol) was added dropwise and the resultant reaction mixture stirred at −78° C. for 2 h. This was then allowed to warm up slowly to RT with continued stirring overnight.", "Next day, the mixture was cooled to an external temperature of −10° C. and excess methanol (2 ml) carefully added to quench the reaction.", "Excess triethylamine (4 ml) was then added slowly to neutralise the HBr given off and the resultant solution stirred for 30 min after which the mixture was then evaporated in vacuo to dryness.", "The residue was dissolved in methanol (10 ml) and acetic acid (0.2 ml) and then heated to reflux.", "Water (20 ml) was gradually added ensuring the resultant mixture remained at reflux after which the mixture was allowed to cool to RT and then cooled in the fridge overnight.", "The solid formed was filtered, washed with water (10 ml) and dichloromethane (2×25 ml) and then dried (vacuum oven, 60° C., 2 h) to yield the title compound (0.18 g, 99%).", "N-Me-NC-m-THPP Physical Data: δH (360 MHz, CD3OD) −2.44 (1H, s, H-21), 3.83 (3H, s, NCH3), 7.27-7.31 (2H, m, CHar), 7.40-7.45 (2H, m, CHar), 7.52-7.54 (4H, m, CHar), 7.63-7.67 (2H, m, CHar), 7.75-7.85 (6H, m, CHar), 7.87 (1H, s, H-3), 8.23 (4H, m, H-7, 12, 13, 18), 8.60 (1H, d, J17, 18 4.7 Hz, H-17), 8.73 (1H, d, J8, 7 4.6 Hz, H-8); λmax (CH3OH): 285 (ε 22 806), 371 (ε 31 554), 457 (ε 87 224), 573 (ε 3 873), 636 (ε 3 411), 846 (ε 10 795); m/z (+ve FAB, NOBA) 693 (M+, trace), (Found: MH+ 693.25018.C45H33N4O4 requires 693.25018).", "Examples of substitution at the 2-N or 21-C are given in Scheme 2 and examples 6-9.The propyl sulfonate derivative is shown as an internal salt, inferred from its property of solubility in both methanol and dichloromethane, unlike the other examples, which are soluble in one or the other only.", "The deprotection of 21-NO2—NC-m-TMPP and 21-Br—NC-m-TMPP can be performed in a similar manner to that shown for other examples.", "The syntheses described below outline substitutions of the tetramethoxy derivative followed by demethylation to the tetrahydroxy compound, however it is noted that these steps may be reversed to yield the equivalent desired product.", "Example 6 2-Aza-21-carba-2-propylsulfonate-5,10,15,20tetra-(3-methoxyphenyl)porphyrin (N—PS—NC-m-TMPP): A mixture of NC-m-TMPP (200 mg, 0.27 mmol), propane sultone (0.96 ml, 10.9 mmol) and triethylamine (0.19 ml, 1.35 mmol) in chloroform (20 ml) was stirred at reflux for 3.5 h after which TLC analysis (100% dichloromethane) indicated that all the starting material had been consumed.", "The solvent was removed in vacuo and water (40 ml) was then added.", "A 2.0M solution of aqueous sodium hydroxide (9 ml) was added to give a resultant solution at pH13-14 and this was left stirring at RT overnight.", "The mixture was then acidified to pH3-4 using acetic acid (3.5 ml) and the resultant solution left stirring at RT for 2 h. The precipitated solid that had formed was filtered, dissolved in methanol (40 ml) and then heated to reflux.", "Water (50 ml) was gradually added ensuring the resultant mixture remained at reflux after which the mixture was allowed to cool to RT and then cooled in the fridge overnight.", "The solid formed was filtered, washed with water (20 ml) and then dried (vacuum oven, 60° C., 3 h) to yield the title compound (0.20 g, 83%).", "N—PS—NC-m-TMPP Physical Data: δH (360 MHz, CD3OD) -2.40(1H, bs, H-21), 1.67 (2H, q, J 6.9 Hz, CH2CH2CH2), 2.05 (2H, t, J 6.4 Hz, NCH2), 4.07 (3H, s, OCH3), 4.15 (6H, s, OCH3), 4.24 (2H, t, J 6.9 Hz, CH2SO331 ), 7.41-7.95 (17H, m CHar and H-3), 8.08 (1H, m, H-8 or 17), 8.10 (1H, d, J 4.7 Hz, H-8 or 17), 8.17 (2H, m, H-12, 13), 8.47 (1H, d, J18, 17 4.9 Hz, H-18), 8.56 (1H, m, H-7); λmax (CH3OH): 296 (ε 22 853), 372 (ε 32 074), 462 (ε 93 000), 573 (ε 4 085), 632 (ε 2 913), 850 (ε 12 077); m/z (+ve FAB, NOBA) 857 (MH+, 10.9%), (Found: MH+ 857.30065.C51H45N4O7S requires 857.30090).", "Example 7 2-Aza-21-arba-2-propylsulfonate-5,10,15,20-tetra-(3-hydroxyphenyl)porphyrin (N—PS—NC-m-THPP): N—PS—NC-m-TMPP (200 mg, 0.23 mmol) was dissolved in dry dichloromethane (20 ml) and the solution was cooled to an external temperature of −78° C. A 1.0M solution of boron tribromide in dichloromethane (2.1 ml, 2.1 mmol) was added dropwise and the resultant reaction mixture stirred at −78° C. for 2 h. This was then allowed to warm up slowly to RT with continued stirring overnight.", "Next day, the mixture was cooled to an external temperature of −10° C. and excess methanol (8 ml) carefully added to quench the reaction.", "Excess triethylamine (10 ml) was then added slowly to neutralise the HBr given off and the resultant solution stirred for 30 min after which the mixture was then evaporated in vacuo to dryness.", "The residue was dissolved in methanol (24 ml) and acetic acid (0.5 ml) then heated to reflux.", "Water (56 ml) was gradually added ensuring the resultant mixture remained at reflux, after which the mixture was allowed to cool to RT and then cooled in the fridge overnight.", "The solid formed was filtered, washed with water (20 ml) and dichloromethane (2×20 ml) and then dried (vacuum oven, 60° C., 2 h) to yield the title compound (0.16 g, 84%).", "N—PS—NC-m-THPP Physical Data: δH (360 MHz, DMSO-D6) −2.40 (1H, s, H-21), 1.36 (2H, bs, CH2CH2CH2), 1.57 (2H, bs, NCH2), 4.16 (2H, bs, CH2SO3−), 7.18-7.72 (16H, m, CHar), 7.96 (1H, s, H-3), 8.05-8.12 (4H, m, H-8, 12, 13, 17), 8.45 (1H, bs, H-18), 8.64 (1H, bs, H-7), 9.80 (2H, bs, OH), 10.10 (2H, bs, OH); λmax (CH3OH): 285 (ε 12 518), 371 (ε 17 268), 462 (ε 43 293), 566 (ε 2 838), 794 (ε 4 195), 853 (ε 5 544); m/z (+ve FAB, NOBA) 801 (MH+, 1.2%), (Found: MH+ 801.23834.C47H37N4O7S requires 801.23830).", "Example 8 2-Aza-21-arba-21-nitro-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (21-NO2—NC-m-TMPP): A solution of sodium nitrite (2.5 g, 36.2 mmol) and hydrochloric acid (37%, 22.2 ml) in water (227.8 ml) was added to a stirred solution of NC-m-TMPP (250 mg, 0.34 mmol) in dichloromethane (150 ml).", "The resultant reaction mixture was stirred vigorously at RT for 1 min after which the solution was neutralised by adding 2.0M aqueous sodium hydroxide solution.", "The organic layer was then separated, washed with water (2×75ml), dried (Na2SO4) and evaporated in vacuo to dryness.", "The residue collected was dissolved in dichloromethane (25 ml) before being absorbed onto 6 g of basic alumina (activity III) and was added to the top of a column (50 mm) of basic alumina (200 g, activity m) eluting with 100% dichloromethane.", "The fractions containing the desired product were combined and the solvent removed in vacuo to dryness to yield the title compound (0.24 g, 89%).", "21-NO2—NC-m-TMPP Physical Data: δH (360 MHz, CDCl3) −3.45 (1-2H, bs, NH), 3.95-3.98 (6H, m, OCH3), 4.00 (3H, m, OCH3), 4.02 (3H, m, OCH3), 7.33-7.44 (4H, m, CHar), 7.62-7.83 (8H, m, CHar), 8.00-8.05 (4H, m, CHar), 8.13 (1H, s, H-3), 8.60-8.84 (4H, m, H-8, 12, 13, 17), 9.20 (1H, d, J18, 17 5.0 Hz, H-18), 9.25 (1H, d, J7, 8 4.9 Hz, H-7); λmax (CH2Cl2): 280 (ε 23 591), 368 (ε 39 387), 472 (ε 166 850), 646 (ε 13 657), 698 (ε 13 040); m/z (+ve FAB, NOBA) 780 (MH+, 7.9%), (Found: MH+ 780.28218.C48H38N5O6 requires 780.28221).", "Example 9 2-Aza-21bromo-21-carba-5,10,15,20-tetra-(3-methoxyphenyl)porphyrin (21-Br—NC-m-TMPP): A solution of NC-m-TMPP (50 mg, 69 μmol) and N-bromosuccinimide (15 mg, 83 μmol) in dichloromethane (5 ml) was stirred for 5 min at RT after which TLC (100% dichloromethane) indicated that all the starting material had been consumed.", "The product was then pre-absorbed onto 5 g of basic alumina (activity III) and was added to the top of a column (20 mm) of basic alumina (30 g, activity III) eluting with 100% dichloromethane.", "Fractions were combined and the solvent evaporated in vacuo to give the title compound (24 mg, 43.3%).", "21-Br—NC-m-TMPP Physical Data: δH (200 MHz, CDCl3) 3.97-4.03 (6H, m, OCH3), 4.08 (3H, s, OCH3), 4.12 (3H, s, OCH3), 7.28-8.07 (17H, m, CHar and H-3), 8.55-8.62 (4H, m, H-8, 12, 13, 17), 8.96 (1H, d, J18, 17 4.5 Hz, H-18), 9.06 (1H, d, J7, 8 4.9 Hz, H-7); m/z (+ve FAB, NOBA) 815 (MH+, trace), 735 (MH+—Br, trace), 613 (MH+—Br—(4×OCH3), 1.3%), 460 (MH+—Br—(4×OCH3)-2Ph, 8.7%).", "Further N-Substituted N-confused Porphyrins In the discussion earlier in this patent, it is stated that the external nitrogen is a convenient attachment point for functionalisation.", "Such groups that can be attached may be designed to promote water solubility in the product, which is a desired attribute in a pharmaceutical, and possibly increase the intensity of the lowest energy Q band.", "The synthesis of some of these compounds is given in Scheme 3.The initial substitution reaction may be aided by the addition of bases such as Et3N, DIPEA or DMAP.", "Water solubilising groups such as m-PEG, carbohydrates or amino acid residues can then be coupled to the derivative obtained.", "Synthesis of N-confused Chlorins and Bacteriochlorins of Types IIIA-D and VA-F from N-confused Porphyrins of Type IIA and N-confused Chlorins and Bacteriochlorins of Types IVA-D and VIA-F from N-confused Porphyrins of Type IIB Porphyrins with imidazole substituents are easily reduced to chlorins and bacteriochlorins via hydrogen transfer catalysis.", "We expected that electron-withdrawing substituents would render the porphyrin macrocycle more susceptible to hydrogen transfer.", "In searching for other systems on which to test this hypothesis, we uncovered the work by Lindsey on N-confused porphyrins.", "We expected that the external nitrogen should similarly make the macrocycle more susceptible to hydrogen transfer envisaging that hydrogen will add specifically across the external 2,3 —N═C— bond in the macrocycle.", "Reduction of the N-confused porphyrins via hydrogen transfer catalysis, using 10% Pd on charcoal or Pd black and formic acid as hydrogen transfer reagent, indicated that with N-confused porphyrins, 10% Pd on charcoal achieves reduction to a chlorin-like species with a broad intense band at 686 nm and an intensity almost 50% that of the B band at 435 nm.", "This is in comparison with m-THPC which has a Q/B band ratio of 15-20%.", "Further work has shown that reduction to the N-confused chlorin occurs with NaCNBH3 in the presence of acid.", "Reduction of the N-confused porphyrins can be performed by using catalytic hydrogen transfer or with agents that supply hydride ions, H−, such as alkali metal boron and aluminium hydrides.", "These preferentially reduce the 2,3 —N═C— bond to produce an N-confused chlorin without further reduction to give an N-confused bacteriochlorin (See Scheme 4).", "The addition of acid to reactions with the above named reagents aids in polarization of the 2,3 —N═C— bond and allows hydride ion attack to occur more readily.", "From the literature, (Whitlock et al.", "J.", "Am.", "Chem.", "Soc.", "1969, 91, 7485), it is indicated that reduction of a porphyrin with diimide (used in the production of the known PDT agent, m-THPC) will proceed by the initial reduction of a peripheral ring —C═C— bond.", "If the 12,13 bond of the N-confused porphyrin is reduced preferentially by this method, then further reduction to an N-confused bacteriochlorin is slow as the opposing ring contains the —C═N— bond, which would not be reduced by this reagent.", "The N-confused chlorin will therefore be the predominant product.", "In the reduction of m-THPP, the bacteriochlorin is produced as an intermediate, which can then be oxidised to the chlorin.", "The N-confused bacteriochlorin will not be formed in this way as we are proposing a stepwise synthesis.", "Thus only the sequential hydride or hydrogen transfer reaction followed by diimide reduction or vice versa will form the N-confused 2,3-12,13 bacteriochlorin (see Scheme 4).", "Example 10 2-Aza-21-arba-5,10,15,20-tetra-(3-methoxyphenyl)dihydroporphyrin (NC-m-TMPC): A mixture of NC-m-TMPP (100 mg, 0.14 mmol) and hydrochloric acid (37%, 0.57 ml, 6.87 mmol) in 15:1 tetrahydrofuran/methanol (10 ml) was added to sodium cyanoborohydride (14.4 mg, 0.23 mmol) with vigorous stirring under an N2 blanket.", "The resultant reaction mixture (pH2-6) was then left for 30 min after which TLC analysis (100% dichloromethane) indicated that all the starting material had been consumed.", "The mixture was then neutralised with 2.0M aqueous sodium hydroxide solution and the product extracted using dichloromethane (2×50 ml).", "The combined organic extracts were washed with water (40 ml), dried (Na2SO4) and concentrated in vacuo to dryness.", "The residue collected was then dissolved in 75% dichloromethane in hexane (3 ml) and added to the top of a column (20 mm) of basic alumina (35 g, activity III) eluting with 75% dichloromethane in hexane.", "Fractions containing the new product were combined and the solvent removed under vacuo to give the title compound (17.9 mg, 17.7%).", "NC-m-TMPC Physical Data: λmax (CH2Cl2): 282 (ε 18 360), 309 (ε 20 567), 421 (ε 47 183), 698 (ε 24 329); m/z (+ve FAB, NOBA) 737 (MH+, 42.4% or 100% when matrix subtracted).", "N-confused chlorins and bacteriochlorins of types IVA-D and VIA-F can also be synthesised from N-confused porphyrins of Type IIIA by a two step process of reduction followed by substitution of the external nitrogen with the appropriate R group (See Scheme 5).", "This provides an alternative route to the desired compounds.", "BIOLOGY The biological efficacy of one of the N-confused porphyrins was tested as detailed below.", "Experimental N-Me-NC-m-THPP was dissolved in dilution vehicle (polyethylene glycol 400:ethanol:water 3:2:5 v:v:v) to produce a stock solution of 0.72 mgml−1.Adult female Balb/c mice bearing the syngeneic colo26 tumour implanted subcutaneously were treated with N-Me-NC-m-THPP at a dose of 2.6 μmol kg−1 (1.8 mg kg−1) by injection of the stock solution at a volume of 2.5 μlg−1 live weight into the tail vein.", "Twenty-four hours after injection of the photosensitiser, an area of 1.5 cm diameter surrounding the tumour was irradiated with broad-spectrum white light derived from a high-pressure xenon lamp (Applied Photo Physics clinical photo-irradiator model UV90, serial 020).", "The power density at the skin surface was 713 mWcm−2 and irradiation was applied for 70 s to give a total light dose of just under 50 Jcm−2.The tumours were assessed 24 h after irradiation, when the animals were sacrificed and the irradiation site examined and the depth of necrosis measured.", "Results are shown in table 1 below.", "In a separate group of mice, the normal tissue damage was assessed by irradiating one ear with 40 Jcm−2 of broad spectrum white light delivered using a fibre bundle from the xenon light source.", "The incident power at the ear surface was 638 mWcm−2 and light was applied for 63 s. Animal ears were irradiated with light 24 h after administration of N-Me-NC-m-THPP.", "The opposing ear served as a control.", "The response was assessed twenty-four hours after irradiation by measuring the ear thickness at three sites on the upper third of the ear using a fixed-force micrometer with a vernier-interpolated resolution of 2 μm and an accuracy of 10 μm (Neill instruments).", "No adverse effects to the animals were noted on injection of N-Me-NC-m-THPP and no subsequent toxic reactions were noted.", "The animals were held in subdued lighting conditions (ambient light ranged from 10 to 300 lux) and no phototoxicity was noted.", "There were no notable responses on irradiation, although some animals gave signs of slight discomfort during light delivery.", "When examined 24 h after irradiation, all of the treated tumours showed extensive signs of photonecrosis.", "In addition, in two of the animals an area of mildly oedematous skin was observed corresponding to the irradiated area In all cases, tumour necrosis extended the full thickness of the tumour.", "The depths of necroses noted were 5.5, 2.5 and 7.0 mm.", "On measurement of the ear thickness, no difference was found between the irradiated and control ears in the N-Me-NC-m-THPP treated animals.", "The mean difference (irradiated-control) was −2 μm with a standard error of 5 μm.", "In the case of animals treated with m-THPC at 0.88 μMkg−1 irradiated at the same light dose 24 h after drug injection, the mean difference in ear thickness (irradiated-control) was 41 μm.", "TABLE 1 Dose Compound (μmol/kg) Tumour necrosis (mm; mean ± SD) N—Me—NC-m-THPP 2.6 5.0 ± 2.3 (n = 3) Ear Swelling (irradiated − control; μm) N—Me—NC-m-THPP 2.6 −2 (±5 Standard Error) m-THPC 0.88 41 The results on tumour necrosis show effective photodynamic therapeutic (PDT) activity for N-Me-NC-m-THPP using broad spectrum white light.", "An improvement in the potency of this effect is anticipated with the use of light selective to the Q band of the N-Me-NC-m-THPP.", "In comparison to literature reported data (Berenbaum et al.", "Lasers in Medical Science 1993, 8, 235-243) on the activity of m-THPP in this model, which shows tumour necrosis of 0.14 mm at an m-THPP dose of 1.61 μmol/kg and using light at 652 nm, this PDT activity of N-Me-NC-m-THPP is very promising.", "Results on photosensitivity, tested by ear swelling, suggest that N-Me-NC-m-THPP shows little or no cutaneous photosensitivity compared to m-THPC.", "This signifies a potentially important therapeutic advantage for N-Me-NC-m-THPP and other N-confused porphyrins, chlorins and bacteriochlorins of the present invention." ] ]

[ [ "Mutated aqp, method for detecting cancer using the same, dna chip having oligonucleotides of said mutated aqp sequence", "The present invention relates to mutation genes of the AQP(aquaporin), a method for detecting cancer using mutations and expressions of the AQP and a DNA chip possessing oligonucleotides of mutated AQP base sequence.", "In case of the present method for detecting cancer and DNA chip using the AQP's mutations and expressions, it is highly accurate, rapid and effective in cancer diagnosis." ], [ "1.A mutant AQP5 gene, in which at least single base was changed from SEQ ID NO: 1.2.A method for diagnosing cancer comprising detecting presence or absence of mutation of AQP5 gene in claim 1 and expression of AQP5, AQPL1, AQP3 and/or AQP 4.3.The method as set forth in claim 2, wherein the cancer comprises lung cancer, stomach cancer, colon cancer, prostatic cancer, and head and neck cancer.", "4.The method as set forth in claim 3, wherein the cancer comprises lung cancer.", "5.The method as set forth in claim 2, wherein presence or absence of mutation of AQP5 gene is detected by at least one method selected from the group consisting of immunohistochemical study, western blotting, dot blotting, ELISA (enzyme-linked immunosorbent assay), RT-PCR, nucleic acid sequencing, restriction enzyme analysis, nothern blotting, Southern blotting, RNase protection assay, in situ hybridization, SSCP (single strand conformational polymorphism) analysis, MSO hybridization (Mutant specific oligonucleotide-hybridization) assay, ARMS (amplification refractory mutation system) and DNA chip (microarray) analysis.", "6.The method as set forth in claim 5, wherein the MSO hybridization assay comprises the steps of: 1) arraying oligonucleotides to nitrocellulose membrane or nylon membrane in parallel; 2) preparing biotin labeled probe DNA by using PCR with oligonucleotide primer labeled by biotin at 5′ end; 3) denaturing biotin labeled probe, followed by hybridization of probe with the membrane in 1) and eliminating unlabelled probe by washing; and 4) treating the washed membrane with alkaline phosphatase-labeled streptavidin, combining the formed biotin labeled hybrid, and inducing the color reaction, in particular comprising the use of strip in hybridization.", "7.The method as set forth in claim 5, wherein the DNA chip analysis comprises the steps of: 1) synthesizing oligonucleotide primer for each base of AQP5 cDNA, modifying 5′ end of primers with chemical linker, spotting the primers onto slide treated by special coating and thus producing oligo DNA chip; 2) preparing target DNAs by PCR amplification of coding sequence of AQP5 followed by fragmentation of the PCR products into nucleotides with 50 to 100-bp in length; 3) adding fragmented PCR products of 2), four fluorescence-labelled dideoxynucleotides and DNA polymerase onto the oligo DNA chip in 1), mixing them, and then performing arrayed primer extension reaction; and 4) analyzing automatically the results of arrayed primer extension reaction in 3) by using 4 color fluorescence DNA scanner.", "8.A DNA chip comprising one of nucleotide sequences of mutant AQP5 gene in claim 1.9.An oligonucleotide chip wherein oligonucleotide primers sequences of FIG.", "20 are arrayed." ], [ "<SOH> BACKGROUND ART <EOH>Neoplastic diseases, including most: particularly the collection of diseases known as cancer, are major cause of mortality and morbidity of human and are the most difficult disease to treat.", "Although medical science and natural science has recently advanced so much, cancer still remains unresolved problem.", "In United States of America, cancer is surpassed only by cardiovascular diseases as the primary cause of adult death, one million and three hundred thousands of new cases of cancer develop yearly and five hundred and fifty thousands of men die of cancer every year.", "This means that one of every 2 or 3 American people falls victim to cancer.", "The four major cancers in United States of America include lung cancer, colorectal cancer, prostate cancer and breast cancer, and the risk of American people to get these 4 major cancer are shown in Table 1 (Bang Y J et al.", "Cancer: Current Diagnosis and Therapy.", "Hanuri Company:Seoul, 1999;69-107) TABLE 1 The risk of American men to get four major cancers (from National Cancer Institute of United States, SEER Data) Risk of getting cancer Risk of dying of Type of cancer Sex (%) cancer(%) Lung cancer Male 8.6 7.1 Female 5.4 4.2 Colorectal Male 6.2 2.6 cancer Female 5.9 2.6 Prostate Male 18.5 3.6 cancer Breast cancer Female 12.6 3.6 The mechanism of development of human cancer is being clarified in more detail owing to advances of molecular biology and genetics; especially human genome project, functional genomics, nanotechnology and bioinformatics.", "Cancer is genetic disease, ie.", "Cancer develops secondary to genetic abnormality.", "Acquired genetic abnormality secondary to chemical carcinogen, UV light, irradiation or virus and hereditary genetic abnormality induces change (ie, mutation) into genetic information (DNA, RNA) of genome.", "When these mutation activate oncogenes and inactivate tumor suppressor genes, cancers may develop.", "Oncogene and tumor suppressor genes play key roles in regulation of signal transduction, cell cycle progression, cellular death and survival, accommodation with neighbor cells and angiogenesis is.", "Oncogenes induce proliferation, survival and escape from death, invasion of adjacent tissues and angiogenesis and thus stimulate development of cancer, whereas, tumor suppressor genes counteract oncogenes and thus inhibit development of cancer (Evan G et al.", "Matter of life and cell death, Science (1998) 281, 1317-1322; Harrington E A et al.", "Oncogene and cell death.", "Curr Opin Genet Dev (1994) 4, 120-129).", "During the past twenty years, many medical scientists have focused on oncogenes and tumor suppressor genes and have tried to find genetic markers of cancer and tumor markers through investigation of oncogenes and tumor suppressor genes.", "Through these research, they have found important genes such as p53, Rb, p16 and other CDK inhibitors, which regulate cell cycle and cellular apoptosis (Macleod K et al.", "Tumor suppressor genes.", "Curr Opin Genet Dev (2000) 10, 81-93; Adams P D et al.", "Negative control elements of the cell cycle in human tumors.", "Curr.", "Opin.", "Cell.", "Biol.", "(1998); 10, 791-797), BRCA1 and BRCA2 which are closely related with hereditary breast cancer and hereditary ovarian cancer (Miki Y et al.", "A strong candidate for the breast and ovarian susceptibility gene BRCA1, Science (1994) 266, 66-71); Wooster R et al.", "Identification of the breast cancer susceptibility gene BRCA2.Nature (1995) 378, 789-792), and APC gene which is closely related with hereditary colorectal cancer (Kinzier K, et al, Lessons from hereditary colorectal cancer, Cell(1996) 87, 159-170).", "These findings, had greatly contributed to progress of cancer research.", "In addition, these research stimulated establishment of many research centers which tested specific gene mutation on a commercial basis, in particular, BRCA1 and BRCA2 in women with high risk of development of breast cancer and ovarian cancer owing to family history (Levine A J. p53, the cellular gatekeeper for growth and division, Cell(1997), 88, 323-331; Frank T S. Laboratory.", "identification of hereditary risk of breast and ovarian cancer, Curr.", "Opin.", "Biotech.", "(1999) 10, 289-294).", "However, ideal genetic marker remains not to be found for acquired solid tumors which constitute most of human cancers.", "In addition, no molecular marker common to all human cancer has been found so far.", "It is p53 gene that shows highest frequency of mutation in all forms of human cancer, but even for p53 gene, the frequency of mutation or deletion is only 30 to 50%, which suggests that p53 is inappropriate for use as a molecular diagnostic marker of human cancer in clinical practice (Levine A J et al.", "p53, the cellular gatekeeper for growth and division, Cell (1997) 88, 323-331).", "Oligonucleotide DNA chip which detects mutation of p53 gene has recently been tested in patients with lung cancer, but only 40% of the lung cancer tissues showed mutation of p53 gene, which indicates limitation of analysis of mutation of p53 gene as a diagnostic tool of lung cancer (Ahrendt S A et al.", "Rapid p53 sequence analysis in primary lung cancer using an oligonucleotide probe array, Proc Natl Acad Sci U.S.A(1999) 96, 7382-7387).", "So far, no marker has been found to be of practical value for the clinical management of lung cancer, stomach cancer, colorectal cancer and breast cancer which form more than 50% of all human cancers.", "Affymetrix company (www.affymetrix.com) has recently manufactured Human Cancer G110 Array, a new type complementary DNA(cDNA) chip which detects expression of about 100 oncogenes and tumor suppressor genes which have been found so far.", "However, it is questionable whether this DNA chip pan detect all human cancer, due to the fact that the genes found so far are at most 5-10% of the genes related to all human cancer.", "Despite progress of surgery, chemotherapy, radiation therapy and immunotherapy, the success rate of treatment of human cancer except some hematologic cancer and childhood malignancies has not been remarkably improved during last several decades.", "The main reason for the poor treatment outcome of human cancer lies in the delayed diagnosis of cancer in advanced status when it has already metastasized and cure is hard to attain rather than limited efficacy of current therapy for cancer.", "Nowadays the prevention of cancer takes a key place in clinical science as does treatment of cancer.", "Primary method of cancer prevention is so called chemoprevention which aims to delay or inhibit multistep development of cancer by change of life style, diet or drugs.", "The chemoprevention is appropriate in particular for asymptommatic people with high risk of cancer because of family history or past medical history of cancer.", "For example, a clinical study of chemoprevention is under way to administer retinoic acid to patients in status of long term remission from lung cancer after therapy.", "However, we still do not exactly know either the etiology of cancer (except smoking) or effective chemopreventive drugs, and we have no reliable marker to identify the efficacy of chemopreventive agents, all of which limit the practical value of chemoprevention of cancer.", "The secondary method of cancer prevention is early detection or screening of cancer.", "The fate of individual cancer patient, ie.", "cure rate and long term survival, is primarily determined by volume and stage of tumor at the time of diagnosis; The cure rate and survival rate is highest among cancers in stage 1 or stage 2.In fact, we can expect cure of cancer only when it is diagnosed in early stage, ie.", "stage 1 and/or stage 2.Therefore, medical society makes every effort to detect cancer from the general public in early stage.", "Screening methods of cancer include inspection (skin, oral cavity, external genitalia, uterine, cervix), palpation (breast, oral cavity, thyroid, anus and rectum, prostate, testicle, uterus, lymph nodes), clinical chemistry tests such as, Papanicolaou smear and tumor markers including serum prostate specific antigen (PSA) or α-feto protein, radiologic study such as barium enema study of colon, chest X ray, and endoscopic examination.", "Table 2 shows the cancer screening methods recommended by American Cancer Society.", "TABLE 2 Cancer screening methods: Guideline recommended by American Cancer Society (1993).", "Target Screening Age of screening Screening Cancer method Sex population frequency Prostate Digital rectal Male 50 years or after yearly cancer examination Serum Male 50 years or after Yearly PSA assay Breast Self Female 20 years or after monthly cancer examination Clinical Female 20-40 years/ Every 3 breast 40 years or years/ examination after Yearly Mammography Female 50 years or after Yearly Colorectal Stool occult Male and 50 years or after Yearly cancer blood test female Colonoscopy Male and 50 years or after Every 3 to female 5 years Uterine Pap smear Female 18 years or after Yearly cervix Pelvix Female 18-40 years/ Every 1-3 cancer examination 40 years or after years/ Yearly Endometrial Female Postmenopausal, Depending biopsy High risk women on doctor's recom- mendation Lung Chest X ray Not recommended as a routine study cancer Sputum cytology The cancer screening tests listed in Table 2 have been shown actually to improve the treatment outcome of target cancers.", "In particular, serum PSA assay is widely used for the screening, diagnosis, follow up after therapy of prostate cancers (Rimer B K et al.", "Cancer Screening.", "In DeVita V T Jr, Hellman S, Rosenberg S A. eds.", "Cancer.", "Principles and Practice of Oncology.", "fifth ed., Lippincott-Rave:Philadelphia, 1997; 619-631).", "Detection of expression of specific gene in blood has recently been used to identify specific cells and diagnosis of specific diseases, especially cancer.", "For example, detection of benign or malignant prostatic epithelial cells which express PSA or prostate specific membrane antigen (PSMA) from blood by using reverse transcription, polymerase chain reaction (RT-PCR) assay has been shown to be of value for the staging of cancer, ie.", "detection of metastatic cancer (which has been called molecular staging) as well as diagnosis of prostatic cancer.", "Presence of cancer cells within blood does not indicate metastasis by itself, but highly suggests metastasis (Katz A E et al.", "Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay; Israeli R S et als.", "Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate specific membrane antigen and prostate specific antigen-based assays.", "Cancer Research(1994) 54: 6306).", "However, no pan-tumor molecular marker has been found so far which show abnormality in most human cancers and thus is of practical value for the diagnosis and staging of cancer in clinical practice.", "Lung cancer ranks the first of all human cancers both in the incidence and death rates in United States of America: About 180,000 new cases of lung cancer develop yearly, about 160,000 patients die of lung cancer and overall 5-year survival rate of patients with lung cancer is only around 10%.", "Most of human lung cancers are bronchogenic carcinomas, which is primarily classified into small cell carcinoma and non-small cell carcinoma.", "The small cell carcinomas are a single type, while the non-small cell carcinomas consist of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioalveolar carcinoma.", "Primary cause of lung cancer is smoking and the amount and duration of smoking is directly correlated with incidence and death rates of lung cancer.", "The risk of getting lung cancer increases twenty-folds and risk of death of lung cancer becomes 13% on smoking 25 cigarettes daily for 10 years.", "Of remark is that the risk of lung cancer increases not only in primary or direct smokers but also in secondary or indirect smokers.", "The screening for lung cancer is indicated both in primary smokers and secondary smokers and also in men who had been exposed to lung carcinogen such as asbestos.", "Classical methods for the screening of lung cancer include chest radiography (simple X ray) and sputum cytology examination, however the former and the latter has a diagnostic sensitivity for lung cancer of only 30% and 40-60%, respectively.", "It is hard for these two studies to detect lung cancer in early stage and to significantly improve treatment outcomes of lung cancer, and for this reason these two studies were excepted from a list of recommended screening tests for cancers by American Cancer Society.", "Owing to lack of effective screening methods, ninety percent of cases of lung cancer are nowadays diagnosed in, advanced status(stage III or IV), in which cases most of the patients die within 2 years after diagnosis and 5 year survival rate is less than five percent despite aggressive chemotherapy and irradiation therapy (Choi S J et al.", "eds.", "Lung: neoplasia and cancer.", "In Current Diagnosis and Therapy.", "Han-Uri Publishing Co.:Seoul.", "1999;323-332).", "In contrast, if lung cancer can be detected by screening study in occult carcinoma status when patient has no symptoms and radiologic study of the lung shows no cancerous lesion, the cure rate of cancer is more than eighty percent.", "In fact some reports showed evidence that treatment outcomes of lung cancer are remarkably improved even by limited classical screening study of chest radiography and sputum cytology examination and that there were significant differences in 5 year survival rate between lung cancers detected by screening study (35%) and those diagnosed by lung cancer-related symptoms (13%) (Berlin N I et al.", "Early lung cancer detection: Summary and conclusions, American Review of respiratory diseases (1984) 30, 565).", "Diagnostic study and follow up study after therapy of lung cancer leaves much room for improvement.", "Accurate diagnosis of lung cancer is in reality not easy.", "It is hard to detect early stage lung cancer by chest X ray and sputum cytology examination and, even after detection of lung mass, it is not easy to differentiate between lung cancer and benign lung mass and between primary lung cancer and metastatic lung cancer.", "Definitive diagnosis of lung cancer is usually made by bronchoscopic biopsy, brush biopsy, bronchoalveolar lavage cytology examination, percutaneous needle aspiration cytology examination, mediastinoscopic biopsy, lymph node biopsy or pleural biopsy, but sometimes requires even open lung biopsy.", "The diagnosis is often ambiguous even after radiologic study and biopsy, in particular for solitary pulmonary nodules with diameter of less than 5 mm as being important in clinic (Ginsberg R J et al.", "Cancer of the lung.", "Section 2.Non-small cell lung cancer.", "In DeVita V T Jr. Hellman S, Rosenberg S A. eds.", "Cancer.", "Principles and Practice of Oncology, 5th ed., Lippincott-Raven: Philadelphia, 1997; 858-910).", "The next step after diagnosis of lung cancer is staging work up, ie., study of extent of cancer.", "The conventional staging methods for lung cancer include: computerized tomography (CT) scan, bronchoscopy, thoracoscopy, mediastinoscopy, and biopsy and cell examination using them, but all of these methods have limited accuracy and endoscopic studies are invasive.", "Lung cancer commonly invades pleura band and thus induce pleural effusion, in which cases, pleural fluid cytology examination and/or pleural biopsy are performed to identify the cause of pleural effusion, but reveals definitive diagnosis in only about half of the cases.", "Therefore, staging method for lung cancer definitely leaves much room for improvement.", "The appropriate follow up study is essential after therapy for lung cancer which can accurately define the results of therapy, detect residual or recurrent cancer in a sensitive and rapid way.", "The current follow Up study of lung cancer include radiologic study such as CT scan and endoscopic examination, but it is almost impossible to detect microscopic residual or recurrent cancer by these study.", "Therefore, novel method for follow up of lung cancer is urgently necessary.", "Appropriate maintenance of membrane water permeability is a fundamental requirement of all living organisms.", "Aquaporin (AQP) is a family of water channel proteins of membranes through which water are transported into and out of cells.", "AQP exists in all type of living organisms which include microorganisms, plants, mammalians.", "Ten types of mammalian AQP, ie., from type 1 to type 10 AQP, have been identified so far, whereas, more than 100 types of AQP exist in plants in which transport of water are more critical for the survival than in mammalians.", "AQP1 was the first type to be isolated in erythrocytes.", "Human type AQP1 was cloned for the first time by one of the present inventors (Moon C et als.", "Cloning of human aquaporin 1 gene, J Biol Chem (1993) 268, 15772-15778).", "Two functional groups of AQP are now being recognized.", "The first, including AQP1, AQP2, AQP4, AQP5, AQP6, AQP8 and AQP10 are permeable only to water, as classically defined.", "A second group, including AQP3, AQP7 and AQP9 are highly permeable to water, but also are permeable by glycerol (King L S et al.", "Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65).", "The present inventors have recently found the evidence that AQP also plays important roles in cell cycle regulation, signal transduction, delayed early response to growth factors, and gas exchange in hypoxic condition.", "AQP proteins exist in cell membranes, and to adapt to water channel function, its structure has six transmembrane domains and five connecting loops (loop A-E).", "The.", "Amino terminal (NH2 terminal) and carboxy terminal (COOH terminal) portion of AQP are located inside cytoplasm.", "Loop B and E of AQP contains signature motif Asn-Pro-Ala, which is called NPA, and adjacent cysteine.", "Two NPA motifs and cysteine combine to become center of the water channel (Walz T et als.", "Three-dimensional electron density map of human aquaporin 1 at 6 A resolution.", "Nature (1997) 387, 624-627; Lee M D et al.", "The human aquaporine-5 gene.", "J. Biol.", "Chem (1996) 271, 8599-8604).", "Each type of 10 mammalian aquaporins has a distinct tissue and cellular distribution and plays a diverse and specific role depending on the type of tissues and cells where it is located.", "AQP1 is located in erythrocytes, kidney, lung, eye, choroid plexus, biliary tract, nonfenestrated endothelia.", "AQP1 is abundant in proximal tubules and descending thin limb of Henle's loop segments, actively reabsorbs most of glomerular, filtrate and thus greatly contributes to concentration of urine.", "AQP2 is located in collecting duct epithelia of kidney, secreted in response to stimulation of antidiuretic hormone and thus contribute to concentration of urine.", "Deficiency of AQP2 produces nephrogenic diabetes insipidus which is characterized by failure to concentrate urine.", "AQP3 is located in renal collecting duct, gastrointestinal tract, airway epithelia, corneal epithelium and brain.", "AQP4 is abundant in glial cells and ependymal cell of brain tissue, but is also located in retina and airway epithelia.", "AQP5 is located in salivary gland, lacrimal gland and lung, in which plays an important role in production of saliva, tear and airway secretions.", "AQP6 is located in proximal tubular epithelia and collecting duct epithelia of kidney and characteristically acts as intracellular water channel and also is involved in regulation of acid base balance.", "AQP7 and AQP8 are expressed in germ cells and sperms.", "AQP9 is abundant in adipocytes (Deen P R T et al.", "Epithelial aquaporins., Current Opinion in Cell Biology.", "(1999) 10, 435-442; King L S et al.", "Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65; Agre P. Aquaporin water channels in kidney.", "J. American Society of Nephrology (2000) 11, 764-777).", "AQP plays important roles particularly in kidney, lung, brain, eye and eythrocytes.", "The lung has exceptionally high epithelial and endothelial permeability.", "Appropriate removal and supply of water in the airway, vascular and interstitial compartments of the lung are essential for normal gas exchange and lung defence.", "AQP is actively involved in the maintenance of liquid layer of surface of airway epithelia, which is essential for normal mucosal ciliary action, and also involved in appropriate supply of water to airway which prevents dehydration of airway and ensures adequate dehydration of expired air.", "Four water channels, including AQP1, AQP3, AQP4 and AQP5 have been indentified in the lung of rats and mice.", "AQP1 (Genebank No.", "NM-000385) is abundant in apical and basolateral membrane, of microvasculature and pleural membrane.", "AQP5 (Genebank No.", "NM-001651) is abundant in apical membrane of type 1 alveolar pneumocytes and secretory cells of airway submucosal gland.", "AQP3 (Genebank No.", "NM-004925) and AQP4 (Genebank No.", "U63623) are expressed in epithelial cells of airway and nasopharynx.", "AQP is also reported to be involved in CO 2 exchange of alveolar cells, which suggest that AQP may act as a gas channel (Nielsen S et al.", "Aquaporin in complex tissue II., Cellular and subcellular distribution in respiratory tract and glands of rat., American J. Physiology (1997) 273, 1549-1561; King L S et al.", "Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat., J. Clin Invest (1996) 97, 2183-2191).", "However, distribution and function of each type of AQP in the human lung remain to be indefinite.", "In addition, role of AQP in human cancer, in particular lung cancer, remains to be indefinite.", "Considering the prior art up to now, there is a need for the development of new tumor markers, which is useful for screening, diagnosis, and follow-up study after treatment for human cancer including lung cancer." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 illustrates expression of aquaporin (AQP) gene in bronchial and airway tissues of adult human by using in situ hybridization methodology.", "In A: antisense probe of aquaporin type 1 (AQP1) was used, In B: sense probe of AQP1 was used, In C: antisense probe of aquaporin type 5 (AQP5) was used, In D: sense probe of AQP5 was used, In E: antisense probe of aquaporin type 3 (AQP3) was used, In F: sense probe AQP3 was used, In G: antisense probe of aquaporin type 4 (AQP4) was used, In H: sense probe of AQP4 was used.", "FIG.", "2 illustrates expression of aquaporin (AQP) gene in bronchial and airway tissues of 17-week old male infant by using in situ hybridization methodology.", "In A and B, antisense probe of AQP1 and sense probe of AQP1 was used for the study of bronchial epithelium and developing bronchiolar structure, respectively, In C and D, antisense probe of AQP1 and sense probe of AQP1 was used for the study of immature alveolar structure, respectively, In E and F, antisense probe of AQP5 and sense probe of AQP5 was used for the study of bronchial epithelium and developing bronchiolar structure, respectively, In G and H, antisense probe of AQP5 and sense probe of AQP5 was used for the study of immature alveolar structure, respectively, FIG.", "3 illustrates expression of aquaporin gene family in bronchial tissues of 3 men with history of smoking as analyzed by reverse transcription polymerase chain reaction (RT-PCR) assay.", "Products of RT-PCR were shown on gel electrophoresis.", "1: AQP1 3: AQP3 4: AQP4 5: AQP5 The sample number indicates serial number of man under study.", "FIG.", "4 illustrates expression of AQP1 gene in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR were identified on gel electrophoresis.", "FIG.", "5 illustrates expression of AQP3 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "6 illustrates expression of AQP4 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "7 illustrates expression of AQP5 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "8 illustrates expression of AQP5 in human lung cancer tissues as analyzed by Nothern blotting.", "SQC1 and SQC2 indicate tissue of squamous cell carcinoma, ADE1, ADE2 and ADE3 adenocarcinoma, BAC1 bronchioalveolar carcinoma, LAG large-cell carcinoma, and SMC1 small cell carcinoma, respectively.", "FIG.", "9 illustrates expression of AQP gene family in human lung cancer tissues as analyzed by in situ hybridization.", "In A-1, antisense probe of AQP1 was used in the analysis of a tissue of squamous cell carcinoma, In A-2, sense probe of AQP1 was used in the analysis of a tissue of squamous cell carcinoma, In B-1, antisense probe of AQP1 was used in the analysis of a tissue of brochioalveolar carcinoma, In B-2, sense probe of AQP1 was used in the analysis of a tissue of bronchioalveolar carcinoma, In C, antisense probe of AQP5 was used in the analysis of a tissue of squamous cell carcinoma, In D, antisense probe of AQP5 was used in the analysis of a tissue of bronchioalveolar carcinoma, FIG.", "10 illustrates detection of AQP expression in sputum of patients with lung cancer and normal man as analyzed by using RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "11 illustrates detection of AQP expression in blood of patients with lung cancer and normal man as analyzed by using RT-PCR.", "Products of RT-PCR were identified on gel electrophoresis.", "FIG.", "12 illustrates the results of nucleic acid sequencing analysis of cDNA of AQP5 which were obtained from normal lung tissues and lung cancer tissues by RT-PCR followed by cloning.", "FIG.", "13 illustrates the results of automated sequencing analysis of cDNA of mutant AQP5 gene which was obtained from bronchoscopic lavage sample of a patient with lung cancer by using RT-PCR followed by cloning.", "FIG.", "14 illustrates the result of automated sequencing analysis of cDNA of mutant AQP5 gene which was obtained by RT-PCR followed by cloning from sputum of a patient with lung cancer.", "FIG 15 illustrates the frequency of mutation of AQP5 gene, which was found in human lung cancer tissues.", "In FIG.", "15 a, the mutation frequency of AQP5 were analyzed depending on exon number.", "In FIG.", "15 b, the mutation frequency of AQP5 were analyzed depending on codon number, FIG.", "16 illustrates an example of detection of mutation of exon 1 of AQP5 by using single strand conformational polymorphism (SSCP) analysis.", "FIG.", "17 illustrates an example of detection of AQP5 mutation by using mutant specific oligonucleotide (MSO) hybridization method.", "FIG.", "18 illustrates an example of detection of mutation of AQP5 by using multiplex PCR.", "LANE 1: mutant AQP5, LANE 2: wild type AQP5.FIG.", "19 illustrates an example of analysis of mutation of AQP5 by using DNA chip of the present invention.", "FIG.", "20 illustrates an list of nucleic acid sequences of sense primer and antisense primer which were arrayed on oligonucleotide chip of the present invention.", "FIG.", "21 illustrates a four-colored image of oligonucleotide chip of AQP5 gene in which each base of adenine (A), cytosine (C), guanine (G) and thymine (T) is shown in different color and thus is easily discriminated.", "FIG.", "22 illustrates an example of test for AQP5 mutation by using oligonucleotide DNA chip and automated nucleic acid sequencing assay.", "In FIG.", "22 a, point mutation of AQP5 was found in the form of heterozygosity of A/G.", "This point mutation was missed on automated sequencing analysis (ABI Prism) of FIG.", "22 b.", "FIG.", "23 .", "illustrates an example of test for AQP5 point mutation by using oligonucleotide chip of the present invention.", "A base was changed to T on analysis of both sense strand and antisense strand.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to mutant aquaporin (AQP) gene, a method for detecting cancer by using the mutant and expression thereof; and DNA chip having oligonucleotides of said mutated AQP sequence.", "BACKGROUND ART Neoplastic diseases, including most: particularly the collection of diseases known as cancer, are major cause of mortality and morbidity of human and are the most difficult disease to treat.", "Although medical science and natural science has recently advanced so much, cancer still remains unresolved problem.", "In United States of America, cancer is surpassed only by cardiovascular diseases as the primary cause of adult death, one million and three hundred thousands of new cases of cancer develop yearly and five hundred and fifty thousands of men die of cancer every year.", "This means that one of every 2 or 3 American people falls victim to cancer.", "The four major cancers in United States of America include lung cancer, colorectal cancer, prostate cancer and breast cancer, and the risk of American people to get these 4 major cancer are shown in Table 1 (Bang Y J et al.", "Cancer: Current Diagnosis and Therapy.", "Hanuri Company:Seoul, 1999;69-107) TABLE 1 The risk of American men to get four major cancers (from National Cancer Institute of United States, SEER Data) Risk of getting cancer Risk of dying of Type of cancer Sex (%) cancer(%) Lung cancer Male 8.6 7.1 Female 5.4 4.2 Colorectal Male 6.2 2.6 cancer Female 5.9 2.6 Prostate Male 18.5 3.6 cancer Breast cancer Female 12.6 3.6 The mechanism of development of human cancer is being clarified in more detail owing to advances of molecular biology and genetics; especially human genome project, functional genomics, nanotechnology and bioinformatics.", "Cancer is genetic disease, ie.", "Cancer develops secondary to genetic abnormality.", "Acquired genetic abnormality secondary to chemical carcinogen, UV light, irradiation or virus and hereditary genetic abnormality induces change (ie, mutation) into genetic information (DNA, RNA) of genome.", "When these mutation activate oncogenes and inactivate tumor suppressor genes, cancers may develop.", "Oncogene and tumor suppressor genes play key roles in regulation of signal transduction, cell cycle progression, cellular death and survival, accommodation with neighbor cells and angiogenesis is.", "Oncogenes induce proliferation, survival and escape from death, invasion of adjacent tissues and angiogenesis and thus stimulate development of cancer, whereas, tumor suppressor genes counteract oncogenes and thus inhibit development of cancer (Evan G et al.", "Matter of life and cell death, Science (1998) 281, 1317-1322; Harrington E A et al.", "Oncogene and cell death.", "Curr Opin Genet Dev (1994) 4, 120-129).", "During the past twenty years, many medical scientists have focused on oncogenes and tumor suppressor genes and have tried to find genetic markers of cancer and tumor markers through investigation of oncogenes and tumor suppressor genes.", "Through these research, they have found important genes such as p53, Rb, p16 and other CDK inhibitors, which regulate cell cycle and cellular apoptosis (Macleod K et al.", "Tumor suppressor genes.", "Curr Opin Genet Dev (2000) 10, 81-93; Adams P D et al.", "Negative control elements of the cell cycle in human tumors.", "Curr.", "Opin.", "Cell.", "Biol.", "(1998); 10, 791-797), BRCA1 and BRCA2 which are closely related with hereditary breast cancer and hereditary ovarian cancer (Miki Y et al.", "A strong candidate for the breast and ovarian susceptibility gene BRCA1, Science (1994) 266, 66-71); Wooster R et al.", "Identification of the breast cancer susceptibility gene BRCA2.Nature (1995) 378, 789-792), and APC gene which is closely related with hereditary colorectal cancer (Kinzier K, et al, Lessons from hereditary colorectal cancer, Cell(1996) 87, 159-170).", "These findings, had greatly contributed to progress of cancer research.", "In addition, these research stimulated establishment of many research centers which tested specific gene mutation on a commercial basis, in particular, BRCA1 and BRCA2 in women with high risk of development of breast cancer and ovarian cancer owing to family history (Levine A J. p53, the cellular gatekeeper for growth and division, Cell(1997), 88, 323-331; Frank T S. Laboratory.", "identification of hereditary risk of breast and ovarian cancer, Curr.", "Opin.", "Biotech.", "(1999) 10, 289-294).", "However, ideal genetic marker remains not to be found for acquired solid tumors which constitute most of human cancers.", "In addition, no molecular marker common to all human cancer has been found so far.", "It is p53 gene that shows highest frequency of mutation in all forms of human cancer, but even for p53 gene, the frequency of mutation or deletion is only 30 to 50%, which suggests that p53 is inappropriate for use as a molecular diagnostic marker of human cancer in clinical practice (Levine A J et al.", "p53, the cellular gatekeeper for growth and division, Cell (1997) 88, 323-331).", "Oligonucleotide DNA chip which detects mutation of p53 gene has recently been tested in patients with lung cancer, but only 40% of the lung cancer tissues showed mutation of p53 gene, which indicates limitation of analysis of mutation of p53 gene as a diagnostic tool of lung cancer (Ahrendt S A et al.", "Rapid p53 sequence analysis in primary lung cancer using an oligonucleotide probe array, Proc Natl Acad Sci U.S.A(1999) 96, 7382-7387).", "So far, no marker has been found to be of practical value for the clinical management of lung cancer, stomach cancer, colorectal cancer and breast cancer which form more than 50% of all human cancers.", "Affymetrix company (www.affymetrix.com) has recently manufactured Human Cancer G110 Array, a new type complementary DNA(cDNA) chip which detects expression of about 100 oncogenes and tumor suppressor genes which have been found so far.", "However, it is questionable whether this DNA chip pan detect all human cancer, due to the fact that the genes found so far are at most 5-10% of the genes related to all human cancer.", "Despite progress of surgery, chemotherapy, radiation therapy and immunotherapy, the success rate of treatment of human cancer except some hematologic cancer and childhood malignancies has not been remarkably improved during last several decades.", "The main reason for the poor treatment outcome of human cancer lies in the delayed diagnosis of cancer in advanced status when it has already metastasized and cure is hard to attain rather than limited efficacy of current therapy for cancer.", "Nowadays the prevention of cancer takes a key place in clinical science as does treatment of cancer.", "Primary method of cancer prevention is so called chemoprevention which aims to delay or inhibit multistep development of cancer by change of life style, diet or drugs.", "The chemoprevention is appropriate in particular for asymptommatic people with high risk of cancer because of family history or past medical history of cancer.", "For example, a clinical study of chemoprevention is under way to administer retinoic acid to patients in status of long term remission from lung cancer after therapy.", "However, we still do not exactly know either the etiology of cancer (except smoking) or effective chemopreventive drugs, and we have no reliable marker to identify the efficacy of chemopreventive agents, all of which limit the practical value of chemoprevention of cancer.", "The secondary method of cancer prevention is early detection or screening of cancer.", "The fate of individual cancer patient, ie.", "cure rate and long term survival, is primarily determined by volume and stage of tumor at the time of diagnosis; The cure rate and survival rate is highest among cancers in stage 1 or stage 2.In fact, we can expect cure of cancer only when it is diagnosed in early stage, ie.", "stage 1 and/or stage 2.Therefore, medical society makes every effort to detect cancer from the general public in early stage.", "Screening methods of cancer include inspection (skin, oral cavity, external genitalia, uterine, cervix), palpation (breast, oral cavity, thyroid, anus and rectum, prostate, testicle, uterus, lymph nodes), clinical chemistry tests such as, Papanicolaou smear and tumor markers including serum prostate specific antigen (PSA) or α-feto protein, radiologic study such as barium enema study of colon, chest X ray, and endoscopic examination.", "Table 2 shows the cancer screening methods recommended by American Cancer Society.", "TABLE 2 Cancer screening methods: Guideline recommended by American Cancer Society (1993).", "Target Screening Age of screening Screening Cancer method Sex population frequency Prostate Digital rectal Male 50 years or after yearly cancer examination Serum Male 50 years or after Yearly PSA assay Breast Self Female 20 years or after monthly cancer examination Clinical Female 20-40 years/ Every 3 breast 40 years or years/ examination after Yearly Mammography Female 50 years or after Yearly Colorectal Stool occult Male and 50 years or after Yearly cancer blood test female Colonoscopy Male and 50 years or after Every 3 to female 5 years Uterine Pap smear Female 18 years or after Yearly cervix Pelvix Female 18-40 years/ Every 1-3 cancer examination 40 years or after years/ Yearly Endometrial Female Postmenopausal, Depending biopsy High risk women on doctor's recom- mendation Lung Chest X ray Not recommended as a routine study cancer Sputum cytology The cancer screening tests listed in Table 2 have been shown actually to improve the treatment outcome of target cancers.", "In particular, serum PSA assay is widely used for the screening, diagnosis, follow up after therapy of prostate cancers (Rimer B K et al.", "Cancer Screening.", "In DeVita V T Jr, Hellman S, Rosenberg S A. eds.", "Cancer.", "Principles and Practice of Oncology.", "fifth ed., Lippincott-Rave:Philadelphia, 1997; 619-631).", "Detection of expression of specific gene in blood has recently been used to identify specific cells and diagnosis of specific diseases, especially cancer.", "For example, detection of benign or malignant prostatic epithelial cells which express PSA or prostate specific membrane antigen (PSMA) from blood by using reverse transcription, polymerase chain reaction (RT-PCR) assay has been shown to be of value for the staging of cancer, ie.", "detection of metastatic cancer (which has been called molecular staging) as well as diagnosis of prostatic cancer.", "Presence of cancer cells within blood does not indicate metastasis by itself, but highly suggests metastasis (Katz A E et al.", "Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay; Israeli R S et als.", "Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate specific membrane antigen and prostate specific antigen-based assays.", "Cancer Research(1994) 54: 6306).", "However, no pan-tumor molecular marker has been found so far which show abnormality in most human cancers and thus is of practical value for the diagnosis and staging of cancer in clinical practice.", "Lung cancer ranks the first of all human cancers both in the incidence and death rates in United States of America: About 180,000 new cases of lung cancer develop yearly, about 160,000 patients die of lung cancer and overall 5-year survival rate of patients with lung cancer is only around 10%.", "Most of human lung cancers are bronchogenic carcinomas, which is primarily classified into small cell carcinoma and non-small cell carcinoma.", "The small cell carcinomas are a single type, while the non-small cell carcinomas consist of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioalveolar carcinoma.", "Primary cause of lung cancer is smoking and the amount and duration of smoking is directly correlated with incidence and death rates of lung cancer.", "The risk of getting lung cancer increases twenty-folds and risk of death of lung cancer becomes 13% on smoking 25 cigarettes daily for 10 years.", "Of remark is that the risk of lung cancer increases not only in primary or direct smokers but also in secondary or indirect smokers.", "The screening for lung cancer is indicated both in primary smokers and secondary smokers and also in men who had been exposed to lung carcinogen such as asbestos.", "Classical methods for the screening of lung cancer include chest radiography (simple X ray) and sputum cytology examination, however the former and the latter has a diagnostic sensitivity for lung cancer of only 30% and 40-60%, respectively.", "It is hard for these two studies to detect lung cancer in early stage and to significantly improve treatment outcomes of lung cancer, and for this reason these two studies were excepted from a list of recommended screening tests for cancers by American Cancer Society.", "Owing to lack of effective screening methods, ninety percent of cases of lung cancer are nowadays diagnosed in, advanced status(stage III or IV), in which cases most of the patients die within 2 years after diagnosis and 5 year survival rate is less than five percent despite aggressive chemotherapy and irradiation therapy (Choi S J et al.", "eds.", "Lung: neoplasia and cancer.", "In Current Diagnosis and Therapy.", "Han-Uri Publishing Co.:Seoul.", "1999;323-332).", "In contrast, if lung cancer can be detected by screening study in occult carcinoma status when patient has no symptoms and radiologic study of the lung shows no cancerous lesion, the cure rate of cancer is more than eighty percent.", "In fact some reports showed evidence that treatment outcomes of lung cancer are remarkably improved even by limited classical screening study of chest radiography and sputum cytology examination and that there were significant differences in 5 year survival rate between lung cancers detected by screening study (35%) and those diagnosed by lung cancer-related symptoms (13%) (Berlin N I et al.", "Early lung cancer detection: Summary and conclusions, American Review of respiratory diseases (1984) 30, 565).", "Diagnostic study and follow up study after therapy of lung cancer leaves much room for improvement.", "Accurate diagnosis of lung cancer is in reality not easy.", "It is hard to detect early stage lung cancer by chest X ray and sputum cytology examination and, even after detection of lung mass, it is not easy to differentiate between lung cancer and benign lung mass and between primary lung cancer and metastatic lung cancer.", "Definitive diagnosis of lung cancer is usually made by bronchoscopic biopsy, brush biopsy, bronchoalveolar lavage cytology examination, percutaneous needle aspiration cytology examination, mediastinoscopic biopsy, lymph node biopsy or pleural biopsy, but sometimes requires even open lung biopsy.", "The diagnosis is often ambiguous even after radiologic study and biopsy, in particular for solitary pulmonary nodules with diameter of less than 5 mm as being important in clinic (Ginsberg R J et al.", "Cancer of the lung.", "Section 2.Non-small cell lung cancer.", "In DeVita V T Jr. Hellman S, Rosenberg S A. eds.", "Cancer.", "Principles and Practice of Oncology, 5th ed., Lippincott-Raven: Philadelphia, 1997; 858-910).", "The next step after diagnosis of lung cancer is staging work up, ie., study of extent of cancer.", "The conventional staging methods for lung cancer include: computerized tomography (CT) scan, bronchoscopy, thoracoscopy, mediastinoscopy, and biopsy and cell examination using them, but all of these methods have limited accuracy and endoscopic studies are invasive.", "Lung cancer commonly invades pleura band and thus induce pleural effusion, in which cases, pleural fluid cytology examination and/or pleural biopsy are performed to identify the cause of pleural effusion, but reveals definitive diagnosis in only about half of the cases.", "Therefore, staging method for lung cancer definitely leaves much room for improvement.", "The appropriate follow up study is essential after therapy for lung cancer which can accurately define the results of therapy, detect residual or recurrent cancer in a sensitive and rapid way.", "The current follow Up study of lung cancer include radiologic study such as CT scan and endoscopic examination, but it is almost impossible to detect microscopic residual or recurrent cancer by these study.", "Therefore, novel method for follow up of lung cancer is urgently necessary.", "Appropriate maintenance of membrane water permeability is a fundamental requirement of all living organisms.", "Aquaporin (AQP) is a family of water channel proteins of membranes through which water are transported into and out of cells.", "AQP exists in all type of living organisms which include microorganisms, plants, mammalians.", "Ten types of mammalian AQP, ie., from type 1 to type 10 AQP, have been identified so far, whereas, more than 100 types of AQP exist in plants in which transport of water are more critical for the survival than in mammalians.", "AQP1 was the first type to be isolated in erythrocytes.", "Human type AQP1 was cloned for the first time by one of the present inventors (Moon C et als.", "Cloning of human aquaporin 1 gene, J Biol Chem (1993) 268, 15772-15778).", "Two functional groups of AQP are now being recognized.", "The first, including AQP1, AQP2, AQP4, AQP5, AQP6, AQP8 and AQP10 are permeable only to water, as classically defined.", "A second group, including AQP3, AQP7 and AQP9 are highly permeable to water, but also are permeable by glycerol (King L S et al.", "Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65).", "The present inventors have recently found the evidence that AQP also plays important roles in cell cycle regulation, signal transduction, delayed early response to growth factors, and gas exchange in hypoxic condition.", "AQP proteins exist in cell membranes, and to adapt to water channel function, its structure has six transmembrane domains and five connecting loops (loop A-E).", "The.", "Amino terminal (NH2 terminal) and carboxy terminal (COOH terminal) portion of AQP are located inside cytoplasm.", "Loop B and E of AQP contains signature motif Asn-Pro-Ala, which is called NPA, and adjacent cysteine.", "Two NPA motifs and cysteine combine to become center of the water channel (Walz T et als.", "Three-dimensional electron density map of human aquaporin 1 at 6 A resolution.", "Nature (1997) 387, 624-627; Lee M D et al.", "The human aquaporine-5 gene.", "J. Biol.", "Chem (1996) 271, 8599-8604).", "Each type of 10 mammalian aquaporins has a distinct tissue and cellular distribution and plays a diverse and specific role depending on the type of tissues and cells where it is located.", "AQP1 is located in erythrocytes, kidney, lung, eye, choroid plexus, biliary tract, nonfenestrated endothelia.", "AQP1 is abundant in proximal tubules and descending thin limb of Henle's loop segments, actively reabsorbs most of glomerular, filtrate and thus greatly contributes to concentration of urine.", "AQP2 is located in collecting duct epithelia of kidney, secreted in response to stimulation of antidiuretic hormone and thus contribute to concentration of urine.", "Deficiency of AQP2 produces nephrogenic diabetes insipidus which is characterized by failure to concentrate urine.", "AQP3 is located in renal collecting duct, gastrointestinal tract, airway epithelia, corneal epithelium and brain.", "AQP4 is abundant in glial cells and ependymal cell of brain tissue, but is also located in retina and airway epithelia.", "AQP5 is located in salivary gland, lacrimal gland and lung, in which plays an important role in production of saliva, tear and airway secretions.", "AQP6 is located in proximal tubular epithelia and collecting duct epithelia of kidney and characteristically acts as intracellular water channel and also is involved in regulation of acid base balance.", "AQP7 and AQP8 are expressed in germ cells and sperms.", "AQP9 is abundant in adipocytes (Deen P R T et al.", "Epithelial aquaporins., Current Opinion in Cell Biology.", "(1999) 10, 435-442; King L S et al.", "Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65; Agre P. Aquaporin water channels in kidney.", "J. American Society of Nephrology (2000) 11, 764-777).", "AQP plays important roles particularly in kidney, lung, brain, eye and eythrocytes.", "The lung has exceptionally high epithelial and endothelial permeability.", "Appropriate removal and supply of water in the airway, vascular and interstitial compartments of the lung are essential for normal gas exchange and lung defence.", "AQP is actively involved in the maintenance of liquid layer of surface of airway epithelia, which is essential for normal mucosal ciliary action, and also involved in appropriate supply of water to airway which prevents dehydration of airway and ensures adequate dehydration of expired air.", "Four water channels, including AQP1, AQP3, AQP4 and AQP5 have been indentified in the lung of rats and mice.", "AQP1 (Genebank No.", "NM-000385) is abundant in apical and basolateral membrane, of microvasculature and pleural membrane.", "AQP5 (Genebank No.", "NM-001651) is abundant in apical membrane of type 1 alveolar pneumocytes and secretory cells of airway submucosal gland.", "AQP3 (Genebank No.", "NM-004925) and AQP4 (Genebank No.", "U63623) are expressed in epithelial cells of airway and nasopharynx.", "AQP is also reported to be involved in CO2 exchange of alveolar cells, which suggest that AQP may act as a gas channel (Nielsen S et al.", "Aquaporin in complex tissue II., Cellular and subcellular distribution in respiratory tract and glands of rat., American J. Physiology (1997) 273, 1549-1561; King L S et al.", "Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat., J. Clin Invest (1996) 97, 2183-2191).", "However, distribution and function of each type of AQP in the human lung remain to be indefinite.", "In addition, role of AQP in human cancer, in particular lung cancer, remains to be indefinite.", "Considering the prior art up to now, there is a need for the development of new tumor markers, which is useful for screening, diagnosis, and follow-up study after treatment for human cancer including lung cancer.", "DISCLOSURE OF INVENTION Based on the background information as summarized in the above, inventors have carried out extensive study and have found that analysis of mutation or expression of AQP is invaluable for the accurate, efficient and rapid detection of cancer and the present invention is based on these findings.", "Therefore, the object of the present invention is to provide information on mutant AQP5 gene by which we can detect cancers.", "It is another object of the present invention to provide a method for detecting cancer in quick, efficient and accurate ways by using analysis of mutation of AQP5 and expression of AQPs.", "It is a further object of the present invention to provide DNA chip (microarray) on which oligonucleotides of AQP5 are arrayed.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 illustrates expression of aquaporin (AQP) gene in bronchial and airway tissues of adult human by using in situ hybridization methodology.", "In A: antisense probe of aquaporin type 1 (AQP1) was used, In B: sense probe of AQP1 was used, In C: antisense probe of aquaporin type 5 (AQP5) was used, In D: sense probe of AQP5 was used, In E: antisense probe of aquaporin type 3 (AQP3) was used, In F: sense probe AQP3 was used, In G: antisense probe of aquaporin type 4 (AQP4) was used, In H: sense probe of AQP4 was used.", "FIG.", "2 illustrates expression of aquaporin (AQP) gene in bronchial and airway tissues of 17-week old male infant by using in situ hybridization methodology.", "In A and B, antisense probe of AQP1 and sense probe of AQP1 was used for the study of bronchial epithelium and developing bronchiolar structure, respectively, In C and D, antisense probe of AQP1 and sense probe of AQP1 was used for the study of immature alveolar structure, respectively, In E and F, antisense probe of AQP5 and sense probe of AQP5 was used for the study of bronchial epithelium and developing bronchiolar structure, respectively, In G and H, antisense probe of AQP5 and sense probe of AQP5 was used for the study of immature alveolar structure, respectively, FIG.", "3 illustrates expression of aquaporin gene family in bronchial tissues of 3 men with history of smoking as analyzed by reverse transcription polymerase chain reaction (RT-PCR) assay.", "Products of RT-PCR were shown on gel electrophoresis.", "1: AQP1 3: AQP3 4: AQP4 5: AQP5 The sample number indicates serial number of man under study.", "FIG.", "4 illustrates expression of AQP1 gene in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR were identified on gel electrophoresis.", "FIG.", "5 illustrates expression of AQP3 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "6 illustrates expression of AQP4 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "7 illustrates expression of AQP5 in human head and neck cancer cell lines and human lung cancer cell lines as analyzed by RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "8 illustrates expression of AQP5 in human lung cancer tissues as analyzed by Nothern blotting.", "SQC1 and SQC2 indicate tissue of squamous cell carcinoma, ADE1, ADE2 and ADE3 adenocarcinoma, BAC1 bronchioalveolar carcinoma, LAG large-cell carcinoma, and SMC1 small cell carcinoma, respectively.", "FIG.", "9 illustrates expression of AQP gene family in human lung cancer tissues as analyzed by in situ hybridization.", "In A-1, antisense probe of AQP1 was used in the analysis of a tissue of squamous cell carcinoma, In A-2, sense probe of AQP1 was used in the analysis of a tissue of squamous cell carcinoma, In B-1, antisense probe of AQP1 was used in the analysis of a tissue of brochioalveolar carcinoma, In B-2, sense probe of AQP1 was used in the analysis of a tissue of bronchioalveolar carcinoma, In C, antisense probe of AQP5 was used in the analysis of a tissue of squamous cell carcinoma, In D, antisense probe of AQP5 was used in the analysis of a tissue of bronchioalveolar carcinoma, FIG.", "10 illustrates detection of AQP expression in sputum of patients with lung cancer and normal man as analyzed by using RT-PCR.", "Products of RT-PCR are shown on gel electrophoresis.", "FIG.", "11 illustrates detection of AQP expression in blood of patients with lung cancer and normal man as analyzed by using RT-PCR.", "Products of RT-PCR were identified on gel electrophoresis.", "FIG.", "12 illustrates the results of nucleic acid sequencing analysis of cDNA of AQP5 which were obtained from normal lung tissues and lung cancer tissues by RT-PCR followed by cloning.", "FIG.", "13 illustrates the results of automated sequencing analysis of cDNA of mutant AQP5 gene which was obtained from bronchoscopic lavage sample of a patient with lung cancer by using RT-PCR followed by cloning.", "FIG.", "14 illustrates the result of automated sequencing analysis of cDNA of mutant AQP5 gene which was obtained by RT-PCR followed by cloning from sputum of a patient with lung cancer.", "FIG 15 illustrates the frequency of mutation of AQP5 gene, which was found in human lung cancer tissues.", "In FIG.", "15a, the mutation frequency of AQP5 were analyzed depending on exon number.", "In FIG.", "15b, the mutation frequency of AQP5 were analyzed depending on codon number, FIG.", "16 illustrates an example of detection of mutation of exon 1 of AQP5 by using single strand conformational polymorphism (SSCP) analysis.", "FIG.", "17 illustrates an example of detection of AQP5 mutation by using mutant specific oligonucleotide (MSO) hybridization method.", "FIG.", "18 illustrates an example of detection of mutation of AQP5 by using multiplex PCR.", "LANE 1: mutant AQP5, LANE 2: wild type AQP5.FIG.", "19 illustrates an example of analysis of mutation of AQP5 by using DNA chip of the present invention.", "FIG.", "20 illustrates an list of nucleic acid sequences of sense primer and antisense primer which were arrayed on oligonucleotide chip of the present invention.", "FIG.", "21 illustrates a four-colored image of oligonucleotide chip of AQP5 gene in which each base of adenine (A), cytosine (C), guanine (G) and thymine (T) is shown in different color and thus is easily discriminated.", "FIG.", "22 illustrates an example of test for AQP5 mutation by using oligonucleotide DNA chip and automated nucleic acid sequencing assay.", "In FIG.", "22a, point mutation of AQP5 was found in the form of heterozygosity of A/G.", "This point mutation was missed on automated sequencing analysis (ABI Prism) of FIG.", "22b.", "FIG.", "23.illustrates an example of test for AQP5 point mutation by using oligonucleotide chip of the present invention.", "A base was changed to T on analysis of both sense strand and antisense strand.", "BEST MODE FOR CARRYING OUT THE INVENTION In the followings are provided detailed description of the present invention.", "First, the inventors investigated expression pattern of aquaporins in bronchial tissues of normal adult humans.", "To identify novel molecular markers of cancer inventors have previously focused on genes, proteins or nuclear transcription factors which play important roles in regulation of cell cycle, signal transduction, survival and death.", "However, we have only found that different genes act in development or proliferation of each cancer (ie.", "clonal heterogeneity) and could not find any pan-tumor marker, which is common to every human cancer.", "We herein had turned our focus to cell membrane and cell surface, and through comparative analysis of expression and nucleic acid coding sequences of membrane proteins in both normal tissue and cancer tissue, we have found that AQP gene is a very unique gene which commonly shows change (mutation and/or abnormal expression) in human cancers, in particular lung cancers.", "As the first step of study, we investigated expression pattern of a various type of AQP in normal lung tissues from both adult and infant human by in situ hybridization analysis, because these have not been previously defined.", "From these studies, we have found that all of AQP 1, AQP3, AQP4 and AQP5 were expressed in bronchial epithelia of both adults and infants and that AQP1 is abundant in pulmonary microvascular endothelial cells and AQP5 in type 1 pneumocytes, respectively (See FIGS.", "1 and 2).", "We also have found by RT-PCR analysis that all of AQP1, AQP3, AQP4 and AQP5 were expressed in bronchial tissues of adults who have never smoked and have no evidence of cancer (FIG.", "3).", "These results indicate that human bronchial epithelia express 4 types of AQP and simultaneous expression of AQP1, AQP3, AQP4 and AQP5 may be a marker of bronchial epithelia.", "In addition to in situ hybridization and RT-PCR, a variety of methods, including immunohistochemical study western blotting, and DNA microarray analysis can be used to test expression of AQPs.", "In the next step of study in the present invention, expression pattern of AQP in human lung cancers was analyzed.", "First, expression of AQP1, AQP2, AQP3, AQP4, AQP5 and AQP6 in human head and neck carcinoma cell lines and lung cancer cell lines were analyzed by RT-PCR, All of the cell lines were found to express AQP1, AQP3 and AQP5, expression level of AQP5 was highest among all AQPs investigated, AQP4 was expressed by 3 of 4 human lung cancer cell lines, and AQP 2 and AQP6 were not expressed in any of the cell lines investigated (See FIGS.", "4, 5, 6 and 7).", "The expression and its level of AQP gene family in a variety of human lung cancer cell lines were investigated by northern blotting analysis, which showed that all of the cell lines tested expressed AQP5 in high level (FIG.", "8).", "In addition, expression pattern of AQP gene family in human lung cancer tissues was analyzed by in situ hybridization, the result of which showed that human lung cancer tissues expressed AQP5, AQP1, AQP3 and/or AQP4 (FIG.", "9).", "In addition, expression profiles of AQP in lung cancer tissue specimens, sample of sputum, bronchoalveolarlavage, pleural fluid and blood of patients with lung cancer were investigated by RT-PCR, the results of which showed that AQP5, AQP3 and AQP1 were clearly expressed in all of the samples investigated (FIG.", "10).", "Messenger RNA (mRNA) of AQP1, AQP3, AQP4 and AQP5 were clearly found in mononuclear cells from blood of lung cancer patients, whereas, only AQP1 was consistently expressed, and AQP3 was rarely expressed, but AQP4 and AQP5 were not expressed in blood mononuclear cells from control people without evidence of lung cancer (FIG.", "11).", "In addition, all of AQP1, AQP3, AQP4 and AQP5 were expressed in cancer cells of stomach, colon and rectum and prostate.", "These results indicate: first, human cancer cells including lung cancer simultaneoudsly express AQP1, AQP3, AQP4 and AQP5; second, mRNA of AQP can be easily detected in not only tissue but also in sample of sputum, bronchoalveolar lavage, pleural fluid, blood and other body fluids; third, simultaneous tests for expression of AQP1, AQP3, AQP4 and AQP5 in lung tissue, sample of sputum, bronchial lavage, pleural fluid and blood can be of value to identify the presence of lung cancer.", "A variety of methods can be used to investigate expression of AQP in cancer cell lines, lung tissues, sputum, bronchoalveolar lavage sample, pleural fluid, blood and other body fluids, which include RT-PCR, RT-PCR-Southern blotting or oligonucleotide hybridization, in situ hybridization, nothern blotting, immunohistochemical study, western blotting, DNA microarray, etc.", "The expression of AQP gene in human lung cancer tissues were investigated by northern blotting analysis and in situ hybridization, which showed that human lung cancer tissues expressed AQP1, AQP3, and AQP5 and especially, expressed AQP5 in high level (See FIGS.", "8 and 9).", "The object of the next study of the present invention was to identify the mutation of AQP in human lung cancer.", "The inventors performed PCR amplification of coding sequence of genomic DNA of AQP1, AQP3, AQP4 and AQP5 from lung cancer tissues and peripheral blood lymphocytes of control population with no evidence of lung cancer, followed by cloning and nucleic acid sequencing analysis of PCR products.", "The sequences of cloned PCR products were comparatively analyzed with that of wild type AQP1 (Genebank No.", "NM-000385), wild type AQP3 (Genebank No.", "NM-004925), wild type AQP4 (Genebank No.", "U63623) and wild type AQP5 (Genebank No.", "NM-001651).", "The results of this comparative study showed that most of the lung cancers tested carried mutation of AQP5 gene in widely variable pattern and that none of the control population carried mutation of AQP5 gene.", "To further confirm these results, functioning domains of AQP5 cDNA, which include most of exon 1, the whole exon 2 and most of exon 3 (from the one hundred forty third base to five hundred ninety third base), were amplified by RT-PCR, and their nucleic acid sequences were analyzed.", "The results of this study again showed that most of lung cancer tissues tested and none of the lung tissues from normal control population carried mutation of AQP5 (See FIGS.", "12, 13 and 14).", "Mutational pattern of AQP in human cancers, in particular lung cancer, were variable, but we could identify major hot spots of AQP5 mutation, which are listed in Table 5a and 5b.", "Remarkably, almost all human lung cancers tested were found to carry mutant AQP5 gene and these AQP5 mutation were concentrated in central 4 domains, including loop B, loop C, loop E and the fourth domain (TM4), all of which play key role in water channel function (See FIG.", "15).", "Therefore, mutation of AQP5, is a promising genetic tumor marker to detect lung cancer.", "In addition, the other human cancers, including prostate cancer, colorectal cancer, and stomach cancer also commonly carry mutation of AQP5, and therefore, mutation of AQP5 can be regarded as a pan-tumor marker.", "The inventors also tested mutation of AQP5 in samples of bronchial lavage, sputum and malignant pleural fluid from patients with lung cancer and found AQP5 mutation in 100% frequency from bronchoscopic ravage, in 96.7% frequency from sputum and in 100% frequency from malignant pleural fluid, respectively.", "In addition to nucleic acid sequencing analysis, a variety of methods can be used to detect mutation of AQP5, which include SSCP (Single strand conformational polymorphism) analysis (See FIG.", "16), MSO (Mutant specific oligonucleotide) hybridization analysis (See FIG.", "17), ARMS (amplification refractory mutation system) analysis (See FIG.", "18 and EXAMPLE 7) and other known methods.", "DNA chip or DNA microarray is a biochip onto which several hundreds to several hundred thousands of DNA fragments are arrayed by using robotic and computer technology.", "For example, DNA chip is a microarray chip onto which a number of DNA fragments are arrayed in extremely high density and is used for a wide variety of genetic study.", "The DNA chip can replace a number of existing methods for genetic study, including Southern blotting, nothern blotting, DNA sequencing analysis and a variety of mutation analysis methods.", "The major difference of DNA chip from methods of classical genetic study are that matrix for arraying genetic materials in DNA chips are sold materials such as glass, but matrix in classical methods are usually nitrocellulose or nylon membrane and that DNA chip makes it possible to analyze many genes simultaneously in a short time (Case-Green S C et al.", "Analyzing genetic information with DNA arrays, Current Opinions in Chemical Biology (1998) 2, 404-410; Lemieux B et al.", "Overview of DNA chip technology, Molecular Breeding(1998), 4, 277-289).", "DNA chip is classified into complementary DNA (cDNA) chip and oligonucleotide chip (oligochip) depending on the type and size of genetic materials to be arrayed on the chip.", "cDNA chip is arrayed by a number of cDNA fragments which are whole or part of open reading frame (with more than 500 bases in length) or EST.", "Oligonucleotide chip is arrayed by oligonucleotides with 15 to 25 bases in length.", "Oligonucleotide chip is highly useful for detection of mutation or polymorphism, and cDNA chip for analysis of gene expression, respectively.", "One of the main objects of the present invention was to make oligonucleotide chip which can detect mutation of AQP5, accurately and efficiently from large number of clinical samples.", "The inventors have designed and produced hybridization type oligonucleotide chip on the basis of mutation profile information of AQP5 in lung cancer which were obtained from nucleic acid sequencing analysis as in EXAMPLE 4.These oligonucleotide chips are based on oligonucleotide probe hybridization principles and can accurately detect mutation of AQP5.With oligonucleotide DNA chips produced as in the above, mutation of AQP5.was analyzed in tissues of lung cancer and normal lung tissues, and in samples of bronchoscopic lavage and sputum from patients with lung cancer and control people without evidence of lung cancer.", "By using the oligonucleotide chip analysis, mutation of AQP5 cDNA was found in all of the samples from lung cancer patients but no mutation was found in any of the samples from normal control group.", "This result strongly suggest that the above oligonucleotide chip is useful to test for mutation of AQP5 (See FIG.", "19).", "The other method invented by us to detect mutation of AQP5 is a novel oligonucleotide chip which interprets nucleic acid sequence by using arrayed primer extension (APEX) reaction.", "This sequencing type oligochip combines both microarray technology and Sanger's dideoxy sequencing analysis technology and thus are called minisequencing chip (Kurg A. et al.", "Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.", "Genet Test (2000) 4, 1-7; Tonisson N et al.", "Arrayed primer extension on the DNA chip: Method and application.", "In Schena M ed.", "Microarray biochip technology.", "Eaton Publishing:Natick,2000; 247-264).", "The basic technology of analysis of AQP5 mutation by using this minisequencing type oligonucleotide chip are as follows: First, oligonucleotide chips were designed and produced.", "The appropriate oligonucleotide primers were designed for each base of sequence of AQP5 cDNA, modified by attaching chemical linker to their 5′ ends, and were arrayed (ie spotted or printed) by using microarrayer machine onto microscopic glass slides, which had been treated by special coating solutions.", "Second, target DNAs were prepared.", "Genomic DNA or cDNA are isolated from clinical samples of cancer or control population, coding sequence of AQP5 are amplified by PCR, and then PCR products are changed into fragments of nucleotides with 50 to 100 base pairs in length.", "Third, APEX reaction was performed on the oligonucleotide chips.", "Fragmented PCR products, each of four dideoxynucleotides (ddATP, ddCTP, ddGTP, ddTTP) which were labeled by different fluorescence and DNA polymerase were placed onto oligonucleotide chips; and then APEX reaction, a variant of Sanger's sequencing reaction, was carried out.", "Fourth, oligonucleotide chips were analyzed after APEX reaction by using 4 color fluorescence DNA scanner and the sequence of each base of coding sequence of AQP5 gene from the target samples were interpreted.", "All the coding sequence of AQP5 can be analyzed automatically and quickly by using software of the scanner, and the equivocal results were corrected.", "The results of AQP5 cDNA mutation analysis by conventional automated nucleic acid sequencing method (ABI PRISM) were comparatively analyzed with those by the above minisequencing type oligonucleotide chip.", "The results of AQP5 cDNA mutation test by using oligonucleotide chip concurred with those by conventional automated sequencing method in 98 percent of sample tested.", "In the remaining 2 percent of samples, oligonucleotide chip detected additional point mutation of AQP5 which were missed by automated sequencing analysis (See FIGS.", "21, 22, and 23).", "The inventors recommend test for expression of AQP1, AQP3, AQP4 and AQP5 as the first step to detect or screen cancer.", "The next step necessary to confirm the presence of cancer is to test for mutation of AQP5.To test for AQP5 mutation, cDNA of AQP5 are PCR-amplified from target DNA or cDNA isolated from clinical samples, PCR products are initially screened by MSO hybridization or ARMS and finally analyzed by oligonucleotide chip and/or automated nucleic acid sequencing method.", "We recommend oligonucleotide chip in the present invention as the best single tool to test for mutation of AQP5.The methods described in the present invention, in particular oligonucleotide chip, can detect mutation of AQP5 from a wide variety of clinical samples, including tissue or cellular specimen, blood, sputum, stool, urine, sputum, cerebrospinal fluid, pleural fluid, peritoneal fluid and lavage samples obtained by endoscopy.", "The methods described in the present invention can be applied to test for extent of cancer in clinical practice as follows: Cells or tissue specimens are taken under the guidance of computerized tomography scan or endoscopy from the body areas where tumor extension is suspected, and blood and pleural fluid are also-taken from patients with cancer.", "RNA are isolated from tissues, cells, blood or pleural fluid, RT-PCR analysis are carried out to identify cells which express all of AQP5, AQP3, AQP4 and AQP1, which is followed by test for AQP5 mutation by using oligonucleotide chip, etc.", "These molecular study make it possible to identify therapeutic response, residual or recurrent cancer after therapy for cancer, including surgery, radiation therapy and chemotherapy.", "The above genetic tests for AQP are also of value to identify the efficacy of chemoprevention of cancer.", "Chemoprevention is indicated when cancer or precancerous lesion of the lung are highly suspected by finding AQP5 mutation in sputum or bronchoalveolar lavage, but no cancer is found on clinical study.", "The AQP5 mutation tests in the present invention can be ideally applied to sputum or bronchoalveolar lavage to identify the outcome of chemoprevention for lung cancer.", "In the following examples, the present invention is described in more detail.", "However, these are only some of examples and the present invention is not limited to these examples: EXAMPLE 1 Analysis of AQP Gene Expression in Normal Lung Tissues by Using in situ Hybridization and RT-PCR Method.", "Bronchial tissue specimen were taken by bronchoscopic brush biopsy from adult human without evidence of lung cancer (some with smoking history) and 17-week old male infant, and were treated, by RNAsol (TEL-TEST, USA), followed by homogenization.", "Wherein, bronchial tissue from adult human with smoking history was obtained from bronchial brush biopsy.", "The mixture was treated by chloroform, 0.2 ml, shaked, placed in 4° C. water bath for 15 minute, followed by centrifuge in 4° C. at 15,000 rpm.", "Two thirds of supernatant after centrifuge was taken to new tube, added by same volume of 2-propanol and were placed in 4° C. water bath for 15 minute.", "The pellet of RNA was obtained after precipitation using centrifugation in 4° C. at 15,000 rpm, washed by 80% ethanol containing diethyl pyrocarbonate (DEPC), dried at room temperature, treated by 50 μl DEPC-deionized water and then purified total RNA was obtained.", "The concentration of RNA was measured by spectrophotometer.", "Complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription (RT) reaction.", "Total RNA was mixed with DEPC-deionized water in 1:10 ratio, placed for 10 minutes in 70° C. water bath, mixed with 10×RT-buffer (500 mM Tris (pH 8.3), 60 mM MgCl2, 400 mM KCl), 0.1 M DTT (Dithiothreitol), 25 mM dNTP, oligo(dT) primer 2μl, and RNAsin (Promega, USA) 1 μl.", "The reaction mixture was incubated for 10 minutes at 37° C., and was added by 100 nits of Superscript II reverse transcriptase (GIBCO BRL, USA), incubated for 1 hour 37° C., and then cDNA was obtained, which was used as the template of the following PCR reaction.", "Sense and antisense oligonecleotide primer were designed and synthesized for each type of AQP as in Table 3.cDNA of each type of AQP was produced by RT, and was amplified by PCR reactions as in the condition listed in Table 4.TABLE 3 Sequence of oligonucleotides which were used as the primer for PCR of each type of aquaporin.", "Target gene of PCR Sense primer sequence Antisense primer sequence AQP1 SEQ ID NO: 2 SEQ ID NO: 3 AQP3 SEQ ID NO: 4 SEQ ID NO: 5 AQP4 SEQ ID NO: 6 SEQ ID NO: 7 AQP5 SEQ ID NO: 8 SEQ ID NO: 9 Beta-actin SEQ ID NO: 10 SEQ ID NO: 11 (control) TABLE 4 PCR condition of cDNA of AQP genes Composition of PCR reaction Reaction condition 10 × Taq buffer 1.Denaturation for 4 minutes at 95° C. 100 mM Tris-Cl 2.Repeat 40 cycles for AQPas follows: 500 mM KCl (beta actin: 25-cycles) 15 mM MgCl 10 seconds at 94° C. 0.01% gelatin 50 seconds at 63° C. Taq polymerase 50 seconds at 72° C. (5 units/μl) 3.Final extension for 10 minutes at 72° C. dNTP 500 uM sense primer (20 pmole) antisense primer (20 pmole) PCR products were visualized on 0.9% agarose gel electrophoresis.", "PCR product of AQP1 was shown as a band with size of 230-bp, AQP3 130-bp, AQP4 220-bp, AQP5 430-bp, and beta-actin 340-bp, respectively.", "Human cDNA of AQP1, AQP3, AQP4 and AQP5 obtained in the way above were introduced into the plasmid pCRII-TOPO (Invitrogen, USA) and this construct was used as a template to generate sense and antisense probes during in vitro transcription reaction.", "During the transcription, non-radioactive labelling of the single strand RNA probe was performed using digoxygenin-UTP (DIG RNA labeling kit, Boehringer Mannheim, USA).", "DIG-labelled RNA probe was mixed with RNAase inhibitor, stored at −80° C. and used for in situ hybridization as follows.", "Paraffin-embedded tissue section with 4 μm thickness were cut onto silane-coated slides (Signia Chemical, USA).", "The sections were deparaffinized in xylene, rehydrated in gradually decreasing concentrations of ethanol from 90% to 50%, and treated with 0.2 N HCl.", "The sections were then treated with protein kinase K for 15 min at 37° C., washed 3 times with 1×PBS, post-fixed in 4% paraformaldehyde for 5 min at room temperature, and re-rinsed with 1×PBS.", "Then they were acetylated in 0.25% acetic anhydride, 0.1M triethanolamine for 10 min.", "They were dehydrated in gradually increasing concentration of ethanol and air-dried prior to hybridization.", "They were prehybridized for 1 hour at 42° C. in hybridization buffer which consist of 20×SSC (3M NaCl, 0.3M sodium citrate, pH 7.0), 50% deionized formamide, 2.5 mg prenatured salmon sperm DNA, 1 g dextran sulfate, 2% 100× Denhart solution (20 g/l Ficoll, 20 g/l polyvinylpyrolidone, 20 g/l bovine serum albumin), 2% DTT and 4 mg of yeast tRNA.", "Hybridization was performed at 42° C. in the hybridization buffer containing 400 ng of probe of AQP1, AQP3, AQP4 and AQP5.The sections were then washed 2×SCC, and were treated with RNase solution (500 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 20 μg/ml RNase A) for 30 min at 37° C. Then the sections were rinsed in buffer 1 (0.1M maleic acid, 0.15M NaCl) for 5 min at room temperature, and then incubated with buffer 2 (2% normal sheep serum, 0.3% Triton X-100) for 30 min also at room temperature.", "Slides were then incubated for 12 hours at 4° C. with an anti-digoxigenin antibody (in 1:500 dilution).", "After two rinses in buffer 1, slides were rinsed shortly in buffer 3 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl, pH 9.5).", "Color reaction was induced by treatment with 5-bromo-4-chloro-3-indoyl phosphate and nitro-blue tetrazolium chloride and then slides were rinsed in buffer 4 (10 mM Tris-HCl, 1 mM EDTA), mounted, were observed under microscopy and alkaline phosphatase present within sections was detected.", "Sections incubated with digoxygenin-labelled sense probe in the same condition were used as negative controls.", "The results of in situ hybridization showed that all of AQP1, AQP3, AQP4 and AQP5 were expressed by bronchial epithelium of normal adults with or without history of smoking as well as normal infant.", "(See FIGS.", "1, 2 and 3).", "EXAMPLE 2 Analysis of AQP Gene Expression in Human Lung and Head and Neck Carcinoma Cell Lines.", "Expression of each type of human AQP gene in human lung carcinoma cell lines and head a neck carcinoma cell lines were analyzed by RT-PCR, Nothern blotting and in situ hybridization assay.", "Human lung cancer cell lines, including H460, H1944, H596 and A549, and head and neck carcinoma cell lines, including 183A, 22B, 17A, 11B were purchased from ATCC company (USA) and these cell lines were examined for AQP expression by using RT-PCR.", "The methods of RT-PCR were as same as in EXAMPLE 1, except the sequences of oligonucleotide primer for PCR of cDNA of AQP5.The sequence of sense primer for AQP5 was as same as SEQ ID NO: 12 and antisense primer SEQ ID NO: 13, respectively.", "On electrophoresis, the PCR product of AQP1 was shown as a band with size of 230-bp, 247-bp for AQP5, and 340-bp for beta-actin, respectively.", "PCR products of AQP were introduced into TA cloning vector and sequenced to confirm each AQP specific sequences.", "On the RT-PCR assay, all of the cell lines studied were found to express AQP5, AQP1 and AQP3, and the expression level of AQP5 was highest of all types of AQP.", "AQP4 was expressed in 3 of 4 lung cancer cell line, including H460, H1944 and A596, and only 11B cell line out of 4 head neck cell lines (See FIGS.", "4, 5, 6 and 7).", "AQP2 and AQP6 were not expressed in any of the cell lines tested.", "Next, AQP gene expression was analyzed by Nothern blotting in cancer cell lines established from a variety of type of human lung cancer.", "The target cell lines included squamous carcinoma cell lines (SC1 and SC2); adenocarcinoma cell lines (ADE-1, ADE-2 and ADE-3), bronchoalveolar carcinoma cell lines (BAC), large cell carcinoma cell line (LAC) and small cell carcinoma cell line (SMC).", "All of these cell lines were purchased from ATCC (USA).", "The conventional method was used for northern blotting assay (Sambrook J et al.", "Molecular cloning, second edition, Cold Spring Harbor Laboratory Press (1989)).", "RNA was extracted from cell lines in the same method as in EXAMPLE 1 except that RNAsol B (Genomed, Germany) was used instead of RNAsol.", "Twenty μg of total RNA were heated for 1 min, mixed with agarose gel buffer for RNA which consist of agarose 1.2 g, 10 ml of 10×MOPS(3-[N-morpholino]propanesulfonic acid) buffer (41.8 g/l MOPS, 3M sodium acetate 16.6 ml, 20 ml of 0.5M EDTA, pH 8.0) and 18 ml of 37% formaldehyde, 100 ml of DEPC-treated deionized water and the mixture were loaded into electrophoresis.", "After electrophoresis, RNA was transferred into nylon membrane by using conventional capillary method (Sambrook J et als.", "Molecular cloning, second edition, Cold Spring Harbor Laboratory Press (1989)) and fixed with UV-crosslinker (1200 J/cm2).", "The same probe as in EXAMPLE 1 was used as the AQP cDNA probe and GADPH cDNA probe was prepared in the same way as in EXAMPLE 1.The cDNA probes for AQP and GADPH were labeled by [α−32P] using the conventional methods (Sambrook J et al.", "Molecular cloning, second edition, Cold Spring Harbor Laboratory Press (1989)).", "Hybridization reaction was performed between the nylon membrane transferred by RNA of lung cancer cell lines and radio labelled probe of AQP and GADPH.", "After hybridization, membrane was washed with washing solutions 1 (50 ml 20×SSC, 5 ml 10% SDS, total volume 500 ml) twice or thrice for 10 min at 42° C., then washed again with washing solution 2 (2.5 ml 20×SSC, 5 ml 10% SDS, DEPC-treated deionized water, total volume 500 ml) twice at room temperature.", "Then membrane was exposed to X-ray film (Kodak, USA), the signals were observed and their density were analyzed by image analyzer.", "The results of northern blotting showed that all of the lung cancer cell lines expressed AQP1, AQP4 and AQP5 regardless of the cell type of each cell line and AQP5 was expressed in highest level (see FIG.", "8).", "In addition, inventors investigated expression of AQP in tissues of a variety of type of human lung cancer by using in situ hybridization to identify the type of cells which express AQP and their level of expression of AQP.", "The same as in EXAMPLE 1 was used for in situ hybridization.", "The results of this study showed that many types of human lung cancer, including squamous cell carcinoma, bronchioalveolar carcinoma and adenocarcinoma, expressed highly AQP5, AQP1 and AQP3 and that in particular, human lung cancer cells expressed AQP5 in high level regardless of type of carcinoma (See FIG.", "9).", "EXAMPLE 3 Analysis of AQP Gene Expression from Lung Tissue, Bronchial Lavage, Sputum, Blood and Pleural Fluid in Lung Cancer and Normal Control by RT-PCR.", "Lung cancer tissues, normal bronchial tissues, samples, of bronchoscopic lavage, sputum, peripheral venous blood and pleural fluid were obtained from patients with lung cancer and benign controls who underwent bronchoscopy but did not show evidence of lung cancer.", "In particular, blood samples were taken from patients with stage 3 or stage 4 lung cancer and pleural fluid from patients with lung cancer accompanied by malignant pleural effusion.", "RNA and DNA were extracted using the same method as in EXAMPLE 1 and cDNA of AQP were amplified by PCR using the same method as in EXAMPLE 1, except that PCR of AQP5 cDNA was performed by the method as in EXAMPLE 2.The PCR products were visualized in 0.9% agarose gel, in which PCR product of AQP1 cDNA was visualized as band with size of 230-bp, AQP3 130-bp, AQP4 220-bp, AQP5 247bp, and beta-actin 340-bp, respectively.", "In addition, dot blotting and Southern blotting were carried out using probes specific to each type of AQP and beta-actin in the conventional way (Sambrook J et al.", "Molecular cloning, second edition, Cold Spring Harbor Laboratory Press (1989)).", "On the above study, mRNA of AQP1, AQP3 and AQP5 were found to present in high level in lung cancer tissues, and samples of sputum, bronchial lavage and pleural fluid from patients with lung cancer (See FIG.", "10).", "With respect to test of blood sample, all of AQP 1, AQP3, AQP4 and AQP5 were found to be expressed in mononuclear cells from patients with lung cancer, whereas, in normal controls, only AQP1 was consistently expressed, AQP4 and AQP5 were not expressed at all and rarely AQP3 was expressed, which may be due to contamination of erythrocytes (See FIG.", "11).", "These results indicate that expression of AQP5, AQP1 and AQP3 is detectable not only in lung cancer tissues but also in samples of sputum, blood, bronchial lavage and pleural fluid from patients with lung cancer.", "Of course, we can also detect information on nucleic acid sequence of AQP from this study.", "EXAMPLE 4 Analysis of Nucleic Acid Sequences of AQP5 Gene in Lung Cancer Tissues and Samples of Bronchoscopic Lavage, Sputum and Blood.", "Total RNA and cDNA were isolated from tumor tissues of 20 patients with lung cancer, and then most of exon 1, the whole exon 2 and most of exon 3 of cDNA of AQP1, AQP3, AQP4 and AQP5 were amplified by PCR followed by cloning by using the same methods as in the previous EXAMPLE 1.In addition, cloned cDNA of AQP were analyzed by automated sequencing method (ABI PRISM), followed by study of presence and location of mutation by using Blast Search Program.", "In addition, lung cancer tissues were obtained from 112 patients with lung cancer and normal bronchial tissues from 105 control populations, respectively, RNA and cDNA were obtained from the tissues and cDNA of AQP5 was amplified by PCR by the same method as in previous EXAMPLE 2.The sequences of four hundred fifty-bp PCR product, which amplified central portion of AQP5 cDNA from the one hundred forty third base to five hundred ninety third base which include most of exon 1, the whole exon 2 and most of exon 3, were analyzed directly by nucleic acid sequencing or initially cloned in pGEM T-easy vector (Promega, USA), followed by nucleic acid sequencing analysis.", "The sequences of each amplified product of AQP5 were compared to normal cDNA sequence of human AQP5 gene (SEQ ID NO: 1), and then the presence or absence and location of mutation of AQP5 were investigated.", "A variety of mutations of AQP5 were found in all of the cDNA samples from lung cancer tissues, whereas, mutation of AQP was rarely found in cDNA samples from normal lung tissues.", "Mutational hot spots of AQP5 are summarized in Table 5a and 5b (See FIGS.", "12 and 15).", "TABLE 5a Mutational pattern of cDNA of AQP5 Gene: Summary of sixty mutational hot spots No.", "Hot Mutation spots Exon No Region Codon No Base No.", "frequency (%) 1 1 TM2 54 162 1.8 2 1 TM2 55 164 1.8 3 1 Loop B 64 192 7.1 4 1 Loop B 66 197 2.7 5 1 Loop B 69 205 2.7 6 1 Loop B 74 221 3.6 7 1 Loop B 78 233 2.7 8 1 Loop B 79 235 14.3 9 1 Loop B 80 238 2.7 10 1 Loop B 83 247 2.7 11 1 Loop B 84 251 3.6 12 1 TM3 91 273 7.1 13 1 TM3 95 283 2.7 14 1 TM3 96 288 2.7 15 1 TM3 97 290 7.1 16 1 TM3 101 303 7.1 17 1 TM3 103 307 7.1 18 1 TM3 109 327 3.6 19 1 Loop C 111 331 7.1 20 1 Loop C 112 334 2.7 21 1 Loop C 112 335 1.8 22 1 Loop C 119 357 3.6 23 1 Loop C 121 363 2.7 24 2 Loop C 122 365 2.7 25 2 Loop C 123 367 1.8 26 2 Loop C 124 371 3.6 27 2 Loop C 126 376 17.9 28 2 Loop C 126 378 2.7 29 2 Loop C 127 381 2.7 30 2 TM4 132 394 2.7 31 2 TM4 135 404 2.7 32 2 TM4 136 407 7.1 33 2 TM4 138 412 1.8 34 2 TM4 140 419 2.7 TABLE 5b Mutational pattern of cDNA of AQP5 Gene: Summary of sixty mutational hot spots(continued) No.", "Hot Mutation spots Exon No Region Codon No Base No.", "frequency (%) 35 2 TM4 142 426 14.3 36 2 TM4 144 431 2.7 37 2 TM4 145 433 2.7 38 2 TM4 146 436 14.3 39 2 Loop D 152 455 2.7 40 2 Loop D 154 460 2.7 41 2 TM5 158 476 3.6 42 2 TM5 163 488 2.7 43 2 TM5 164 491 2.7 44 2 TM5 166 498 2.7 45 2 TM5 168 502 7.1 46 2 TM5 169 506 2.7 47 2 TM5 175 524 2.7 48 3 Loop E 179 535 3.6 49 3 Loop E 179 536 7.1 50 3 Loop E 181 543 2.7 51 3 Loop E 182 544 1.8 52 3 Loop E 184 551 2.7 53 3 Loop E 184 552 2.7 54 3 Loop E 185 553 1.8 55 3 Loop E 186 558 3.5 56 3 Loop E 187 562 3.5 57 3 Loop E 189 565 2.7 58 3 Loop E 189 567 14.3 59 3 Loop E 190 569 2.7 60 3 Loop E 191 573 7.1 The results of analysis of mutation of AQP5 cDNA from lung cancer tissues are summarized as follows: First, mutations of AQP5 were characteristically found in multiple bases in each sample with a mean of 2.9 mutant bases per single cDNA sample from lung cancer tissue.", "Second, mutations of AQP5 in lung cancer were widely scattered from one hundred sixty second base (after A of start codon) to five hundred seventy third base.", "Third, mutational hot spots were identified.", "Mutation of AQP5 were particularly prevalent in loop B (codon number 62 to 86 or base number 184 to 258), loop C (codon number 110 to 130, base number 328 to 390), loop D (codon number 151 to 157 or base number 451-471), loop E (codon number 179-204, base number 535 to 612) and transmembrane domain™ between the loop, all of which are important area of water channel structure.", "About ninety percent of samples from lung cancer showed mutation in the above four areas, and in only ten percent of the sample, mutation was found outside of these four areas (See FIG.", "15).", "In addition, the inventors investigated mutation of AQP5 in samples of bronchoscopic lavage, sputum, malignant pleural fluid and blood from patients with lung cancer.", "On the analysis of cases in which cDNA of AQP5 was adequately amplified from the samples, AQP5 mutation was found in all of bronchoscopic lavage sample, 96.7% of sputum sample and all of blood samples, respectively (See FIGS.", "13 and 14).", "EXAMPLE 5 Test for Mutation of AQP5 in Lung Cancer Tissues, Lung Cancer Cell Lines, Samples of Bronchial Lavage, Sputum and Blood by using SSCP (Single Strand Conformational Polymorphism) Analysis.", "The SSCP is based on the principles that single-stranded DNA has a tendency to fold up and form complex structures stabilized by intramolecular bonding, ie.", "base-paring hydrogen bonding and the electrophorectic mobilities of such structures on denaturing gel depend on not only on their chain lengths but also on their conformations, which are dictated by the DNA sequence, ie.", "even single base difference makes mobility shift.", "For SSCP, PCR or RT-PCR is performed using primers specific to mutation site and PCR products are loaded on a denaturing polyacrylamide gel electrophoresis, and after silver staining, mutation can be detected by observing mobility difference of a specific PCR product from the wild type pattern.", "SSCP is adequately sensitive for detecting mutation in DNA fragments up to 200-bp long.", "SSCP for AQP5 were performed as follows: First, Exon 1, 2 and 3 of AQP5 were amplified by RT-PCR of RNA obtained from lung cancer tissues, lung cancer cell lines, and sample of bronchial lavage, sputum and blood from patients with lung cancer and normal controls.", "The sequences of oligonucleotide primers are as follows: sense primer for exon 1, SEQ ID NO:16; antisense primer for exon 1, SEQ ID NO: 17; sense primer for exon 2, SEQ ID NO: 18; antisense primer for exon 2, SEQ ID NO:19; sense primer for exon 3, SEQ ID NO: 20; antisense primer for exon 3, SEQ ID NO: 21.Each PCR was performed in 25 μl reaction mixture containing 50 ng of target DNA, 30 ng of each primer, 67 mM Tris-HCl (pH 8.8), 1 mM MgCl2, 100 μM dNTP, 16 mM (NH4)2SO4, 0.45% Triton-X 100, 200 mg/ml gelatin and 0.5 U Taq polymerase.", "After initial denaturation for 4 min at 94° C., the above reaction mixture was subjected to 35 cycles of amplification with 30s at 94° C., 1 min at 62° C., and 1 min at 72° C. The PCR products were mixed with deionized water to make 7 μl aliquot and then were mixed with 8 μl of loading buffer (0.5% dextran, 95% formamide).", "These mixtures were denatured by incubation for 3 min at 95° C., chilled on ice for 1 min, and then were loaded on 12% polyacrylamide gel electrophoresis.", "After applying silver staining to the gel, mutant DNA samples were easily identified by difference in mobility from normal AQP5 cDNA (See FIG.", "12).", "EXAMPLE 6 Test for Mutation of AQP5 Gene by MSO (Mutant Specific Oligonucleotide)-Hybridization Method.", "Mutant specific oligonucleotide (MSO) hybridization is a form of reverse blotting: Oligonucleotide probes for wild type gene and mutant type gene are immobilized on nitrocellulose or nylon filter (membrane), and this filter is hybridized with radio- or biotin-labelled PCR products.", "MSO hybridization distinguishes between wild type DNA and mutant type DNA by detecting difference in hybridization intensity.", "The inventors modified classical method of MSO to make novel MSO method to test for mutation of AQP5, which were performed as follows: 1) Oligonucleotide probes were synthesized based on mutated sequences of AQP5 which were identified from lung cancer tissues as in EXAMPLE 4.These oligonucleotides were immobilized on nylon filters.", "2) The exon 1, 2 and 3 of AQP5 were PCR-amplified in the same condition as in EXAMPLE 4, except that oligonucleotide primers were labeled by biotin at their 5′ end.", "3) The biotin-labeled PCR products of AQP5 we denatured and hybridized, with nylon membrane for 30 min at 58° C. The unbound DNA was removed by washing solution twice for 20 min.", "at RT and once for 10 min at 58° C. 4) The hybridized nylon membrane was incubated with streptavidin-labeled alkaline phosphatase for 30 min at RT and then was treated by BCIP/NBT chromogen, which induced color reaction.", "The presence or absence of mutation of AQP5 can be identified by observed color reaction at specific site.", "The results of MSO hybridization were comparatively analyzed with those of automated nucleic acid sequencing.", "On MSO hybridization of DNA samples which had been confirmed to carry mutation in two sites of AQP5, two bands with strong color reaction were found, which represented double mutation of AQP5 (See FIG.", "17).", "EXAMPLE 7 Test for Mutations of AQP5 Gene by ARMS (Amplification Refractory Mutation System or Allele-Specific Amplification) Method.", "The ARMS method is based on the principle that mismatch between the 3′ end of the primer and the template DNA will result in its inability during DNA amplification, ie.", "fail to produce product on PCR (Newton, C R et al.", "Analysis of any point mutant in DNA.", "The amplification refractory mutation system (ARMS).", "Nucleic Acid Res (1989) 17:2503-2561).", "With information on specified mutation, ARMS can distinguish between mutant type and wild type by specifically amplifying mutant or normal DNA by using set of primer specific to the normal sequence and mutant sequence.", "The ARMS for AQP5 was performed as follows: First, five oligonucleotide primers were synthesized based on information on mutational hot spots of AQP5 in lung cancer which were acquired in EXAMPLE 4.PCR was performed in 50 μl reaction mixture containing 5 μl of 10× buffer (25 mM Tris-acetate (pH 7.8), 100 mM potassium acetate, 1 mM DTT), 5 μl of DMSO, 3 μl of 25 mM dNTP, 4 μl of 25 mM MgCl2, 2.5 U of Taq polymerase (Promega), 50 ng of each target DNA and 12.5 pmol of each primer.", "After initial denaturation for 6 min at 94° C., reaction mixtures were subjected to 35 cycles of PCR with 30 sec at 94° C., 30 sec at 53° C., and 4 min at 65° C., followed by a final extension step of 7 min at 65° C. Presence of mutant DNA was identified by electrophoresis of PCR products on 2% Metaphor gel (FMC company, USA).", "Mutation of AQP5 gene was easily detected by observation of PCR products under mutant specific condition as compared with negative control sample (See FIG.", "18).", "EXAMPLE 8 Test for AQP5 Mutation by using DNA Chip (Hybridization Type Oligonucleotide Microarray) The inventors had designed and produced hybridization type oligonucleotide chip which can scan all of the mutation of AQP5 as follows: First, we had designed and synthesized about 400 different types of oligonucleotide probes which were 20-bp long and contain not only wild type AQP sequence as well as sequences of all the mutant type AQP as were found in EXAMPLE 4 and listed in Table 5a and 5b.", "These probes were modified by attaching amine at their 5′ end and were spotted onto silanated glass slide (Telechem, USA) with spotting buffer (2×SSC, pH 7.0).", "After spotting, slides were dried to stimulate binding of oligonucleotide probes, and were washed by 0.2% SDS for 2 min and then deionized water to remove unbound oligonucleotide probes.", "These slides were denatured by heating for 2 min at 95° C., and washed with blocking solution(1.0 g NaBH4, 300 ml of PBS (pH 7.4), ethanol 100 ml) for 15 min, 0.2% SDS solution for 1 min, and finally with deionized water for 2 min, and then were dried at room temperature to produce final oligonucleotide chip ready for use.", "Next, fluorescence-labeled target DNA (AQP5 cDNA) was prepared as follows: 1 μl of RNA was prepared from each sample as mentioned in EXAMPLE 2, mixed with oligo d(T)15-mer primer and incubated for 5 min at 70° C., and for 5 min at 4° C. The reverse transcription (RT) reaction mixture was prepared by mixing the above mixture of RNA and oligo d(T) primer with 1 μl of 25 mM dATP, dGTP, dTTP, 1 μl of 1 mM dCTP (Roche, USA), 21 μl of 1 mM Cy3-dCTP or 1 μl of Cy5-dCTP(NEN), 20 U of RNase inhibitor (Roche), 100 U of M-MLV reverse transcriptase (Roche), and 2 μl of 10× first strand buffer in a total volume of 20μl.", "This RT reaction mixture was incubated for 2 hours at 38° C. Unbound nucleotides were removed by ethanol precipitation and then fluorescence-labeled cDNA of AQP were obtained.", "Finally, hybridization reaction was performed on oligonucleotide chip and was analyzed by fluorescence scanner.", "Hybridization of the oligonucleotide DNA chip with fluorescence labeled cDNA fragments as prepared in the above was accomplished in UniHyb hybridization solution (TeleChem) for 4 hours at 42° C. The slides were washed twice with SSC solution for 5 min at RT, and air-dried.", "Then the slides were inserted into ScanArray 5000 fluorescence scanner (GSI Lumonics), scanned and scanning results were analyzed by ImaGene software (BioDiscovery, USA) (See FIG.", "19).", "EXAMPLE 9 Production of Sequencing Type Oligonucleotide Microarray and Testing for AQP5 Mutation by using this DNA Chip.", "1) Design and Production of Oligo Chip The oligonucleotide primers are designed so that each base in the AQP cDNA is analyzed by two unique 25-mer oligonucleotides, one for sense and one for antisensestrand.", "The oligonucleotide primers were designed depending on the wild type sequence of AQP5 cDNA with their 3′ ends one base upstream of the base to be identified.", "These oligonucleotide primers are spotted (arrayed) onto chips and will react with cDNA or genomic DNA of AQP5 gene of the subject.", "It is important to consider secondary structure or GC contents on design of oligonucleotide primers, because secondary structure or high GC content induce self-priming and interfere with annealing to the DNA sample.", "To prevent this, about 15% of oligonucleotide s required modification of internal base sequence.", "Oligonucleotides prepared in this way were tested by APEX reaction and scanning analysis.", "Oligonucleotide primers which did not work well required modification for 2 to 5 times.", "Finally the present inventors have established complete set of oligonucleotide primers (both sense and antisense) for AQP5, the sequences of which are listed in FIG.", "20.The oligonucleotide primers were modified by attaching chemical linker to their 5′ ends.", "The linker is an amino linker with 12 carbon arm.", "This linker makes oligonucleotide primers to bind to glass surface firmly.", "Sequencing reaction(APEX reaction) occurs via 3′ end of the oligonucleotide primers.", "The oligonucleotide primer with amino linkers at their 5′ ends were purchased from MWG (Germany).", "The raw material of DNA chip is microscopic glass slide which is 24×60 mm in size and 0.13-0.16 mm in thickness and were purchased from Menzel (Germany).", "To activate the slide surface for tight chemical binding of oligonucleotide primers, the slides were coated in advance before spotting as follows: Glass slides were washed in Alconox solution, sequentially washed and sonicated in deionized MilliQ water, acetone, MilliQ water, 2M NaOH/95% ethanol solution, MilliQ water, and acetone.", "Finally, the slides were was placed in 1% silane solution (380 ml of acetone, 16 ml of water, 3 ml of 3-aminopropyltrimethoxysilane), washed in aceton/95% ethanol, and dried.", "The slides were stored in 0.2% 1,4-phenylene-diisothiocyanate/10% pyridine-dimethylformamide solution, rinsed in MeOH, acetone, and 95% ethanol, and were dried by centrifugation.", "Oligonucleotide primers of AQP5 were spotted onto the glass slide prepared as above by using GMS-417 arrayer (GMS, USA).", "The process of spotting was performed depending on the guide of software of GMS 417.The present method of design and production is just one of the examples.", "The design and manufacture of sequencing type oligonucleotide chip can be freely modified and supplemented.", "2) Preparation of Samples DNA and RNA were purified from patient's sample by conventional method and RNA was reverse transcribed into cDNA.", "Genomic DNA or cDNA of AQP5 were amplified by PCR as follows: PCR was performed in 50 μl reaction mixture containing 5 μl of 10× reaction buffer, 5 μl of 25 mM MgCl2, 5 μl of 2.5 mM dNTP (20% dUTP), 2 μl of each primer, 0.5 μl of cDNA, 1 μl of Taq DNA polymerase, and 29.5 μl of water.", "After initial denaturation for 5 min at 95° C., reaction mixtures were subjected to 2 cycle of PCR with 20 sec at 95° C., 30 sec at 64° C., for 30 sec at 72° C., respectively, followed by 30 cycles of PCR with 20 sec at 95° C., 30 sec at 58° C., 30 sec at 72° C., and 4 min at 65° C., followed by a final extension step of 7 min at 72° C. Oligonucleotide primers for the above PCR were sense primer with SEQ ID NO: 22 for exon 1 and antisense primer with SEQ ID NO: 23 for exon 1, sense primer with SEQ ID NO: 24 for exon 2 and antisense primer with SEQ ID NO: 25 for exon 2, sense primer with SEQ ID NO: 26 for exon 3 and antisense primer with SEQ ID NO: 27 for exon 3, respectively.", "PCR products were purified by conventional method including ammonium acetate/cold ethanol precipitation, ethanol treatment and centrifuge.", "Purified PCR products were fragmented to approximately 50-100 bp nucleotides in length as follows.", "PCR products were mixed with 0.5 □l Epicentre UNG (1 unit/l), 0.5 □l USB sAP (1 unit/l) and 2 □l 10× Epicentre buffer solution and this mixture was incubated for 1 hour at 37° C., and heated for 10 min at 95° C. Electrophoresis was performed to confirm appropriate fragmentation of PCR products.", "3) APEX Reaction Target DNA produced by PCR and fragmented as above were added onto oligonucleotide array on glass and APEX reaction was performed as follows.", "APEX reaction is a kind of Sanger's sequencing reaction.", "APEX reaction was performed in a reaction mixture containing 5-10 μl of single stranded PCR product, 0.8 μl of 50M Texas-Red ddATP, 0.8 μl of 50M Cy3-ddCTP, 50M Cy5-ddUTP, 0.8 μl of 50M Fluorescein-ddGTP, and 150M Cy3-ddCTP, thermosequnase and 10× thermosequnase reaction buffer.", "The reaction mixture was denatured for 5 min at 95° C., added onto glass slide, covered by parafilm and incubated for 25 min at 58° C. Then parafilm was removed, slides were washed with boiling water, and were analyzed by fluorescence scanner.", "4) Analysis of Nucleotide Sequences The final step after APEX reaction is scanning analysis of nucleotide sequences of AQP5 from sample in oligo chip by using fluorescence scanner.", "Here, GENORAMA fluorescence DNA scanner (Asper, Estonia) was used, which is a 4-channel microarray fluorescence image system with 4 color lasers and CCD detector.", "The sequence of each base of AQP5 can be interpreted in an automated and quick way by using Genorama 3.0 genotyping software (Asper, Estonia), 5) Results About two hundred cDNA samples from a various type of human cancer and peripheral blood lymphocytes of normal population were comparatively analyzed by both oligonucleotide chip analysis and automated sequencing.", "The results of mutation test by using oligonucleotide chip concurred with those by automated sequencing in 98% of cases analyzed.", "In the remaining 2% of samples, oligonucleotide chip detected additional point mutation of AQP5, which were missed by automated sequencing analysis (See FIG.", "21, 22, and 23).", "INDUSTRIAL APPLICABILITY As described hereinbefore, the method described in the present invention to detect AQP5 mutation including sequencing type oligonucleotide chips, is highly accurate, quick, easy and thus invaluable for cancer diagnosis." ] ]

[ [ "Controlled rheology polypropylene heterophasic copolymers", "This invention relates to the use of a cyclic ketone peroxide of half-life time larger than one second at a temperature of 225° C., for producing a controlled rheology polypropylene heterophasic copolymer of melt index MI2 larger than 15 g/10 min, having simultaneously a very high impact resistance and a high flexural modulus." ], [ "1.A polymer composition produced by the process of degrading a polypropylene (co)polymer with a cyclic ketone peroxide, said polymer composition characterized by an Izod notched impact strength for melt flow indices greater than 15 g/10 min that is at least 50% higher than the Izod notched impact strength for melt flow indices greater than 15 g/10 min of a comparative polymer composition produced by degrading said propylene (co)polymer with a linear peroxide under the same conditions of degrading.", "2.The polymer composition of claim 1 produced by degrading a polypropylene (co)polymer that looses its impact strength when the melt flow index reaches a threshold value that increases with decreasing extrusion temperature.", "3.The polymer composition of claim 1 produced by degrading said polypropylene (co)polymer in the presence of a cyclic ketone peroxide having at least two peroxide groups.", "4.The polymer composition of claim 3 produced by degrading said propylene (co)polymer with 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane.", "5.The polymer composition of claim 3 wherein the propylene (co)polymer is degraded by extrusion at a temperature of from 160° C. to less than 200° C. 6.The polymer composition of claim 1 produced by degrading a polypropylene heterophasic copolymer, containing from 5 to 20 wt % of ethylene.", "7.The polymer composition of claim 6 produced by degrading a polypropylene heterophasic copolymer containing from 9 to 15 wt % of ethylene.", "8.The polymer composition of claim 6 produced by degrading a copolymer that looses its impact strength when the melt flow index reaches a threshold value that increases with increasing amount of ethylene.", "9.The polymer composition of claim 1 characterized by an Izod notched impact strength that is at least twice that of the Izod notched impact strength of said comparative polymer composition.", "10.The polymer composition of claim 1 characterized by a flexural modulus that is at least 30 Mpa higher than the flexural modulus of said comparative polymer composition.", "11.A method of degrading a polypropylene (co)polymer which comprises extruding said polypropylene (co)polymer with a cyclic ketone peroxide to form a solid product of said polypropylene (co)polymer having a melt index greater than 15 g/10 min with an Izod notched impact strength that is at least 50% higher and a flexural modulus that is at least 30 Mpa higher than the Izod notched impact strength and flexural modulus of a corresponding polypropylene (co)polymer degraded with a linear peroxide under similar conditions.", "12.The method of claim 11 wherein said extrusion is carried out at an extrusion temperature of 200° C. or less.", "13.The method of claim 11 wherein said cyclic ketone peroxide has a half-life of more than one second at a temperature of 225° C. 14.The method of claim 11 wherein said cyclic ketone peroxide has a half-life of from two to 10 seconds at a temperature of 225° C. 15.The method of claim 11 wherein said extrusion is carried out at a temperature of from 160° C. to less than 200° C. 16.The method of claim 15, wherein the polypropylene (co)polymer is a polypropylene heterophasic copolymer containing from 5 to 20 wt % of ethylene.", "17.The method of claim 11 wherein said polypropylene (co)polymer is a polypropylene heterophasic copolymer containing ethylene in an amount within the range of 9-15 wt %.", "18.The method of claim 17 wherein said polypropylene heterophasic copolymer contains from 11 to 14 wt % ethylene.", "19.The method of claim 11 wherein the extrusion temperature is within the range of 160-190° C. 20.The method of claim 11 wherein said cyclic ketone peroxide is 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane." ], [ "The present invention relates to polypropylene heterophasic copolymers modified with cyclic ketone peroxides in order to better control their impact strength.", "Several processes for increasing the impact strength of polypropylene (co)polymers are known in the art, for example, by modifying said (co)polymers with elastomeric modifiers or with peroxides.", "Where elastomeric modifier is used to modify the (co)polymers, it can be added in either of the following ways: reactor polymerisation of polypropylene heterophasic copolymers.", "These polypropylene heterophasic copolymers exhibit typical heterophasic morphology consisting of ethylene propylene bipolymer spherical domains dispersed in a semi-crystalline polypropylene matrix.", "This material consists generally of three components: a polypropylene homopolymer, a rubbery ethylene propylene bipolymer and a crystalline ethylene-rich ethylene propylene bipolymer.", "The amount and properties of the three component material are controlled by the process conditions.", "The mechanical properties of the final product are influenced for example by: 1.the molecular weight, molecular weight distribution and tacticity of the propylene homopolymer matrix; 2.the molecular weight and molecular weight distribution of the ethylene propylene rubber phase; 3.the ethylene/propylene ratio of the ethylene propylene rubber phase; 4.the content and dispersion of the optional ethylene rich ethylene propylene bipolymer; 5.the size and distribution of the rubber phase domains; 6.the melt viscosity ratio of the propylene matrix and rubber phase components.", "Melt blending polypropylene (co)polymers with elastomeric modifiers to prepare polypropylene heterophasic copolymers.", "Elastomers such as ethylene propylene rubber (EPR) or ethylene propylene diene monomer (EPDM) provide improved impact behaviour.", "The impact resistance of these compositions depends upon the content, the composition and the morphology of the elastomeric modifier.", "Both methods have been described for example in: Polypropylene, structure, blends and composites.", "Volume 2—Copolymers and blends.", "Edited by J. Karger-Kocsis, Published in 1995 by Chapman § Hall.", "WO-95/11938 discloses a process of modifying (co)polymers by contacting them with a peroxide compound containing an activated unsaturated group and an acid group in the presence of a polymer reinforcing material, or prior to the addition of a polymer reinforcing material.", "The primary object of that invention was to modify (co)polymers in order to introduce an adhesion promoting functional group and to improve their properties.", "The resulting modified (co)polymers have improved impact strength, flexural strength, tensile strength and elongation at break, increased melt flow index and the other properties equal those of the unmodified impact (co)polymers.", "WO-97/49759 discloses a process for enhancing the melt strength of a propylene (co)polymer by the steps of: mixing an initiator with the propylene (co)polymer at a temperature below the decomposition temperature; then heating the mixture above the initiator decomposition temperature in order to decompose the initiator before the polymer has melted and in order to react the radicals created by the decomposition with the polymer.", "WO-96/03444 discloses a process for modifying (co)polymers by contacting these with an organic peroxide, some of said peroxide being decomposed.", "Cyclic ketone peroxides have been found particularly efficient in the modification processes.", "They have been employed in the degradation of polyolefins, the cross-linking of polyolefins, the dynamic cross-linking of blends of elastomers and thermoplastic polymers, the grafting of monomers onto polymers, or the functionalisation of polyolefins.", "The resulting modified (co)polymers had a larger melt flow index, a lower weight average molecular weight and a narrower molecular weight than the starting (co)polymers, while keeping an adequate melt strength.", "WO-00/23434 discloses a composition comprising a cyclic ketone peroxide and a phlegmatizer having a 95% boil-off point falling in the range of 220-265° C. Preferably, the peroxide is a cyclic ethyl ketone peroxide and a single phlegmatiser is used.", "U.S. Pat.", "No.", "4,707,524 discloses the use of peroxides that do not decompose to tert-butyl alcohol and have a half-life in the range of 1 to 10 hours at 128° C. for controlling the molecular weight and molecular weight distribution of polypropylene.", "WO-96/03397 discloses a transportable, storage stable ketone peroxide composition which comprises 1 to 90 wt % of one or more cyclic ketone peroxides and 10 to 99 wt % of one or more diluents selected from the group consisting of liquid phlegmatisers for the cyclic ketone peroxides, plasticisers, solid polymeric carriers, inorganic supports, organic peroxides and mixtures thereof.", "WO-96/20247 discloses cross-linked polymer compositions of propylene-ethylene copolymer and ethylene-α-olefin copolymer prepared by melting and kneading the constituents in the presence of a radical forming agent, a cross-linking agent and eventually a peroxide inhibitor.", "These compositions were characterised by a high impact strength and a high flexural modulus.", "EP-0,208,330 discloses a propylene polymer composition with increased whitening resistance and increased impact strength, obtained by addition of an ester, in the presence of a peroxide, during extrusion.", "None of these prior art documents discloses polypropylene heterophasic copolymers having simultaneously a melt flow index MI2 larger than 15 g/10 min and increased impact strength, while keeping adequate rigidity.", "It is an aim of the present invention to provide polypropylene heterophasic copolymers exhibiting simultaneously high melt flow index and high impact strength.", "It is another aim of the present invention to provide polypropylene heterophasic copolymers with very high impact resistance over a large range of temperatures.", "It is a further aim of the present invention to obtain polypropylene heterophasic copolymers with controlled rheology.", "It is yet another aim of the present invention to obtain a material with an optimal balance of stiffness, impact strength and melt flow.", "This invention discloses a polypropylene (co)polymer degraded with a cyclic ketone peroxide characterised in that it retains an Izod notched impact strength for melt flow indices larger than 15 g/10 min that is at least 50% higher than that of a polypropylene (co)polymer degraded with a linear peroxide under similar conditions.", "Preferably, the impact strength of the degraded polypropylene (co)polymer of the present invention retains an Izod notched impact strength that is twice as large as that of the prior art resins.", "This invention also discloses the use of cyclic ketone peroxide, to degrade a polypropylene (co)polymer, for producing a controlled rheology material of melt index MI2 larger than 15 g/10 min, said impact propylene copolymer having simultaneously an impact resistance that is at least 50% higher and a flexural modulus that is 30 Mpa higher than those of the polypropylene (co)polymers degraded with linear peroxides under similar conditions.", "The half-life time of the cyclic ketone peroxides of the present invention is typically longer than one second at a temperature of 225° C., preferably it is of from 2 to 10 seconds at a temperature of 225° C., and most preferably, it is about 4 seconds at a temperature of 225° C. The half-life time of peroxide is defined as the time required to decompose one half of the molecules at a given temperature, and thus a less reactive peroxide has a longer half-life time.", "A longer half-life time has two favourable consequences: 1.the peroxide decomposes more slowly; there is thus more time for mixing with the polymer melt in the extruder resulting in a more homogeneous material; 2.there is at any time a lower radical concentration, reducing the probability of side reactions.", "Reducing the extrusion temperature increases the half-life time of the peroxide.", "The melt index MI2 is measured using the method of standard test ISO 1133 at 230° C. and under a load of 2.16 kg, the flexural modulus is measured using the method of standard test ISO 178 and the impact strength is the Izod notched impact strength measured according to the methods of standard test ISO 180.The process for preparing a controlled rheology polypropylene heterophasic copolymer by degrading a polypropylene with a cyclic ketone peroxide, comprises the steps of: either a) Reactor polymerising a polypropylene heterophasic copolymer; b) Extruding the polypropylene heterophasic copolymer of step a), with said cyclic ketone peroxide and optionally with one or more filler(s), in an extruder, at a temperature sufficient to maintain the copolymer in the molten state; Or c) Extruding a polypropylene (co)polymer with said cyclic ketone peroxides, and optionally, with one or more elastomeric modifier(s) and/or one or more filler(s), in an extruder, at a temperature sufficient to maintain the copolymer in the molten state.", "The specific group of cyclic ketone peroxide of half-life time longer than one second at a temperature of 225° C., can be represented by either of the general formulae: Wherein R1-R10 are independently selected from the group consisting of hydrogen, C1-C20 alkyl, C3-C20 cycloalkyl, C6-C20 aryl, C7-C20 aralkyl, C7-C20 alkaryl, which groups may include linear or branched alkyl moieties; and each of R1-R10 may be optionally substituted with one or more groups selected from hydroxy, C1-C20 alkoxy, linear or branched C1-C20 alkyl, C6-C20 aryloxy, halogen, ester carboxy, nitrile, and amino.", "Preferably, the peroxide is a cyclic peroxide containing at least two peroxide goups, and most preferably, it is 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane.", "The latter molecule has three peroxide groups and a relatively small number of carbon atoms and thus a level of active oxygen of the order of 18.16 wt %.", "The treatment of a polypropylene with peroxide generally produces a modified polymer by creation of functional groups.", "Peroxide radicals can cause chain scission and/or cross-linking, resulting in an increase of the melt flow index.", "It must be noted however that increasing the degradation ratio causes a decrease of the flexural modulus.", "The amount of peroxide necessary to carry out the invention depends upon the chemical nature of the peroxide, upon the starting melt flow index and upon the desired final melt flow index: it is directly proportional to the final melt flow index.", "Melt flow index of from 2 to 70 g/10 min have been obtained, but the efforts of the present invention are focused on products having a melt flow index larger than 15 g/10 min.", "The main departure from the strength and stiffness behaviour of prior art materials occurs for resins having a melt flow index above 15 g/10 min.", "In a preferred embodiment of the present invention, the polypropylene heterophasic copolymer is prepared by copolymerising propylene with ethylene in the proportions of from 5 to 20 wt % of ethylene and 95 to 80 wt % of propylene.", "The copolymerisation is effected in two reactors as follows: a) the catalyst and propylene are charged into a first loop reactor equipped with a circulation pump, at a temperature of from 60 to 80° C. and under a pressure of from 35 to 40 bars, using the liquid monomer as a suspension vehicle, in order to produce a homopolymer of propylene on the surface of the catalyst grains; b) the polymer-coated catalyst grains are transferred to one or more secondary gas phase reactors with a fluidised bed and ethylene is added in order to produce an ethylene-propylene rubber.", "The polypropylene heterophasic copolymer so obtained has a typical heterophasic morphology consisting of ethylene-propylene bipolymer spherical domains dispersed in a semi-crystalline polypropylene matrix.", "These materials generally consist of three components: a propylene homopolymer, a rubbery ethylene-propylene bipolymer and a crystalline ethylene-rich ethylene-propylene bipolymer.", "The amount and properties of the components are controlled by the process conditions and the physical properties of the resulting material are correlated to the nature and amount of the three components.", "In the present invention, the preferred amount of ethylene is of from 9 to 15 wt % and more preferably, it is from 11 to 14 wt %.", "The polypropylene heterophasic copolymer is then extruded in an extruder with a cyclic ketone peroxide and with one or more optional fillers, such as glass fillers, talc, calcium carbonate or clay minerals.", "The cyclic ketone peroxide has a half-life time longer than one second at a temperature of 225° C. The extrusion is carried out at a temperature sufficient to maintain the material in a molten state.", "In the examples carried out with the preferred peroxide of the present invention, the extrusion temperatures are from 160° C. up to less than 200° C., preferably from 160 to 190° C. The resin obtained after degradation of the polypropylene (co)polymer at low temperature exhibit an excellent impact performance.", "That result is totally unexpected as it is generally known in the art to work at temperatures higher than 200° C. with long half-life time peroxides, in order to compensate for their low reactivity level.", "It must be noted in addition that the resins prepared according to the present invention retain higher impact strength than prior art resins, for extrusion temperatures higher than 200° C. The Izod notched impact strength of the final resin depends upon the amount of ethylene present in the polypropylene heterophasic copolymer: it increases with increasing amounts of ethylene.", "The rigidity, on the contrary, decreases with increasing amounts of ethylene, thereby imposing an upper limit to the amount of ethylene incorporated into the copolymer.", "It is further observed, that the final resins obtained according to the present invention, when extruded at cold temperature, retain an Izod notched impact strength at 23° C. above 40 kJ/m2, for melt flow indices ranging from 15 to 40 g/10 min and for an ethylene content of from 9 to 15 wt % in the polypropylene heterophasic copolymer.", "For an ethylene content in the polypropylene heterophasic copolymer larger than 12 wt % and an extrusion temperature of at most 200° C., the impact strength of the compositions according to the present invention remains above 40 kJ/m2 for melt flow indices up to 70 g/10 min.", "Throughout this disclosure, cold extrusion temperature is understood as a temperature ranging from the temperature at which all components are in the molten state up to a temperature of less than 200° C. In addition, it is also observed that both the extrusion temperature and the percentage of ethylene contained in the polypropylene heterophasic copolymer have an influence on the behaviour of the Izod notched impact strength as a function of melt flow index.", "Decreasing the extrusion temperature or increasing the amount of ethylene results in final resins that retain the impact properties at values of the melt flow index larger than 40 g/10 min.", "It is thus possible, playing with these two parameters to tailor the desired final resins.", "The copolymers of the present invention are used in several applications that require simultaneously a melt flow index larger than 15 g/10 min, high impact strength and high flexural modulus such as for example: crates, ice cream containers, yoghurt beakers, storage bins, suitcases, lids, pails, technical parts, garden articles, automotive parts, batteries, thin wall packaging, medical waste containers and compounds.", "Compounds are particularly valuable as they allow the production of articles with less or no elastomeric modifiers thereby allowing reduction of cost and processing time.", "LIST OF FIGURES FIG.", "1 represents a plot of the Izod notched impact strength at 23° C., expressed in kJ/m2, as a function of the melt flow index, expressed in g/10 min, for an ethylene content in the polypropylene heterophasic copolymer of 11.3 wt % and for an extrusion temperature of 200° C. FIG.", "2 represents a plot of the Izod notched impact strength at 23° C., expressed in kJ/m2 as a function of the melt flow index, expressed in g/10 min, for an ethylene content in the polypropylene heterophasic copolymer of 11.3 wt % and for extrusion temperatures of 160° C. and of 200° C. FIG.", "3 represents a plot of the Izod notched impact strength at 23° C., expressed in kJ/m2, as a function of the melt flow index, expressed in g/10 min, for an ethylene content in the polypropylene heterophasic copolymer of 13.2 wt % and for an extrusion temperature of 200° C. FIG.", "4 represents a plot of the Izod notched impact strength at 10° C., expressed in kJ/m2, as a function of the melt flow index, expressed in g/10 min, for an ethylene content in the polypropylene heterophasic copolymer of 13.2 wt % and for an extrusion temperature of 200° C. FIG.", "5 represents a plot of the Izod notched impact strength at 23° C., expressed in kJ/m2, as a function of the melt flow index, expressed in g/10 min, for ethylene contents in the polypropylene heterophasic copolymer of 11.3 and of 13.2 wt %, and for an extrusion temperature of 200° C. EXAMPLE 1 Several samples have been prepared using as starting material a polypropylene heterophasic copolymer having a melt flow value MI2 of 2 g/10 min and an ethylene content of 11.3 wt %.", "The polypropylene heterophasic copolymer has been extruded in a single-screw Gloenco extruder, at a temperature of 200° C., with various amounts of the cyclic peroxide 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane in a 41.3% solution of Isopar M diluent and having 7.5 wt % of active oxygen, in order to obtain the desired melt flow index for the finished material.", "The formulation of these materials contains in addition Irganox and Irgafos as antioxidants, 400 ppm of calcium stearate, 3500 ppm of talc and 2000 ppm of glycerol monostearate (GMS) as antistatic agent.", "The data are summarised in Table I.", "TABLE I Cyclic peroxide consumption (ppm) Final MFI (g/10′) 350 7.2 700 12 900 24.7 1380 37.9 1810 56 2110 73.4 The flexural modulus has been measured at 23° C. using the method of standard test ISO 178 and the Izod notched impact strength has been measured at 23° C. using the method of standard test ISO 180.The results are summarised in Table II and in FIG.", "1.COMPARATIVE EXAMPLES The same polypropylene heterophasic copolymer as that used hereabove has been extruded with various amounts of 2,5-bis(tert-butylperoxy)-2,5-dimethylhexane sold by Akzo Nobel.", "Chemicals B.V. under the trade-name Trigonox 101 under the same conditions as in Example 1.The amounts of peroxide have been adjusted in order to produce finished materials with comparable melt flow indices of respectively 8, 12, 27.2, 38.2, 63.5 and 74, as those of the samples according to the invention.", "The amounts of peroxide consumed are respectively 250, 500, 800, 1060, 1390 and 1650 ppm.", "The flexural modulus and Izod notched impact strength are also presented in Table II and FIG.", "1 for comparison.", "TABLE II Perox.", "101* cycl* 101 cycl 101 cycl 101 cycl 101 cycl 101 cycl MFI 8 7.2 12 12 27.2 24.7 38.2 37.9 63.5 56 74 73.4 g/10′ Flex.", "1160 1216 1050 1120 1040 1065 1000 1036 965 1000 945 995 Mod.", "Mpa Izod 56.5 60.7 19 54 20.7 51 13.2 21.1 12.7 15.1 11.7 14.3 23° C. kJ/m2 *101 = Trigonox 101 and cycl = 3,6,9-triethyl-3,6,9-trimethyl-1,4,7-triperoxonane.", "From Table II and FIG.", "1, it appears that the material produced according to the present invention has a flexural modulus that is larger by about 40 MPa than that of the material produced with Trigonox 101 for melt indices larger than 15 g/10 min.", "From Table II and FIG.", "1, it is observed that the Izod notched impact strength at 23° C., of the material produced according to the present invention, quite unexpectedly does not decrease sharply for a melt flow index larger than 15 g/10 min as does the material prepared with Trigonox 101.It remains fairly high up to a value of the melt index of about 40 g/10 min.", "Above that value, it remains significantly higher than that of the comparative samples.", "EXAMPLE 2 The Izod notched impact strength has been measured, at the temperatures 23, 10 and −20° C., for melt indices of 12, 25 and 40, for extrusion temperatures of 160, 180 and 200° C. and for two peroxides.", "The controlled rheology polypropylene heterophasic copolymer samples according to the present invention have been prepared with an ethylene content of 11.3 wt % and with the same cyclic ketone peroxide as that used in example 1.The comparative examples have been prepared with the linear peroxide sold by Akzo Nobel Chemicals B.V. under the name Trigonox 101.The results are summarised in Table III and in FIG.", "2.TABLE III Izod notched impact strength (kJ/m2) at 23° C., 10° C. and −20° C. Ex.", "T. = 200° C. Ex.", "T. = 180° C. Ex.", "T. = 160° C. Peroxide MFI 23 10 −20 23 10 −20 23 10 −20 101 12 19 na 6 45* 13 6 47* 13 6 101 25 20 na 8 32** 10 6 24** 10 6 101 40 13 na 7 14 8 5 14 9 5 cyclic 12 54* na 7 51* 43* 6 52* 42* 7 cyclic 25 51* na 7 48* 13 6 46* 13 6 Cyclic 40 21 na 8 45* 11 6 45* 11 6 na: not available *no break **no break of some samples It can be concluded from these results that the material prepared according to the present invention has an impact strength that is superior to that of the comparative samples, in all cases.", "The results are particularly remarkable at low extrusion temperatures of 160 and 180° C. EXAMPLE 3 Controlled rheology resins were prepared based on the MI2=2 g/10 min (ISO 1133) reactor polymerised polypropylene heterophasic copolymer with an ethylene content of 13.2%.", "Anti-oxidants, calcium stearate (400 ppm), nucleation (talc 3500 ppm) and antistatic agents (glycerol monostearate (GMS) 90% 2000 ppm) were added during extrusion on a single screw Gloenco extruder.", "Two different peroxide were used: 2,5-di-tert-butyl-2,5-dimethylhexyl peroxide sold by Akzo Nobel Chemicals B.V. under the trade-name Trigonox 101 and 3,6,9-Triethyl-3,6,9-Trimethyl-1,4,7-triperoxonane.", "The degradation ratios were of 6, 12.5 and 20 and the extrusion temperature was 200° C. Melt flow index, flexural modulus and Izod notched impact strength are reported in Table IV.", "The Izod notched impact strength results are also summarised on FIGS.", "3, 4 and 5 for temperatures of +23° C. and +10° C. respectively.", "TABLE 4 Tr.", "101 cyclic Tr.", "101 cyclic Tr.", "101 Cyclic Tr.", "101 Cyclic Property Start MFI 12 MFI 12 MFI 25 MFI 25 MFI 40 MFI 40 MFI 70 MFI 70 MFI (g/10 min) 2.4 11.3 11.3 24.3 23.0 39.0 39.2 70 70 Flexural Modulus 1130 1040 1035 990 995 925 980 (MPa) Izod Notched +23° C. 57.7* 49.4* 54.2* 43.1* 48.7* 14.5 41.7* 14 41 (kJ/m2) Izod Notched +10° C. 50.1* 16.0 48.3** 10.3 27.4** 12.1 11.9 (kJ/m2) Izod Notched −20° C. 7.5 6.4 6.6 5.6 6.2 5.7 4.9 (kJ/m2) *no-break **no-break of some samples.", "All the resins produced with both the linear and cyclic peroxides at an extrusion temperature of 200° C. and for melt flow index of from 12 to 25 g/10 min show ‘no break’ at room temperature.", "This in contrast to the results obtained in the previous example prepared by degrading a polypropylene heterophasic copolymer with a lower ethylene content.", "A higher ethylene content (13.2 vs. 11.3%) thus drastically improves the impact performance of polypropylene heterophasic copolymers.", "Changing the peroxide type from linear (Trigonox 101) to cyclic improves the ‘no break’ performance of the final resin with a melt flow index of 70 g/10 min at a temperature of 23° C. The resins having a melt flow index of 12 and of 25 g/10 min produced with the cyclic ketone peroxide also show excellent impact performance at a temperature of 10° C. From these examples, it can be concluded that the cyclic ketone peroxide offers an important mechanical advantage over the linear peroxide Trigonox 101.It is possible to produce better flow materials that keep their impact strength for a melt flow index as high 70 g/10 min and show ‘no-break’ from an Izod notched impact test at room temperature.", "The materials also show a better impact performance at other temperatures.", "In almost all cases the flexural modulus is higher.", "In conclusion, the resins produced according to the present invention exhibit an improved balance of stiffness, impact, strength and flow properties.", "The materials produced according to the present invention are thus particularly useful for preparing articles that require simultaneously high melt flow and good impact strength.", "Indeed high melt flow material is easier and faster to process, particularly in injection moulding, thus allowing shorter cycle time and reduction of the walls' thickness while keeping an acceptable stiffness and impact strength." ] ]

[ [ "Droplet deposition apparatus", "Droplet deposition apparatus comprises a plurality of fluid chambers (2), each fluid ejection chamber being defined in part by at least one wall (11) actuable by an electrical signal to effect droplet ejection from that chamber.", "The apparatus provides means (16) for cyclically supplying electrical signals to the walls (11) for actuation thereof, means (60) for measuring, within a period between the application of successive electrical signals to the walls, a temperature dependent electrical property of a wall of a fluid chamber to provide a signal having a magnitude dependant on the temperature of fluid in the fluid chambers, and means for adjusting the magnitude of the actuating electrical signals depending on the magnitude of the temperature dependant signal." ], [ "1.Droplet deposition apparatus comprising: a plurality of fluid chambers; for each fluid chamber; piezoelectric actuator means actuable by an electrical signal to effect droplet ejection from that chamber; means for cyclically supplying electrical signals to each said actuator means for actuation thereof; means for measuring, within a period between the application of successive electrical signals to a said actuator means, a temperature dependent electrical property of said actuator means to provide a signal having a magnitude dependant on the temperature of fluid in a fluid chamber associated with said actuator means; and means for adjusting the magnitude of the actuating electrical signals depending on the magnitude of the temperature dependant signal.", "2.Droplet deposition apparatus according to claim 1, wherein the supply means is arranged to supply electrical signals to the actuator means at a frequency in the range from 4 to 5 kHz.", "3.Droplet deposition apparatus according to claim 2, wherein the supply means is arranged to supply electrical signals at a frequency of 4.2 kHz.", "4.Droplet deposition apparatus according to any preceding claim, wherein the period has a duration of 240 μs.", "5.Droplet deposition apparatus according to any preceding claim wherein the temperature dependent electrical property is electrical capacitance.", "6.Droplet deposition apparatus according to any preceding claim, wherein said actuator means comprise piezoelectric material extending over the major part of a wall of a respective said chamber, each actuable channel wall being deformable upon the application of an actuating electrical signal to eject fluid from a fluid chamber.", "7.Droplet deposition apparatus according to claim 6, wherein the measuring means comprises a measuring circuit comprising two transistors connected in series for receiving a measuring voltage at an input thereof, one side of the wall being connected to a common output of the transistors, the other side of the wall being connected to an output from the circuit, and means connected to the output for measuring the rate of decay of the voltage at the output to provide the signal having a magnitude dependant on the temperature of fluid in the fluid chamber.", "8.Droplet deposition apparatus according to claim 7, wherein a 5V supply is connected to the input to provide the measuring voltage.", "9.Droplet deposition apparatus according to any of claims 6 to 8, wherein the piezoelectric material is such that application of the actuating electrical signal deforms it in shear mode to generate an acoustic pressure wave in the fluid chamber and thereby eject the fluid.", "10.Droplet deposition apparatus according to any of claims 6 to 9, wherein the piezoelectric material is disposed along the sides of each fluid chamber.", "11.A method of operating droplet deposition apparatus comprising a plurality of fluid chambers and, for each fluid chamber, piezoelectric actuator means actuable by an electrical signal to effect droplet ejection from that chamber, the method comprising the steps of: cyclically supplying electrical signals to each said actuator means for actuation thereof; measuring, within a period between the application of successive electrical signals to a said actuator means, a temperature dependent electrical property of said actuator means to provide a signal having a magnitude dependant on the temperature of fluid in the fluid chamber associated with said actuator means; and adjusting the magnitude of the actuating electrical signals depending on the magnitude of the temperature dependant signal.", "12.Droplet deposition apparatus substantially as herein described with reference to the accompanying drawings.", "13.A method of operating droplet deposition apparatus substantially as herein described with reference to the accompanying drawings." ], [ "The present invention relates to a droplet deposition apparatus such as, for example, a drop-on-demand inkjet printer.", "In particular the invention is concerned with a printer or other droplet deposition apparatus in which an acoustic pressure wave is generated by an electrical signal to eject a droplet of fluid (e.g.", "ink) from a chamber.", "The apparatus may have a single such droplet ejection chamber, but more typically has a printhead with an array of such chambers each with a respective nozzle, the printhead receiving data-carrying actuating electrical signals which provide the power necessary to eject droplets from the chambers on demand.", "Each chamber is bounded by a piezoelectric element which is caused to deflect by the actuating electrical signal, thereby generating the acoustic pressure wave which ejects the droplet.", "Reference is made to our published specifications EP 0277703, U.S. Pat.", "No.", "4,887,100 and WO91/17051 for further details of typical constructions.", "These specifications describe arrangements in which piezoelectric material is in a “chevron” configuration, in which a longitudinal side of the chamber is bounded by piezoelectric material having oppositely-poled regions extending longitudinally of the chamber, so that application of the electrical signal deforms both regions of the material in the same direction and into a chevron shape, when viewed in cross-section.", "Such a configuration is described in the context of an “end-shooter” print-head in EP 0277703, in which the nozzle is at the end of elongated chamber and the piezoelectric material is disposed along the sides of the chamber.", "Alternatively or in addition, the printhead can be configured as a “side shooter” as described in WO91/17051 in which the nozzle is instead disposed in one of the long sides of the chamber which is not bounded by piezoelectric material.", "Both of these designs provide significant reductions in the drive voltage for a given droplet ejection performance.", "During printing, heat is generated by, for example, the drive circuitry providing the actuating electrical signals to the piezoelectric material.", "This heat dissipates into the ejection chambers and heats up the ejection fluid therein.", "This gives rise to a decrease in the viscosity of the ejection fluid.", "Such variations in the viscosity of the ejection fluid can give rise to variations in droplet ejection velocity and consequent dot placement errors in the printed image.", "Furthermore, as described in WO97/35167, hysteresis losses resulting from actuation of the piezoelectric material can cause an increase in the temperature of the ink in the ejection channels.", "In extreme cases, this temperature increase can be local to the active channel and the neighbouring channels only.", "We have discovered that it is desirable to monitor the temperature of the droplet ejection fluid during printing and adjust the magnitude of the actuating signals in response to the monitored temperature.", "One known technique is to mount a thermistor on the external surface of the printhead in the proximity of a piezoelectric element, the thermistor being electrically connected to the drive circuitry.", "Any temperature increase in the location of the thermistor thus causes a reduction in a resistance value of the drive circuitry, which is used to reduce the magnitude of the actuating electrical signals applied to the piezoelectric element.", "However, the thermal insulation provided between the thermistor and the piezoelectric element by the casing of the printhead and the glue layer attaching the thermistor to the casing results in a difference between the temperature at the thermistor and the temperature of the droplet ejection fluid.", "This difference can be substantial if there are fast temperature changes in the printhead during printing, as there is a slow reactance of the drive circuitry to the temperature changes in the ejection fluid.", "The preferred embodiment of the present invention seeks to solve these and other problems.", "In one aspect, the present invention provides droplet deposition apparatus comprising a plurality of fluid chambers, for each fluid chamber, piezoelectric actuator means actuable by an electrical signal to effect droplet ejection from that chamber, means for cyclically supplying electrical signals to each said actuator means for actuation thereof, means for measuring, within a period between the application of successive electrical signals to a said actuator means, a temperature dependent electrical property of said actuator means to provide a signal having a magnitude dependant on the temperature of fluid in a fluid chamber associated with said actuator means, and means for adjusting the magnitude of the actuating electrical signals, for example, the amplitude and/or duration, depending on the magnitude of the temperature dependant signal.", "The inventors of the present application have realised the importance of ensuring that any temperature sensor should be in direct contact with the ejection fluid during printing.", "The inventors have also realised that any such temperature sensing should not interfere with the standard printing operations or printing speed of the printhead.", "As the temperature sensing takes place wholly within a period between application of successive electrical signals, this can ensure that the temperature sensing does not interfere with the actuating electrical signals or reduce printing speed.", "In one embodiment, the supply means is arranged to supply electrical signals to the actuating means at a frequency in the range from 4 to 5 kHz, preferably 4.2 kHz.", "The period may have a duration of 240 μs.", "In one embodiment, the time taken to measure the electrical property takes 42 μs, significantly less than the period of 240 μs between actuation.", "In a preferred embodiment, the temperature dependent electrical property is electrical capacitance.", "With reference to FIG.", "1, the inventors of the present application have found, and verified experimentally, that the capacitance of the piezoelectric actuator of a fluid chamber is a substantially linear function of temperature.", "As a consequence, the magnitude of the temperature dependent signal can be directly proportional to the temperature of the ink.", "Said actuator means preferably comprises piezoelectric material extending over the major part of a wall of a respective said chamber, each actuable channel wall being deformable upon the application of an actuating electrical signal to eject fluid from a fluid chamber.", "Thus, in a preferred embodiment the present invention provides droplet deposition apparatus comprising a plurality of fluid chambers, each fluid ejection chamber being defined in part by at least one wall actuable by an electrical signal to effect droplet ejection from that chamber, means for cyclically supplying electrical signals to the walls for actuation thereof, means for measuring, within a period between the application of successive electrical signals to the walls, a temperature dependent electrical property of a wall of a fluid chamber to provide a signal having a magnitude dependant on the temperature of fluid in the fluid chambers, and means for adjusting the magnitude of the actuating electrical signals, for example, the amplitude and/or duration of the actuating electrical signals, depending on the magnitude of the temperature dependant signal.", "The apparatus preferably comprises means for shaping the temperature dependent signal to provide a temperature dependent voltage signal for superimposition by the adjusting means on the actuating electrical signals.", "The shaping means may adopt any suitable arrangement according to whether that signal varies linearly or non-linearly with temperature.", "In one embodiment the measuring means comprises a measuring circuit comprising two transistors connected in series for receiving a measuring voltage at an input thereof, one side of the wall being connected to a common output of the transistors, the other side of the wall being connected to an output from the circuit, and means connected to the output for measuring the rate of decay of the voltage at the output to provide the signal having a magnitude dependant on the temperature of fluid in the fluid chambers.", "In order to prevent excessive heating of the wall during measurement, a 5V supply may be connected to the input to provide the measuring voltage.", "Preferably, the piezoelectric material is such that application of the actuating electrical signal deforms it in shear mode to generate an acoustic pressure wave in the fluid ejection chamber and thereby eject the fluid.", "In a preferred arrangement, the piezoelectric material is disposed along the sides of each fluid chamber.", "The droplet deposition apparatus can take either an “end-shooter” or “side shooter” configuration.", "Alternatively, piezoelectric material may be disposed at the back of each fluid chamber, as described in our published specification WO00/16981, so that application of an actuating signal to the piezoelectric material causes it to move towards or away from the nozzle of the ejection chamber, thereby generating the required acoustic pressure wave for fluid ejection.", "The present invention also provides droplet deposition apparatus including an array of fluid ejection chambers, each fluid ejection chamber comprising means for ejecting a droplet therefrom in response to an electrical actuating signal, comprising means exposed to fluid in the chambers to provide a signal dependent on the temperature of that fluid, and means responsive to the temperature dependent signal for adjusting the actuating electrical signals.", "Preferably, each fluid ejection chamber is defined in part by at least one wall actuable by an electrical signal to effect droplet ejection from that chamber, the apparatus comprising means for utilising a temperature dependent electrical property of the wall to provide the signal.", "The present invention also provides a method of operating droplet deposition apparatus comprising a plurality of fluid chambers and, for each fluid chamber, piezoelectric actuator means actuable by an electrical signal to effect droplet ejection from that chamber, the method comprising the steps of: cyclically supplying electrical signals to each said actuator means for actuation thereof; measuring, within a period between the application of successive electrical signals to a said actuator means, a temperature dependent electrical property of said actuator means to provide a signal having a magnitude dependant on the temperature of fluid in the fluid chamber associated with said actuator means; and adjusting the magnitude of the actuating electrical signals depending on the magnitude of the temperature dependant signal.", "An embodiment of the invention will now be described with reference to the accompanying drawings, in which:— FIG.", "1 shows the variation of capacitance with temperature for an actuable wall of a fluid chamber; FIG.", "2 is a perspective view of an end-shooter chevron printhead; FIG.", "3 is a section through the printhead of FIG.", "2; FIG.", "4 illustrates the charging curve of a capacitor; FIG.", "5 illustrates the arrangement of a measuring circuit used to provide a signal indicative of the temperature of fluid in the printhead; FIG.", "6 illustrates in block diagram form a test board including the measuring circuit; and FIG.", "7 illustrates the output of the measuring circuit.", "Referring first to FIG.", "2, a planar array, drop-on demand ink jet printer according to an embodiment of the present invention comprises a printhead 10 formed with a multiplicity of parallel fluid chambers or channels 2, nine only of which are shown and the longitudinal axes of which are disposed in a plane.", "The channels 2 are closed by a cover (not shown) which extends over the entire top surface of the printhead.", "The channels are of end-shooter configuration, terminating at corresponding ends thereof in a nozzle plate 5 in which are formed nozzles 6, one for each fluid ejection channel 2.Fluid, such as ink 4, is ejected on demand from the fluid ejection channels 2 in the form of droplets 7 and deposited on a print line 8 of a print surface 9 between which and the printhead 10 there is relative motion normal to the plane of the channel axes.", "The printhead 10 has a planar base part 20 in which the channels 2 are cut or otherwise formed of a PZT piezoelectric material so as to extend in parallel rearwardly from the nozzle plate 5.The channels 2 are long and narrow with a rectangular cross-section and have opposite side walls 11 which extend the length of the channels.", "The side walls 11 of the fluid ejection channels 2 are provided with electrodes (not shown) extending along the length of the channels whereby the side walls are displaceable in shear mode transversely relatively to the channel axes along substantially the whole of the length thereof, to cause changes of pressure in the ink in the channels 2 to effect droplet ejection from the nozzle.", "The channels 2 connect at their ends remote from the nozzles, with a transverse channel (not shown) which in turn connects with an ink reservoir (not shown) by way of pipe 14.Electrical connections (not shown) for activating the side walls 11 of the fluid ejection channels are made to an LSI chip 16 on the base part 20.Typically, a chip 16 is connected to up to 32 separate electrodes for supplying electrical signals thereto for displacement of the associated side walls of the fluid ejection channels 2, and therefore it is usual for a plurality of chips 16 to be provided for supply of actuating electrical signals to the side walls of all of the channels in the array.", "However, the number of electrodes to which a chip is connected can, of course, be modified as required.", "As shown in FIG.", "3, the channel side walls 11 have oppositely-poled regions so that application of an electric field deflects them into a chevron shape.", "The array incorporates displaceable side walls 11 in the form of shear mode actuators 15, 17, 19, 21 and 23 sandwiched between base and top walls 25 and 27 and each formed of upper and lower wall parts 29 and 31 which, as indicated by arrows 33 and 35, are poled in opposite senses normal to the plane containing the channel axes.", "The inner walls of the fluid ejection channels 2 are covered by respective electrodes 37, 39, 41, 43 and 45.Thus, when a voltage is applied to the electrode of a particular channel, say electrode 41 of the channel 2 between shear mode actuator 19 and 21, whilst the electrodes 39 and 43 of the channels 2 on either side of that of electrode 41 are held to ground, an electric field is applied in opposite senses to the actuators 19 and 21.By virtue of the opposite poling of the upper and lower wall parts 29 and 31 of each actuator, these are deflected in shear mode into the channel 2 therebetween in chevron form as indicated by broken lines 47 and 49.An impulse is thus applied to the ink 4 in the channel 2 between the actuators 19 and 21 which causes an acoustic pressure wave to travel along the length of the channel and eject an ink droplet 7 therefrom.", "During printing, heat is generated by, for example, the chip 16.This heat dissipates into the fluid chambers 2 and increases the temperature of the ink 4, which gives rise to a decrease in the viscosity of the ink 4.Such variations in the viscosity of the ink can result in variations in droplet ejection velocity and consequent dot placement errors in the printed image.", "To seek to avoid such errors, in the present droplet deposition apparatus the temperature of the ink is monitored during printing.", "This enables the magnitude of the actuating signals applied to the walls 11 of the fluid ejection chambers 2 to be adjusted in response to the monitored temperature so as to compensate for the decrease in the viscosity of the ink.", "In the present apparatus a temperature dependent electrical property of an actuable side wall 11 is used to monitor the temperature of ink 4 during printing.", "As the walls 11 are in direct contact with the ink 4, any rapid changes in the temperature of the ink 4 can be detected and acted upon quickly.", "With reference to FIG.", "1, the inventors of the present application have found, and verified experimentally, that the capacitance of the walls 11 of a channel 2 is a substantially linear function of temperature.", "As a consequence, the magnitude of the temperature dependent signal can be directly proportional to the temperature of the ink.", "FIG.", "4 shows a standard charging curve for a capacitor.", "With reference to FIG.", "5, a measuring circuit 60 is used to provide a signal having a magnitude dependant on the temperature of ink in the channels 2.The circuit 60 comprises two input resistors 62,64 each connected to the gate of a respective one of a pair of transistors 66,68 connected in series.", "The wall of a channel 2 is represented at 70 as a capacitance C to be measured, the capacitor 70 being connected at one side thereof to the commonly-connected drains of transistors 66,68 and at the other side thereof to a first output resistor 72.A second output resistor 74 is connected to the source of transistor 68.A 5 volt input is supplied to the source of transistor 66, and an output 76 is connected to the other side of the capacitor 70.The measuring circuit is sufficiently simple to be implementable in an ASIC mounted on the printhead, for example, as part of the chip 16.FIG.", "6 illustrates a test board 80 carrying the measuring circuit 60, a power supply 82, a controller 84 and a comparator circuit 86.The output of the measuring circuit 60 is supplied to the comparator circuit 86.The output at contact 76 is illustrated in FIG.", "7 which shows a curve representing either the charging current Ic of the capacitor 70, or the voltage Vo at contact 76, as a function of time t. It will be seen that the current or voltage increases sharply then decays to zero before going negative.", "The capacitance of the capacitor 70 is proportional to the decay or charging time, t(ch).", "The comparator circuit 86 is arranged to measure this time.", "The controller 84 can be used to set the comparator 86 to measure the decay to a predetermined percentage, such as 96%.", "It has been found by the applicant that the charging/decay time is shorter than the time between activations of a channel 2, so that the measurement of capacitance of the wall of an active channel 2 can be made.", "There is thus no interference with a printing operation.", "For example, for a 200 dots per inch printhead operating at a frequency of 4.2 kHz, a measurement of wall capacitance can be made in 42 microseconds, which is well within the period of 240 microseconds between activations of a channel.", "Faster measurement is also possible for faster printheads.", "Any variation of the wall capacitance, due to deviation of the temperature of the ink 4 from room temperature, varies the decay or charging time, t(ch), in response to which the comparator circuit 86 outputs a signal indicating the temperature of the ink in the channels 2.The signal is subsequently shaped to enable the signal to be superimposed on the actuating electrical signals supplied to the wall.", "This in turn modifies the velocity of the droplets ejected from the ejection channel 2 so as to avoid drop placement errors.", "As mentioned above, in the preferred embodiment a single chip 16 supplies actuating electrical signals to up to 32 electrodes only, and thus controls the ejection of droplets from a group of up to 32 channels.", "Therefore, a plurality of chips 16 are typically provided, each controlling the ejection from a respective channel group.", "In one embodiment, the capacitance of one of the walls of each of the groups is measured regularly by a respective chip 16, and the magnitude of the actuating channels supplied to the walls of the channels in that group is adjusted accordingly.", "Thus, by measuring the capacitance of each 32nd wall of the array, the magnitude of the actuating electrical signals can be varied across the array in dependence on the actuation sequence.", "To increase temperature sensitivity across the array, the number of walls in each group may be reduced.", "An advantage of the invention is that, using active channels, temperature homogeneity across a multichannel printhead can be measured.", "A further advantage is that the measuring circuit is sufficiently simple to be implementable in an ASIC mounted on the printhead, e.g.", "as part of the chip 16.Also, the use of a 5 volt supply means that there is no additional heating of the printhead even when measurements are made every second.", "It will be understood that the present invention has been described above purely by way of example, and modifications of detail can be made within the scope of the invention.", "For example, although the present invention as been described with reference to an “end-shooter” printhead, it is equally applicable to a “side shooter” or any other form of printhead.", "Furthermore, any suitable means may be employed for detecting the capacitance, or other suitable electrical property, of the walls of the actuable channels.", "For example, a digital detection circuit may be employed in order to avoid problems associated with the generation of noise during detection of the chosen electrical property Each feature disclosed in this specification (which term includes the claims) and/or shown in the drawings may be incorporated in the invention independently of other disclosed and/or illustrated features." ] ]

[ [ "Method of culturing mesenchymal stem cells", "This invention provides a novel method of culturing mesenchymal stem cells whereby a remarkably larger number of mesenchymal stem cells can be obtained compared with the conventional culture methods.", "This culture method is characterized by adding to a medium a fibroblast growth factor (FGF) as a substance which stimulates the proliferation potency of the mesenchymal stem cells while retaining the pluripotency thereof and prolongs the life.", "According to the method, mesenchymal stem cells can be cultured at least over 30 generations and, moreover, about 105 to 106 times as much as cell count in the conventional culture methods can be obtained." ], [ "1.A method of culturing mesenchymal stem cells, comprising culturing said mesenchymal stem cells in a medium that contains a substance that stimulates the proliferation of said mesenchymal stem cells.", "2.The method of claim 1, wherein the substance is a fibroblast growth factor.", "3.The method of claim 2, wherein the fibroblast growth factor is added to the medium at a concentration of 0.04 to 10 ng/ml.", "4.The method of claim 3, wherein the fibroblast growth factor is added to the medium at a concentration of 0.1 to 1 ng/ml.", "5.The method of claim 11, wherein the mesenchymal stem cells are derived from a human.", "6.Mesenchymal stem cells obtained by the method of claim 1 or 2, wherein the mesenchymal stem cells retain their pluripotency.", "7.The mesenchymal stem cells of claim 6, wherein the mesenchymal stem cells have the potential to differentiate into chondrocytes.", "8.", "(canceled) 9.The mesenchymal stem cells of claim 6, wherein the mesenchymal stem cells retain their pluripotency in subculture for at least 15 generations.", "10.The mesenchymal stem cells of claim 6, wherein the mesenchymal stem cells retain their pluripotency in subculture for at least 16 days.", "11.The method of claim 1, wherein the mesenchymal stem cells are derived from a mammal.", "12.The method of claim 2, wherein the fibroblast growth factor is fibroblast growth factor 1 or fibroblast growth factor 2.13.The mesenchymal stem cells of claim 6, wherein the mesenchymal stem cells have the potential to differentiate into osteoblasts." ], [ "<SOH> BACKGROUND TECHNOLOGY <EOH>Tissues of a vertebrate, especially mammalian tissues, contain stem cells which have the ability to regenerate cells and tissues in cell regeneration system, for example, to regenerate cells and tissues lost by trauma, disease or aging.", "Stem cells therefore are found within the tissue or in other tissues that serve as stem cell reservoirs.", "Bone marrow and periosteum are the major stem cell reservoirs.", "Bone marrow is the major source of adult hematopoietic stem cells, which are undifferentiated pluripotent cells.", "It is known that all blood cells (e.g.", "erythrocyte, leukocyte, lymphocyte, etc.)", "are differentiated from the hematopoietic stem cells.", "Many researches have been made on cultivation and differentiation of hematopoietic stem cells, and many substances (growth factors) have been reported which are useful for culturing hematopoietic stem cells and for differentiating and inducing to various blood cells (components).", "On the other hand, bone marrow, periosteum and the like contain other stem cells than hematopoietic stem cells, i.e.", "mesenchymal stem cells, which play an important role for regenerating the mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose and stroma.", "Mesenchymal stem cells proliferate as undifferentiated cells and retain the pluripotency, by which can differentiate into such as adipocytes, chondrocytes or osteoblasts.", "The stem cells can easily be isolated from adult bone marrow and periosteum, and show a stable phenotypic cell form.", "For example, the mesenchymal stem cells derived from bone marrow retain the character of monolayer in vitro cultivation (Pittenger M. F. et al., Science 284, 143-147, 1999).", "It was therefore proposed to culture mesenchymal stem cells in large amounts and to differentiate and induce thus obtained undifferentiated cells into cartilage tissue or bone tissue, which can be used for transplantation therapy.", "Cultivation of bone marrow-derived mesenchymal stem cells was attempted as the first step.", "The conventional culture methods however cannot produce sufficient amounts of mesenchymal stem cells because the proliferation of said stem cells stops or becomes extremely slow around 15 th generation.", "There is further problem that the differentiation potential to chondrocytes or osteoblasts is lost.", "In order to induce cartilage or bone tissue from the mesenchymal stem cells and to use them for transplantation therapy, large amounts of said stem cells are required as a start material." ], [ "<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG.", "1 a graph which shows the cell growth in FGF-added group by [cell number/days in culture].", "FIG.", "2 is a graph which shows the cell growth in FGF-added group by [generation number/days in culture].", "FIG.", "3 is a microphotograph of the tissue before chondrogenic differentiation by the addition of bFGF.", "FIG.", "4 is a microphotograph of the tissue after chondrogenic differentiation by the addition of bFGF.", "FIG.", "5 contains graphs showing the cell growth of the rabbit mesenchymal stem cells (derived from ilium (I), derived from tibia (T)) (A, B) in the presence of bFGF (1 ng/ml) or absence, and human mesenchymal stem cells (C).", "FIG.", "6 is to illustrate the chondrogenic differentiation potential of the mesenchymal stem cells by FGF, contains a series of microphotographs of cell pellets cultured in the presence of FGF (A-C) and absence (D), after 2 days (A), 4 days (B) and 8 days (C, D) from chondrogenic differentiation, and a series of microphotographs of cell pellets of 3, 6, 9 or 12 generations subcultured in the presence of FGF (E-H).", "FIG.", "7 is to illustrate the chondrogenic differentiation potential of the mesenchymal stem cells subcultured in FGF(+) or FGF(−), and shows the amount of glycosaminoglycan content (A), alkaline phosphatase activity (B) and expression of cartilage specific genes (type II collagen, type X collagen) (C).", "FIG.", "8 is to illustrate the osteogenic differentiation potential of the mesenchymal stem cells which subcultured in FGF(+) or FGF(−), and shows the calcium content of the mesenchymal stem cells seeded at a high density or low density (A, B), alkaline phosphatase activity (C), and expression of bone specific genes (bone sialoprotein (BSP), osteopontin (OP) and osteocalcin (OC)) (D).", "FIG.", "9 is to illustrate the adipogenic differentiation potential of the mesenchymal stem cells subcultured in FGF(+) or FGF(−), and shows microphotographs of the cells differentiated into adipocyte in FGF(+) (A) and FGF(−) (B), and expression of adipocyte specific gene (PPAR-γ2) (C).", "detailed-description description=\"Detailed Description\" end=\"tail\"?" ], [ "THE TECHNICAL FIELD This invention relates to a novel method of culturing mesenchymal stem cells and to the mesenchymal stem cells with the pluripotency obtained by the method.", "The mesenchymal stem cells of the invention mean the undifferentiated mesenchymal cells with differentiation potential at least into chondrocytes or osteoblasts.", "BACKGROUND TECHNOLOGY Tissues of a vertebrate, especially mammalian tissues, contain stem cells which have the ability to regenerate cells and tissues in cell regeneration system, for example, to regenerate cells and tissues lost by trauma, disease or aging.", "Stem cells therefore are found within the tissue or in other tissues that serve as stem cell reservoirs.", "Bone marrow and periosteum are the major stem cell reservoirs.", "Bone marrow is the major source of adult hematopoietic stem cells, which are undifferentiated pluripotent cells.", "It is known that all blood cells (e.g.", "erythrocyte, leukocyte, lymphocyte, etc.)", "are differentiated from the hematopoietic stem cells.", "Many researches have been made on cultivation and differentiation of hematopoietic stem cells, and many substances (growth factors) have been reported which are useful for culturing hematopoietic stem cells and for differentiating and inducing to various blood cells (components).", "On the other hand, bone marrow, periosteum and the like contain other stem cells than hematopoietic stem cells, i.e.", "mesenchymal stem cells, which play an important role for regenerating the mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose and stroma.", "Mesenchymal stem cells proliferate as undifferentiated cells and retain the pluripotency, by which can differentiate into such as adipocytes, chondrocytes or osteoblasts.", "The stem cells can easily be isolated from adult bone marrow and periosteum, and show a stable phenotypic cell form.", "For example, the mesenchymal stem cells derived from bone marrow retain the character of monolayer in vitro cultivation (Pittenger M. F. et al., Science 284, 143-147, 1999).", "It was therefore proposed to culture mesenchymal stem cells in large amounts and to differentiate and induce thus obtained undifferentiated cells into cartilage tissue or bone tissue, which can be used for transplantation therapy.", "Cultivation of bone marrow-derived mesenchymal stem cells was attempted as the first step.", "The conventional culture methods however cannot produce sufficient amounts of mesenchymal stem cells because the proliferation of said stem cells stops or becomes extremely slow around 15th generation.", "There is further problem that the differentiation potential to chondrocytes or osteoblasts is lost.", "In order to induce cartilage or bone tissue from the mesenchymal stem cells and to use them for transplantation therapy, large amounts of said stem cells are required as a start material.", "DISCLOSURE OF THE INVENTION This invention aims to provide a method of culturing mesenchymal stem cells which can produce mesenchymal stem cells in remarkably larger amounts compared with conventional culture methods.", "This invention also aims to provide mesenchymal stem cells retaining the pluripotency obtained by the method.", "As a result of intensive research for solving the above-mentioned problems, it has been found that when mesenchymal stem cells are cultured in a medium containing fibroblast growth factor (FGF), they can be subcultured over 25 generations, based on which finding the invention was completed.", "This invention therefore provides a method of culturing mammalian mesenchymal stem cells which is characterized by adding to a medium a substance which stimulates the proliferation potency of mesenchymal stem cells.", "In this culture method, FGF is useful as the substance which stimulates the proliferation potency of mesenchymal stem cells.", "The concentration of FGF in the medium is usually from 0.01 to 100 ng/ml, preferably from 0.04 to 10 ng/ml, more preferably from 0.1 to 1 ng/ml.", "In the method of this invention, FGF regardless of its origin is applicable so long as it stimulates the proliferation potency of mammalian mesenchymal stem cells.", "FGF derived from the mammal such as FGF-1 (aFGF) or FGF-2 (bFGF) is desirable.", "Human bFGF and bovine bFGF are on the market and easily available.", "FGFs derived from other mammals can also be used in the invention because the receptor is common.", "Various FGFs including bFGF have been used like other growth factors for culturing various cells, for example, hematopoietic stem cells (Japanese KOKAI (JP-A) Hei-11(1999)-103856) and chondrocytes (Y. Kato & D. Gospodarowicz, J.", "Cell Biol., vol 100, 477-485, 1985).", "It has not been known however that FGF is remarkably effective in retaining the pluripotency of mesenchymal stem cells derived from bone marrow or periosteum, extending their in vitro life span and increasing their passage number.", "Mesenchymal Stem Cells Mesenchymal stem cells of this invention are defined to be derived from bone marrow and/or periosteum and to be undifferentiated pluripotent cells which can differentiate into mesenchymal tissues such as adipose tissue, cartilage tissue or bone tissue.", "Mesenchymal stem cells are extractable from any bone marrow or periosteum which contains them.", "It is desirable to extract them from femur, tibia or pelvis (ilium) because of availability in large amounts and simplicity of extraction.", "In cases of other mammals than human it is also possible to extract mesenchymal stem cells from ilium, tibia and the like.", "In order to extract bone marrow-derived mesenchymal stem cells, any conventional method used for medical treatment and the like can be used.", "In order to extract stem cells from bone marrow of other mammals than human by a laboratory-method, both ends of bone (e.g.", "femur, tibia) are cut, the inside of bone is washed by a medium suitable for culturing mesenchymal stem, cells, and mesenchymal stem cells can be obtained from the emerged medium.", "A practical procedure is as follows: (1) The both ends of rabbit femur or tibia are cut.", "Subsequently the inside of bone marrow is washed by a medium (e.g.", "DMEM medium) to which antibiotics (penicillin, streptomycin, etc.)", "and heparin can optionally be added.", "In the case of human ilium, bone marrow liquid is extracted from the ilium bone marrow.", "(2) The medium used for washing is centrifuged for about 3 minutes at 300×g to precipitate bone marrow cells.", "The obtained cells which are diluted with a suitable medium (e.g.", "DMEM medium containing 10% FBS) to a suitable concentration are seeded onto a cell culture plate.", "They are cultured for three days to produce bone marrow-derived mesenchymal stem cells that adhere on the culture plate.", "In order to extract the stem cells from the periosteum, a well-known method can be used [M. Iwasaki et al., Endocrinology 132, 1603-1608 (1993); J. Fang & B. K. Hall, Developmental Biol.", "180, 701-712 (1996); S. Bahrami et al., The Anatomical Record 259, 124-130 (2000)].", "An example of method is given in the following.", "(1) Periosteum is exposed by exfoliating soft tissues such as connective tissue and the like from rabbit femur or tibia, cut in a suitable size and extracted.", "The extracted periosteum is chopped finely and treated with collagenase at 37° C. (2) Cells are subsequently isolated by pipetting or other suitable method and collected by filtration or centrifugal separation.", "Thus obtained cells are counted, diluted with a suitable medium (e.g.", "DMEM medium containing 10% FBS) to a suitable concentration and seeded onto a cell culture plate.", "They are cultured for three days to produce periosteum-derived mesenchymal stem cells that adhere on the culture plate.", "By such method, mesenchymal stem cells of about 104-105 cell counts can be obtained.", "Although thus obtained mesenchymal stem cells can directly be seeded and cultured, it is common to use them after culturing for 10-20 days in a suitable medium.", "The cells can be cryo-preserved.", "Method of Culturing Mesenchymal Stem Cells The stem cells obtained as mentioned above are cultured in a medium which is suitable to culture the cells and contains bFGF in an amount of 0.01 to 100 ng/ml, preferably 0.04 to 10 ng/ml, more preferably 0.1 to 1 ng/ml, for example, 1 ng/ml.", "Cultivation can be carried out under conditions suitable for culturing mammal cells, usually in the presence of 5% CO2 at 37° C. An example of cultivation is given in the following.", "(3) The mesenchymal stem cells adhering on the cultivation plate as mentioned above are cultured in a suitable medium (e.g.", "DMEM medium containing 10% FBS) in the presence of 5% CO2 at 37° C. (4) The medium is renewed every 3 days.", "bFGF will be added from day 5 to the medium in an amount of 1 ng/ml.", "The subculture of the stem cells can be performed by a suitable method known in the field of cell culture.", "For example, cells are collected from the plate of the primary culture which are close to confluence, seeded in a suitable medium containing bFGF and cultured under the similar conditions as primary culture.", "When cells approach confluence, they are subcultured.", "An example of subculture is given in the following.", "(5) The primary culture mentioned in above (4) becomes close to confluence in around 10 days.", "This plate is treated with trypsin (e.g.", "0.05%)+EDTA (e.g.", "0.2 mM).", "Cells are collected from the plate and counted.", "(6) The cultured stem cells are seeded at a density of 5×103 cells/cm2 in a medium containing bFGF (1 ng/ml), cultured and subcultured before cells become confluent.", "(7) Subculture is performed by repeating the procedure of above (6).", "According to the culture method of the invention the proliferation of mesenchymal stem cells continues to 15 generations, preferably 25 generations, more preferably over 30 generations, or for more than 16 days, preferably more than 20 days, more preferably more than 30 days, further preferably more than 40 days and most preferably more than 50 days to produce extremely high numbers of stem cells.", "Thus cultured stem cells can be differentiated into various mesenchymal tissues such as cartilage tissue or bone tissue.", "In order to obtain chondrocytes (tissue) from the cultured mesenchymal stem cells, the stem cells are treated with trypsin, isolated by centrifugal separation etc., transferred to a suspension culture system, an agarose culture system or an alginic acid culture system, and cultured to induce chondrocytes in a medium suitable for chondrogenic differentiation, for example, a medium described by M. F. Pittenger et al., Science 284, 143-147, 1999.In order to obtain osteocytes (tissue) from the mesenchymal stem cells, the stem cells are treated with trypsin, isolated by centrifugal separation etc.", "and cultured to induce osteocytes in a medium suitable for osteogenic differentiation, for example, a medium described by M. F. Pittenger mentioned above.", "The invention is explained more in detail by examples by which the invention is not restricted.", "BEST MODE FOR WORKING THE INVENTION EXAMPLES Example 1 Cultivation of Mesenchymal Stem Cells 1) Collection of Mesenchymal Stem Cells 1-1) Collection of Stem Cells from Bone Marrow Femurs and tibias of four-week-old rabbits were freed from muscles, ligament and the like, and extracted.", "The both ends were cut off.", "The inside of bone marrow was washed with DMEM medium (containing 32 unit/ml penicillin, 50 μg/ml streptomycin and 6000 units/ml heparin).", "The emerged medium was subjected to centrifugal separation (at 300×g, for 3 minutes) to precipitate stem cells.", "They were diluted with DMEM medium containing 10% FBS, seeded at 2×108 cells (containing erythrocytes)/10 cm culture plate and cultured for three days in the presence of 5% CO2 at 37° C. to produce adhering cells (about 2000 cells/culture plate), which were used as the bone marrow-derived mesenchymal stem cells.", "1-2) Collection of Stem Cells from Periosteum Femurs and tibias of four-week-old rabbits were freed from soft tissues such as connective tissue to expose periosteum.", "The periosteum was excised and extracted in a size of about 5×5 mm.", "After removing soft tissues, the periosteum was chopped finely and treated with 0.1% collagenase for 120 minutes at 37° C. Subsequently, cells were liberated by pipetting, collected by filtering and then washed 3 times with PBS.", "The cells were diluted with DMEM medium containing 10% FBS, counted and seeded at 2×105 cells/10 cm culture plate.", "The cells were cultured for 3 days to produce adhering cells which were used as periosteum-derived mesenchymal stem cells.", "2) Cultivation of Mesenchymal Stem Cells The mesenchymal stem cells extracted from bone marrow were cultured in DMEM medium containing 10% FBS, during which the medium was renewed every 3 days.", "bFGF was added in case of periosteum-derived cells from 2nd day, in case of bone marrow-derived cells from 5th day to the medium in an amount of 1 ng/ml, and no bFGF was added to control group.", "The both groups approached confluence around 10th day.", "The plates were treated with 0.05% trypsin+0.2 mM EDTA for 5 minutes to isolate the grown stem cells.", "After counting the number of cells by Coulter counter (Z1 single, produced by Coulter Company), the cells were seeded at a density of 5000 cells/cm2.bFGF was also added in the subculture in an amount of 1 ng/ml.", "The cultivation results of mesenchymal stem cells of bFGF-added group and control group are shown in graphs of FIG.", "1 (cell number/days in culture) and FIG.", "2 (generation number/days in culture).", "With the conventional culturing method (control group) in which no bFGF was added, the cell growth was active up to 10th day from the start of culturing (around 15 generations) but then stopped.", "On the other hand with the culturing method of this invention, the cell growth continued even after 30th day (about 30 generations) although the cell growth speed lowered in some degree around 20th day.", "The mesenchymal stem cells produced by the culturing method of this invention in 50 days was 105 times the cell count of the control group (see FIG.", "1 and FIG.", "2).", "Example 2 Chondrogenic Differentiation The mesenchymal stem cells cultured in Example 1 (bFGF added-group and control group, 16th day from the start of culturing) were treated with 0.05% trypsin+0.2 mM EDTA for 5 minutes to isolate mesenchymal stem cells.", "Then 2.5×105 cells were transferred to 15 ml test tube (made of polypropylene) and subjected to centrifugal separation at 1000×g for 5 minutes to remove DMEM medium containing FBS.", "The precipitated cells were mixed with a chondrogenic differentiation medium having the following composition and cultured in centrifugation tubes.", "Chondrogenic Differentiation Medium High glucose α-MEM medium 10 ng/ml TGFβ1 100 nM Dexamethasone 50 μg/ml Ascorbic acid-2-phosphate 100 μg/ml Sodium pyruvate ITS-plus 6.25 μg/ml Transferrin 6.25 μg/ml Insulin 6.25 ng/ml Selenic acid 5.33 μg/ml Linoleic acid 1.25 mg/ml Bovine serum albumin The mesenchymal stem cells were cultured in the above-mentioned medium in the presence of 5% CO2 at 37° C. After 24 hours from the start of culturing, the cells formed spherical pellets.", "Renewing the medium every 2 days the cultivation was continued for 14 days.", "The slides of the pellet cultures before cultivation and after cultivation were prepared and stained with Toluidine Blue.", "Judging from the shape of cell mass and the stainability of proteoglycan, an extracellular matrix, it was confirmed with the bFGF-added group that the undifferentiated state was observed before the induction (FIG.", "3) but the cartilage tissue induction was observed after the cultivation (FIG.", "4).", "On the other hand chondrocytes were not observed with the control group.", "Cell shape of the mesenchymal stem cells before chondrogenic differentiation was retained with bFGF-added group, but fibroblast-like cells were observed with the control group.", "These results are summarized in the following table.", "TABLE 1 Retention of Chondrogenic shape of stem differentiation cells before Chondrogenic Ratio induction differentiation (%) bFGF-added ++ +++ 100 group Control − − 0 group Example 3 Osteogenic Differentiation The mesenchymal stem cells (bFGF added-group, control group) were collected on 16th day after cultivation as in Example 2 and transferred into an osteogenic differntiation medium having the following composition.", "Osteogenic Differentiation Medium αMEM medium 10% FBS 100 nM Dexamethasone 10 mM β-Glycerophosphoric acid 50 μg/ml Ascorbic Acid 2-phosphate The stem cells were cultured in the above-mentioned medium in the presence of 5% CO2 for 12 days at 37° C., renewing the medium every 2 days.", "When the cells after cultivation were stained by Alizarin Red, the calcification was observed with bFGF added-group.", "On the other hand with the control group the calcification was remarkably low compared with the bFGF-added group.", "The results are summarized in the following table.", "TABLE 2 Osteogenic differentiation bFGF-added +++ group Control group + Example 4 Effects on Proliferation of Various Mesenchymal Stem Cells The bone marrow-derived mesenchymal stem cells from rabbit ilium and tibia were obtained as in Example 1.The bone marrow-derived mesenchymal stem cells from human tibia were obtained from BioWhittaker Inc. (Walkersville, Md.).", "The rabbit-derived mesenchymal stem cells were seeded in at 2×108 cells (containing erythrocyte)/10 cm cultivation plate.", "The human-derived mesenchymal stem cells were seeded at 103 cells/cm2.They were cultured in the presence of bFGF (1 ng/ml) or absence.", "Subculture was performed by seeding the rabbit-derived mesenchymal stem cells at 5×103 cells/cm2 and the human-derived mesenchymal stem cells at 103/cm2 (see Example 1).", "Cultivation was carried out twice for each mesenchymal stem cells independently from each other.", "The results are shown in FIG.", "5.As shown in FIG.", "5, the addition of bFGF increased the growth rate and the life span of both mesenchymal stem cells derived from rabbit ilium (I) and tibia (T) (see A and B).", "The mesenchymal stem cells derived from human tibia showed an increase of growth rate and an extension of life in human serum-containing medium (see C).", "The same results were obtained also with mesenchymal stem cells derived from dog ilium (results are not shown).", "Example 5 Verification of the Chondrogenic Differentiation Potential of Cultured Mesenchymal Stem Cells 1) Histochemical Analysis The mesenchymal stem cells extracted from a four-week-old rabbit were grown in the presence of bFGF (FGF(+)) or absence (FGF(−)), subcultured 3 times, transferred to the chondrogenic differentiation medium used in Example 2, cultured for 2, 4 or 8 days and stained with Toluidine Blue.", "The differentiation into chondrocytes was not observed in 2 days from the bFGF chondrogenic differentiation (FIG.", "6-A) but spherical cells (chondrocytes) were observed on the surface of pellets in 4 days (FIG.", "6-B).", "In eight days all mesenchymal stem cells of FGF(+) became spherical (FIG.", "6-C).", "On the other hand, mesenchymal stem cells of FGF(−), chondrogenic differentiation was notably late as compared with the cells of FGF(+) (FIG.", "6-D).", "The mesenchymal stem cells of FGF(+) which were passaged 3, 6, 9 or 12 times were transferred to the chondrogenic differentiation medium, cultured for 16 days and stained with Toluidine Blue.", "Although a cartilage-like tissue was observed in almost all cells of 3rd -9th passage cultures, the amount of cartilage proteoglycan, which was stained with Toluidine Blue, decreased with the increase in the passage number (FIG.", "6-E˜G).", "Only a part of cells of 12th passage turned into chondrocytes (FIG.", "6-H).", "The results indicate that although FGF, promotes chondrogenic differentiation of mesenchymal stem cells but the differentiation potential decreases with the increase of passage.", "2) Biochemical Analysis and Analysis With Marker Genes The mesenchymal stem cells subcultured to 3, 6, 9 or 12th generation in FGF(+) or FGF(−) were tested for glucosamine content and alkaline phosphatase activity and analyzed for the amount of mRNAs of type II collagen and type X collagen, which are molecular markers of cartilage.", "(1) Method The glycosaminoglycan content was measured by the method of Bitter and Muir (Anal.", "Biochem.", "4 (1962), 330-334).", "Alkaline phosphatase activity in cultured cells was measured by the method of Bessey (J. Biol.", "Chem.", "164 (1946), 321-329).", "RT-PCR was performed to detect mRNA as follows.", "RT-PCR Total RNA was extracted from cultured cells using ISOGEN (Nippon Gene, Tokyo, Japan).", "The first-strand cDNA was synthesized using SUPERSCRIPT II RNase H-reverse transcriptase (Life Technologies, Inc. Rockville, Md.)", "from 1 μg of total RNA.", "Using synthesized cDNA as a template, PCR amplification was carried out under the following PCR conditions: denaturation at 94° C. for 30 seconds; the primer extension at 65° C. for 1.5 minutes in 25 or 30 cycles.", "Thus obtained PCR products were separated by 1% agarose gel electrophoresis and detected by ethidium bromide staining.", "The following set of primers was used.", "Primers Rabbit type II collagen 5′-CATACCGGTAAGTGGGGCAAGACTG-3′ (SEQ ID NO: 1) and 5′-TGCCCAGTTCAGGTCTCTTA-3′: (SEQ ID NO: 2) Rabbit type X collagen: 5′-CCCAACACCAAGACACAGTT-3′ (SEQ ID NO: 3) and 5′-ATCACCTTTGATGCCTGGCT-3′ (SEQ ID NO: 4) GAPDH 5′-GTCAAGGCCGAGAATGGGAA-3′ (SEQ ID NO: 5) 5′-GCTTCACCACCTTCTTGATG-3′ (SEQ ID NO: 6) (2) Result The amount of glycosaminoglycan, which is a cartilage matrix, of the mesenchymal stem cells in FGF(+) and FGF(−) cultures decreased with the increase in the passage number.", "However the amount of glycosaminoglycan of the mesenchymal stem cells in FGF(+) cultures was remarkably higher as compared with FGF(−) cultures (FIG.", "7-A).", "Similarly the alkaline phosphatase activity of FGF(+) and FGF(−) cultures decreased with the increase in the passage number, while the alkaline phosphatase activity of FGF(+) cultures was remarkably higher as compared with FGF(−) cultures (FIG.", "7-B).", "The amount of mRNA of type II collagen and type X collagen, which are marker molecules of chondrocyte, of FGF(+) and FGF(−) cultures decreased with the increase of passage number, while FGF(+) cultures contained higher amount of mRNA of type II collagen and type X collagen than FGF(−) cultures (FIG.", "7-C).", "These results indicate that FGF reinforces notably the chondrogenic differentiation potential of mesenchymal stem cells in vitro and represses the decrease in the differentiation potential along with passage.", "Example 6 Verification of Osteogenic Differentiation Potential of Cultured Mesenchymal Stem Cells The osteogenic differentiation potential of mesenchymal stem cells of FGF(+) or FGF(−) subcultures was verified in the following.", "(1) Method Human mesenchymal stem cells were seeded at a high density (5000 cells/cm2) or a low density (1000 cells/cm2), subcultured in the presence of FGF or absence up to 3, 6 or 9 passages, and subsequently transferred to osteogenic differentiation medium described in Example 3 and cultured.", "Thus obtained cultures were analyzed for calcium content, alkaline phosphatase activity as well as the amount of mRNAs of bone sialoprotein (BSP), osteopontin and osteocalcin, which are genes characteristic of osteoblast.", "The alkaline phosphatase activity in cell cultures was measured as in Example 5.The calcium content was measured according to the method of Gitelman (Anal.", "Biochem.", "18 (1967), 521-531).", "The amount of mRNAs of bone sialoprotein (BSP), osteopontin and osteocalcin was analyzed by RT-PCR method described in Example 5 using a set of the following primers.", "Primers Human bone sialoprotein (BSP): 5′-CATTTTGGGAATGGCCTGTG-3′ (SEQ ID NO: 7) and 5′-ATTGTCTCCTCCGCTGCTGC-3′ (SEQ ID NO: 8) Human osteopontin: 5′-CTAGGCATCACCTGTGCCATACC-3′ (SEQ ID NO: 9) and 5′-CAGTGACCAGTTCATCAGATTCATC-3′ (SEQ ID NO: 10) Human osteocalcin: 5′-CCACCGAGACACCATGAGAG-3′ (SEQ ID NO: 11) and 5′-CCATAGGGCTGGGAGGTCAG-3′ (SEQ ID NO: 12) GAPDH: 5′-GTCAAGGCCGAGAATGGGAA-3′ (SEQ ID NO: 5) and 5′-GCTTCACCACCTTCTTGATG-3′ (SEQ ID NO: 6) (2) Results When the cells were seeded in a high density, the calcium content and alkaline phosphatase activity showed no significant difference regardless of the passage number, or of FGF(+) or FGF(−) (FIG.", "8-A, C).", "However, when cells were seeded in a low density, the calcium content of FGF(+) was higher than FGF(−), and decreased along with passage number (FIG.", "8-B).", "The amount of mRNAs of bone sialoprotein (BSP), osteopontin and osteocalcin of FGF(+) and FGF(−) cultures showed no significant difference between 3rd generation and 6th generation, while the mRNA levels of FGF(−) mesenchymal stem cell cultures were decreased at 9th generation (FIG.", "8-D).", "These results indicate that FGF represses the decrease in osteogenic differentiation potential of mesenchymal stem cells in vitro.", "Comparative Example Verification of Adipogenic Differentiation Potential of Cultured Mesenchymal Stem Cells The adipogenic differentiation potential of mesenchymal stem cells of FGF(+) or FGF(−) subcultures was verified in the following.", "Cells subcultured under FGF(+) or FGF(−) up to 4, 6, or 9 passages were transferred to the following adipogenic differentiation medium and cultured for 25 days to differentiate the mesenchymal stem cells into adipocytes.", "Adipogenic Differentiation Medium αMEM medium (high glucose) 10% FBS 1 μM Dexamethasone 0.2 mM 3-Isobutyl-methyl-xanthine The differentiation into adipocytes was detected by Oil Red staining and RT-PCR analysis of mRNA of PPAR-γ2, a differentiation marker molecule of adipocyte.", "RT-PCR was carried out as described in Example 5 using the following set of primers.", "Primers Human PPAR-γ 2: 5′-CATTCTGGCCCACCAACTT-3′ (SEQ ID NO: 13) and 5′-CCTTGCATCCTTCACAAGCA-3′ (SEQ ID NO: 14) GAPDH: 5′-GTCAAGGCCGAGAATGGGAA-3′ (SEQ ID NO: 5) and 5′-GCTTCACCACCTTCTTGATG-3′ (SEQ ID NO: 6) The mesenchymal stem cells of FGF(+) and FGF(−) subcultures were stained with Oil Red at the same level (FIG.", "9-A, B).", "mRNA level of PPAR-γ2, a differentiation marker of adipocyte, was almost the same up to 9th passage.", "The results indicate that the existence or absence of FGF does not influence the differentiation of mesenchymal stem cells into adipocyte.", "INDUSTRIAL APPLICABILITY The culture method of this invention enables to culture mesenchymal stem cells over 30 generations in contrast to the conventional culture method in which cell growth stopps around 15 generations.", "Available number of cell increases about 105 to 106-fold compared with the conventional culture methods.", "It is also possible to prevent the decrease in pluripotency along with the passage.", "Since the mesenchymal stem cells cultivated by this invention retain the differentiation potential to chondrocyte and osteoblast and have prolonged life span, they can provide large amounts of chondrocytes or osteocytes for transplantation of cartilage tissue or bone tissue.", "This is very important for the transplantation treatment of cartilage or bone.", "BRIEF DESCRIPTION OF DRAWINGS FIG.", "1 a graph which shows the cell growth in FGF-added group by [cell number/days in culture].", "FIG.", "2 is a graph which shows the cell growth in FGF-added group by [generation number/days in culture].", "FIG.", "3 is a microphotograph of the tissue before chondrogenic differentiation by the addition of bFGF.", "FIG.", "4 is a microphotograph of the tissue after chondrogenic differentiation by the addition of bFGF.", "FIG.", "5 contains graphs showing the cell growth of the rabbit mesenchymal stem cells (derived from ilium (I), derived from tibia (T)) (A, B) in the presence of bFGF (1 ng/ml) or absence, and human mesenchymal stem cells (C).", "FIG.", "6 is to illustrate the chondrogenic differentiation potential of the mesenchymal stem cells by FGF, contains a series of microphotographs of cell pellets cultured in the presence of FGF (A-C) and absence (D), after 2 days (A), 4 days (B) and 8 days (C, D) from chondrogenic differentiation, and a series of microphotographs of cell pellets of 3, 6, 9 or 12 generations subcultured in the presence of FGF (E-H).", "FIG.", "7 is to illustrate the chondrogenic differentiation potential of the mesenchymal stem cells subcultured in FGF(+) or FGF(−), and shows the amount of glycosaminoglycan content (A), alkaline phosphatase activity (B) and expression of cartilage specific genes (type II collagen, type X collagen) (C).", "FIG.", "8 is to illustrate the osteogenic differentiation potential of the mesenchymal stem cells which subcultured in FGF(+) or FGF(−), and shows the calcium content of the mesenchymal stem cells seeded at a high density or low density (A, B), alkaline phosphatase activity (C), and expression of bone specific genes (bone sialoprotein (BSP), osteopontin (OP) and osteocalcin (OC)) (D).", "FIG.", "9 is to illustrate the adipogenic differentiation potential of the mesenchymal stem cells subcultured in FGF(+) or FGF(−), and shows microphotographs of the cells differentiated into adipocyte in FGF(+) (A) and FGF(−) (B), and expression of adipocyte specific gene (PPAR-γ2) (C)." ] ]

[ [ "Arrangement in a distributed control system for increasing the availability of data and/or control commands", "There is a need for increasing the availability of data and/or control commands in a distributed control system for one or more machines, vehicles and/or processes.", "The control system comprises or is controlled by a transmitting node (1) or transmitter unit and has two or more receiving nodes (13, 14, 15, 16).", "The transmitting nodes and receiving nodes are connected to each other via wire [sic] radio links.", "The receiving nodes are located at a distance from each other and are connected to a few-wire digital link in a wired system for forwarding of the said data and/or control commands to the executing devices.", "The control [sic] node transmits the said data and/or the control commands in different directions towards the receiving nodes which are arranged at a distance from each other.", "Receiving nodes with reception that is essentially not subject to interference are arranged to be able to connect to the few-wire link in parallel and simultaneously with a receiver unit or units which have links that are connected to the few-wire link.", "In another case, only those receiver unit or units are connected that have reception links that are not subject to interference, while the receiver unit of units with links that are subject to interference do not participate in the transmission of the message on the few-wire link." ], [ "1.An arrangement for increasing availability of data and/or control commands (D) in a distributed control system (CAN) for one or more machines (2″) (also vehicles) and/or processes (2″), the control system being influenced by or comprising at least one transmitting node (1) or transmitter unit, and also comprising two or more receiving nodes (13, 14, 15, 16), and where the transmitting node or transmitter unit and the receiving nodes can be connected or are connected to each other via wireless links (radio links 5, 6, 7, 8) and where the receiving nodes are located at a distance (A, B) from each other and are connected to a few wire digital (21) link in a wired system for forwarding the said data and/or control commands to executing devices (41) for, for example, functions, measurements, etc, characterized in that the transmitting node or transmitter unit is arranged to send the said data and/or control commands in parallel and simultaneously in different directions (9, 10, 11, 12) towards the receiving nodes, and that in the event of wireless links that are essentially not subject to interference between the transmitting node or transmitter unit and the receiving nodes, the latter are arranged to connect in parallel and simultaneously for parallel and simultaneous transmission of the said data and/or control commands to the few-wire link and thereby to the executing device or devices (41) concerned, and in the event of wireless link or links that are essentially subject to interference between the transmitting node or transmitter unit and the receiving node or nodes concerned, the latter are prevented from making their connections to the few-wire link, whereby the transmission of the said data and/or control commands to the few-wire link and respective executing device in this case is effected by the remaining receiver devices with wireless link or links that are essentially not subject to interference.", "2.An arrangement according to claim 1, characterized in that the transmitting node (1) or transmitter unit sends the said data and/or control commands to the receiving nodes (13, 14, 15, 16) on different frequencies.", "3.An arrangement according to claim 2, characterized in that data and/or the control commands are incorporated in or form messages (27, 27′, 27″, 27′″) with system identification (ID, C), data (D) and data length codes and control codes (CRC and CH).", "4.An arrangement according to any one of the preceding claims, characterized in that the distributed system (CAN) consists of or comprises a system of the control area system type where the receiving nodes (13, 14, 15, 16) are synchronized with each other or coordinated with the connection or arbitration functions of the few-wire link (21), with the result that they simultaneously obtain access to the few-wire link via messages received from the transmitting node or transmitter unit that are essentially or completely not subject to interference and that in the event of absent coordinated connections on account of messages (27) from the transmitting node (1) or transmitter unit essentially subject to interference or absent, they obtain information from the few-wire link concerning cancelling further attempts to connect to the few-wire link after the receiving node or receiving nodes with link or links that are essentially not subject to interference have completed the transmission of the message on the few-wire link.", "5.An arrangement according to any one of the preceding claims, characterized in that, in the event of its message from the transmitting node or transmitter unit being essentially subject to interference or absent, the respective receiving part is arranged to receive or detect all or parts of the message that was transmitted on the few-wire link (21) from the receiving unit or receiving units with received message or messages that are essentially not subject to interference, and that, in the event of such received part of the message or received message, it is arranged to prevent continued attempts to access the few-wire link (21).", "6.An arrangement according to any one of the preceding claims, characterized in that, in the event of a predetermined level of interference in its received message, the respective receiving node is arranged to generate an internal cancellation signal (i2) or cancelling which prevents the connection of the receiving node to the few-wire link (21).", "7.An arrangement according to any one of the preceding claims, characterized in that, in the event of interference in their received messages above a predetermined value, the receiving nodes are arranged to effect an arrangement between the receiving nodes which selects the receiver unit that at the time of reception has reception which is the least subject to interference, and that only the receiving node thereby selected obtains access to the few-wire link for transmission of its received message on this link.", "8.An arrangement according to any one of the preceding claims, characterized in that the transmitting node consists of or comprises a service unit provided with a computer, and that the receiver units are located in a vehicle (2′), with the executing devices in the vehicle controlling function executors, for example in the form of door mirrors, driver's seat, sunroof, etc.", "9.An arrangement according to any one of the preceding claims, characterized in that the transmitting node or transmitter unit is fixed, that is allocated a predetermined geographical position.", "10.An arrangement according to any one of claims 1-9, characterized in that the transmitting node or transmitter unit is movable and constitutes a mobile unit for example incorporated in the general communication mobile system (GSM).", "11.An arrangement according to any one of the preceding claims, characterized in that the nodes or units in the system are arranged for message forms that exist in association with Bluetooth.", "12.An arrangement according to any one of the preceding claims, characterized in that the receiving nodes are synchronized with each other with regard to connection and arbitration functions that occur in conventional control area systems.", "13.An arrangement according to any one of the preceding claims, characterized in that the receiver units operate with and are synchronized or coordinated with each other by means of time functions incorporated in the control area system (CAN).", "14.An arrangement according to any one of the preceding claims, characterized in that the system has two types of link, wireless links and wired links, where by wired links is meant such links as have wires, leads, etc, and/or optical connections comprising opto links.", "15.An arrangement according to any one of the preceding claims, characterized in that the system can operate with two, three or more steps, where in a first step a transmission is carried out from wireless transmitter to wireless receiver and from wireless receiver to wired link and in the case with three steps from wired link to wireless transmitter(s), from wireless transmitter to wireless receiver(s) per channel, and from wireless receiver(s) to wired link.", "16.An arrangement according to any one of the preceding claims, characterized in that the wireless transmitters each transmit on a separate channel (channel frequency), where a channel is characterized in that when transmitting each transmitter has access to a part of the available bandwidth in the ether which is exclusive to the system.", "17.An arrangement according to claim 16, characterized in that the exclusivity can be an allocated frequency in a time interval, for example as in the known Bluetooth protocol, IEEE 802.11 for jumping frequency or a correlation code (as in IEEE 802.11 Direct Sequence Spread Spectrum).", "18.An arrangement according to any one of the preceding claims, characterized in that a transmitter and one or more receivers can operate on one and the same channel during one and the same time interval.", "19.An arrangement according to any one of the preceding claims, characterized in that units in the form of g-nodes, that is nodes that are connected both to the respective wireless network and the respective wired network, are arranged to operate as gateways, where they receive a complete message on the wireless links in question and ascertain in a known way that it has been correctly received by means of check code, error-correcting code, etc, after which the message is able to be sent on the wired links concerned.", "20.An arrangement according to any one of the preceding claims, characterized in that the respective message has an identity in the respective medium which is unique at least at the time of transmission, which identity can be a bit code or can consist of a particular time slot in a scheduled system or a combination of these functions.", "21.An arrangement according to claim 19 and 20, characterized in that the identity is the same or different for media involved, where in the case where they are different, respective g-nodes are arranged to know the association between the identifies.", "22.An arrangement according to any one of the preceding claims 17-21, characterized in that the Bluetooth protocol is used in a modified form as a protocol, inasmuch as several slaves are allocated the same time slot for the reception in question, that a field bus message (CAN message in the form of LLC data frame) is arranged for generation in a computer (microcomputer) which is equipped with a radio interface of the Bluetooth type.", "23.An arrangement according to any one of the preceding claims 17-22, characterized in that a CAN message is arranged for packaging as data in a Bluetooth message which is sent by the computer (microcomputer) and that reception of this message is carried out in two or more g-nodes.", "24.An arrangement according to any one of the preceding claims 17-23, characterized in that the g-nodes that received the CAN message [error-]free according to the Bluetooth protocol's error-detection mechanisms are able thereafter to transmit the CAN message on the CAN bus.", "25.An arrangement according to any one of the preceding claims 19-24, characterized in that in the case where several g-nodes commence their transmission synchronized to the same frame (Strat of Frame=SOF), simultaneous transmission of the message from the g-nodes takes place bit by bit, which is made possible as the messages are identical.", "26.An arrangement according to any one of the preceding claims 19-25, characterized in that in the case where any one or more of the g-nodes can not synchronize with the message that the first g-node transmits, they change over to receiving nodes in accordance with the CAN protocol, which means that the respective g-node concerned will receive a message which is identical to the one the respective g-node was in the process of transmitting on the bus, and that when the respective g-node has ascertained that such is the case, the g-node ceases to send its message in question, which thus means that there is parallel redundancy for signalling from the computer (the microcomputer) to the CAN network.", "27.An arrangement according to any one of the preceding claims 19-26, characterized in that in the opposite direction there is serial redundancy where the respective g-nodes are each allocated a time slot for transmission and information about which CAN identifier identifies the CAN message in question which is arranged to be sent to the computer/processor (microcomputer PC), where the respective g-node receives at the same time the message on the CAN bus in accordance with the CAN protocol.", "28.An arrangement according to any one of the preceding claims 19-27, characterized in that the respective message which is arranged to be sent to the computer/processor (PC) is arranged for packaging as data in a Bluetooth message and for dispatch in the respective time slot, where the computer (PC) receives one or more identical messages from the CAN system.", "29.An arrangement according to any one of the preceding claims 19-28, characterized in that information about which receiver has received the strongest signal is able to be produced, where the radio part or parts of the g-nodes provide a value for the signal strength (signal strength indicator, SSI), for example a measurement value 0-255, that the CAN identifier is divided into at least three fields, where one contains the value 255 minus the SSI value, one field indicates which g-node is sending the message, and one field indicates that it is one (of several) messages from the computer/processor (PC).", "30.An arrangement according to any one of the preceding claims 19-29, characterized in that the said SSI value−255 is applied in the CAN identifier, where the g-node that [has] the highest signal strength gains access to the bus if all the anodes commence signalling at the same time, which information about which g-node has the strongest signal and the relevant value of this is thus indicated in the CAN identifier and is thus available to all nodes which require this information.", "31.An arrangement according to any one of the preceding claims, characterized in that the signal strength in combination with indication of bit errors can be used together with other measures, for example change of transmitter, and/or amended propagation diagram from the transmitter or transmitters, etc, which can be relevant in the case when the signal comes via several different signal paths (a so-called multipath situation).", "32.An arrangement according to any one of the preceding claims, characterized in that in the case with one or more mobile transmitters, in a first position these can have contact with a first number of receivers, for example two receivers, thereafter with a second number of receivers, for example three receivers, and thereafter again with the first number of receivers, for example the said two receivers, that the transmission reliability is thereby able to be ensured by the P-presentation function, where the SSI-presentation provides an indication of where the mobile transmitter is located.", "33.An arrangement according to claim 32, characterized in that the combination of a mobile transmitter and a fixed network can consist of a car that is in a garage, a car that is at a service station, an ignition key (or corresponding unit that can comprise a similar function, for example a mobile phone, identity card, etc) for a vehicle.", "34.An arrangement according to any one of the preceding claims, characterized in that a unit that is not yet connected (for example a mobile unit) can listen on a fixed frequency on which time messages will appear and on which the unit that is not yet connected is able to synchronize itself to the network on the messages that are still to be sent to maintain the global time in the system, and that the messages can contain identification data for the system in addition to the said messages." ], [ "The present invention relates to an arrangement for increasing the availability of data and/or control commands in a distributed control system for one or more machines, vehicles and/or processes.", "The control system is thereby able to be controlled by or comprises at least one transmitting node or transmitter unit.", "The control system also comprises two or more receiving nodes.", "The transmitting node or transmitter unit and the receiving nodes are able to be connected together or are connected together via wireless links, by which is meant here principally radio links.", "The receiving nodes are situated geographically separated from each other and are connected to a few-wire digital connection in a wired system for forwarding the said data and/or control commands to executing devices for the functions, measurements, etc, in question.", "The invention is associated with the invention according to the Swedish patent application 0001148-6.Reference is made to this application and to the references mentioned in this application.", "Reference is also made to EP 513137 and 470199.All the references relate to the same Applicant as the present patent application.", "In connection with so-called field busses, that is to systems of the type CAN (Controller Area Network) and CAN Kingdom (developed by the Applicant of the present patent application), there is a need for availability to be able to be extended or increased in various practical situations.", "With wireless links, in particular radio links that are used in connection with Bluetooth, there can be cases when the link for various reasons is subject to interference between the transmitting and receiving nodes or units.", "The invention relates, among other things, to solving this problem.", "It is essential that proposed measures can be incorporated in the functions of the utilized systems, so that these do not need to be modified and allocated new functions.", "The invention also solves this problem and proposes, for example, a technically simple and also economically advantageous solution in association with field busses of the said type.", "The principle characteristics of the new invention can be considered to be, among other things, that the control node or control unit is arranged to transmit the data and/or control commands in question in parallel and simultaneously in various directions to the geographically-separated receiving nodes.", "The invention is also characterized in principle by two different functional cases.", "In the first functional case, the wireless links between the transmitting nodes or transmitter units and the receiving nodes are essentially not subject to interference.", "In this case, the receiving nodes are arranged to be connected in parallel and simultaneously to the few-wire link.", "Using the parallel and simultaneous connection, the receiver units transmit the said received data and/or control commands in parallel and simultaneously to the few-wire link and thereby to the executing device or devices concerned, which devices execute the functions in question in the distributed control system.", "In the second functional case, the wireless link or links between the transmitting node or transmitter unit and one or more associated receiving nodes are essentially subject to interference.", "The receiving node or nodes are thereby prevented from making their connection or connections to the few-wire link.", "In this second functional case, the transmission of the said data and/or control commands to the few-wire link and respective executing device is carried out using only the receiver device or devices that have wireless link or links that are essentially not subject to interference.", "In a preferred embodiment, the control node or control unit transmits on different frequencies to the said receiving nodes.", "Messages with the said data and/or control commands are thus transmitted on the said different frequencies.", "In a preferred embodiment, the distributed control system consists of one or more systems of the control area system type (=CAN), where the control nodes are synchronized with each other or coordinated with the connection and arbitration functions of the few-wire link.", "This means that they simultaneously obtain access to the few-wire link in the event of messages that are essentially not subject to interference being received from the transmitting node or unit.", "In the absence of coordinated connections, due to messages from the transmitting node or unit being essentially subject to interference or absent, the respective receiving node obtains information via the few-wire link concerning cancelling further attempts to connect to the few-wire link after the receiving node with the link that is essentially not subject to interference has concluded the transmission of the message on the few-wire link.", "The respective receiver unit is thus preferably arranged, in the event of a message from the transmitting node or transmitter unit being essentially subject to interference or absent, to receive or detect all or parts of the message that was transmitted on the few-wire link from the receiver unit or receiver units with message(s) that are essentially not subject to interference.", "The respective receiver unit with messages subject to interference or absent will thus, upon the reception of the said transmitted part of the message or upon the reception of the transmitted message, inhibit continued attempts to access the few-wire link.", "The said data and/or control commands can be incorporated in or can form messages with control system identification, data and data link codes and control codes.", "The respective receiving node can be arranged in the event of a predetermined level of interference in its received message to generate an internal cancelling signal that prevents the connection of the receiving node to the few-wire link.", "The receiving node can also be arranged, in the event of interference to messages above a predetermined value, to implement an arrangement between the receiving nodes that selects the receiving node which at the time of the reception has the reception which is subject to the least interference and can be arranged so that only the receiving node thereby selected obtains access to the few-wire link for transmission of its received message upon this.", "Further developments of the invention will be apparent from the following subsidiary claims.", "What is proposed above has advantages, for example, in association with service functions and service installations for cars, where the car or corresponding vehicle is used or set up indoors or outdoors and where various interference phenomena can arise, for example, associated with movements of other adjacent vehicles, movements in working tools, movements of personnel, etc.", "In association with CAN systems, existing connection and arbitration functions can be utilized.", "Existing types of message can likewise be utilized.", "A currently preferred embodiment of an arrangement that has the significant characteristics of the invention will be described below with reference to the attached figures, in which FIG.", "1 shows schematically and in principal diagram form, a transmitting node or transmitter unit which transmits information in question to receiver units in a CAN system and where the receiving nodes transmit or forward all or parts of the received information to the distributed control system (CAN), FIG.", "2 shows an enlargement of receiving nodes operating in parallel which, in the event of wireless links not subject to interference, receive in parallel and in a coordinated way the messages received from the transmitting node or transmitter unit, FIG.", "3 shows in principal diagram form coordinated and simultaneous transmission of the messages received in the receiver units on the system's few-wire link or bus, and forwarding to the executing device incorporated in or connected to one or more nodes in the CAN system, FIG.", "4 shows in principal diagram form, the system according to FIG.", "1, but where the wireless transmissions to the receiving nodes are partly or completely not subject to interference to one or more of the receiving nodes and are partly subject to interference or are absent to other nodes, FIG.", "5 shows enlarged and in principal diagram form, receiving nodes in the system according to FIG.", "4, FIG.", "6 shows in principal diagram form that only receiving nodes with radio links from the transmitting node or transmitter unit that are not subject to interference effect a connection to the few-wire link for forwarding to the executing device in question in a node connected to the few-wire link, FIG.", "7 shows in an enlarged and principal diagram form, a receiving node with wireless radio link from the transmitting node or transmitter unit in question subject to interference, FIG.", "8 shows in diagrammatic form, a concrete example of the construction of a message with an SSI value part, a g-node identifier part, a PC message node and a data part, FIG.", "9 shows in principal and from above in a horisontal view vehicles/cars in a garage, and FIG.", "10 shows schematically units arranged in a Bluetooth arrangement.", "There can be two types of link incorporated in the system, wireless links and wired links.", "Wired links also include optical connections via opto links.", "The transmission of messages can be carried out in two steps (a) and three steps (b): a) 1.From wireless transmitter to wireless receivers 2.From wireless receivers to wired link b) 1.From wired link to wireless transmitter(s) 2.From wireless transmitter to wireless receivers per channel 3.From wireless receivers to wired link.", "The wireless transmitters each transmit on their own “channel”.", "A channel is characterized in that, when transmitting, each transmitter has access to a part of the available bandwidth in the ether which is exclusive to the system.", "The exclusivity can be an allocated frequency in a time interval, for example as in the known Bluetooth protocol or IEEE 802.11 for jumping frequency or a correlation code as in IEEE 802.11 Direct Sequence Spread Spectrum.", "A transmitter and one or more receivers can operate on one and the same channel during one and the same time interval.", "Nodes which are connected both to the wireless network and to the wired network are here designated g-nodes.", "They act as “gateways”, that is they receive a complete message on the wireless link and ascertain in a known way that it has been correctly received, by means of check codes, error-correction codes, etc.", "Thereafter the message is transmitted on the wired link.", "Each message has an identity on the respective medium which is at least unique for that transmission occasion.", "The identity can be a bit code or can consist of a particular time slot in a scheduled system or a combination of these methods.", "The identity can be common or different for the two media.", "If they are different, the association between the identities is known by the respective g-node.", "Such an association can be made in a plurality of ways, some of which are described in the patent referred to above.", "In one embodiment, the Bluetooth protocol can be modified so that several slaves are allocated the same time slot for reception.", "A CAN message (here an LLC data frame) is generated in a PC (computer/micro-computer/processor) equipped with a radio interface of the Bluetooth type.", "The CAN message is packaged as data in a Bluetooth message which is sent by the PC and received by two or more g-nodes.", "The g-nodes that have received the CAN message error-free according to the Bluetooth protocol's error-detection mechanisms thereafter send out the CAN message on the CAN bus.", "If several g-nodes commence their transmission synchronized to the same Strat of Frame (SOF), they will simultaneously send the message bit by bit, which will work because the messages are identical.", "If any g-node or g-nodes can not synchronize with the message that the first g-node sends, then they change over into receiving nodes in accordance with the CAN protocol.", "They will then receive a message that is identical to the one they were in the process of sending.", "When they have ascertained that such is the case, they can refrain from sending the message.", "In this way, a parallel redundancy is achieved for signalling from the PC to the CAN network.", "In the opposite direction, serial redundancy can be achieved.", "Each g-node is allocated a time slot for transmission and information about which CAN identifier identifies the CAN messages that are to be sent to the PC.", "At the same time, they obtain the messages on the CAN bus in accordance with the CAN protocol.", "The messages that are to be sent to the PC are packaged as data in a Bluetooth message that is sent in the respective time slot.", "The PC then receives one or more identical messages from the CAN system.", "This method is here designated “P-presentation”.", "If information is required about which recipient has received the strongest signal, the following procedure can be applied.", "It assumes that the radio part of the g-nodes provides a value for the signal strength, (signal strength indicator, SSI), for example a measurement value 0-255.The CAN identifier is then divided into at least three fields: One which contains the value 255−the SSI value, one which indicates which g-node is sending the message and one which indicates that it is one (of several) messages from the PC.", "Now if the SSI value−255 is inserted first in the CAN identifier, the g-node that has the highest signal strength will gain access to the bus if all the g-nodes commence signalling simultaneously.", "The information about which g-node has the strongest signal and the actual value of this is thus to be found in the CAN identifier and is available to all nodes that can use this information.", "In an example of organisation of CAN identifiers by signal strength, the identity can be used of up to 16 g-nodes and the identity of up to 64 messages from the PC.", "More information about how CAN identifiers can be constructed to give the required characteristics is to be found in the specification for the known CAN-Kingdom protocol.", "This method is called “SSI-presentation”.", "In the event of a so-called multipath situation, messages with errors can be obtained, in spite of the signal strength being high.", "The messages with errors can be detected by the error-detection mechanism in the protocol as bit errors in the message.", "If the error persists, the conclusion can be drawn that there is a multipath situation and measures can be implemented in the form of a change of transmitter, transmitter position, amendment of propagation diagram, etc.", "The invention can be used in connection with mobile transmitters.", "In the first position a transmitter has contact with two receivers, then three, thereafter two.", "Maximal transmission reliability is ensured in a simple way by means of P-presentation.", "Using SSI-presentation, it is also possible to obtain a good idea of where the mobile transmitter is located.", "Examples of combinations of a mobile transmitter and a fixed network can be a car in a garage, a car at a service station, an ignition key (or corresponding item that can comprise a similar function, for example a mobile phone, identity card, etc,) for a vehicle.", "In FIG.", "1 a distributed control system of the control area network type is provided by CAN (or CAN KINGDOM).", "The system comprises or interacts with a transmitting node or transmitter unit 1 which can consist of a computer-based node or unit of a known type.", "The node or unit 1 can consist of a mobile unit which in a known way is connected to or incorporated in a mobile system, for example an ordinary mobile system which is incorporated in or connected to the general or public telecommunications network.", "The node or unit 1 can be arranged to be fixed, which can be the case for service units associated with car repair workshops.", "A car repair workshop installation is indicated by 2 in FIG.", "1.The transmitting node or transmitter unit 1 can also be arranged to be mobile, where reference is made to the movement of the mobile units in the said telecommunications and/or data system which is symbolized by 3 in FIG.", "1.The transmitting node or transmitter unit 1 is thereby of the type that can effect transmission of radio messages.", "The node or unit is thereby provided with one or more antennas 4.The node or unit 1 shown in FIG.", "1 is of the type that establishes wireless connections, in this case radio links 5, 6, 4, 7, 8, etc.", "Alternatively, the transmitting node or transmitter unit 1 can consist of two or more transmitting nodes or transmitter units.", "The transmission of the messages or information in question on the different frequencies is carried out in different directions which are symbolized by 9, 10, 11 and 12 in FIG.", "1.The transmission is carried out to the receiver units 13, 14, 15, 16, etc.", "These receiver units are provided with antennas 17, 18, 19 and 20 respectively.", "The receiver units are thus tuned for frequencies in the respective link and the frequencies are preferably selected in the free ISM band, where the 2.45 GHz or 5.8 GHz band can be used.", "The receiver units 13, 14, 15 and 16 are connected in a known way to a few-wire digital link which is normally used in the control area system in question.", "Further nodes 22, 23, 24, 25, 26, etc, are also connected to the few-wire link or the bus 21.The link 21 can be relatively long and the different receiving nodes 13, 14, 15, and 16 and said additional nodes 22, 23, 24, 25 and 26 are geographically separated.", "The transmission from the transmitting node or transmitter unit to the receiver unit is in this case digital and the links operate with slot functions.", "The respective message can comprise identification bits and code bits in a so-called “Header”, data bits D, and code bits and control bits in a so-called “Tail”.", "Such a message is indicated by 27 in FIG.", "1.The message in question is transmitted via the link 5.Corresponding messages are transmitted via the links 6, 7, 8, etc.", "The few-wire link 21 and the nodes connected to it also operate digitally.", "The nodes or units incorporated in the system are mutual time-clearances in the present application and in the said Swedish application 0001148-6.A vehicle is also indicated in FIG.", "1 and is designated 2′.", "Reference is hereby made to the said Swedish patent applications, which is also the case concerning the said machines and/or processes.", "The units or components incorporated in a process are indicated by 2″ in FIG.", "1.In FIG.", "1 the geographical distances between the receiver units 13 and 14 and the receiver units 14 and 15 are indicated by A and B respectively.", "FIG.", "2 shows how messages 27, 27′, 27″, etc, which are received by the receiver units 13, 14, 15 via the links 5, 6, 7, and 8, are downloaded in parallel and simultaneously in the receiver units 13, 14, 15, 16, etc.", "In the present case half of the downloading function has been implemented, that is the first parts 27a, 27′a, 27″a, etc, have been downloaded in the respective units 13, 14, 15, etc.", "Continued downloading of the parts 27b, 27′b, 27″b of the message will be carried out in the continued downloading procedure.", "This downloading of the message in the receiver units 13, 14, 15, 16, etc, is carried out in a known way and reference is made among other things to the references listed above.", "The coordinated downloading function can be synchronized using the time functions in the system.", "The messages to the various receiver units are constructed in a corresponding or similar way.", "FIG.", "3 shows among other things that the messages 27, 27′, 27″, etc, transmitted in parallel from the transmitting node or the transmitter unit 1 (FIG.", "1) via the links 5, 6, 7, 8, etc, have completed being downloaded at the nodes 13, 14, 15, 16, etc.", "These nodes have essentially the same construction, at least concerning the said receiving functions and transmitting and interacting functions respectively via the few-wire link 21.In accordance with the concept of the invention, in the case shown in FIG.", "1 with links that are not subject to interference between the transmitting node or transmitter unit and the receiving nodes 13, 14, 15, 16, there is parallel connection of the receiver units to the few-wire link.", "Using this connection, the said messages 27, 27′, 27″ or parts thereof are transmitted via known means 13a, 14a, 15a, etc, to the few-wire link 21.The parallel and simultaneous connection to the link 21 is shown by i1, i′, i″.", "As a result of the parallel and simultaneous transmission of the messages to the few-wire link 21, a number of messages are obtained on the few-wire link which in principle are laid one on top of each other.", "In FIG.", "3, this has been symbolized by the frames or blocks 28, 29, 30, 31, etc, which refer to the messages 27, 27′, 27″ and 27′″ respectively.", "In FIG.", "3, for the sake of clarity the blocks or frames 28, 29, 30 and 31 have been shown laid out in a common plane (the plane of the paper).", "The overlaying of each other by the messages has been symbolized by arrows 32, 33 and 34.The frames or blocks appearing at the link 21 have also been illustrated by reference arrows 35, 36, 37, 38, etc, to the various receiver units.", "The messages thus appearing on the few-wire link are received in the additional receiving nodes, of which one receiving node 39 has been shown in FIG.", "3.The additional receiving node 39 in question receives all or parts 40 of the said message which is sent on the link 21 by the units 13, 14, 15, 16, etc, The node 39 utilizes the received information or the received message in a known way in order to control the executing device 41.The direction of reception is indicated by 42 and the reception signals are indicated by i′″.", "The unit 39 comprises a processing device 43 which processes or selects information content from the received message 40 or part of the message 40 for forwarding to the executing device 41.The construction of the message can thereby be of the kind that comprises an identification part ID and a code part C. In addition, there is a data part and code and control parts CRC and CH respectively.", "The parts ID and C form the “Header” and the parts CRC and CH form the “Tail”.", "In accordance with the above, the transmitting node or transmitter unit (see FIG.", "1) consists of or comprises a mobile unit in the mobile system GSM which is provided with Bluetooth function.", "In a corresponding way, the distributed control system operates with message constructions that correspond to or are obtained from the said mobile system.", "Messages can in principle be sent in the opposite direction, but this two-way case does not concern the present invention and will therefore not be described here in greater detail.", "FIG.", "4 shows the construction according to FIG.", "1 with the transmitting node or transmitter unit 1 and the receiving nodes 13, 14, 15 and 16 and the additional units 22, 23, 24, 25 and 26.In this case, only the link 5 is established, so that there is a radio link that is essentially not subject to interference between the node or unit 1 and the receiver unit 13.A block 44 blocks the radio link 6 so that there is no connection to the node 14.The radio link 7 passes a block 45 which brings about a considerable interference in the radio link in question to the node 15.The link 8 is reflected by a block 46 so that no other radio link or links reach the receiver unit or receiver units 16 either.", "Thus, in this case, it is only the receiving node 13 that can forward the message to the few-wire link 21 and thereby to the executing devices in additional receiving nodes.", "FIG.", "5 shows that the receiving node 13 can download the message 27 in accordance with the above.", "The nodes 14 and 16 have in this case no radio link with the transmitting node or transmitter unit 1 according to FIG.", "4.The receiving node 15 receives a message that is essentially subject to interference 27″ via the radio link 6.FIG.", "6 shows that only the receiver unit 13 transmits the message 27 to the link 21.Other receiving nodes are not connected to the few-wire link by the circuits 14a, 15a, etc, not being activated for access to and transmission on the link 21.Like other receiving nodes, the said receiver units 14, 15, 16 detect the message transmitted by the node 13 on the few-wire link 21.The arbitration function in the system can thereby be selected so that when the nodes 14, 15, 16 detect all or parts of the message 27 that was transmitted by the node 13 on the link 21, [they] are prevented from making further attempts to connect to the few-wire link after the unit 13 has sent the message 27.The detected or received part or the detected or received message can thereby cause a circuit 14b, 15b, etc, in the receiving nodes 14, 15, 16 to generate a cancellation signal that prevents the unit 14, 15, 16, etc, in question from attempting to make further subsequent connections.", "FIG.", "6 also shows a signal i2 which is a selection signal that selects the receiving node which has the link which is the least subject to interference in the case where all the receiver units 13, 14, 15, 16, etc, have links that are subject to interference.", "Only the selected link is thereby connected to the few-wire link.", "The signal i2 is shown only symbolically and is generated in all the receiver units 13, 14, 15, 16, etc.", "FIG.", "7 shows that the node 15 received a message 27′ that is essentially subject to interference, and that circuits 15a prevent the transmission of that message on the link 21.FIG.", "7 also shows a cancellation signal i1 which, for example, is generated in a circuit 15b [and] prevents the unit 15 from making further attempts to connect to the link 21 after the unit 13 (see FIG.", "6) has sent its message 27 in question, not subject to interference, on the link 21.FIG.", "8 shows an example of a concrete construction of a message.", "The construction is the one described above, in which the SSI value is indicated by 28, the g-node identifier part by 29, the PC message number part by 30 and a subsequent part (the data part) by 31.In this case, the number of g-nodes is thus 16 and the number of PC message numbers is 64.In the above, a case has been described where the node 13 obtained reception via a radio link not subject to interference and the units 14, 15, 16, etc, had radio links that were absent or essentially subject to interference.", "Of course, two or more receiving nodes can operate in parallel with radio links not subject to interference, while one or more nodes have radio links essentially subject to interference or absent.", "It can be seen thereby that the system is highly insensitive to various interference phenomena that can arise in association with the radio links.", "A unit that is not yet connected can listen on a fixed frequency where it knows that time messages will appear.", "These can, in addition to the time, also contain a header that identifies the system.", "In this way, the unit that is not yet connected can synchronize itself to the network on the messages which are due to be sent in order to maintain the global time in the system.", "FIG.", "9 shows that one mobile transmitter 32 in a car in a first position 33 of the transmitter area is in contact with or covers two receivers 34 and 34a of the garage arrangement.", "In a second position 33′ of the transmitter area this one covers three receivers 33, 35 and 36.In a third position 33″ covers two receivers 35 and 36.In the shown way the transmitter area can cover different numbers of receivers at its movement into the garage.", "In the parked positions the transmitter areas 37, 38 and 39 can cover different numbers of receivers.", "Thus, the area 37 covers two, the area 38 covers one and the area 39 covers three.", "Thus, respective area get contacts with the receivers in varying way when the car moves and the cars are parked, respectively.", "FIG.", "10 refers to the above mentioned embodiment with a number of slaves allotted the same time slot.", "A PC-unit 40 includes arrangement (Tool/SW) for dealing with CAN messages or CAN DATA and arrangement 42 for CAN/Bluetooth wrapping and CAN Higer Layer Protocol.", "Via member 43 the omnidirectional antenna arrangement 44 transmits BT-Packets 45, 45′, 45″ in the same time slot in different directions 46, 46′, 46″.", "Units or mobiles 47, 47′ and 47″ receives the BT-Packets via their antennas and each unit is arranged for Bluetooth reception and having the corresponding arrangement for Bluetooth/CAN wrapping, CAN Higher Layer Protocol, CAN, and a CAN driver for attaining CAN messages to or from a bus 49, to which different units 50 are connected by means of wires 51.In the disclosed embodiment units 47 and 47″ receives the BT Packets 45 and 45″.", "The unit 47 does not receive the BT Packets owing to disturbances or another reason.", "The Bluetooth system creates its communication data, and the CAN-system create its own data.", "The invention is not limited to the embodiments described above by way of example, but can be modified within the scope of the following claims and invention concept." ] ]

[ [ "Method and device for the measurement of chemical and/or biological samples", "A device for the measurement of chemical and/or biological samples, in particular by means of luminescence spectroscopy, comprises an irradiation unit (12), a sample receiver (10), at least one optical unit (30) and a detector unit (40).", "Electromagnetic radiation of various wavelength ranges and/or polarizations are led from the sample (10) from the irradiation unit (12).", "The color marker in the sample (10), which contains at least one color marker, is stimulated into producing luminescence and gives off light.", "The emitted light is led by means of an optical unit to a detector unit.", "The light emitted by the color markers is detected by detectors (42, 44, 46, 48) in the detector unit.", "According to the invention, the measurement results may be improved on the irradiation unit (12) generating a pulsed irradiation.", "The irradiation unit is thus preferably controlled by a control unit (18) in such a way that the irradiation pulses impinge on the sample (10) in a temporal sequence." ], [ "1.A method for measuring chemical and/or biological samples by means of spectroscopic or microscopic methods, in particular using luminescence spectroscopy, wherein: the sample including at least one marker in a measuring volume is irradiated with electromagnetic radiation of at least two different wavelength ranges and/or polarizations, the excited marker emits radiation in an emission wavelength range, and the emitted radiation is detected by at least one detector, characterized in that the electromagnetic radiation used to excite the marker is pulsed in at least two different wavelength ranges and/or polarizations and the radiation pulses of the individual wavelength ranges and/or polarizations impinge on the sample with a temporal offset.", "2.The method of claim 1, wherein a relative movement between the measuring volume and the marker, in particular between the measuring volume and a single marker molecule, occurs in a relative movement period and the electromagnetic radiation used to excite the marker is pulsed in at least one wavelength range and/or one polarization such that at least two radiation pulses hit the marker within the relative movement period in which the measuring volume contains the marker.", "3.The method of claim 1 or 2, wherein the marker, in particular a single marker molecule, diffuses through the measuring volume during a diffusion period and the electromagnetic radiation used to excite the marker is pulsed in at least one wavelength range and/or one polarization such that at least two radiation pulses hit the marker within the diffusion period.", "4.The method of claim 1 or 2, wherein the marker is stationary and a relative movement between the measuring volume and the marker occurs by moving the measuring volume and/or by moving the sample receiver containing the marker.", "5.The method of one of claim 1, wherein a radiation pulse is generated only after the excitation of the marker, excited by a previous radiation pulse of a different wavelength range and/or a different polarization, has substantially decayed.", "6.The method of one of claims 1-5, wherein the radiation of the individual wavelength ranges and/or polarizations is emitted in repetitive sequence.", "7.The method of one of claims 1-6, wherein the sample includes two markers, each marker being excited by one wavelength range and/or one polarization, respectively.", "8.The method of one of claims 1-7, wherein a red and/or a green color marker are used together with red and/or green excitation light, the green excitation light has a wavelength range preferably of 480 to 550 nm, more preferred of 485 to 535 nm, and the red excitation light has a wavelength range preferably of 630 to 690 nm, more preferred of 635 to 655 nm.", "9.Device for measuring chemical and/or biological samples by means of spectroscopic or microscopic methods, in particular by luminescence spectroscopy, comprising a irradiation unit (12) for generating electromagnetic radiation in at least two different wavelength ranges and/or polarizations, a sample receiver (10) for holding the sample including at least one marker, a detector unit (40) comprising at least one detector (42-48) for detecting the radiation emitted by the sample, and at least one optic unit (30) directing the radiation from the irradiation unit (12) to a measuring volume in the sample receiver (10) and/or directing the radiation emitted by the sample to the detector unit (40), characterized in that the irradiation unit (12) generates radiation pulsed in at least two different wavelength ranges and/or polarizations and the radiation pulses of the individual wavelength ranges and/or polarizations are temporally offset.", "10.The device of claim 9, wherein a relative movement between the measuring volume and the marker, in particular between the measuring volume and a single marker molecule, occurs in a relative movement period and the irradiation unit generates radiation that is pulsed such that at least two radiation pulses hit the marker within the relative movement period in which the measuring volume contains the marker.", "11.The device of claim 9 or 10, wherein the marker, in particular a single marker molecule, diffuses through the measuring volume during a diffusion period and the irradiation unit (12) generates radiation pulsed in at least one wavelength range and/or one polarization such that at least two radiation pulses hit the marker within the diffusion period.", "12.The device of one of claims 9-11, wherein it comprises means for moving the measuring volume and/or the sample receiver containing the marker.", "13.The device of at least one of claims 9-12, wherein the irradiation unit is connected to a control unit (18), preferably a mode coupler, for generating the radiation pulses.", "14.The device of claim 13, wherein the control unit (18) controls the radiation pulses such that a radiation pulse is generated only after the excitation of the marker, excited by a previous radiation pulse of a different wavelength range and/or a different polarization, has substantially decayed.", "15.The device of claims 13 or 14, wherein the control unit (18) controls the radiation pulses such that the radiation pulses of the individual wavelength ranges and/or polarizations are emitted in repetitive sequence.", "16.The device of one of claims 9-15, wherein the irradiation unit (12) comprises at least two radiation sources, in particular lasers (14, 16), for generating different wavelength ranges and/or polarizations.", "17.The device of claim 12, wherein the irradiation unit (12) comprises a common control unit (18) for all radiation sources (14, 16).", "18.The device of claim 17, wherein the control unit (18) is connected to the radiation sources (14, 16) through a respective trigger wire (20, 22), the time interval between the radiation pulses being defined by the length of the trigger wires (20, 22).", "19.The device of one of claims 16-18, wherein the irradiation unit (12) comprises two radiation sources (14, 16), one radiation source (14) generating red light and the other (16) generating green light, and wherein the green light has a wavelength range preferably of 480 to 550 nm, more preferred of 485 to 535 nm, and the red light has a wavelength range preferably of 630 to 690 nm, more preferred of 635 to 655 nm.", "20.The device of one of claims 9-19, wherein the irradiation unit (12) comprises only one non-polarized pulsed radiation source (14), the irradiation unit (12) additionally comprising (a) a beam splitter and, in each beam path established, a polarizing filter as well as a component for combining the beam paths, or (b) a polarizing beam splitter for establishing two beam paths of different polarizations as well as a component for combining the beam paths, or (c) a rapidly rotating polarizing filter in an unsplit beam path.", "21.The device of one of claims 9-20, wherein the irradiation unit (12) comprises two polarized pulsed radiation sources (14, 16) of the same wavelength range but of opposite polarization.", "22.The device of one of claims 9-21, wherein the detector unit (40) comprises only one combination detector connected to an evaluating unit (52), the evaluating unit (52) evaluating the radiation emitted by the at least one marker separately due to the time interval.", "23.The device of one of claims 9-22, wherein the detector unit (40) comprises two detectors (42, 44), in particular one for detecting the light emitted by red color markers and one for detecting light emitted by green color markers.", "24.The device of one of claims 9-23, wherein the detector unit (40) comprises a beam splitter (50), arranged before the detectors (42-48), for generating two beams (54, 56) of different polarization and each beam (54, 56) is detected by at least one detector (42, 44, 46, 48).", "25.The device of at least one of claims 9-24, wherein the marker, in particular a single marker molecule, has a characteristic fading time during which only it emits radiation, and the irradiation unit (12) generates radiation that is pulsed in at least one wavelength range and/or polarization such that at least two radiation pulses hit the marker within the fading period.", "26.The method of at least one of claims 1-8, wherein the marker, in particular a single marker molecule, has a characteristic fading time during which only it emits radiation, and the electromagnetic radiation used to excite the marker is pulsed in at least one wavelength range and/or one polarization such that at least two radiation pulses impinge on the marker within the fading period." ], [ "The invention relates to a method and device for the measurement of chemical and/or biological samples, in particular with the use of luminescence spectroscopy.", "A known method for the measurement of such samples is the screening of the samples.", "In screening, the interaction between two bio-molecules in the presence of a test substance is studied.", "The bio-molecules can be, for example, the pairs of ligand-receptor, substrate-enzyme, protein-protein, or protein-DNA.", "A measurable signal may be produced either by the bio-molecules themselves, or, as in most cases, sample markers have to be bound to the bio-molecules.", "As markers, substances are employed that produce signals based on radio activity, luminescence or absorption.", "When color markers are used that produce luminescence, the color markers are excited by electromagnetic radiation, e.g.", "suitable laser light, so that the electromagnetic radiation lifts an electron to a higher energy level and the color marker gives off light, i.e.", "luminesces, by the electron returning to its original energy level.", "The probability of the return of the electron to its original energy level, and thus of the emission of luminescence, is exponentially distributed overtime.", "The mean duration of the exited state is therefore also referred to as the luminescence life.", "Since, in most cases, luminescent markers have but a slight influence on the interactions between bio-molecules and are extremely sensitive compared to other markers, the use of luminescent color markers is particularly advantageous.", "Information on reactions between two bio-molecules are obtained by establishing a relation between the change in the light emitted by the color markers and the reaction of the bio-molecules.", "In an example of a measuring method, a target molecule is first exposed to a fluorescent reagent as a luminescent reagent adapted to bind to the target molecule.", "If, for example, the intensity of the fluorescence changes during the binding, it can be used to quantify the binding.", "In another experiment, the target is exposed to both the fluorescence-marked reagent and a single substance.", "If a binding between the substance and the target occurs, the fluorescence-marked reagent is separated from the target.", "Thus, the ratio of bound and free marked molecules changes.", "In turn, this entails a change in the emission of fluorescence by the sample and it can be assessed whether and in how far a substance binds to the target.", "In order to increase the amount of information to be acquired during a measurement, a plurality of markers, in particular two color markers, may be used.", "Depending on the excitation energy of the two color markers, two electromagnetic radiation sources, e.g.", "lasers, of different wavelengths are employed to excite the color markers.", "For example, a red and a green color marker is used in combination with a red and a green laser.", "When red and green color markers were used, it has been found that, when a red laser was used together with a green laser, the intensity of the light emitted by the red color marker is less than the intensity of the red color markers, when the same is irradiated exclusively with red laser light.", "This loss of intensity entails a loss of information and leads to falsified results.", "This phenomenon can also be found when color markers of different colors are used.", "It is the object of the invention to provide a method and device for the measurement of chemical and/or biological samples, in particular with the use of luminescence spectroscopy or microscopy, by which improved results can be obtained.", "In particular, it is the object of the invention, to achieve an improvement of the measured results for commonly used red and green color markers.", "The object is solved according to the invention with the method of claim 1 and the device of claim 15, respectively.", "According to the invention, an improvement of the measured results can be achieved by pulsing the electromagnetic radiation used to test the samples at least in a wavelength range and/or at least one polarization.", "A measuring volume of the sample including at least one color marker is irradiated with electromagnetic radiation of at least two different wavelengths/wavelength ranges and/or polarizations, the radiation being pulsed in at least one wave-length/wavelength range and/or at least one polarization.", "Specifically, it is possible to provide for a relative movement between color marker and measuring volume during a relative movement time; in this case, it is desirable to pulse the radiation such that at least two radiation pulses impinge on the color marker per relative movement time.", "Pulsating one or a plurality of wavelength ranges and/or one or a plurality of polarizations, is preferably effected such that at least two radiation pulses impinge on the color marker within a time during which the marker diffuses through the measuring volume.", "The measuring volume, which in high-throughput screening preferably corresponds to the focal point of an irradiation optic, is preferably smaller than 10−9 l, in particular smaller than 10−12 l. A particle, such as a molecule, provided with a color marker diffuses through the measuring volume.", "According to the invention, the time interval between successive pulses is chosen such that a particle diffusing through the measuring volume is hit by at least two radiation pulses and the color marker is excited to luminesce at least twice.", "In this case, the relative movement time corresponds to the diffusion time of the marker through the measuring volume.", "In another embodiment, a stationary marker (e.g.", "a marked immobilized macro-molecule, a marked sedimented or adherent cell, etc.)", "may be examined, the relative movement being effected in particular by a stepped movement of the measuring volume.", "This scanning process can be performed either by moving the sample with the exciting beam path being stationary or by scanning the sample by varying the position of the exciting beam path.", "In this case, the relative movement time corresponds to the time required to move the measuring volume along the marker or to move the marker through the measuring volume by moving the sample.", "The scanning could also be continuous, this embodiment eventually corresponding to a step-wise scanning in infinitesimally small steps.", "Of course, any intermediate form is conceivable between these extremes, such as scanning slowly diffusing markers or using a parallelized optic configuration (e.g.", "by using a plurality of optical fibers (a fiber bundle)) to simultaneously produce several measuring volumes.", "Typical diffusion times of particles through the measuring volume are within the range of 50 μs to 100 ms, in particular 2 ms to 10 ms.", "The interval between successive radiation pulses is preferably chosen as short as to have the individual particles preferably be hit by at least 100 radiation pulses even with diffusion times that short.", "It is most preferred to have at least 1,000 and in particular at least 5,000 radiation pulses hit during the diffusion through the measuring value.", "Because of the high number of radiation pulses, the electrons of the color markers are raised to higher energy levels several times, therefore giving off photons several times while falling back to their original level.", "The more light is emitted from a color marker bound to a particle, such as a molecule, the more information can be acquired within the diffusion time.", "This allows for an increase in the expressiveness of the information obtained.", "Similarly, it is also desirable to provide a high number of radiation pulses during the other above-mentioned possible relative movement times.", "In one embodiment of the present method, such a radiation pulse could be sub-pulses as they are known from multi-photon excitation, in particular dual-photon excitation, to excite a luminescence emission by the color marker.", "When using electromagnetic radiation, for example, with two different wave-length ranges, for example, such as red and green laser light, the red or green laser light continuously irradiates the sample and the respective other laser light is pulsed.", "When using red and green laser light, preferably the green laser light is continuous and the red laser light is pulsed.", "The luminescence signal caused by a pulsed excitation has a characteristic course in time, the intensity decreasing over time after one excitation to the next excitation.", "The course in time of a pulsed excitation is thus known with sufficient exactness.", "The excitation and the emission of the color markers excited by the other wavelength range have a continuous course in time.", "Both luminescence signals emitted by the sample are thus discernible by their different courses in time.", "It is thus possible to significantly improve the measured results by pulsing a light source.", "Since laser light of different wavelengths is emitted and only one of both laser light sources has to be pulsed, when two laser light sources are used, this is a simple and economic modification of the device required to practice the method.", "Specifically, it is advantageous in the above example to pulse the red laser, since green pulsed lasers are still very costly today.", "Otherwise, a combination of all wavelengths tuned to the color markers to be examined is possible, the use of at least two different wavelengths between 350 nm and 800 nm being preferred.", "Moreover, in another embodiment of the present method, the use of UV laser diodes would be possible.", "Another advantage of the present method is that only one detector is needed to detect the two luminescence signals emitted by the sample.", "Of course, the present invention can also be applied for more than two different wavelength ranges.", "It is likewise possible to use different polarizations of the radiation exciting the color markers, instead of using different wavelength ranges.", "Similarly, an improvement of the measured results can be achieved with the above present method if only one color marker is employed, since the same is excited differently by radiation of different wavelength ranges and/or different polarizations, for example, so that different sets of information can be obtained.", "Among other things, the invention is based on the fact that a red color marker excited by red laser light could be destroyed by green laser light.", "The destruction is caused by an electron of the red color marker is raised to a higher energy level by the red laser light and is further excited by the green laser light.", "This excitation by the green laser leads to an increase in the probability of the destruction of the red color marker, e.g.", "by ionization.", "In most cases this destruction is irreversible, i.e.", "the color marker can no longer be excited to emit luminescence and is lost for further measurement.", "This causes a deterioration in the measuring signal.", "This is also true for color markers of other colors.", "In a particularly preferred manner, a further inventive improvement of the measuring signal can be achieved by pulsing the radiation used to examine the samples, such as the laser light, and by using electromagnetic radiation having at least two different wavelength ranges and/or polarizations that are given off in a deferred manner.", "Here, the pulsing is effected in at least two wavelength ranges and polarizations, respectively.", "The electromagnetic radiation of different wavelength ranges and/or different polarizations thus impinges successively on the sample.", "For example, the risk of a destruction of the color marker is reduced by the fact that the radiation pulse that could destroy the color marker impinges at a moment in time in which it highly probable that the excited electron has already returned to its original state.", "By pulsing the electromagnetic radiation in all wavelength ranges and/or both polarizations, further improvements of the measured results can be obtained.", "An improvement of the measured results can be obtained by the above inventive method even if only a single color marker is present in the sample.", "This color marker is excited differently by the electromagnetic radiation of different wavelength ranges and/or different polarizations.", "For example, different polarizations can be detected in the emitted radiation, allowing to draw conclusions on the rotational properties of the color markers.", "The terms color substance marker/color marker/marker refer to both a marker supplied to the sample (such as rhodamine, oxazine, cyanine or another color substance) and a marker inherent to the substance to be examined, i.e.", "substances that preferably have luminescent properties, such as certain bio-polymers.", "The terms color substance marker/color marker/marker also refer to other substances that may be examined using spectroscopic or microscopic methods such as Raman scattering.", "Luminescence particularly also includes fluorescence and phosphorescence.", "The term wavelength range used herein also includes, besides wider excitation wavelength ranges typically extending over a plurality of nanometers, more discrete wavelengths.", "In particular, it may be desired to provide a monochromatic excitation for at least two different wavelengths.", "In the following, the invention will first be discussed with reference to the use of electromagnetic radiation with different wavelength ranges.", "For a better understanding, this is done with reference to an example including a red and a green color marker, each being excited by red and green laser light, respectively.", "According to the invention, the laser light, i.e.", "the red and the green laser light in the example discussed, is pulsed to excite the color markers.", "In addition, the laser light pulses of the individual wavelength ranges are deferred in time relative to each other.", "Due to the time-synchronized temporal offset of the pulses relative to each other, the sample is always hit only by either a red or a green laser light pulse at any moment.", "Even with an extremely small interval between the green laser light pulse and the red laser light pulse, substantially fewer red color markers are destroyed.", "This increases the intensity or the count rate of the light given off by the color markers.", "Thus, the measuring results can be improved significantly.", "According to the invention, when using red and green color markers, first, one or a plurality of light pulses of the red laser light and, subsequently, one or a plurality of light pulses of the green laser light are directed to the sample.", "A time gap exists between the last red light pulse and the first green light pulse.", "The time span is chosen such that the excitation of the red color markers has substantially decayed again so that the electrons of the red color marker are not further excited from a high energy level by the green laser light pulse and the red color markers will not be destroyed in the course.", "Preferably, in the present method, a laser light pulse is generated only after the substantial decay of the excitation of the color marker excited by previous laser light pulse of a different wavelength range.", "Thus, for example, the green laser light pulse is directed to the sample only when the excitation of the color markers excited by the red laser light pulses has decayed substantially.", "Preferably, the next laser light pulse is fired on the sample only hen the excitation of the previously excited color marker has decayed by at least 90%, more preferably by at least 95%, most preferably by at least 98%.", "Hereby, the measuring results obtainable are significantly improved.", "The necessary temporal offset of two successive light pulses of different wavelength ranges depends on the luminescence life of the color marker used.", "For a color marker with a life of 3 ns, the necessary temporal offset is at least 2 ns, preferably at least 7 ns.", "For a life of 1 ns, it is at least 0.7 ns, preferably at least 2.3 ns.", "Pulses with different intensities can be used, which is also true for non-luminescent excitation.", "When a red and a green laser light are used, the distance between a green laser light pulse following a red laser light pulse and a red pulse is crucial, since a green laser light pulse emitted too early or simultaneously with the red laser light pulse could cause the destruction of the red color marker.", "However, the temporal offset between a red laser light pulse following a green laser light pulse and a green pulse is of no importance, since the green color marker is not destroyed by the red laser light pulse.", "In this case, it should merely be ensured that both pulses do not occur at the same time.", "The pulse length of the individual radiation pulses impinging on the measuring volume is preferably smaller than 1 ns.", "In particular, the radiation pulses are smaller than 500 ps and, more preferred, smaller than 300 ps.", "The pulse length specifically depends on the time deferment of successive pulses.", "Here, as stated above, it has to be made sure, according to the invention, that the excitation of a color marker has substantially decayed.", "When modern lasers are employed, it is even possible to achieve pulse widths smaller than 10 ps.", "For fluorescence excitation, the pulse frequency of the laser light preferably is 20-100 MHz, more preferably 60-80 MHz.", "The pulse frequency of the individual laser light ranges are preferably identical so that the distances between the successive laser light pulses of different wavelength ranges and/or polarizations remain constant over time.", "Preferably, the sequence of the laser light pulses of the individual wavelength ranges and polarizations is repetitive.", "For example, when three lasers with different wavelength ranges are used, a light pulse is sent to the sample first by the first, then by the second and thereafter by the third laser, whereafter the first laser again emits a pulse, followed by the second laser, and so on.", "When two lasers are used, e.g.", "a red and a green laser, the laser light pulses are preferably generated alternately.", "According to the above described preferred embodiments of the invention with reference to the use of laser light and the laser light pulses generated according to the invention, the same inventive effect is caused when other electromagnetic radiation, such as radiation in the invisible wavelength range, is used.", "Corresponding radiation pulses have the same effect as laser light pulses.", "Likewise, it is possible with the above described embodiments, to use a sample with only one color marker.", "This one color marker is excited differently by radiation pulses of different wavelength ranges, the emitted radiation being different or possibly being different depending on the wavelength ranges of the exciting radiation.", "It is also possible to employ different pulse frequencies of common multiples (e.g.", "40 MHz and 80 MHz).", "A constant offset in time between the two laser light pulses would still be ensured.", "This is advantageous, if one of the two lasers, e.g.", "the red laser, has less power.", "Since the intensity of the luminescence emission is proportional to the excitation power, a pulse frequency of the green laser (e.g.", "40 MHz) reduced by half with respect to the red laser (e.g.", "80 MHz) can result in a comparable intensity of the luminescence emission by the red and the green color marker.", "Despite the lower excitation power, the red color marker is excited twice as often.", "In particular, the invention also provides for detection with the use of Raman scattering instead of luminescence effects (luminescence spectroscopy and luminescence microscopy).", "In this case, excitation sources with particularly high repetition rates can be used because of the instantaneous emission of the Raman photons.", "Here, it is particularly advantageous, to exploit surface-amplified Raman emission, since the effective cross section and, thus, the number of emitted photons is particularly high.", "The conventional methods for exploiting the surface-amplified Raman emission, such as the metallizing of surfaces, especially particle surfaces, with silver, can be applied here.", "The present method using two or more pulsed electromagnetic beams of different wavelength ranges, offers a plurality of possible applications in the field of transient spectroscopy, such as the transient absorption spectroscopy (TRABS).", "For example, when using two pulsed laser light sources, all color markers are excited by the first laser light pulse.", "The second, deferred laser light pulse selectively saturates or photo-destroys certain color markers or fluorescent impurities.", "Hereby, a controlled lowering of fluorescence signals can be achieved and specific color markers may be made preferred or discernible.", "It is possible to employ a plurality of deferred pulses in different wavelength ranges, instead of time pulses with different wavelength ranges, the respective wavelength ranges being selected such that certain color markers are saturated or photo-destroyed.", "Results, corresponding to those obtained with the use of different wavelength ranges, may also be obtained with different polarizations of the electromagnetic beams.", "For example, instead of using red and green lasers, similar measuring results can be obtained with the use of vertically and parallel polarized light that also allows to make statements on the substance examined.", "The wavelength range of green laser light used preferably ranges from 480-550 nm, more preferred 485-535 nm.", "The wavelength range of a red laser preferably ranges from 630-690 nm, more preferred 535-655 nm.", "Preferred possible green laser light sources are high-quality argon ion lasers with monochromatic excitation at 488 nm, 466 nm, 502 nm, 515 nm, 528 nm, or Nd:YAG Lasers with monochromatic excitation at 492 nm or 532 nm.", "Red laser light sources preferably are krypton lasers with monochromatic excitation at 647 nm, such as red laser diodes available for various excitation wavelengths.", "Often applied methods using two color markers with two lasers of different colors are coincidence analyses discussed here with reference to fluorescence.", "Here, it can be determined in how far the color markers occur simultaneously or separately, i.e.", "to what degree they are bound to a common reagent or to two separate reagents.", "This makes use of the fact that in case of a simultaneous occurrence the fluorescent light of both colors is detected at substantially the same time, while in case of a separate occurrence, the detection of the fluorescent light of both colors is randomly distributed over time.", "Again, the example of red an green may serve to explain this.", "A special case of coincidence analysis is the cross correlation analysis.", "Here, the fluctuations over time of the fluorescent light of one color marker, Fgreen(t), are registered by a second detector detecting those of the other color marker Fred(t).", "The cross correlation function G(tc) is calculated based on these fluctuation traces.", "G ⁡ ( t c ) = < F green ⁡ ( t ) ⁢ F red ⁡ ( t + t c ) > < F green ⁡ ( t ) > < F red ⁡ ( t ) > Eq .", "⁢ 1 (t: measuring time, tc: correlation time, < .", ".", ".", ">: averaging over t) A cross correlation function not equal to zero will only be obtained, if the fluorescent lights of both color markers are connected (“correlated”) with respect to time.", "This is true when they are bound to a common reagent.", "The amplitude of the cross correlation function, G(tc=0), allows for a direct statement on the concentration, Cgreen+red, of this twice color-marked reagent compared to the concentrations, Cgreen and Cred, of the once marked reagents (Cgreen and Cred can be determined by other analytic methods).", "G ⁡ ( t c = 0 ) = C green + red ( C green + C green + red ) ⁢ ( C red + C green + red ) Eq .", "⁢ 2 By the already mentioned destruction of the color markers, an undesired reduction of the concentrations, Cgreen+red, of the twice color marked reagent (and of Cred, the red marked reagent) and, thus, a decrease of the cross correlation amplitude, G(tc=0) occurs.", "Such an analysis of a biological system by cross correlation would lead to falsified results and underestimate the actual biological concentration of the twice color marked reagent, Cgreen+red.", "Therefore, the invention provides for a temporal offset between the red and the green laser light pulses.", "By preventing the red color marker from being destroyed, this disadvantageous influence on the correlation amplitude is canceled out and a true cross correlation or coincidence analysis is obtained.", "Another problem with measuring methods using two color markers with two lasers of different colors is the cross-talk of the luminescence signal of both color markers.", "This will be explained with reference to fluorescence.", "The absorption and emission spectra of fluorescent colors are comparatively wide, i.e.", "they extend over a relatively large wavelength range, and they may overlap.", "This may cause the following problems that do not allow for an unambiguous attribution of the fluorescent light to a particular color marker or excitation laser—e.g., they could lead to falsifications in the cross correlation function.", "These will again be explained with reference to red and green: i) the fluorescent light of the red color marker may also be excited by the green laser (in a restricted manner)—the red fluorescent light excited by the green laser and the red laser, respectively, overlap; ii) a (small) part of the fluorescent light of the green color marker overlaps with the red fluorescent light (“crosstalk”)—an overlapping of the fluorescent light emitted by the green and the red color markers occurs on the detector for the red radiation; iii) the fluorescent light of the red color marker may be generated not only by the red laser, but also—through resonance energy transfer—by the green color marker excited by the green laser—similar to the case i), an overlapping of the red fluorescent lights excited by the red laser and, indirectly through energy transfer, by the by the green laser.", "It is the intention of the invention, to prevent this crosstalk of the fluorescence signals by deferring the red and green laser light pulses relative to each other.", "The fluorescent light excited by the green and the red laser can then be separated with respect to time and allows for an unambiguous attribution, as can be explained with reference to the three problem cases: after the green laser light signal, only the portion of the green color marker (suppression of the “crosstalk”) or of the red color marker in the red fluorescent light, the red color marker being excited directly or indirectly by energy transfer.", "If, after decay of this fluorescence, the red laser light pulse follows, the red fluorescent light only contains portions of the red color marker excited directly by the red laser.", "If, however, the green laser is timed to the decay of this fluorescence, the fluorescent light can clearly be attributed to the color markers or the exciting lasers.", "Thus, an uncompromised cross correlation analysis becomes possible, for example.", "A further inventive application of the temporal offset between two laser light pulses is the detection of different polarizations of the luminescent light—in the following referred exclusively to fluorescent light—of a color marker by only one detector.", "In conventional measuring methods for detecting different polarizations of fluorescent light, the color marker is excited by a laser polarized in a certain plane X and the polarization portion of the fluorescent light parallel and vertical to X is registered either simultaneously on two different detectors with the help of a polarization beam splitter, or, separated in time (several seconds), on a single detector by a timed switching of the transmission direction of a polarization filter.", "This allows to draw conclusions on the rotational properties of the examined color markers.", "It is the idea of this invention, to generate time-separated fluorescence signal pulses (decay pulses) of different polarization by two time-separated laser pulses polarized in the plane X and in the plane Y perpendicular thereto (this is possible even with only one laser when split).", "If the detection is done on a single detector with a polarization filter with a constant transmission direction, a comparable detection of the fluorescent light component parallel and vertical to the polarization plane of the laser may be performed: the transmission direction of the polarization filter is assumed to b the plane X; if the first laser light pulse is emitted with a polarization also in the plane X, the portion of the fluorescent light polarized in parallel to the polarization plane of the laser is then detected; after the decay of this fluorescence, the second laser pulse is emitted with a polarization in the plane Y vertical to the plane X; thereafter, only the portion of the fluorescent light that is polarized perpendicular to the plane X of the laser is detected; after its decay, another laser pulse is emitted with a polarization in the plane X; etc.", "This detection of the two polarization portions is done almost simultaneously since the delay between the two laser light pulses is restricted only to the decay of the fluorescent light which is within the range of the fluorescence life of the color markers, typically between 1-4 ns.", "This “simultaneous” detection of both polarizations can therefore be achieved with little material effort using only one detector and one laser, so that it is of great interest for any application employing fluorescence anisotropy and polarization measurement.", "In a similar manner, a delay between the red and the green laser allows for a “simultaneous” detection of the red and the green luminescence signal on only one detector with a delay in the range of ns.", "Thus, the present method allows for a two color cross-correlation analysis with only one detector.", "To obtain particularly good measuring results when using different wavelength ranges and/or polarizations, it is preferred to employ a high-sensitive confocal microscope.", "Another advantageous possible application of the present method lies with analyses employing fluorescence resonance energy transfer (FRET).", "In FRET analyses of chemical and/or biological samples, advantage is taken of the fact that the excitation energy of a substance (donor), previously excited with a particular wavelength, can cause luminescence of another substance (acceptor).", "The acceptor is thus additionally or exclusively excited by energy emitted from the donor (energy transfer).", "The effectiveness of this energy transfer is extremely dependent on the spatial distance and the spatial orientation of the donor and the acceptor, so that variations in the distance between these two substances can be studied very effectively using FRET.", "Due to the primary excitation of the donor, the same could in principle emit luminescent light.", "However, this is attenuated or nonexistent (quenched) by the energy transfer and can thus no longer be detected.", "Immediate changes in the donor substance that would normally be discovered immediately by its luminescent light can now be observed only indirectly through changes in the FERT effectiveness.", "As stated above, this depends from further factors (such as the donor-acceptor distance).", "The possibility to additionally observe the luminescent light from the donor would allow a distinction between the various effects.", "If, when practicing the present method, first a sample is pulsed or continuously irradiated with light ot the acceptor excitation wavelength, a saturation of the corresponding acceptor color markers, i.e.", "an excitation of most of those color markers, is obtained.", "A light pulse of a second wavelength (time deferred with respect to the light of the first wavelength, if the same is pulsed) excites the donor color marker which in turn indirectly excites the acceptor color marker through FRET.", "Since many of the acceptor color markers have already been excited (saturated) by the light of the first wavelength, the FRET excitation by the donor color marker is less.", "The inventive use of the pulsation of one of both wavelength ranges or the delay of the pulses of both wavelength ranges is advantageous in that less quenching of the luminescence of the donor color marker is effected.", "It is avoided that a large portion of the luminescent light of the donor color marker is invisible, since its excitation energy is used up by the FRET excitation of the acceptor color marker.", "By suitably selecting the temporal offset or the power of the light of the first wavelength, a different extent of saturation of the acceptor color marker is obtained, and thus an optimum suppression of the FRET quenching or the FRET signal is set.", "A further possible application of the present method lies with an improved use of a FRET analysis employing FRET cascades.", "As indicated above, the effectiveness, and thus the observability of FRET, strongly decreases with the distance between donor and acceptor and disappears at a maximum distance (typically about 100 nm).", "In FRET cascades, a plurality of color markers is provided in a sample, so that this maximum possible distance can be increased.", "For example, a green color marker is excited by a green laser as the first donor (donor 1).", "Through FRET, the same excites a yellow color marker as the second donor (donor 2) which, through another FRET excitation, in turn excites a third, red color marker as an acceptor to emit luminescence signals.", "Thus, the distance variations between the donor 1 and the acceptor can be detected over an even larger region, since the energy transfer from donor 1 to the acceptor passes over donor 2 (FRET cascade) and, for example, the maximum distance can be used twice.", "A disadvantage of the method thus practiced is the insecurity whether a change in the acceptor luminescence signal has been caused by a change in the distance, and thus a FRET change, between donor 1 and donor 2, or by a change in the distance, and thus a FRET change, between donor 2 and the acceptor.", "Using two temporal offset green and yellow pulsed light sources exciting only donor 1 or donor 2, respectively, the FRET effectivities and the distances between donor 1 and donor 2 can selectively be analyzed by the green laser and, for the donor 2 and the acceptor, by the yellow laser.", "Such, possibly even longer, FRET cascades are thus substantially improved by the use of electromagnetic radiation pulsed in at least one wavelength range.", "In all fields of application of the invention, the possibility of varying the temporal offset of both laser pulses is advantageous.", "In this manner, a temporal offset optimal for the improvement of the measuring results (e.g.", "less photo destruction, more optimal cross-correlation, better signal-noise ratio) can be found.", "Moreover, it becomes possible, by systematic studies, i.e systematic changes in the temporal offset in different measurements, to characterize the mutual effects of processes and/or states (e.g.", "photo destruction, transient absorption, excited state of the color marker).", "The present device serves to generate electromagnetic radiation pulsed in at least one wavelength range and/or one polarization.", "Preferably, a pulsating radiation is effected in at least two wavelength ranges and/or polarizations.", "Specifically, the device is constructed such, according to the invention, that the radiation pulses of the individual wavelength ranges and/or polarizations are temporally offset with regard to each other.", "Pulsating the at least one wavelength range and/or the at least one polarization is preferably effected such that at least two radiation pulses impinge on the color marker within the diffusion period in which a color marker diffuses through a measuring volume.", "The same is true for the other relative movement periods described before in connection with the method.", "The number of pulses and their length preferably correspond to the magnitudes described above with reference to the method.", "Such a device comprises a irradiation unit, a sample receiver, a detector unit and at least one optic unit.", "The sample receiver serves to hold a chemical and/or biochemical sample containing at least one color marker in order to perform luminescence spectroscopy.", "The detector unit serves to detect the radiation emitted by the sample.", "With the help of the optic units, the radiation is directed from the irradiation unit to the sample receiver and/or the radiation from the sample is directed to the detector unit.", "The present device may be configured such that the light is directed from the irradiation unit to the sample receiver and the radiation from the sample is directed to the detector unit using the same optic unit, the same being arranged above or below the sample receiver.", "It is further possible to design the device such that the light from the irradiation unit is directed to the sample receiver by an optic unit located above the sample receiver and the radiation emitted by the sample is directed to the detector unit using another optic unit arranged below the sample receiver.", "The irradiation unit configured according to the invention generates radiation in at least two different wavelength ranges and/or two different polarizations.", "Preferably, a laser unit is used as the irradiation unit.", "Here, two or more lasers are used, each producing laser light of another wavelength range and/or another polarization, at least one of the lasers being operated in a pulsed manner.", "The laser light pulses of the individual wavelength ranges and/or polarizations are mutually offset with regard to time, if, as preferred, a pulsation of both wavelength ranges or polarizations is effected.", "Thereby, as described above with reference to the present method, a significantly better measuring result is obtained.", "Preferably, the laser unit comprises a control unit that is connected with one or all lasers.", "Here, a separate control unit may be provided for each individual laser, the individual control units being interconnected through a common control.", "Preferably, a single control unit is provided as a mode coupler for all lasers of the laser unit.", "A first laser is controlled through this control unit.", "The second and any further laser is preferably connected to the single control unit via a trigger wire.", "Because of the signal propagation times that depend on the length of the trigger wire, the laser light pulses, the laser light pulses of the second and any further laser can be delayed automatically with respect to those of the first laser.", "Other practical possibilities to obtain a delay between the lasers are path length differences between the different beam paths, or other electric components changing the signal propagation times.", "The control unit of the present invention can preferably be constructed as described above with respect to the present method, so that, for example, a laser light pulse reaches the sample only when the excitation of a color marker, excited by the previous laser light pulse of a different wavelength range and/or another polarization, has substantially decayed.", "Further, the control unit allows for a control of the sequence of the individual lasers as well as of the time intervals between the laser light pulses.", "In a preferred embodiment of the detector unit, the detector unit only comprises one combination detector connected to an evaluating unit.", "This single detector detects all luminescent light pulses given off by the individual color markers in the sample.", "Since the luminescent light pulses arrive at the detector with an offset in time, due to the delay of the radiation pulses exciting the color markers, the individual detected values can be attributed by the evaluating unit to the corresponding color markers and/or polarizations they come from.", "By combining the information on the sequence of the radiation pulses and the sequence of the detector signals in the evaluating unit, it is possible to sort the signals in red and green signals or parallel and perpendicularly polarized signals.", "In another preferred embodiment, the detector unit comprises two detectors, one detector being for detecting the light emitted from the red color markers, the other being for detecting the light emitted from the green color markers.", "This ensures that no falsification of the results occurs due to errors by the evaluating unit.", "Further, the evaluating unit may be arranged in a simpler manner.", "When providing three or more color markers, the corresponding number of detectors can be provided.", "In order to acquire additional information, the a polarizing beam splitter may be arranged downstream of the detectors.", "A light beam coming from a certain color marker is thus split into two beams of different polarization.", "Further information on the sample, e.g.", "rotation properties, can be taken from these two beams.", "In particular, following the above described method, each of these detectors can measure different colors in succession.", "Thus, it is possible to measure more than one color in both polarizations using two detectors.", "By gating, i.e.", "the synchronous turning off of a detector during a laser pulse, the measuring method may further be improved.", "Besides the already known suppression of prompt stray light and the resulting increase in the signal-noise ratio, the detection of the luminescence emission caused by a laser pulse can be suppressed specifically.", "When using a plurality of pulsed lasers of different wavelength ranges and/or different polarizations, this allows for a specific studying of transient states and processes.", "Preferably, the device is designed as a high-precision confocal microscope.", "Preferably, the detecting unit comprises electronics with which to perform cross-correlation measurement, for example.", "The use of a confocal optical structure is preferred also because of the high resolution in the direction Z, i.e.", "along the optical axis, and of the good signal-noise ratio.", "However, any non-confocal optical measuring systems can be used for the practice of the present method.", "When using the present device with differently polarized radiation and only one detector, the irradiation unit is preferably modified as follows: (1) The radiation from a single non-polarized radiation source, such as a non-polarized laser, is split into two beams by a beam splitter which are polarized differently by a polarization filter and which are later on united again.", "The delay between the two differently polarized bemas is realized by path length differences in the two beam paths.", "(2) The radiation from a single non-polarized radiation source, such as a non-polarized laser, is split into two beams of different polarization by a polarizing beam splitter and afterwards united again.", "The delay between both differently polarized beams is obtained by path length differences in both beam paths.", "(3) Generating two radiation pulses of different polarization using a single non-polarized radiation source can also be achieved with a fast rotating polarizing filter in a non-split beam path.", "Here, the speed of rotation of the polarizing filter is adjusted to the pulse frequency of the radiation source so that the individual radiation pulses alternately have a different polarization direction.", "(4) Two polarized pulsed radiation sources of the same wavelength ranges but opposite polarizations are tuned to each other or triggered, as in the two-color approach, such that their pulses reach the sample successively with a delay.", "Whereas all embodiments of the device have the advantage that only a single detector is required to register the differently polarized luminescent light portions, the embodiments (1)-(3) even require only a single pulsed radiation source.", "The present invention, as described before with reference to the method, is advantageously developed especially by the suitable radiation device.", "By coupling in two temporally offset radiation pulses of different wavelength ranges, e.g.", "red and green, and by using suitable optical filters, luminescent light of different wavelength ranges, e.g.", "read and green, can be detected separately and almost “simultaneously” with one detector, as described above.", "Thus, methods such as coincidence analysis or two-color cross-correlation measurement can be performed with only a single detector.", "This is not possible according to prior art.", "With the present invention and the present device, the following measuring methods can be performed: spectrometry, multi-photon excitation (e.g.", "in two photon operation), laser scanning excitation, near field spectroscopy, Raman and Rayleigh scattered light applications, FIDA (fluorescence intensity distribution analysis) applications, two-dimensional FIDA applications, coincidence analysis and fluorescence life measurements.", "One may also use parallel confocal systems, such as Nipkow discs, line scanners, or PAM arrangements.", "Preferably, gated CCDs, CIDs, CMOSs or a plurality of CCDs, CIDs, or CMOSs are used that measure different signal portions through color splitters or polarizers.", "With the present method it is also possible to measure changes in the conformation of a particle if the particle is marked with at least two markers and/or shows intrinsic luminescence at at least two locations (intrinsic markers).", "In particular, the particle is marked at defined locations with exactly two molecules.", "Here, the excitation is effected in alternating polarization.", "In an arrangement allowing the simultaneous detection of two colors and two polarization directions (for example, four detectors measuring the sample volume at the same time), the polarization of the two differently colored excitation pulses may be randomly polarized with respect to each other, as long as the ratio of the polarization directions is constant in time.", "This also includes changing polarization directions, as long as they occur periodically or quasi-periodically.", "When excitation photons hit the markers in a defined polarization direction, the absorption and thus the intensity of the emitted luminescence depends on the orientation of the markers with respect to each other.", "This dependence shows in the relationship between the respective detected polarizations.", "With fast rotating particles or particle portions or markers, an arrangement with four detectors, since otherwise the contributions from the two markers are mixed.", "Specifically, it is thus possible to track bistable states of molecular rotation axes, such as cis-trans rearrangements or chair rearrangements of ring structures.", "More complex applications of this method also include the breaking or the forming of cysteine bridges between protein units or folding leaflet arrangements.", "Specifically, the detection signal can be overlaid by luminescence life effects that can also be used for quantifying.", "In this application, it may be preferred to set the concentration of the sample such that, on average, less than one particle is in the measuring volume.", "It may also be preferred to fix the particles on a surface, for example, the surface of other particles, in particular nano particles, or on a planar surface, in particular in the form of arrays.", "The present method further allows for a measurement of coded beads on the basis of particles referred to as “quantum dots” in prior art or similar particles.", "To this avail, different excitation wavelengths are emitted successively and the luminescence intensity or life of the particles is measured.", "Since the particles described could be adjusted exactly with respect to their luminescence properties, i.e.", "their excitation and emission wavelengths, for example, through size and material selection, it is possible by combining, e.g., three excitation colors, three emission colors and three luminescence lives to differentiate between two to the power of nine=512 particles.", "Thus, the particles described can be used directly without having to build combinations thereof into beads as is necessary in prior art systems.", "Again, it may be preferred to set the concentration of the sample such that, on average, less than one particle is in a measuring volume.", "It may also be preferred to fix the particles on a surface, such as on that of other particles, in particular nano particles, or on a planar surface, in particular in the form of arrays.", "The present method and the present device are particularly suitable for pharmaceutic active substance search (screening), for identifying and characterizing pharmaceutically relevant substances and molecules, for identifying analytes in diagnostic applications, for genome analysis or for cleaning and concentrating substrates.", "The following is a detailed description of the invention with reference to test results and a preferred embodiment of the device.", "In the figures: FIG.", "1 schematically illustrates a preferred embodiment of the present device, FIGS.", "2-4 illustrate diagrams of the fluorescence intensities of a purely red color substance solution irradiated with a different light pulse delay of a red and a green laser, FIG.", "5-7 illustrate diagrams of the fluorescence decay in a red and a green detection channel in tests described with reference to FIGS.", "2-4, and FIG.", "8 shows a diagram in which the graph of cross-correlation measurements is illustrated.", "The preferred embodiment of the present device illustrated in FIG.", "1 comprises a sample receiver 10.This sample receiver 10 is schematically illustrated as a single container holding the sample to be examined.", "The sample receiver may be micro- or nano-titer plates, for example.", "In the example shown, a laser unit 12 serving as the irradiation unit comprises an argon laser 14 generating green laser light with a wavelength of 496 nm.", "A second laser 16 is a red laser diode, generating laser light with a wavelength of 635 nm.", "Both lasers 14, 16 are operated in a rapid pulsed mode.", "When using the device of FIG.", "1 to carry out the tests described in connection with FIGS.", "2-8, the pulse frequency was 73 MHz.", "The argon laser 14 is controlled through a mode coupler 18 connected to the argon laser 14 via a wire 20.Through the mode coupler, an exact pulse frequency is generated.", "The mode coupler 18 is further connected to the red laser diode 16 via a trigger wire 22.By providing the mode coupler as a common trigger unit, the pulse frequency of both lasers 14, 16 is identical.", "Due to the length of the trigger wire 22, the pulses generated by the two lasers 14, 16 are delayed with respect to each other.", "The delay is due to the signal propagation time of the control signals from the mode coupler 18 to the laser 16.The light beams emitted by the two lasers 14, 16 are combined by a dichroitic beam splitter 24 so that they pass along an identical beam path.", "However, since the laser pulses are delayed with respect to each other, no overlapping of the individual pulses occurs.", "The laser light bundled by the dichroitic beam splitter 24 is directed towards the sample receiver 10 by a dichroitic mirror 26 and focused through an objective 28 into the sample held in the sample receiver 10.The objective 28 and the dichroitic mirror 26 are already parts of an otic unit 30.The optic unit 30 further comprises a tube lens 34 and a pinhole diaphragm 36.The light emitted by the color markers contained in the sample passes through the objective 28, the dichroitic mirror 26 and the tube lens 34 by which it is focused on the pinhole diaphragm 36.This is a typical arrangement of a confocal microscope where portions of the light are canceled out by the pinhole diaphragm 36.In the embodiment of the device illustrated, a detector unit 40 comprises four optical filters 32, four detectors 42, 44, 46, 48 as well as a polarizing beam splitter 50 and an evaluating unit 52.The beams passing the pinhole diaphragm 36 are split by the polarizing beam splitter 50 into a beam 54 with parallel polarized light and a beam 56 with perpendicularly polarized light.", "The beam 54 is split into two beams 60, 62 by a dichroitic beam splitter 58, one of the beams including the light given off by the red color marker and the other beam including the light given off by the green color marker.", "Correspondingly, the other polarized beam 56 is split into a red and a green beam 66, 68 by a second dichroitic beam splitter 64, which are detected by the detectors 46 and 48, respectively.", "The optic filters 32 filter out edge portions of the emitted light, for example, which do not come from the color markers but, for example, from the material of the sample receiver 10.The beams 60, 62, 66, 68 detected by the detectors 42, 44, 46, 48 are transformed into electric signals and transferred to the evaluating unit 52 which typically is a PC adapted to the device.", "The evaluating unit determines the type of reaction that has occurred in the sample.", "Instead of directing the laser light to the sample and to direct the light emitted from the sample to the detector unit using a single optic unit, two optic units may be used.", "The present device may be arranged such that the light from the irradiation unit to the sample receiver and the radiation emitted from the sample are directed to the detector unit using the same optic unit which may be located above or below the sample receiver.", "It is further possible to design the device such that the light from the irradiation unit is directed to the sample receiver by an optic unit located above the sample receiver, and that the radiation emitted from the sample is directed to the detector unit through another optic unit arranged below the sample receiver.", "Instead of the irradiation unit with two lasers 14, 16 illustrated in FIG.", "1, a irradiation unit with only a single light source may be used.", "To establish beam paths with two different wavelength ranges, a beam splitter is provided behind the light source, which decouples 50%, for example, of the light generated from the beam path, irrespective of the frequency of the light.", "This may be an inclined mirror, for example, that covers 50% of the beam path.", "Due to path length differences, a delay in time may be obtained between the two beam paths established.", "Here, only a single pulsed light source is required.", "To cause different wavelength ranges in both beam paths, a unit for changing the wavelength is provided in one of the beam paths.", "This may be a frequency doubling means or a frequency multiplier, for example.", "Further, an OPA may be provided.", "This is a non-linear crystal causing a frequency shift.", "Likewise, a Raman shifter may be provided to shift the wavelength range in one of the two beam paths.", "A corresponding device with only a single light source may also be used when two beam paths with different polarizations are to act on the sample.", "Again, the beam path generated by the light source is split and a delay in the pulsed single light source is caused by the path length differences.", "To make a polarization change in one of the beam paths, a polarization filter is provided, for example, in the beam path as a unit for changing the polarization.", "In the measurements depicted in FIGS.", "2-7, a purely red color substance solution has always been examined.", "This is the color substance cyanin 5 (Cy5) dissolved in water in a concentration of 5 nM.", "The fluorescence life of Cy5 in water is 0.7 ns.", "FIGS.", "2-4 each illustrate the count rate of the detector over time.", "In all three tests, the sample was irradiated exclusively with the red laser within the first 5 s, with the red and the green laser in the time between 5s and 15 s, and again exclusively with the red laser between 15 and 20 s. in all three measurements, the frequency of the two pulsed lasers was 73 MHz.", "In the measurement illustrated in FIG.", "2, no pulse offset between the lasers was effected in range from 5-15 s win which both the red and the green laser were on.", "Thus, the read and green laser light pulses hit the sample and the red color marker simultaneously.", "It is obvious from the diagram (FIG.", "2) that the count rate decreases largely in the range from 5-15 s. in those ranges, where the green laser was not on, i.e.", "in the range from 0-5 s and the range from 15-20 s, the count rate is significantly higher.", "This illustrates the destructive influence of the green laser on the red color markers.", "In the measurement depicted in FIG.", "3, a pulse offset between the red and the green laser of 2 ns was set in the time section from 5-15 s. the pulses from the green laser always occurred 2 ns after those of the red laser, the two laser light pulses alternately hitting the sample.", "As is obvious from the diagram (FIG.", "3), the count rate in the range from 5-15 s is significantly higher than in the diagram of the measurement taken first (FIG.", "2).", "Thus, even within a temporal offset of 2 ns, a certain decay of the excitation of the red color marker has occurred (about 94% for Cy5), so that a significantly lower number of red color markers has been destroyed by the green laser.", "The effect of the present method is especially obvious from FIG.", "4.In this measurement, a pulse offset of 10 ns was set in the range from 5-15 s. Here, the diagram shows no deviation of the count rate for the single ranges.", "Thus, it may be assumed that even after 10 ns approximately all previously excited red color markers have returned to their original state.", "In FIGS.", "5-7, the count rate of a red and a green detection channel is depicted over time.", "The detection channels are the corresponding detectors in connection with the evaluating unit.", "FIG.", "5 corresponds to the measurement in FIG.", "2, FIG.", "6 corresponds to the measurement in FIG.", "3, and FIG.", "7 corresponds to the measurement in FIG.", "4.The solid line represents the fluorescence caused by the red laser, while the broken line represents the fluorescence caused by the green laser.", "The measurements depicted in FIGS.", "5-7 were made in the time from 5-15 s (FIGS.", "2-4), i.e.", "when both lasers were on.", "In FIG.", "5, there is no delay between the red and green laser light pulses, in FIG.", "6, the delay is 2 ns as in FIG.", "3, and in FIG.", "7, the delay is 10 ns as in FIG.", "4.FIGS.", "5-7 clearly show the shift of the maximum fluorescence signals of the red and the green detection channel with respect to each other, due to the pulse offset.", "With a pulse offset of 10 ns, the two detection channels can clearly be separated from each other.", "For example, this allows for the use of a single detector for both color markers, since it is known at which moment light signals from which color marker reach the detector.", "The test illustrated in FIG.", "8 is a measurement of a double-stranded oligo-nucleotide marked with Cy5 and rhodamine green, dissolved in water in a concentration of about 1 nM.", "The oligo-nucleotide has a length of 66 base pairs and prevents energy transfer because of the distance between the two color markers.", "A cross-correlation measurement of the red and green fluorescent light was performed.", "Again, the red and green lasers were pulsed and adapted to be shifted in time.", "The lower curve is the course of the cross-correlation for overlying laser light pulses, i.e.", "for laser light pulses without delay.", "In the upper curve, the laser light pulses were mutually offset.", "From equation 2, the upper curve yields a concentration, Cgreen+red=1 nM, of twice marked oligo-nucleotides, whereas the reduced amplitude of the lower curve leads to a lesser concentration, Cgreen+red=0.6 nM.", "Due to the pulse offset, no photo destruction occurs and the cross-correlation analysis leads to less falsified results." ] ]

[ [ "Enhanced virtual navigation and examination", "Virtual navigation (2255) and examination of virtual objects are enhanced using methods of insuring that an entire surface to be examined has been properly viewed.", "A user interface (FIG.", "23) identifies regions which have not been subject to examination and provides a mechanism (2250) to route the user to these regions in the 3D display.", "Virtual examination is further improved by the use of measuring disks (905) to enhance quantitative measurements such as diameter, distance, volume and angle.", "Yet another enhancement to the virtual examination of objects is a method of electronic segmentation, or cleaning, which corrects for partial volume effects occurring in an object." ], [ "1.A method for enhancing a virtual examination of an object comprising: marking voxels in an image dataset as viewed as those voxels are presented on a user display; identifying voxels not marked as viewed after the completion of an initial examination; providing a user display identifying all regions of voxels identified which have a size larger than a predetermined threshold.", "2.The method of claim 1, wherein the user display identifying all regions is a 2D planar projection of a 3D closed object.", "3.The method of claim 2, wherein the 2D planar projection provides a display of a graphical user interface, wherein when a user selects a region of unviewed voxels, a display of the 3D closed object at the selected region is presented.", "4.The method of claim 2, wherein the 2D planar projection is a Mercator projection of a lumen shaped object onto a 2D plane.", "5.The method of claim 1, wherein the user display identifying all missed regions is a tabular list of the missed regions.", "6.The method of claim 1, wherein the marking of voxels as viewed is a binary marking representing that the voxel was displayed.", "7.The method of claim 1, wherein the marking of voxels as viewed is a multi-level marking corresponding to the level of participation of the voxel when displayed.", "8.A method for quantitative measurement of physical parameters of a virtual object comprising placing at least one measuring disk at a location along a centerline of a virtual object, wherein the measuring disk defines a plane aligned perpendicular to the centerline of the object.", "9.The method of claim 8, wherein the at least one measuring disk has a radius corresponding to the minimum radius of the object at the point of placement and wherein the diameter of the measuring disk represents the diameter of the object.", "10.The method of claim 8, wherein the at least one measuring disk comprises two measuring disks, wherein the two measuring disks are placed at desired points on the centerline, between which a distance along the centerline is measured.", "11.The method of claim 8, wherein the at least one measuring disk comprises two measuring disks, wherein the two measuring disks are placed at desired points on the centerline and the plane of the measuring disks and object boundary define a volume to be measured.", "12.The method of claim 8, wherein the at least one measuring disk comprises three measuring disks located along the centerline of an object, the three measuring disks defining the endpoints and vertex of an angle to be measured on the object.", "13.A method of electronically cleaning a virtual object comprising: defining a plurality of intensity value ranges representing corresponding material type classifications in the image data; identifying boundary regions between at least a portion of the material type classifications in the image data; defining at least one set of intersection conditions which correspond to expected intersection regions; casting segmentation rays from the boundary regions to detect regions which satisfy any of the at least one set of intersection conditions; and performing intersection region specific correction on the detected regions.", "14.The method of claim 13, further comprising the step of volumetric contrast enhancement.", "15.The method of claim 13, wherein the step of identifying boundary regions further comprises: selecting a material type for segmentation; identifying seed voxels in the regions of the selected material type; and applying region growing from the seed voxels until a boundary between the selected material type and another material type is detected.", "16.The method of claim 14, wherein the virtual object is a colon and wherein the plurality of material types include air, soft tissue, stool and fluid.", "17.The method of claim 16, wherein the intersection regions include air-soft tissue, air-stool and air-fluid regions.", "18.The method of claim 17, wherein upon detecting an air-soft tissue region, the intersection specific correction includes labeling at least a portion of the voxels of the ray as colon wall and labeling a portion of the voxels of the ray as mucosa.", "19.The method of claim 18, wherein upon detecting an air-fluid region, the intersection specific correction includes labeling the voxels of the region as air voxels.", "20.The method of claim 19, wherein upon detecting an air-soft tissue region, the intersection specific correction includes labeling at least a portion of the voxels of the ray as colon wall and labeling a portion of the voxels of the ray as mucous.", "21.The method of claim 17, wherein upon detecting an air-stool region, the intersection specific correction includes reconstructing the voxels in the region by labeling at least a portion of the stool voxels as fluid voxels and labeling a portion of the voxels of the ray as mucosa.", "22.A method for displaying a region of interest of a virtual object with an increased field of view comprising mapping the 3D voxel representation to a 2D display screen using an equal angular spaced projection.", "23.A method for coordinating the display of a 3D image and one or more corresponding 2D images comprising: displaying a 3D image of a region; displaying a moveable slice marker on the 3D image; and displaying a 2D image corresponding to the position and orientation of the slice marker.", "24.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 23 wherein the 2D image is one of a sagital, a coronal, an axial and a centerline-normal cross sectional image.", "25.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 23 wherein the slice marker is presented by adjusting the color of the voxels in the 3D image corresponding to the intersection of the corresponding 2D image.", "26.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 23 wherein the 2D image has an associated user interface for changing both the slice marker position and the current 2D image.", "27.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 23 wherein there are a plurality of selectable 2D images and a corresponding plurality of slice markers.", "28.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 27 wherein the plurality of 2D images are selected from the group including sagital, coronal, axial and centerline-normal cross sectional images.", "29.A method for coordinating the display of a 3D image and one or more corresponding 2D images of claim 27 further comprising displaying a navigation marker in a displayed 2D image corresponding the current navigation position in the 3D image." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The use of 3D imaging techniques for exploring virtual representations of objects is known.", "For example, medical image data can be transformed into a 3D virtual representation of an organ, such as the colon, to allow a thorough examination of the region.", "In the case of conventional optical colonoscopy, the field of view available is generally directed only along the direction of navigation through the colon.", "As illustrated in FIG.", "1 , because of the folded nature of the colon lumen, this can result in a substantial portion of the colon not being visible during optical colonoscopy.", "Of course, in virtual colonoscopy, the viewer can change navigation direction and can also orient the virtual camera in any direction.", "While this flexibility allows the radiologist or other viewer to explore the entire surface of the colon, it is often difficult or impossible for a viewer to know whether a particular region has been explored.", "Thus, improved navigational aids would be desirable.", "In addition to being used to view a virtual object, the 3D virtual environment can also be used to perform quantifiable analysis, such as distance and volume measurements in a region of interest.", "In present systems, such measurements generally involve the use of manually outlining a region for volume measurements.", "In addition, 3D ray-surface intersection have been used to perform length, diameter and angular measurements.", "Such techniques tend to be time consuming or computationally expensive.", "Thus, improvements for performing quantitative measurements in a virtual environment are desired.", "An additional problem which is encountered during virtual examination is removing extraneous material from the object which is subject to examination.", "For example, in the case of virtual colonoscopy, it is necessary to remove stool and fluid from the colon to expose the surface of the colon wall.", "The traditional pre-imaging protocols for effecting colon cleansing are unpleasant and impose a deterrent to patients from obtaining such examination.", "Although electronic cleansing techniques are known, these techniques still encounter difficulties in properly removing material, such as in regions of intersection of two or more material types.", "Thus, improved electronic cleansing of a virtual object would be beneficial in such virtual examination applications." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, a method for enhancing virtual examination of an object provides for insuring that a virtual examination is thorough.", "The method includes marking voxels in an image dataset as viewed when those voxels are presented on a user display as part of a rendered image.", "By marking viewed voxels, it is then possible to identify the unmarked voxels upon completion of an initial examination.", "Once the unviewed regions are identified, a user display identifying all regions of voxels identified which have a size larger than a predetermined threshold is provided.", "Preferably, the user display is a 2D planar projection of a 3D closed object.", "For example, a colon lumen can be “unraveled” into a 2D planar surface to exposes the entire inside surface of the colon.", "The 2D planar projection can provide the display for a graphical user interface, which allows the user to select a region of unviewed voxels such that a display of the 3D closed object at the selected region is presented for examination.", "A further method in accordance with the present invention provides for quantitative measurement of physical parameters of a virtual object.", "This method includes placing at least one measuring disk at a location along a centerline of a virtual object.", "The measuring disk is a fiducial marker which defines a plane that is aligned perpendicular to the centerline of the object.", "By using a single measuring disk, diameter measurements can be taken.", "Using two measuring disks, a distance along the centerline and the volume of a region can be measured.", "With three measuring disks, angular measurements can be performed.", "Yet another method for enhancing a virtual examination includes electronically cleaning a virtual object.", "This method includes defining a number of intensity value ranges which represent corresponding material type classifications in the image.", "At least one set of intersection conditions which correspond to expected intersection regions are also defined.", "The intersection conditions are generally defined based on observed, repeatable characteristics of the intensity profile in the intersection regions.", "Boundary regions between at least a portion of the material type classifications are then identified in the image data.", "From the boundary regions, segmentation rays are cast towards neighboring voxels to detect regions which satisfy the defined intersection conditions.", "Once identified, intersection region specific correction is performed on the detected regions.", "Volumetric contrast enhancement can also be performed to further resolve any remaining partial volume effects.", "Boundary regions can be identified by selecting a material type for segmentation; identifying seed voxels in the regions of the selected material type; and then applying region growing from the seed voxels until a boundary between the selected material type and another material type is detected.", "In the case of virtual colonoscopy, the virtual object is a colon and the material type classifications include air, soft tissue, and fluid-stool-bone.", "In this case, intersection regions include air-soft tissue, air-stool and air-fluid regions.", "Upon detecting an air-soft tissue region, the intersection specific correction labels at least a portion of the voxels of the ray as colon wall and labels a portion of the voxels of the ray as mucosa.", "Upon detecting an air-fluid region, the intersection specific correction labels voxels of the region as air voxels.", "Upon detecting an air-stool region, the intersection specific correction includes reconstructing the voxels in the region by labeling at least a portion of the stool voxels as fluid voxels and labeling a portion of the voxels of the ray as mucosa.", "Also in accordance with the present invention is a method for coordinating the display of a 3D image and one or more corresponding 2D images.", "This method includes displaying a 3D image of a region along with displaying a moveable slice marker on the 3D image.", "A 2D image corresponding to the position and orientation of the slice marker is also displayed.", "If a different 2D image is selected, such as by manipulating a user interface, the marker position in the 3D image changes accordingly.", "The 2D images are generally cross-sectional slices such as sagital, a coronal, axial or centerline-normal cross sectional images." ], [ "The subject matter of this application was funded in part by the National Institute of Health, contract number CA79180 and the U.S. Office of Naval Research, grant number N000149710402.From these grants, the U.S. government may have certain rights to the invention.", "FIELD OF THE INVENTION The present invention relates generally to computer-based three dimensional visualization and more particularly relates to improved methods for virtual navigation and examination including identifying regions which have been missed during navigation and electronic cleansing.", "BACKGROUND OF THE INVENTION The use of 3D imaging techniques for exploring virtual representations of objects is known.", "For example, medical image data can be transformed into a 3D virtual representation of an organ, such as the colon, to allow a thorough examination of the region.", "In the case of conventional optical colonoscopy, the field of view available is generally directed only along the direction of navigation through the colon.", "As illustrated in FIG.", "1, because of the folded nature of the colon lumen, this can result in a substantial portion of the colon not being visible during optical colonoscopy.", "Of course, in virtual colonoscopy, the viewer can change navigation direction and can also orient the virtual camera in any direction.", "While this flexibility allows the radiologist or other viewer to explore the entire surface of the colon, it is often difficult or impossible for a viewer to know whether a particular region has been explored.", "Thus, improved navigational aids would be desirable.", "In addition to being used to view a virtual object, the 3D virtual environment can also be used to perform quantifiable analysis, such as distance and volume measurements in a region of interest.", "In present systems, such measurements generally involve the use of manually outlining a region for volume measurements.", "In addition, 3D ray-surface intersection have been used to perform length, diameter and angular measurements.", "Such techniques tend to be time consuming or computationally expensive.", "Thus, improvements for performing quantitative measurements in a virtual environment are desired.", "An additional problem which is encountered during virtual examination is removing extraneous material from the object which is subject to examination.", "For example, in the case of virtual colonoscopy, it is necessary to remove stool and fluid from the colon to expose the surface of the colon wall.", "The traditional pre-imaging protocols for effecting colon cleansing are unpleasant and impose a deterrent to patients from obtaining such examination.", "Although electronic cleansing techniques are known, these techniques still encounter difficulties in properly removing material, such as in regions of intersection of two or more material types.", "Thus, improved electronic cleansing of a virtual object would be beneficial in such virtual examination applications.", "SUMMARY OF THE INVENTION In accordance with the present invention, a method for enhancing virtual examination of an object provides for insuring that a virtual examination is thorough.", "The method includes marking voxels in an image dataset as viewed when those voxels are presented on a user display as part of a rendered image.", "By marking viewed voxels, it is then possible to identify the unmarked voxels upon completion of an initial examination.", "Once the unviewed regions are identified, a user display identifying all regions of voxels identified which have a size larger than a predetermined threshold is provided.", "Preferably, the user display is a 2D planar projection of a 3D closed object.", "For example, a colon lumen can be “unraveled” into a 2D planar surface to exposes the entire inside surface of the colon.", "The 2D planar projection can provide the display for a graphical user interface, which allows the user to select a region of unviewed voxels such that a display of the 3D closed object at the selected region is presented for examination.", "A further method in accordance with the present invention provides for quantitative measurement of physical parameters of a virtual object.", "This method includes placing at least one measuring disk at a location along a centerline of a virtual object.", "The measuring disk is a fiducial marker which defines a plane that is aligned perpendicular to the centerline of the object.", "By using a single measuring disk, diameter measurements can be taken.", "Using two measuring disks, a distance along the centerline and the volume of a region can be measured.", "With three measuring disks, angular measurements can be performed.", "Yet another method for enhancing a virtual examination includes electronically cleaning a virtual object.", "This method includes defining a number of intensity value ranges which represent corresponding material type classifications in the image.", "At least one set of intersection conditions which correspond to expected intersection regions are also defined.", "The intersection conditions are generally defined based on observed, repeatable characteristics of the intensity profile in the intersection regions.", "Boundary regions between at least a portion of the material type classifications are then identified in the image data.", "From the boundary regions, segmentation rays are cast towards neighboring voxels to detect regions which satisfy the defined intersection conditions.", "Once identified, intersection region specific correction is performed on the detected regions.", "Volumetric contrast enhancement can also be performed to further resolve any remaining partial volume effects.", "Boundary regions can be identified by selecting a material type for segmentation; identifying seed voxels in the regions of the selected material type; and then applying region growing from the seed voxels until a boundary between the selected material type and another material type is detected.", "In the case of virtual colonoscopy, the virtual object is a colon and the material type classifications include air, soft tissue, and fluid-stool-bone.", "In this case, intersection regions include air-soft tissue, air-stool and air-fluid regions.", "Upon detecting an air-soft tissue region, the intersection specific correction labels at least a portion of the voxels of the ray as colon wall and labels a portion of the voxels of the ray as mucosa.", "Upon detecting an air-fluid region, the intersection specific correction labels voxels of the region as air voxels.", "Upon detecting an air-stool region, the intersection specific correction includes reconstructing the voxels in the region by labeling at least a portion of the stool voxels as fluid voxels and labeling a portion of the voxels of the ray as mucosa.", "Also in accordance with the present invention is a method for coordinating the display of a 3D image and one or more corresponding 2D images.", "This method includes displaying a 3D image of a region along with displaying a moveable slice marker on the 3D image.", "A 2D image corresponding to the position and orientation of the slice marker is also displayed.", "If a different 2D image is selected, such as by manipulating a user interface, the marker position in the 3D image changes accordingly.", "The 2D images are generally cross-sectional slices such as sagital, a coronal, axial or centerline-normal cross sectional images.", "BRIEF DESCRIPTION OF THE DRAWING FIG.", "1 is a pictorial view of a cross section of a colon lumen illustrating regions of visible surfaces during conventional optical colonoscopy; FIG.", "2 is a simplified block diagram of a method for determining regions missed during navigation; FIG.", "3 is a pictorial diagram illustrating a planar representation of lumenal object mapped to a 2D plane to improve the visualization of missed regions of an object.", "FIG.", "4 is a pictorial view of a cross section of a colon lumen illustrating a virtual camera being brought to a selected “missed patch” for viewing.", "FIG.", "5 is a pictorial diagram illustrating an exemplary use of “measuring disks” for performing quantitative measurements in virtual visualization applications.", "FIG.", "6 is a pictorial diagram illustrating an exemplary use of measuring disks in order to determine the diameter of a region of interest; FIG.", "7 is a pictorial diagram illustrating an exemplary use of measuring disks in order to determine the length of a region of interest; FIG.", "8 is a pictorial diagram illustrating an exemplary use of measuring disks in order to determine the volume of a region of interest; FIG.", "9 is a pictorial diagram illustrating an exemplary use of measuring disks in order to determine the angle of curvature of a region of interest; FIG.", "10 is a graph which illustrates an exemplary intensity profile for CT image data of a colon; FIG.", "11 is a flow chart which illustrates an overview of the present segmentation ray electronic segmentation process; FIG.", "12 is a graph illustrating an intensity histogram of representative CT data of a colon; FIG.", "13 is a graph illustrating an intensity profile at an intersection of air and soft tissue voxels within a colon; FIG.", "14 is a graph illustrating the intensity profile of an intersection of air and fluid voxels within a colon; FIG.", "15 is a graph illustrating the intensity profile of an intersection of air and stool voxels within a colon; FIG.", "16 is a simplified 2D graphical representation of casting segmentation rays from boundary voxels to locate regions of intersection; FIG.", "17 is a graphical representation of the classification of voxels on a ray which detects an air-soft tissue intersection; FIG.", "18 is a graphical representation of the classification of voxels on a ray which detects an air-fluid intersection; FIG.", "19 is a graphical representation of the classification and reconstruction of voxels on a ray which detects an air-stool intersection; FIG.", "20 is a graph illustrating an exemplary transfer function which can be used to map voxels from an original intensity to a reconstructed intensity during a volumetric contrast enhancement operation; FIG.", "21 is a diagram illustrating the projection of image rays in normal perspective projection; FIG.", "22 is a diagram illustrating the projection of image rays using equal angular spacing to increase the field of view; and FIG.", "23 is a pictorial representation of an exemplary user interface for a virtual examination system illustrating the use of moving cross section marking.", "Throughout the figures, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components or portions of the illustrated embodiments.", "Moreover, while the subject invention will now be described in detail with reference to the figures, it is done so in connection with the illustrative embodiments.", "It is intended that changes and modifications can be made to the described embodiments without departing from the true scope and spirit of the subject invention as defined by the appended claims.", "DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Many systems are known for performing 3D visualization, navigation and examination.", "These systems generally provide for image data acquisition, such as an MRI scanner, and a computer processing platform for transforming the 2D image data into a useful 3D virtual representation of the imaged object.", "For example, such a system and methods are described in U.S. Pat.", "No.", "5,971,767 to Kaufman et al.", "entitled “System and Method for Performing a Virtual Examination,” which is hereby incorporated by reference in its entirety.", "The present invention relates to improvements in such systems which enhance the ability of the user to examine and quantify parameters of the object being examined in the virtual environment.", "As illustrated in FIG.", "2, a method for enhancing a virtual examination allows a user to quickly identify areas which have not been examined and to be brought to those areas in order to complete a full examination of a virtual object.", "This process involves marking those areas which have been viewed during navigation (step 205) and using this marked data to identify those regions which have not been viewed (step 210).", "Once the unviewed regions have been identified, this information can be presented to the viewer in various forms.", "(Step 215).", "The process of marking areas which have been viewed (step 205) can be performed by storing, in computer readable media, an auxiliary volume buffer that is updated by marking those portions of the volume which are presented to the user on a display during image rendering of the main image data.", "The marking process can be a binary marking process, designating voxels as viewed versus not viewed.", "Alternatively, the marking of those regions which have been viewed can be a quantitative marking process which captures a measure of how the given voxel was used during the navigation.", "For example, a numeric value proportional to a particular voxels participation while being displayed can be stored.", "When navigation is complete, the auxiliary volume is analyzed to identify voxels which have not been marked as viewed.", "When an unmarked voxel is identified, a region growing algorithm can be used to determine the boundaries of the “missed patch” in which the seed voxel belongs.", "This process is repeated until substantially all of the voxels which make up the surface of the object have been tagged as either viewed or as part of a missed patch.", "In the case of a continuous marking application, the user can define a value in the marking range as the stop value for region growing.", "The missed patch of continuously marked voxels can then be displayed as a gray scale or multi-color region which signifies the level of use of the voxels in the missed patch.", "In many virtual display environments, the object being examined, such as a virtual colon, has properties, such as folds and curves, which make it difficult to observe certain areas of the object during normal navigation and examination.", "Thus, in displaying regions marked as unviewed, it is desirable to “unravel” a lumen shaped object, such as the colon, by mapping the lumen to a 2D planar representation, such as illustrated in FIG.", "3.There are a number of known techniques for mapping a 3D surface, such as tube or sphere, to a 2D space which can be used to perform the present invention.", "Many of these techniques, such as Mercator, oblique, cylindrical and stereographic mapping have been used extensively in the field of map making, where surfaces of the globe must be represented on a planar map.", "It is known, however, that each method introduces some level of distortion in 2D representation.", "Thus, the 2D display, while of value in enhancing the visibility of missed patches 305, 310, 315, is not suitable for other purposes in visualization and examination.", "Instead, it is preferred that the 2D display provides the user with an effective way to visualize and select a missing patch 305 using a graphical user interface 325.By selecting a particular missed patch, the system can then automatically bring the user to a display of the original 3D volume where the virtual camera is directed to the missed patch, as illustrated in FIG.", "4.As a user selects the missed patches and examines these regions, the region can be marked as viewed and removed from the 2D representation of missed patches on the object surface.", "As an alternative to displaying the missed patches on a 2D map, the missed patches can be identified in a list of coordinates or as a series of selectable bookmarks in tabular form.", "The user can then sequentially view the designated regions or can manually select the various regions from the list of bookmarks.", "In either case, the user is presented with an indication of regions which were not viewed during initial examination thereby allowing the viewer to accomplish 100 percent viewing of the surface of interest.", "In certain applications of 3D visualization, it is desirable to not only view a region, but also to perform quantitative measurements in a region of interest.", "An example of this can be found in the context of evaluating the aorta for abdominal aortic aneurysm (AAA) treatment.", "In this regard, it is clinically important to determine the volume and position of an identified thrombosis to determine the extent of the disease and in treatment planning, such as stent design and placement.", "The present invention uses a concept referred to as measuring disks to facilitate such quantitative measurements.", "A measuring disk is a flat plane which intersects the surface of a virtual object as its travels along a centerline about the disk origin, while remaining perpendicular at the point of local centerline intersection.", "An example of a number of measuring discs placed within a lumen is illustrated in FIG.", "5.While the use of a disk shape is preferred, it is not necessary.", "For example, any other shape which acts as a fiducial marker on the center line and projects perpendicularly therefrom can generally be used.", "The radius of a measuring disk is adaptive in that it conforms to the dimensions of the structure in which it resides.", "Based on user preference, the disk can have a circular radius equal to the maximum interior radius of the lumen or the minimum interior radius of the lumen at each point along the centerline.", "In the event the minimum interior radius is preferred, the measuring disk will be fully contained within the virtual object with the possibility of small gaps occurring in places larger than this minimum.", "If the maximum radius is preferred, portions of the disk may extend through the surface of the object being examined.", "To perform repeatable, quantitative measurements, a user will place one or more measuring disks along the centerline of the object at selected points of interest.", "This is generally an interactive process which is performed using a graphical user interface, such as a computer mouse controlling a pointer on a computer display, to position the disks within the object which has been rendered translucent or transparent on the display.", "As illustrated in FIG.", "6, the diameter of a lumen at a given point 600 can be determined by the placement of a single measuring disc 605 at this location.", "As illustrated in FIG.", "7, length measurements can be simplified by placing a first measuring disk 705 at a first location of the lumen and a second disk 710 at a second location of the lumen.", "The length along the segment of the centerline 715 between measuring disks 705, 710, rather than the Euclidean length between the points, is determined and used as the actual length of the segment of interest.", "This provides accurate measurements of length for objects which may be tortuous, such as regions of the colon and aorta.", "Referring to FIG.", "8, the volume of a region can also be determined using two measuring disks.", "In this case, a volume is contained by the boundary 805 of the region of interest and measuring disks 805, 810.The volume can be determined in this captured region can be determined by counting the number of voxels contained in the captured region and multiplying this number by the equivalent volume of a single voxel.", "FIG.", "9 illustrates the use of three measuring disks to determine of angle of curvature of an object of interest.", "The three disks define two endpoints 905, 910 and the vertex of an inner angle and an outer angle.", "The disks can be positioned independently along the lumen to variably define the endpoints and vertex of interest.", "The angle can be defined as an inner angle, which is defined by the intersection of rays 920, 925 which join the origins of the disks 905, 915 and 910, 915.The angle can also be defined as an outer angle.", "In this case rays 930, 935 is projected from the origin of each end point disk 905, 910 along a tangent of the centerline towards the vertex.", "The origins of the three measuring disks are used to define a common plane on which to project the rays.", "In this way, the rays projected from the endpoints are guaranteed to converge.", "Electronic Cleansing Using Segmentation Rays.", "A common problem in virtual (and conventional) examination of objects, such as the colon, is the removal of residual material from the object.", "In the case of the colon, colonic fluid and stool within the colon may mask polyps if not removed.", "Traditionally, removal of such stool and fluid requires significant pre-imaging protocols which require the ingestion of large quantities of fluids and contrast agents and pre-imaging enema's to complete colonic cleansing.", "As disclosed in U.S. application Ser.", "No.", "09/343,012, filed on Jun.", "29, 1999, entitled “System and Method for Performing a Three-Dimensional Virtual Segmentation and Examination,” which is hereby incorporated by reference in its entirety, electronic cleansing techniques, such as image segmentation, can be used to effectively clean a virtual object.", "The present invention introduces an improved method for segmentation and digital cleansing of a virtual object, such as a colon, which provides effective treatment for areas which exhibit a partial volume effect which prevents removal by traditional segmentation.", "FIG.", "10 is a graph which illustrates an exemplary intensity profile for CT image data of a colon.", "In this intensity graph, there are three distinct plateau levels signifying soft tissue 1005, air 1010 and fluid voxels 1015.The high intensity of the fluid is the result of ingestion of a contrast agent prior to imaging.", "While these regions are readily distinguishable using traditional image segmentation techniques, the sloped parts of the graph which represent the interface of these regions are difficult to classify.", "These voxels are considered partial volume effect (PVE) voxels 1020 since the intensity is effected by a mix of voxels of the two regions forming the intersection.", "Because PVE voxels are difficult to classify using segmentation, the boundary between regions is often distorted and may not accurately reflect the region of interest.", "For example, alliasing effects of segmentation may result in abrupt borders which remove intermediate layers of material such as colonic mucosa.", "However, such intermediate regions may be valuable diagnostic aids and should be preserved in the segmented image.", "The present invention introduces the notion of segmentation rays which are projected from boundary voxels to identify regions of intersection using predetermined intersection characteristics.", "When a region of intersection is identified, intersection specific adjustments are performed to resolve ambiguities resulting from the partial volume effect.", "FIG.", "11 is a flow chart which illustrates an overview of the present segmentation ray electronic segmentation process.", "In step 1105, the image data is classified into a predetermined number of categories or bins based on expected intensity value.", "For example, in the intensity histogram of representative CT data of a colon illustrated in FIG.", "12, regions designated as air (AIR) 1205, soft tissue (ST) 1210 and fluid, stool, bone (FSB) 1215 can be defined by ranges of intensity values.", "In addition, there are generally one or more regions of partial volume effect, such as PV1 1220 and PV2 1225.The classifications are generally assigned by the system or the user based on prior knowledge of the object being imaged and the expected intensity values for the material being imaged.", "The unique intensity profiles for the different intersections between regions are studied to determine a set of characteristics which can be stored for later use.", "In step 1110, seed points are located in those regions which are to be segmented.", "For example, in the case of electronic colon cleansing, it is desirable to identify and select seed voxels in AIR regions which are located within the colon.", "In this case, an effective technique is to identify a voxel which has an intensity in the FSB intensity region and then examine those voxels located directly above it.", "If an FSB voxel has an AIR voxel immediately above it, that AIR voxel is within the colon and can be stored as a seed voxel.", "This condition results from the fact that gravity forces the fluid to collect in the lower portions of the colonic folds and establish a smooth horizontal interface surface with the air which is within the colon.", "For FSB voxels outside the colon, such as bone, these voxels will be bordered by soft tissue, not air voxels, and the described condition will not be satisfied.", "The process of detecting the seed voxels can be implemented by traversing each vertical scan-line in the image data to determine if the condition, i.e., AIR above FSB, is satisfied.", "If so, the AIR voxel is stored as a seed point in a suitable data structure, such as an array.", "When the seed point detection evaluation is complete, region growing can be used from each seed to determine the boundary between AIR and other regions.", "(Step 1115).", "Region growing can be performed using a naive dilate-and growing strategy.", "This process can be made more efficient by maintaining a data structure for those voxels whose neighbors have not been analyzed.", "In a preferred naive dilate-and growing method, a first seed voxel is removed from the data structure and its neighbors are inspected.", "If a non-masked AIR voxel is found, this voxel is stored.", "If a masked AIR voxel is detected, this voxel is ignored.", "If a non-AIR voxel is found, this voxel is marked as a boundary voxel and is stored in a separate data structure (BV queue).", "After all neighbors have been evaluated, the voxel is masked to indicate that it has been processed.", "This process is repeated for all seed voxels.", "To identify neighboring non-masked voxels a 6-connection analysis (six neighbors evaluated) can be used.", "However, for evaluating boundary conditions a 26-connection (26 neighbors about the current voxel) is preferred to improve accuracy.", "From the boundary voxels identified in step 1115, segmentation rays can be cast to determine areas which satisfy predetermined sets of intersection criteria which indicate various corresponding regions of intersection (step 1120).", "This step involves prior evaluation of the intensity profiles at exemplary regions of intersection and isolating a set of characteristics which differentiate the respective intersection regions.", "An example of this process will be described in the context of the expected intersection regions of the colon.", "It will be appreciated, that the particular sets of intersection characteristics are application specific and will depend on a number of variables, such as the materials being imaged.", "In the case of the region growing operation described above in the context of virtual colonoscopy, the AIR regions within a colon are identified and boundaries of intersection between AIR and other regions determined.", "These intersections will represent either an AIR-ST intersection, and AIR-fluid intersection, or an AIR-stool intersection.", "By characterizing these intersections using unique properties observed in the intensity profiles of such regions of intersection, specific corrections can be applied to remove the partial volume effect.", "For example, FIG.", "13 is a graph illustrating an intensity profile at an AIR-ST intersection.", "This profile is characterized by: a gradual increase in the gradient as intensities transition from air, through PV1, to the soft tissue intensity and, possibly, to the PV2 region 1310 as well; after the first negative or zero gradient 1305, the intensity value remains in the ST range, the intensities do not enter the FSB region; and no more than one PV2 region voxel 1310 is present before a negative or zero gradient.", "FIG.", "14 is a graph illustrating the intensity profile of an AIR-fluid intersection region.", "This intersection is characterized by: a sharp rise in the gradient as the intensity values move from air to FSB; the presence of three or less PV1 voxels encountered between the AIR and FSB regions; and the voxels following the first negative gradient remain in the FSB intensity range.", "FIG.", "15 is a graph illustrating the intensity profile of an AIR-stool intersection.", "This intersection is characterized by: a sharp rise in the gradient as the intensity values move from air to PV2 or FSB; and after the first negative gradient, the intensity values are in the PV2 region.", "For each of these intersection regions, AIR-ST, AIR-fluid, and AIR-Stool, the intersection is said to be found when the above conditions are satisfied.", "Thus, intersection detection requires testing the cast segmentation rays to determine if any of the sets of intersection conditions are met.", "Ray casting is performed at each of the identified boundary voxels.", "The rays are cast outwards in a direction to each of the 26-connected neighbors which are not the same type as the seed voxel (in this case, all neighbors which are not AIR voxels).", "A simplified 2D representation of the ray casting of step 1120 is illustrated in pictorial form in FIG.", "16.In FIG.", "16, the boundary voxels are marked B and the AIR voxels are marked A.", "As the segmentation rays are extended, they are tested to determine if one of the sets of intersection conditions are met.", "If one of the predetermined sets of intersection conditions are met, a respective correction process, described below, can be applied.", "If no set of intersection conditions are satisfied within a predetermined ray distance, that ray is dropped.", "The intersection correction processes are predetermined to correct for the expected errors introduced by the partial volume effect.", "As illustrated in the graph of FIG.", "17, for a detected AIR-ST intersection, the last two voxels of the ray are designated as colon wall voxels and the remaining voxels on the ray are marked as mucosa voxels.", "While this is not intended to accurately distinguish between colon wall and mucosa, the inner contour of the colon wall which will show any polyps is accurately preserved.", "As illustrated in the graph of FIG.", "18, for voxels in the AIR-fluid intersection, these voxels are removed by assigning the voxels an intensity in the AIR region.", "This operation continues the process of removing fluid voxels from the colon interior.", "For the AIR-stool intersection, the objective is to remove the stool voxels while preserving and correcting the position of the underlying mucosa voxels.", "This can be accomplished by moving the stool voxels along the ray and moving the mucosa voxels into their correct position as shown in the graph of FIG.", "19.After the intersection region specific corrections have been applied, all of the partial volume voxels in the PV1 region, and some in the PV2 region are removed.", "What remains are high intensity voxels in the FSB range and partial volume voxels on the Fluid-ST intersection.", "Volumetric contrast enhancement of step 1130 can then be applied to correct these last conditions.", "The volumetric contrast enhancement applies the transfer function illustrated in the graph of FIG.", "20 to increase the contrast between the ST region and the FSB region which is to be removed.", "Thus by mapping the original intensity of voxels to the reconstructed intensity (Y axis), such as by the use of a look up table, the voxels in the Fluid-ST intersection close to the ST intensity are moved into the ST region and are maintained whereas the voxels in the intersection closer to the FSB region or are moved into the FSB region for removal.", "The graph of FIG.", "20, while preferred, is but one example of a transfer function which can be used to effect the desired contrast enhancement and it will be appreciated that other curves, even a straight line having a predetermined slope, can also be employed.", "Fish-Eye Projection Imaging In conventional computer graphics simulations, a perspective mapping is used to project 3D voxels onto the 2D image plane of the computer display.", "As illustrated in FIG.", "21, perspective projection results in rays from a limited field of view being projected from the source into equally spaced positions on the image surface.", "This results in a minimally distorted image which closely resembles that which would be observed by a conventional camera.", "In certain cases, however, it would be desirable to provide a user with a wider field of view, i.e., >60 degrees.", "However, as the field of view widens, the image tends to become distorted.", "Further, a field of view of 180 degrees cannot be achieved with normal perspective projection.", "The present application of equidistant fish eye projection can be used in virtual examinations to provide an increased field of view which retains critical features, such as the spherical nature of polyps being examined in virtual colonoscopy.", "As illustrated in FIG.", "22, the objective is to project the image rays along an equal angular spacing from the origin.", "The projection of a voxel (x,y,z) onto the image plane is performed by computing two angles (φ, θ) relative to the forward viewing direction and the right vector respectively.", "The voxel is then mapped to image coordinates (u,v) by a polar mapping: u=φ*cos θ v=φ*sin θ Conversely, an image coordinate (u,v) is converted to a ray by first computing the angles (φ, θ) by: φ=sqrt((u−uc)ˆ2+(v−vc)ˆ2) θ=a tan((v−vc)/(u−uc)) A ray is computed by then rotating the view forward ray about the view up direction by φ and then rotating again by θ about the view forward direction.", "The resulting ray is then cast through volume in the similar manner to the usual volume rendering process.", "The preceding computations can be made more efficient by pre-computing the two angles (φ, θ) for the entire image or even the corresponding rotation matrices.", "The present methods can be integrated as improvements to known systems and methods for performing 3D visualization, navigation and examination.", "Such systems and methods are described, for example, in U.S. Pat.", "No.", "5,971,767 to Kaufman et al.", "entitled “System and Method for Performing a Virtual Examination,” which is hereby incorporated by reference in its entirety.", "The present methods can be performed on any number of computer processing platforms, including personal computer systems.", "For example, an IBM compatible Wintel PC operating under the Windows2000 operating system and having a single 1 GHZ CPU is generally suitable for such applications.", "Moving Cross-Sectional Marking An additional improvement with respect to virtual navigation and examination is the ability to navigate in a 3D environment while being able to cross-reference the current navigation to one or more 2D images.", "Referring to FIG.", "23, the present invention provides for the display of markers in a 3D environment and the concurrent display of one or more 2D images corresponding to the marker position.", "In the 3D display 2205 of a colon, slice marker 2210 corresponds to the sagital cross sectional image 2215, slice marker 2220 corresponds to the coronal cross sectional image 2225 and slice marker 2230 corresponds to the axial cross sectional image 2235.The axial, coronal and sagital images are reformatted 2D images which are aligned with the axes of the patient.", "In addition, slice marker 2240 corresponds to cross sectional image 2245 which is aligned perpendicularly to the centerline of the object.", "This view is referred to as a centerline-normal cross section.", "As illustrated in FIG.", "23, each reformatted cross sectional image has an associated scroll bar 2250.This provides a graphical user interface for positioning the respective marker to a region of interest in the 3D image, and vice versa, such that the 2D image and 3D image can synchronously display a selected region of interest.", "As the scroll bar, or other user interface, is moved, the 2D image changes to display the reformatted 2D image at the newly selected position.", "In addition, the marker in the 3D position is moved to reflect the selected cross sectional image which is being currently being displayed.", "Thus, visual coordination is achieved among disparate views and viewing modes.", "In addition to the slice markers in the 3D image window, it is preferable if each cross sectional window has a navigation marker 2255 which corresponds to the current navigation position and viewing direction in the 3D object.", "While in FIG.", "23 four different cross sectional images, and associated slice markers, are shown, it is preferred that each of these images can be selected or deselected by the user.", "Generally, only one cross sectional image will be selected by a user during any given operation.", "The slice markers 2210, 2220, 2230, 2240 are preferably displayed with a color or translucency which allows them to be differentiated in the 3D display without obscuring the content of the 3D image window.", "As the shape of a virtual object is perceived by variations in shading, it is preferable to display the slice markers by altering the chrominance values (color) of the voxels corresponding to the slice markers and not the luminance values of such voxels.", "Coloration of the slice markers can be achieved by modifying the base voxel color within the volume rendering module.", "During volume rendering, the distance from each 2D slice plane of each visible voxel is computed.", "This is performed by direct computation of the plane equation for each voxel of each plane.", "The plane equation is expressed as: Ax+By+Cz+D=distance Where A,B,C and D are the plane equation coefficients which define the 2D slice location in three dimensions.", "The current voxel location is given by (x,y,z).", "Voxels precisely on the plane have a distance value equal to zero, while voxels at some distance from the plane have a non-zero value proportional to the distance from the plane, when the coefficients are pre-normalized.", "Thus, computing the plane equation for a voxel results in that voxel's distance from the plane.", "A voxel is considered part of the plane when the absolute value of the distance from the plane is less than a predetermined threshold value.", "If the voxel is considered within the plane, the base color will be modified during volume rendering.", "Although the present invention has been described in connection with specific exemplary embodiments, it should be understood that various changes, substitutions and alterations can be made to the disclosed embodiments without departing from the spirit and scope of the invention as set forth in the appended claims." ] ]

[ [ "Photocatalyst and process for purifying gas effluent by photocatalytic oxidation", "The photocatalyst based on a composite WO3—SiC/TiO2 semiconductor and subjected to radiation whose wavelength is at least partly less than 400 nm gives photocatalytic oxidation of volatile organic compounds and leads to their total mineralisation into CO2 and H2O.", "The process for the photocatalytic purification of industrial, agricultural or domestic gaseous effluent may be conducted at room pressure and temperature.", "Its conversion rate is high and stable." ], [ "1.Photocatalyst containing at least two coupled semiconductor compounds, characterized in that one of said semiconductors is titanium dioxide TiO2, and the other is silicon carbide, SiC, and in that it also contains tungsten trioxide, WO3.2.Photocatalyst according to claim 1, characterized in that it contains between 10 and 60%, preferably between 15 and 25% SiC (weight percentages).", "3.Photocatalyst according to either of claims 1 or 2, characterized in that it contains a quantity of WO3 corresponding to 10 to 50% of a theoretical monolayer relative to the BET specific surface area of TiO2 particles.", "4.Photocatalyst according to any of claims 1 to 3, characterized in that it contains TiO2 having a BET specific surface area of between 40 and 60 m2/g, and in that it contains between 1 and 5%, preferably between 1.5 and 4% WO3 (weight percentages).", "5.Photocatalyst according to any of claims 1 to 4, characterized in that it contains SiC having a BET specific area of between 1 and 600 m2/g, preferably between 10 and 100 m2/g.", "6.Process for preparing a mixed WO3—SiC/TiO2 photocatalyst, comprising at least a first step during which the TiO2 and SiC are simultaneously deposited on a carrier, and a second step during which the SiC/TiO2 deposit is impregnated with a solution containing at least one WO3 precursor.", "7.Process according to claim 6, characterized in that during the first step a first suspension is prepared from TiO2 powder and a second suspension from SiC powder, the two suspensions are mixed, poured onto a carrier and the solvent is evaporated.", "8.Process according to either of claims 6 and 7, characterized in that the WO3 precursor solution is an aqueous solution of (NH4)10W12O41.5H2O.", "9.Process according to any of claims 6 to 8, characterized in that it also comprises a third step, during which calcination is conducted at a temperature of preferably between 300 and 500° C. 10.Use of a photocatalyst according to any of claims 1 to 5, in a process for purifying effluent containing volatile organic compounds by photocatalytic oxidation, said process comprising: a) adding the effluent to a reactor containing said photocatalyst, b) irradiating the photocatalyst with radiation of which at least part of the light power is emitted along a wavelength of less than 400 nm and preferably less than 360 nm, so that at least part of the volatile organic compounds contained in said effluent is decomposed by oxidation; c) evacuating the gaseous reaction mixture from the reactor.", "11.Use of a photocatalyst according to any of claims 1 to 5 in a process for inactivating microorganisms contained in a gaseous flow by photocatalytic oxidation, said process comprising: a) adding a gaseous flow to a reactor containing said photocatalyst, b) irradiating the photocatalyst by radiation of which at least part of the light power is emitted along a wavelength of less than 400 nm and preferably less than 360 nm, so that at least part of the microorganisms contained in said gas flow are decomposed by oxidation, d) evacuating the gas flow from the reactor.", "12.Use according to either of claims 10 or 11, characterized in that said process is conducted at a temperature of between 15 and 30° C. 13.Use according to any of claims 10 to 12, characterized in that said process is conducted at atmospheric pressure.", "14.Use according to any of claims 10 to 13, characterized in that said light power is derived at least in part from sun radiation." ], [ "<SOH> TECHNICAL FIELD OF THE INVENTION <EOH>The present invention concerns the purification of effluent, at room temperature, containing volatile organic compounds (VOCs) by means of a composite WO 3 —SiC/TiO 2 semiconductor that is irradiated.", "The photocatalyst so formed is illuminated by radiation, whose wavelength is at least partly less than 400 nm.", "Photocatalytic oxidation of the pollutants leads to total mineralisation into CO 2 and H 2 O." ], [ "TECHNICAL FIELD OF THE INVENTION The present invention concerns the purification of effluent, at room temperature, containing volatile organic compounds (VOCs) by means of a composite WO3—SiC/TiO2 semiconductor that is irradiated.", "The photocatalyst so formed is illuminated by radiation, whose wavelength is at least partly less than 400 nm.", "Photocatalytic oxidation of the pollutants leads to total mineralisation into CO2 and H2O.", "PRIOR ART Volatile organic compounds (VOCs) are used or produced in numerous industrial or domestic activities.", "They are therefore to be found in soils, water and in the air.", "This raises several environmental and public health problems.", "Firstly, some VOCs are considered to cause an olfactory nuisance.", "And a major part of VOCs are considered to be cancerogenic or mutagenic.", "Finally, the emission of VOCs into the atmosphere is connected with the possible photochemical production of oxidants via the reaction involving VOCs and NOx in the presence of light.", "Therefore, these reactions lead to an increase in tropospherical ozone which is toxic for man, degrades harvests and is involved in the formation of acid rain.", "Some VOCs are also involved in the decrease in the stratospherical ozone layer and may contribute towards global warming.", "Since a high number of VOCs may be oxidized, the heterogeneous catalysis of oxidation could be used for their destruction in effluent.", "But in general these catalysts operate at high temperatures, which carries disadvantages since provision must be made for heating and temperature regulation devices.", "It would be simpler if it were possible to conduct reactions at room temperature, that is to say typically between 15 and 30° C. For the possible destruction of VOCs at room temperature, consideration has been given to the photocatalytic oxidation of gaseous phase organic compounds, using TiO2-based catalysts, whether coupled or not with other semiconductors.", "By way of example, the photocatalytic oxidation of acetone with a pure TiO2 catalyst or a mixed TiO2/ZrO2 catalyst at 77° C., prepared in thin layer form using a sol-gel method was described by M. E. Zorn et al in the article “Photocatalytic oxidation of acetone vapor on TiO2/ZrO2 thin films” published in Applied Catalysis B: Environmental, vol.", "23, p. 1-8 (1999).", "Photocatalysts of TiO2/Pt type have been used to decompose ethanol at a temperature in the region of 200° C. (see J. C. Kennedy and A. K. Datye, Journal of Catalysis, vol.", "179, p. 375-389 (1998).", "A mixed photocatalayst of TiO2/CdS type was tested for the decomposition of phenol, 2-chlorophenol and pentachlorophenol in liquid phase (see N. Serpone et al.", "“Exploiting the interparticle electron transfer process in the photocatalysed oxidation of phenol, 2-chlorophenol and pentachlorophenol: chemical evidence for electron and hole transfer between coupled semiconductors”, Journal of Photochemistry and Photobiology A: Chemistry, vol.", "85, p. 247-255 (1995)).", "German patent application DE 40 23 995 A1 describes semiconductor photocatalysts containing titanium oxide, zinc titanate or oxide, optionally coated with metals such as Pt, Pd, Ir, Rh, Rn, Os, Zn or Ba.", "This document suggests using these photocatalysts to purify the air inside motor vehicles.", "The chief advantage of photocatalysis is that the energy required for oxidation reactions is supplied by direct absorption of light rather than by thermal heating.", "The photocatalysts used for this purpose are semiconductors having a band gap (optical gap) typically lying between 3 and approximately 4 eV corresponding to light irradiation in the near UV spectral region.", "In general, photocatalytic reactions on TiO2 are oxidation-reduction reactions and comprise several main steps: Adsorption of reagents, of the organic pollutant in particular, Production of electron-hole pairs through absorption of photons derived from UV radiation, Spatial separation of electron-hole pairs and migration to the surface of the photocatalyst, Redox reactions of electrons and holes with the species adsorbed on the surface: reduction of an electron acceptor by the electron, oxidation of an electron donor by the hole; Desorption of reaction products.", "Most often, the electron acceptor which is reduced by the electron is oxygen.", "The holes may combine directly with the VOCs.", "But the photocatalytic oxidation of VOCs may also proceed via radicals, such as OH and O. Heterogeneous photocatalysis using TiO2-based catalysts has several advantages: i) TiO2 is relatively low cost, ii) it is not necessary to add other reagents (other than air and the VOC), iii) the process can be conducted at room temperature and atmospheric pressure, iv) in general the reaction products are limited to CO2 and H2O.", "But TiO2-based photocatalysts also suffer from several disadvantages set forth below.", "Problem Raised Photocatalyst activity in the prior art is limited to the yield of the photocatalytic process itself, and by the too high adsorption of reaction products such as CO2 or possible intermediate oxidation products which may block part of the active sites, leading to degradation of catalyst activity.", "Also, the use of a photocatalyst according to the prior art does not always lead to complete VOC mineralisation: with known photocatalysts containing TiO2 it is observed in particular that when the VOC concentration is high, partial oxidation products are formed of which some are toxic.", "The problem which this invention attempts to solve is therefore to propose a TiO2-based photocatalyst, with improved and stable yield, for destroying volatile organic compounds in gas effluent by oxidation.", "Subjects of the Invention The applicant found that the use of a new formulation of photocatalysts containing composite semiconductors of WO3—SiC/TiO2 type bring an improvement in phenomena of reagent adsorption, product desorption and space separation of electron-hole pairs.", "Therefore, the photocatalytic activity is increased and stabilised compared with known TiO2-based photocatalysts.", "One first subject of the present invention is a photocatalyst containing at least two coupled semiconductor compounds, characterized in that one of said semiconductors is titanium dioxide TiO2 and the other is silicon carbide SiC, and in that it also contains tungsten trioxide WO3.A second subject of the present invention is a preparation method for a WO3—SiC/TiO2 photocatalyst, in two steps, one first step for the simultaneous depositing of TiO2 and SiC on a carrier, and a second step to impregnate said deposit with a solution containing at least one WO3 precursor.", "Said precursor is then converted into WO3 by calcination.", "A third subject of the invention is the use of said photocatalyst in a purification process by photocatalytic oxidation of gas effluent containing volatile organic compounds.", "DESCRIPTION OF THE FIGURES FIG.", "1 is a diagram of a reactor used for tests.", "The following reference numbers are used: Main air inlet 1 Dispatcher 2 Dry air duct 3 Wet air duct 4 VOC duct 5 Saturator containing liquid pollutant 6 Bubble chamber containing liquid water 7 Mixer 8 Photocatalytic reactor 9 Ultraviolet ray lamp 10 Gaseous phase microchromatograph 11 Valves 12 FIGS.", "2 to 10 relate to tests.", "Along the Y-axis they show the conversion rate (percent) of the chosen volatile organic compound, and along the X-axis the test time (in seconds for FIGS.", "2 to 9, and in hours for FIG.", "10), for the different formulations of photocatalysts according to the invention or of the prior art.", "DETAILED DESCRIPTION OF THE INVENTION The photocatalyst of the invention contains at least two coupled, semiconductor compounds.", "Their band gap (optical gap) preferably lies between 3.0 and 3.2 eV.", "One of said semiconductors is titanium dioxide, the other SiC.", "The essential characteristic of the photocatalyst of the invention is that it also contains tungsten trioxide.", "In one advantageous embodiment of the invention, the photocatalyst according to the invention contains between 10 and 60% and preferably between 15 and 25% SiC (weight percentages).", "In another advantageous embodiment, it contains a quantity of WO3 corresponding to 10 to 50% of a theoretical monolayer on TiO2 particles.", "Said TiO2 advantageously has a BET specific surface area of between 40 and 60 m2/g and contains between 1 and 5% WO3, preferably between 1.5 and 4% WO3 (weight percentages).", "The photocatalyst of the invention may be prepared using a two-step process: a process for preparing a mixed WO3—SiC/TiO2 photocatalyst, comprising at least one first step during which TiO2 and SiC are simultaneously deposited on a carrier, and a second step during which the SiC/TiO2 deposit is impregnated with a solution of a WO3 precursor.", "This process forms the second subject of the present invention.", "The first step of said process consists of simultaneously depositing TiO2 and SiC.", "During this first step, a first suspension is prepared from TiO2 powder, and a second suspension from SiC powder, the two suspensions are mixed, poured onto a carrier and the solvent is evaporated.", "As an example of TiO2 powder, a commercially available powder with a BET specific surface area in the order of 50 m2/g is suitable.", "Silicon carbide may be chosen from among different SiC materials, in particular those having a BET measured specific surface area of between 1 and 600 m2/g.", "The applicant found that a SiC with a specific surface area of between 10 and 100 m2/g, and more particularly between 20 and 50 m2/g, gives good results.", "Such materials may be prepared for example using the synthesis methods described in the following patents: EP 0 313 480, EP 0 440 569, U.S. Pat.", "No.", "5,217,930, EP 0 511 919, EP 0 543 751 and EP 0 543 752.According to these methods, materials of varied sizes and forms can be synthesized, that is to say in the form of rods, monoliths, extrudates, grains or tubes.", "These types of SiC give good results, but other forms of SiC may also be used within the scope of this invention.", "The desired volumes of each of the two suspensions are sampled and mixed.", "Advantageously, the quantity of deposited TiO2 is chosen so as to obtain a theoretical coating of 1 mg/cm2 on the carrier.", "The suspension so prepared is poured onto the carrier and spread homogeneously by heating until complete evaporation of the water.", "It is preferable to oven dry at 120° C. for 30 minutes.", "In one preferred embodiment, the carrier is an inner wall of the reactor in which the photocatalyst is used.", "During the second step of said process, the TiO2/SiC deposit is impregnated with a solution containing at least one WO3 precursor.", "This may be made with an aqueous solution of the precursor salt (NH4)10W12O41.5H2O.", "In one advantageous embodiment of the invention, the impregnation protocol and heating of the solution to evaporation is the same as for the first step, including oven drying at 120° C. for 30 minutes.", "Then calcination is conducted under a flow of air at a temperature of between 300 and 500° C., for example 420° C., for approximately 1 h. The reactor material under the present invention may be of any type, provided that it is inert.", "For example tubes in polypropylene, carbon fibre or glass fibre may be used, or even glass or quartz.", "The use of a material transparent to ultraviolet radiation (quartz for example) is advantageous if the source of UV radiation is located outside the reactor, or if sunlight is used.", "But it is also possible to consider other radiation passing means, such as a window in material transparent to ultraviolet rays.", "The photocatalyst of the invention may be used in a purification process by photocatalytic oxidation for effluent containing volatile organic compounds.", "This process comprises: a) adding the gaseous effluent to a reactor containing a photocatalyst of the invention; b) irradiating said photocatalyst with radiation of which at least part of the light power is emitted along a wavelength of less than 400 nm and preferably less than 360 nm, so that at least part of the volatile organic compounds contained is said effluent is decomposed by oxidation; c) evacuation of the gaseous reaction mixture from the reactor.", "Photocatalytic oxidation of volatile organic compounds (VOCs) is advantageously conducted as a continuous process at room temperature and atmospheric pressure.", "This makes it possible to use the photocatalyst of the invention directly with the effluent, for example industrial, agricultural or domestic effluent without any particular pre-treatment.", "According to one particular embodiment, the process of the invention comprises the addition of oxygen and/or water vapour before they enter the reactor or the simultaneous adding of oxygen and/or water vapour to the reactor.", "The effluent entering the reactor may be derived directly from industrial, agricultural or domestic processes, or they may result from a pre-treatment of such effluent.", "According to one preferred embodiment of the process of the invention, industrial, agricultural or domestic effluent is added which already contains a sufficient quantity of oxygen and water vapour without any addition.", "An effluent-purifying device containing volatile organic compounds which may be used within the scope of this invention comprises at least: a reactor containing a photocatalyst of the invention; a source of ultraviolet radiation; means for adding the gaseous effluent to be purified; means for evacuating the reaction products.", "The UV radiation source is advantageously in the form of one or more tubular UV lamps emitting radiation, characterized in that at least part of its light power is emitted along a wavelength of less than 400 nm and preferably less than 360 nm.", "In one preferred embodiment, the photocatalyst is deposited on the light-receiving wall of the photocatalytic reactor, and the UV lamp is arranged inside the photocatalytic reactor.", "The gaseous effluent circulates in tangential manner between the outer wall of the UV tubes and the inner wall of the reactor.", "The distance between these two walls is adjusted so as to optimise contact between the gaseous flow and the catalyst surface while minimising load loss.", "A ring-type coaxial reactor with the UV lamp arranged inside is suitable for performing the present invention, but this embodiment does not limit the present invention.", "Different reactor geometries and configurations may be considered.", "Similarly, the photocatalyst of the invention may be deposited on various carriers.", "In another embodiment of the invention, the light power is supplied at least in part by sunlight.", "In this case, it is possible not to provide the reactor with a technical source of UV radiation, such as an appropriate lamp, the source of UV radiation being the sun.", "In this case (and in the case in which the technical source of IV radiation is located outside the reactor), the photocatalytic reactor must be provided with means to allow the sun rays to pass; these means may be a window in appropriate transparent material which allows the sunlight to pass, or the reactor may be built using such appropriate transparent material.", "If sunlight is used, this light may be concentrated and/or focused using optic devices known to persons skilled in the art.", "According to the applicant's findings, to ensure optimum decomposition of effluent containing volatile organic compounds, it is advantageous to adjust the chemical composition of the photocatalyst in relation to the nature, and in particular the polarity of the chief organic molecule it is wished to destroy, and in relation to the specific surface area of the TiO2 used.", "For most VOCs, such as methylethylketone, a WO3 content of between 1.0% and 5.0% (weight percentages) is suitable.", "This optimum content may be determined by persons skilled in the art by means of a simple routine experiment: a TiO2 with a large specific surface area will require a higher WO3 content than a TiO2 with a low specific surface area.", "A WO3 content that is too high risks masking the TiO2 which will reduce the efficacy of the catalyst, since part of the light is absorbed by the WO3.By way of example, for a Tio2 with a BET specific surface area of 50 m2/g, since for WO3 the theoretical monolayer corresponds on average to 0.21 g/100 m2 of carrier [see Y. C. Xie, Y. Q. Tang in Advances in Catalysis, vol.", "37 page 1 (1990)], the optimum WO3 content lying between 0.1% and 5.0% therefore corresponds to approximately 10 to 50% of the theoretical monolayer.", "The optimum SiC content, which is less critical however, and the type of SiC initially used may also be determined by means of a simple routine experiment.", "The process may be used for the purification of industrial, agricultural or domestic effluent.", "By way of example gaseous effluent derived from a tannery, which causes a definite olfactory nuisance, was successfully treated using the process of the invention.", "In this effluent, the chief VOCs were methylethylketone and butyl acetate.", "Treatment was conducted at room temperature (i.e.", "at a temperature typically lying between 15 and 30° C.) and at room pressure (i.e.", "atmospheric pressure) which is advantageous since the reactor can then be of very simple, sturdy design.", "A flow of industrial gas waste of up to 10 000 m3/h was successfully treated in this manner in a prototype reactor.", "The applicant also found that with the photocatalyst of the invention it is possible to disinfect the gaseous flow passing through it; therefore it can be used to inactivate microorganisms such as bacteria or viruses contained in the air.", "The invention will be better understood with the help of the examples which are not, however, of a limitative nature.", "EXAMPLES In these examples, all percentages concerning the chemical composition of the photocatalyst are weight percentages.", "A diagram of the reactor used for tests is shown in FIG.", "1.The photocatalytic reactor 9 was of ring type.", "The source of ultraviolet radiation 10 was a tubular lamp mounted coaxially inside the reactor; the radiation wavelength was centred on 350 to 360 nm.", "The main air inlet 1 is divided into three ducts by means of a dispatcher 2.The flow rate in each of the three ducts, dry air 3, wet air 4 and VOC 5 is fixed by means of a mass flowmeter.", "The air is saturated with water vapour when passing through a bubble chamber containing liquid water 7 and with VOCs by means of a saturator containing liquid pollutant 6.After fixing the flow rates in each duct and therefore the VOC concentrations and relative humidity, the entire flow passes through a mixer 8.By means of valves 12 the reaction mixture can then be directed either to the catalyst 9 irradiated by UV radiation 10, followed by gaseous phase microchromatography for analysis of the reaction products, or directly onto the microchromatograph for analysis of the initial gaseous composition.", "Different formulations of the WO3—SiC/TiO2 catalysts were tested, by varying their relative WO3 and SiC concentrations.", "The SiC was extruded, washed, oven dried, calcined at 700° C. and then crushed.", "The TiO2 used was a commercially available powder (supplier: Prolabo) and the SiC was obtained according to the method described in patent EP 0 543 751 A1, whose invention permits the synthesis of metal carbides and silicon having a high specific surface area by causing a volatile oxide of silicon, SiO, to react with carbon of high specific surface area at a temperature of between 900 and 1400° C. in a stream of inert gas.", "Different experiments, denoted 1, 2, 3, 4, 5, 6, 7 8 and 9 were performed, all with humidity contents of 50%.", "The control volatile organic compound was methylethylketone (MEK) to the proportion of 1500 ppm (weight ppm).", "The tests were conducted in a continuous reaction mixture for times ranging from 40 min to 18 hours.", "Experiments 1, 2, 3 and 4 concern the influence of the nature of the different TiO2, WO3 and SiC materials.", "Experiment 1 was conducted on a TiO2 catalyst alone (FIG.", "2).", "Experiment 2 was conducted on a 3.6% WO3/TiO2 catalyst (FIG.", "3).", "Experiment 3 was conducted on a 20% SiC/TiO2 catalyst (FIG.", "4).", "Experiment 4 was conducted on a 1% WO3-20% SiC/TiO2 catalyst (FIG.", "5).", "Experiments 5, 6 and 7 examine the influence of the tungsten oxide content.", "Experiment 5 was conducted on a 2% WO3-20% SiC/TiO2 catalyst (FIG.", "6).", "Experiment 6 was conducted on a 3.5% WO3-20% SiC/TiO2 catalyst (FIG.", "7).", "Experiment 7 was conducted on a 4.7% WO3-20% SiC/TiO2 catalyst (FIG.", "8).", "Experiment 8 concerned the influence of SiC content.", "It was conducted using the 3.6% WO3-30% SiC/TiO2 catalyst (FIG.", "9).", "Experiment 9 is a study of the stability of the composite catalyst in a reaction mixture.", "It was conducted using a 3.5% WO3-20% SiC/TiO2 catalyst (FIG.", "10) after more than 18 hours in a reaction mixture.", "Even though the initial conversion was approximately 60% on TiO2 alone (FIG.", "2), this catalyst is not very efficient in destroying the pollutant, since it rapidly deactivates in time in a reaction mixture, to arrive at conversion rates in the region of 10%.", "The addition of 3.6% WO3 to TiO2 does not improve the photocatalytic performance of the system (FIG.", "3).", "The use of the composite semiconductor catalyst, 20% SiC/TiO2 leads to a slight improvement compared with TiO2 alone (experiment 3, FIG.", "4) since conversion rates of approximately 20% are achieved after approximately 1000 to 2000 seconds.", "On the other hand, the addition of WO3 and SiC to TiO2 (FIGS.", "5, 6, 7, 8 and 9) results in a considerable increase in VOC photooxidation, depending upon WO3 and SiC content.", "It was found that the optimum weight percentage of SiC is 20% and that of WO3 is 3.5%.", "Therefore the optimum formulation of the photocatalyst to decompose methylethylketone is 3.5% WO3-20% SiC/TiO2.Under these conditions, after three minutes start-up, 80% of the pollutant is converted to CO2 and H2O.", "By increasing the relative proportions of SiC and WO3, this activity is reduced.", "Experiment 9 (FIG.", "10) shows the performance of the 3.5% WO3-20% SiC/TiO2 catalyst in a continuous flow of methylethylketone for more than 18 hours.", "It is observed that there is no deactivation of the catalyst, which corresponds to a substantial improvement compared with the prior art.", "Additional tests, conducted under the same conditions as above, were able to show that a photocatalyst with 50% SiC and 3.4% WO3 gives a conversion rate over approximately 2 000 seconds in the order of 40%, and a photocatalyst with 8% SiC and 3.5% WO3 gives a result that is substantially equivalent to the result observed in experiment 3." ] ]

[ [ "Method of deinking waste paper using cellulase without lowering paper strength and method of evaluating the same", "A deinking method using a cellulase which enables the load upon the environment to be reduced; and an evaluation method whereby a cellulase effective in deinking and the effective addition level of the cellulase can be appropriately determined.", "By proposing pulp-swelling activity (PSA) that elevates the water-retention value of pulp after pulp is reacted with cellulase, it becomes possible to select an enzyme that is effective in cellulase-deinking treatment without lowering the paper strength and to determine the effective enzyme dosage.", "Namely, a method of deinking waste paper is provided wherein the selection of an effective cellulase and the optimization of the enzyme dosage with the use of PSA as an indication make it possible to minimize the cellulase addition level and lessen the amount of the deinking agent." ], [ "1.A deinking method for waste paper characterized by the use of cellulase exhibiting a pulp-swelling activity (PSA).", "2.The method of claim 1, characterized by being conducted with a cellulase of a dosage level wherein the pulp-swelling activity (PSA) is 25 units (25U) or more.", "3.The method of claim 1, characterized by being conducted with cellulase of a dosage level wherein the pulp-swelling activity (PSA) exhibits a maximum value.", "4.The method of claim 1, in which cellulase is endoglucanase.", "5.The method of claim 4, in which endoglucanase is SCE3 derived from Trichoderma genus, NCE4 derived from Humicola genus, or RCEI derived from Rhizopus genus.", "6.An evaluation method for obtaining a cellulase enzyme and an enzyme dosage that are effective in deinking of waste paper without lowering the paper strength, characterized in that, after reacting cellulase with pulp, the degree of swelling of the pulp is determined.", "7.The method of claim 6, characterized in that, after reacting pulp with cellulase for 60 minutes at an optimal pH and optimal temperature of the enzyme used at a pulp consistency of 1% (w/v), a change in a water retention value (WRV) of the pulp is measured.", "8.The method of claim 6 or 7, characterized in that, using 0.5 grams of oven-dried pulp as a substrate, cellulase is reacted for 60 minutes under conditions of an optimal pH and optimal temperature at a pulp consistency of 1% (w/v), and then a pulp-swelling activity (PSA) is determined taking cellulase activity that increases the water retention value (WRV) of pulp by 1% as 10 units (10U).", "9.The method of claim 2, characterized by being conducted with cellulase of a dosage level wherein the pulp-swelling activity (PSA) exhibits a maximum value.", "10.The method of claim 2, in which cellulase is endoglucanase.", "11.The method of claim 3, in which cellulase is endoglucanase.", "12.The method of claim 7, characterized in that, using 0.5 grams of oven-dried pulp as a substrate, cellulase is reacted for 60 minutes under conditions of an optimal pH and optimal temperature at a pulp consistency of 1% (w/v), and then a pulp-swelling activity (PSA) is determined taking cellulase activity that increases the water retention value (WRV) of pulp by 1% as 10 units (10U)." ], [ "<SOH> BACKGROUND ART <EOH>Deforestation is cited as a cause of global warming and the reduction of carbon-dioxide emissions is the focus of attention as a global environmental problem.", "Forest resources are utilized in contemporary industries in the forms of wood, pulp, and paper.", "Various methods are being studied to reduce the amount of deforestation in order to prevent global warming.", "In the field of paper, efforts are being made worldwide to increase the rate of waste paper recycling.", "Waste paper deinking is largely established as a method of waste paper recycling.", "In current methods, alkalis such as sodium hydroxide and sodium silicate, oxidative bleaching agents such as hydrogen peroxide, chelating agents, and agents such as surfactants are added to the waste paper to accelerate the release of ink from the pulp fiber.", "Thereafter, a washing technique and/or flotation technique is used to carry out deinking to separate the pulp and ink.", "However, due to the variety of raw materials used in waste paper and the variety of printing techniques, deinking by the conventional deinking techniques is becoming problematic.", "Further, in order to solve problems concerning adhesion of foreign matter (stickies), fiber damage, effluent load and the like, neutral deinking is attracting attention as a deinking method that is kinder to the environment.", "In order to carry out neutral deinking more effectively, enzymatic treatments utilizing cellulase and the like are being studied.", "A number of proposals have been put forth concerning deinking methods for waste paper using cellulase derived from fungi or bacteria.", "Kao Corp. proposes a deinking agent containing cellulase in JP Patent Publication (Unexamined Application) No.", "59-9299, however there is no disclosure therein regarding the putative lowering of paper strength due to the action of the cellulase.", "In JP Patent Publication (Examined Application) No.", "3-57235, Honshu Paper Co., Ltd. proposes a deinking technique for waste paper in which enzymatic treatment using alkali-resistant cellulase is performed at the same time as treatment with an alkaline deinking agent or after treatment with the deinking agent.", "In Japanese Patent No.", "2805313, Oji Paper Co., Ltd. discloses a deinking technique in which treatment is conducted with a deinking agent after enzymatic treatment that contains cellulase.", "Further, in Japanese Patent No.", "3042718, Novo Nordisk A/S attempts to find an enzyme component that can perform effective deinking by producing monocomponent cellulase.", "However, none of the above methods succeeded in providing an effective means for selecting an enzyme that realizes, at a low cost, a deinking effect that reduces the amount of the chemical agents that constitute an environmental burden or lower the strength (intensity) of waste paper in deinking.", "Although various studies are being conducted, the rate of diffusion of enzymatic deinking is extremely low.", "Up to now, studies have been conducted on the application of cellulases to areas such as elimination of vessel pick, improvement of paper machine runnability and drainage, and deinking of waste paper.", "However, because of the high cost of cellulases and the fact that the action and effects of cellulases is not clear, the current situation is that cellulase has not been offered for practical use in areas other than elimination of vessel pick.", "Furthermore, because cellulase is an enzyme that degrades cellulose fiber, it has been pointed out that a decrease in the yield and the strength (intensity) of waste paper pulp after deinking is prone to occur.", "Meanwhile, in deinking of waste paper, because the objects of effective deinking involve detachment and saponification of printing ink and pulp swelling, alkaline chemical agents and the like are used that amount to as much as several percent of the dry pulp weight.", "Further, as pulp is normally treated by being suspended in water of a volume that is equivalent to several dozen times the weight of the dry pulp, a vast amount of alkaline effluent is generated.", "Thus, because chemical agents are used in the treatment to neutralize the effluent, the recovery treatment is one that involves a heavy environmental burden.", "There is a need for development of an environmentally friendly deinking method that improves the current deinking technology that creates a heavy environmental burden.", "Waste paper recycling is technology that indirectly protects forest resources and contributes to conservation of the global environment.", "It is anticipated that by further improvements such as reducing the environmental burden caused by waste effluent and decreasing the usage amount of chemical agents, the environmental adaptability of waste paper recycling will be enhanced.", "Thus, it was expected that enzymatic deinking would be applied as a technique to effectively remove ink at a neutral pH.", "However, its application has been postponed due to problems concerning decreases in the strength (intensity) of waste paper pulp and the cost of enzymes.", "Accordingly, the object of the present invention is to provide a cellulase deinking method for waste paper without lowering paper strength, that is implemented using an economically feasible enzyme amount that reduces the amount of deinking agents." ], [ "TECHNICAL FIELD The present invention relates to a method of deinking waste paper that lessens the load on the environment.", "The invention further relates to a method that evaluates cellulase that is effective in deinking whereby the paper strength (intensity) is not reduced and a usage amount of deinking agents is lowered, and an effective dosage of the cellulase.", "BACKGROUND ART Deforestation is cited as a cause of global warming and the reduction of carbon-dioxide emissions is the focus of attention as a global environmental problem.", "Forest resources are utilized in contemporary industries in the forms of wood, pulp, and paper.", "Various methods are being studied to reduce the amount of deforestation in order to prevent global warming.", "In the field of paper, efforts are being made worldwide to increase the rate of waste paper recycling.", "Waste paper deinking is largely established as a method of waste paper recycling.", "In current methods, alkalis such as sodium hydroxide and sodium silicate, oxidative bleaching agents such as hydrogen peroxide, chelating agents, and agents such as surfactants are added to the waste paper to accelerate the release of ink from the pulp fiber.", "Thereafter, a washing technique and/or flotation technique is used to carry out deinking to separate the pulp and ink.", "However, due to the variety of raw materials used in waste paper and the variety of printing techniques, deinking by the conventional deinking techniques is becoming problematic.", "Further, in order to solve problems concerning adhesion of foreign matter (stickies), fiber damage, effluent load and the like, neutral deinking is attracting attention as a deinking method that is kinder to the environment.", "In order to carry out neutral deinking more effectively, enzymatic treatments utilizing cellulase and the like are being studied.", "A number of proposals have been put forth concerning deinking methods for waste paper using cellulase derived from fungi or bacteria.", "Kao Corp. proposes a deinking agent containing cellulase in JP Patent Publication (Unexamined Application) No.", "59-9299, however there is no disclosure therein regarding the putative lowering of paper strength due to the action of the cellulase.", "In JP Patent Publication (Examined Application) No.", "3-57235, Honshu Paper Co., Ltd. proposes a deinking technique for waste paper in which enzymatic treatment using alkali-resistant cellulase is performed at the same time as treatment with an alkaline deinking agent or after treatment with the deinking agent.", "In Japanese Patent No.", "2805313, Oji Paper Co., Ltd. discloses a deinking technique in which treatment is conducted with a deinking agent after enzymatic treatment that contains cellulase.", "Further, in Japanese Patent No.", "3042718, Novo Nordisk A/S attempts to find an enzyme component that can perform effective deinking by producing monocomponent cellulase.", "However, none of the above methods succeeded in providing an effective means for selecting an enzyme that realizes, at a low cost, a deinking effect that reduces the amount of the chemical agents that constitute an environmental burden or lower the strength (intensity) of waste paper in deinking.", "Although various studies are being conducted, the rate of diffusion of enzymatic deinking is extremely low.", "Up to now, studies have been conducted on the application of cellulases to areas such as elimination of vessel pick, improvement of paper machine runnability and drainage, and deinking of waste paper.", "However, because of the high cost of cellulases and the fact that the action and effects of cellulases is not clear, the current situation is that cellulase has not been offered for practical use in areas other than elimination of vessel pick.", "Furthermore, because cellulase is an enzyme that degrades cellulose fiber, it has been pointed out that a decrease in the yield and the strength (intensity) of waste paper pulp after deinking is prone to occur.", "Meanwhile, in deinking of waste paper, because the objects of effective deinking involve detachment and saponification of printing ink and pulp swelling, alkaline chemical agents and the like are used that amount to as much as several percent of the dry pulp weight.", "Further, as pulp is normally treated by being suspended in water of a volume that is equivalent to several dozen times the weight of the dry pulp, a vast amount of alkaline effluent is generated.", "Thus, because chemical agents are used in the treatment to neutralize the effluent, the recovery treatment is one that involves a heavy environmental burden.", "There is a need for development of an environmentally friendly deinking method that improves the current deinking technology that creates a heavy environmental burden.", "Waste paper recycling is technology that indirectly protects forest resources and contributes to conservation of the global environment.", "It is anticipated that by further improvements such as reducing the environmental burden caused by waste effluent and decreasing the usage amount of chemical agents, the environmental adaptability of waste paper recycling will be enhanced.", "Thus, it was expected that enzymatic deinking would be applied as a technique to effectively remove ink at a neutral pH.", "However, its application has been postponed due to problems concerning decreases in the strength (intensity) of waste paper pulp and the cost of enzymes.", "Accordingly, the object of the present invention is to provide a cellulase deinking method for waste paper without lowering paper strength, that is implemented using an economically feasible enzyme amount that reduces the amount of deinking agents.", "DISCLOSURE OF THE INVENTION As described above, in waste paper deinking using cellulase, it is desirable that the deinking effect be obtained using a cost-effective amount of enzyme without lowering the paper strength (intensity).", "Therefore, we tested a fraction of each cellulase component of cellulases derived from fungi, and evaluated the activity of the obtained enzyme components towards pulp, the waste paper deinking action, and the intensity characteristics of the enzyme-treated waste paper.", "Surprisingly, we found that by allowing an extremely small level of cellulase components comprising a marked amount of endoglucanase to act on pulp, the conventionally known action of cellulase of degrading cellulose to yield cellulose degradation products such as glucose hardly occurred, and the degree of swelling, which is indicated by the water retention value of the pulp, increased.", "Specifically, we found that the cellulose fiber constituting the pulp was hydrated to cause the pulp to swell.", "Conventionally, pulp swelling is an effect that has been found in sodium hydroxide and liquid ammonia, which are alkaline chemical agents, however this phenomenon had not been found with regard to cellulase.", "As a result of further concentrated studies, we found that, with regard to pulp swelling and waste paper deinking effects produced by cellulase, a favorable correlation exists in the kinds of cellulase effective in waste paper deinking and the kinds of cellulase having a swelling action.", "As a result, we found that, surprisingly, fungi-derived endoglucanase expresses waste paper deinking activity in a dosage level that is lower than the enzyme dosage used in treatment reported up to now, and also that the intensity of waste paper that underwent enzymatic deinking compared favorably to the intensity of the waste paper prior to the enzymatic treatment.", "Further, when we compared deinking activity exerted by endoglucanase with that of sodium hydroxide, sodium silicate, and hydrogen peroxide, which are deinking agents that are currently in widespread use, we found that cellulase reduces the usage amount of these chemicals, or exhibits an effect that adequately substitutes for these chemicals.", "Thus we succeeded in completing the present invention.", "Namely, by evaluating pulp-swelling activity (PSA) that is determined by measuring an increase in the water retention value of pulp according to the present invention, it is possible to determine an effective enzyme and an effective enzyme usage level for a target waste paper deinking application.", "The present invention relates to a method of deinking waste paper characterized by the use of cellulase that exhibits pulp-swelling activity (PSA).", "The present invention further relates to: a method characterized by being conducted with cellulase of a dosage level wherein pulp-swelling activity (PSA) is 25 units (25U) or more; a method characterized by being conducted with cellulase of a dosage level wherein pulp-swelling activity (PSA) exhibits a maximum value; a method wherein the cellulase is endoglucanase; and the above method wherein endoglucanase is SCE3 derived from Trichoderma genus, NCE4 derived from Humicola genus, or RCEI derived from Rhizopus genus.", "The present invention also relates to an evaluation method for obtaining a cellulase enzyme and an enzyme dosage that are effective in deinking of waste paper without lowering the paper strength.", "The present invention further relates to: an evaluation method characterized by measuring the degree of swelling of pulp after reacting cellulase with pulp; an evaluation method characterized by measuring a change in the water retention value (WRV) of pulp after reacting pulp with cellulase for 60 min at the optimal pH and optimal temperature of the enzyme used at a pulp consistency of 1% (w/v); and to an evaluation method characterized by determining pulp-swelling activity (PSA), wherein cellulase activity that increases the water retention value (WRV) of pulp by 1% is taken as 10 units (10U) after reacting cellulase for 60 min under conditions of a pulp consistency of 1% (w/v), optimal pH, and optimal temperature, employing 0.5 g of oven-dried pulp as a substrate.", "Cellulase used in the present invention is characterized by containing endoglucanase.", "More specifically, cellulase containing monocomponent endoglucanase or specific endoglucanase components in a large amount or the like can be used, but a method of manufacturing the enzyme preparation is not limited.", "Further, the origin of endoglucanase contained in the cellulase may be any organism that produces endoglucanase, such as fungi (Trichoderma genus, for example T. reesei or T. viride; Humicola genus, for example H. insolens; Acremonium genus, for example A. cellulolyticus; Rhizopus genus, for example R. oryzae; Mucor genus, for example M. circinelloides; Phycomyces genus, for example P. nitens; and the like) or bacteria (Bacillus genus, Pseudomonas genus, Clostridium genus, and the like) or the like.", "Further, an enzyme derived from a transformant in which a gene of the above endoglucanases is introduced can be used, and the origin is not limited by the present description.", "Cellulase is an enzyme that degrades a water-insoluble cellulose substrate by the synergistic action of a number of kinds of enzyme, and P-glucosidase, endoglucanase, and cellobiohydrolase are known components thereof.", "For example, known components of endoglucanase derived from Trichoderma genus include EG I to EG IV, and known components of endoglucanase derived from Humicola genus include EG I to EG V, and all of these can be utilized in the method of the present invention.", "Further, each component of cellulase may be classified into a sugar hydrolase family as proposed by Hennrissat et al.", "In recent years, it has become known that families in which endoglucanase (EC.3.2.1.4) is present include 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, 61, 74 and the like.", "For example, it is known that endoglucanase II derived from Trichoderma viride is classified into family 5, and endoglucanase V derived from Humicola insolens is classified into family 45.Commercially available cellulase is mainly produced as a mixture of the above three components by a microorganism such as bacteria or fungi.", "By devising the conventional culture conditions it has been possible to somewhat alter the composition ratio of enzyme components, but imparting a major alteration has proved difficult.", "However, because the selection and expression of a specified gene has become possible as a result of progress in genetic engineering techniques in recent years, the possibility has increased of being able to isolate and identify a specific cellulase component effective in treatment of paper pulp and, furthermore, to manufacture a highly expressing clone to create a novel enzyme that offers excellent cost efficiency.", "The present inventors already provide cellulase derived from Trichoderma genus, cellulase derived from Humicola genus, cellulase derived from Rhizopus genus, and the like, as the above kind of cellulase derived from fungi.", "Similar cellulases are also provided by Genencor Int.", "Inc., Novo Nordisk A/S, Iogen Corp., and the like.", "Meanwhile, KAO Corp. provides Bacillus genus cellulase as cellulase derived from bacteria.", "In the present invention, it is also possible to use these cellulases.", "Preferable examples to be used herein include SCE3, which is endoglucanase derived from Trichoderma genus, NCE4, which is endoglucanase derived from Humicola genus, RCE1, which is endoglucanase derived from Rhizopus genus, and the like, which are already provided by the present inventors.", "In the present invention, as endoglucanase we selected SCE3 disclosed in WO 98/54332 as cellulase derived from Trichoderma genus and NCE4 disclosed in WO 98/03640 as cellulase derived from Humicola genus, and conducted a comparison of the deinking activity by means of the following experiments.", "Method for Determining Pulp-Swelling Activity (PSA) The pulp-swelling activity (PSA) as proposed by the present invention was determined in the following manner.", "0.5 g of oven-dried pulp that was disintegrated for 20 min by a standard disintegrator as described in JIS (Japanese Industrial Standard) P-8209 was suspended in a buffer solution having the optimal pH for the enzyme to be used in evaluation to bring the pulp consistency to 1% (w/v), and the enzyme was then added thereto and reacted at the optimal temperature for 60 min.", "To measure the degree of swelling of the pulp after reaction, the water retention value (WRV), which is one indicator of the degree of swelling, was measured according to the water retention value (WRV) measurement method for pulp described on p. 73 of “KAMI PARUPU NO SHIKENHOU (A method for testing paper pulp)” (edited and published by The Japan Technical Association of the Pulp and Paper Industry, 1995).", "The degree of swelling (water retention value) was also measured for untreated pulp in the same way.", "The water retention value of the pulp treated with enzyme was compared with the water retention value of the untreated pulp, and enzyme activity that increased the water retention value by 1% was taken as 10 units (10U), and the unit was defined as pulp-swelling activity (PSA).", "The pulp-swelling activity (PSA) is not necessarily proportional to the added level of enzyme, however, when the level of added enzyme is too small PSA decreases as the pulp does not swell, and when the level of added enzyme is too large PSA decreases because the cellulose fibers of the pulp are excessively cut and water cannot be contained among the fibers.", "Therefore, PSA exhibits a peak (maximum value) at an enzyme dosage within a certain range.", "Because the deinking effect is enhanced as pulp-swelling activity (PSA) increases, a preferable enzyme for carrying out the present invention is an enzyme that exhibits a higher numerical value for PSA even when the added level of the enzyme is small.", "Specifically, a preferable enzyme is one exhibiting a pulp-swelling activity (PSA) value of 25 units (25U) or more per 0.5 g of oven-dried pulp.", "Further, when the added amount of enzyme used exhibits a pulp-swelling activity (PSA) value of 25 units (25U) or more, as previously described, it is preferable that the PSA value is in the vicinity of its maximum, and the range thereof suitably changes according to the enzyme used.", "In the present invention, the optimal pH and optimal temperature of an enzyme can be determined by the following procedure.", "Buffer solutions used herein in determining the optimal pH are as follows: for pH 4 to 5.5, a 50 mM acetate buffer; for pH 6 to 8, a 50 mM phosphate buffer; and for pH 9, a 50 mM glycine-sodium hydroxide buffer.", "0.5 ml of solution containing enzyme is added to 0.5 ml of buffer solution containing 2% of CMC (carboxymethylcellulose, manufactured by Tokyo Kasei Kogyo Co., Ltd.) dissolved therein, and the mixture is then incubated at 30 to 70° C. for 30 min.", "Subsequently, the concentration of reducing sugar produced in the obtained reaction solution is determined by a 3,5-dinitrosalicylic acid method (DNS method).", "More specifically, 3.0 ml of DNS reagent is added to 1.0 ml of reaction solution 30 min.", "after reaction, the mixture is incubated for 5 min in a boiling water bath, diluted with 8.0 ml of distilled water, and the absorbancy at 540 nm is then determined.", "A calibration curve is then created using glucose solution diluted in a stepwise manner, and the amount of reducing sugar produced in the enzyme reaction solution is determined using glucose conversion.", "Activity is calculated by taking as 1 unit an enzyme level that produces reducing sugar corresponding to 1 μmol of glucose in 1 min.", "The DNS reagent can be prepared in accordance with descriptions in the literature (for example, “SEIBUTSU KAGAKU JIKKENHOU 1—KANGENTOU NO TEIRYOUHOU (Biochemical experimental method 1—Quantitative method of reducing sugar)” p. 19-20, 1981, Fukui Sakuzou, GAKKAI SHUPPAN CENTER).", "Deinking Activity of Cellulase The deinking activity of cellulase was determined as described below when using waste newspaper cut into 3 cm-square pieces as the raw material waste paper.", "Waste paper pulp was obtained by disintegrating 24 g of oven-dried raw material waste paper at 50° C. for 20 min using a standard disintegrator as described in JIS P-8209.Cellulase was added to the waste paper pulp for which the pH was adjusted by buffer solution or sulfuric acid, and enzyme reaction was conducted for 1 hour at 50° C. After the reaction, distilled water at 50° C. was added to the waste paper pulp to bring to 4.5 L, the mixture was provided to an experimental flotator (FW model flotation test apparatus, manufactured by KYOUSIN SANGYOU CO., LTD.), a deinking agent (Lipotol LH-350, manufactured by NIKKA Chemical Co., Ltd.) of 0.1 to 0.5% (w/v) of the oven-dried pulp was added thereto, and flotation was conducted for 10 min.", "The pulp was then recovered, washed with distilled water and dehydrated, diluted with distilled water to bring to a pulp consistency of 0.15% (w/v), and made into paper using a handsheets machine according to a method described in JIS P-8209 to obtain a hand-made paper.", "For comparison, the same procedure was performed on waste paper pulp that was not treated with cellulase to obtain a sample untreated with cellulase.", "The brightness of the hand-made paper was determined using a reflectance measuring instrument in accordance with JIS P-8123.Measurement was similarly performed for the sample untreated with cellulase and the value obtained by subtracting the brightness of the sample untreated with cellulase from the brightness of the cellulase treated sample was defined as A brightness (deinking activity).", "When the deinking activity of individual components of cellulase is compared, although superior deinking activity can not be found for cellobiohydrolase, which is considered to promote degradation from the terminus of cellulose, at an added enzyme level in the range of 0.001 to 0.1% (w/w) with respect to oven-dried pulp.", "However, for endoglucanase, which is considered to randomly degrade the cellulose chain, deinking activity can be found at the low added enzyme level of 0.01% (w/w) or less.", "Further, when enzymatic treatment is carried out at an excessive level such as 0.1% (w/w), the deinking effect declines.", "In the method of waste paper deinking using cellulase, the added level of cellulase is shown by the cellulase activity or the cellulase weight with respect to the weight of normal dry pulp.", "Regarding the added amount of cellulase, any amount can be added as long as it is within the range of amounts that can exert a deinking effect.", "However, normally the minimum amount adequate for exhibiting effective deinking activity is added due to cost considerations.", "When an enzyme dosage that can exert a deinking effect is represented by the pulp-swelling activity (PSA) proposed herein, it can be defined as a cellulase dosage that exhibits activity of 25U or more with respect to 0.5 g of oven-dried pulp.", "Further, regarding the level of added cellulase, when representing enzyme activity using hydroxymethyl cellulose (HEC) as a substrate, if an enzyme amount that reduces 1 nmol of substrate with respect to 1 g of oven-dried pulp is defined as 1 nkat/g pulp, a cellulase amount within the range of 0.01 nkat/g pulp to 10000 nkat/g pulp can be added.", "Preferably, an added level is 100 nkat/g pulp or less, and more preferably 10 nkat/g pulp or less.", "In the common deinking processes, after suspending waste paper in water and disintegrating it using a disintegrating apparatus such as a pulper, deinking agents such as sodium hydroxide, sodium silicate, hydrogen peroxide or chelating agents are added thereto and aging is performed.", "The waste paper is then deinked by subjecting it to flotation or washing it together with a deinking agent that is a surfactant to remove printing ink.", "Preferably, the cellulase treatment according to the method of waste paper deinking provided by the present invention is employed at the same time as the disintegration step or at the time of aging after the disintegration step instead of the conventionally used deinking agents.", "At that time, in order to exert cellulase activity at its maximum, the pH can be adjusted using a quantity of acid or alkali, such as sulfuric acid or sodium hydroxide, such that the waste paper pulp is maintained at the optimal pH of the cellulase, and addition of a buffer solution is also possible.", "Waste paper subjected to cellulase deinking may be any normally used waste paper, and preferable examples thereof include waste newspaper, magazine waste paper, office waste paper, and the like.", "BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in further detail by means of examples.", "However, the examples are not intended to limit the scope of the present invention.", "EXAMPLE 1 Determination of Pulp-Swelling Activity (PSA) of Trichoderma Genus-Derived Endoglucanase II (EGII) and Cellobiohydrolase I (CBHI) 0.5 g of oven-dried waste newspaper that was disintegrated for 20 min using a standard disintegrator (TAPPI Pulp Disintegrator, manufactured by Toyo Seiki Seisaku-sho, Ltd.) was suspended in a 50 mM citrate buffer solution (pH 5) to bring the pulp consistency to 1% (w/v).", "Purified EGII or purified CBHI prepared from commercially available Meicelase TP 60 (Meiji Seika Kaisha, Ltd.) based on the method of Rahkamo, L. et al.", "(Cellulose, 3, 153-163 (1996)) was then added thereto at 0.02% (w/w) with respect to the oven-dried pulp.", "After reaction at 50° C. for 60 min, the degree of pulp swelling was determined based on the method for measuring the water retention value (WRV) of pulp described on p. 73 of “KAMI PARUPU NO SHIKENHOU (A method for testing paper pulp)” (edited and published by The Japan Technical Association of the Pulp and Paper Industry, 1995).", "Pulp-swelling activity (PSA) was determined by comparing the water retention value of pulp treated with enzyme with the water retention value of untreated pulp, and taking enzyme activity that increased the water retention value by 1% as 10 units (10U).", "The results are shown in Table 1.The results in Table 1 show that under the above reaction conditions, although cellobiohydrolase exhibits no pulp-swelling action, endoglucanase exhibits a pulp-swelling action that increases the water retention value of pulp.", "TABLE 1 Enzyme PSA [U] Untreated — EGII 35 CBHI 0 EXAMPLE 2 Deinking Test Twenty-four grams of oven-dried waste newspaper cut into 3 cm-square pieces was disintegrated using a standard disintegrator as described in JIS P-8209 (TAPPI Pulp Disintegrator, manufactured by Toyo Seiki Seisaku-sho, Ltd.) at 50° C. for 20 min.", "The obtained waste paper pulp was adjusted to pH 5 with a 50 mM citrate buffer solution, and cellulase was then added thereto to bring to respective dosage of 0.001, 0.01, and 0.1% (w/w) with respect to the oven-dried pulp, and enzyme reaction was conducted for 1 hour at 50° C. The cellulases used were CBHI, i.e., cellobiohydrolase, and SCE3 (described in WO 98/54332), in which the endoglucanase component is enhanced, of Trichoderma genus, for which the optimal pH is in the vicinity of pH 5; and NCE2 (described in JP Patent Publication (Unexamined Application) No.", "8-126492), in which cellobiohydrolase is enhanced, and NCE4 (described in WO 98/03640), in which the endoglucanase component is enhanced, of Humicola genus, for which the optimal pH is in the vicinity of pH 6.After reaction, distilled water at 50° C. was added to the waste paper pulp to bring to 4.5 L, the mixture was then provided to an experimental flotator (FW model flotation test apparatus, manufactured by KYOUSIN SANGYOU CO., LTD.), and a deinking agent (Lipotol LH-350, manufactured by NICCA Chemical Co., Ltd.) of 0.3% (w/v) of the oven-dried pulp was added thereto, and flotation was conducted for 10 min.", "Thereafter, the pulp was recovered using a 140 mesh sieve, washed with distilled water and dehydrated.", "It was then diluted with distilled water to bring to a pulp consistency of 0.15% (w/v), and a hand-made paper was then prepared using a handsheets machine by the method described in JIS P-8209.For comparison, the same procedure was performed on waste paper pulp that was not treated with cellulase to obtain a sample untreated with cellulase.", "The brightness of the hand-made paper was determined using a reflectance measuring instrument (spectrophotometer CM5251, manufactured by Minolta) in accordance with JIS P-8123.The difference between the brightness of the cellulase treated sample and the brightness of the sample untreated with cellulase was defined as A brightness.", "Further, the PSA was determined in the same manner as in Example 1.The tensile strength and tear strength were determined in accordance with JIS P-8113 and JIS P-8116, respectively, and the tensile index and tear index were calculated.", "Tables 2 and 3 show the results of measurement of the deinking effect of cellobiohydrolase and endoglucanase of Trichoderma genus and Humicola genus.", "The results in Table 2 show that no deinking effect was observed for cellobiohydrolase which does not have a swelling action.", "Further, the results of Table 3 showed that, in the low dosage level of 0.01% (w/w) or less of oven-dried pulp weight in which PSA is 25U or more, endoglucanase having a pulp swelling action has a deinking effect without causing a significant decrease in intensity.", "In addition, it was found that when enzymatic treatment is conducted at the excessive level of 0.1% (w/w), the deinking effect is decreased or the intensity declines.", "Furthermore, when we analyzed the intensity of waste paper treated with endoglucanase of the low dosage of 0.01% (w/w) of oven-dried pulp weight in which PSA is 25U or more, an action of cellulase that lowers the paper strength that was observed in the conventional cellulase deinking treatment (0.1% treatment in Table 3) was hardly observed, indicating that the negative effect regarding intensity caused by cellulase treatment had been overcome.", "TABLE 2 Enzyme/added Δ PSA level Brightness Brightness [U] Untreated (pH 5) 43.3 — — CBHI/0.001% 43.1 −0.2 0 CBHI/0.01% 43.2 −0.1 0 CBHI/0.1% 42.7 −0.6 0 Untreated (pH 6) 43.5 — — NCE2/0.001% 42.2 −1.3 0 NCE2/0.01% 43.4 −0.1 0 NCE2/0.1% 43.5 0 0 TABLE 3 Tensile Enzyme/added Δ PSA index Tear index level Brightness Brightness [U] [NM/g] [mNm2/g] Untreated (pH 5) 43.3 — — 27.5 6.4 SCE3/0.001% 44.0 0.7 29 28.2 6.4 SCE3/0.01% 47.0 3.7 97 26.3 6.0 SCE3/0.1% 45.0 1.7 19 23.8 4.2 Untreated (pH 6) 43.5 — — 27.8 6.2 NCE4/0.001% 46.0 2.5 32 28.2 6.2 NCE4/0.01% 44.7 1.2 56 28.4 6.1 NCE4/0.1% 44.6 1.1 8 25.2 5.8 EXAMPLE 3 Comparison of Conventional Chemical Deinking and Enzymatic Deinking We investigated the effect of enzymatic deinking with regard to lowering the usage amount of chemical deinking agents, taking the standard usage amount of conventionally used deinking agents to be 1.5% (w/w) sodium hydroxide+3% (w/w) sodium silicate+0.5% (w/w) hydrogen peroxide with respect to oven-dried pulp.", "In the same manner as in Example 2, waste paper pulp subjected to enzymatic treatment with SCE3 of 0.01% (w/w) was treated with deinking agents of usage amounts graded in three steps, i.e., the usage amounts were ¼, ½, and the equivalent of the above standard usage amount, respectively.", "Treatment with the agents was performed by immersing the waste paper pulp in the agents for 60 min at 50° C. Thereafter, the brightness was determined in the same manner as in Example 2.The results are shown in Table 4.The Δ brightness of untreated+agents (section of experiment with no enzymatic treatment and use of the standard amount of deinking agents) and the Δ brightness of SCE3+½ agents (section of experiment with a step of treatment with SCE3 of 0.01% and use of ½ the standard amount of deinking agents) were roughly equal.", "Thus, it was found that use of cellulase treatment in deinking treatment makes it possible to decrease the usage amount of deinking agents by approximately {fraction (1/2)}.", "TABLE 4 Δ Treatment condition Brightness Brightness Untreated 43.5 — Untreated + agents 49.1 5.6 SCE3 44.7 1.2 SCE3 + ¼ agents 45.0 1.5 SCE3 + ½ agents 48.6 5.1 SCE3 + agents 51.1 7.6 INDUSTRIAL APPLICABILITY The present invention enables the evaluation and selection of cellulase having effective pulp swelling activity in waste paper deinking, and provides a low cost method of deinking waste paper that is kind to the environment in which the amount of deinking agents used is reduced without lowering paper strength, using an enzyme dosage that is lower than the dosage used conventionally.", "All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.", "Also, it will be readily understood by a person skilled in the art that miscellaneous variations and modifications of the present invention are possible without deviating from the technical ideas and scope of the invention as disclosed in the attached claims.", "The present invention is also intended to encompass such variations and modifications." ] ]

[ [ "Cleaning device and container containing a single dosage of cleaning material", "The present invention relates to a cleaning device (1, 1′) especially for cleaning surfaces, as toilet bowls and the like, having a cleaning head (3, 3′) and a handle (2, 2″).", "The cleaning device further comprises delivering means operable, in use, to deliver a single dosage of cleaning material (17), preferably contained in a small container, into or next to the cleaning head (3, 3′) and to release the cleaning material (17) from the cleaning head (3, 3′).", "Thereby exactly the amount of cleaning material needed for the cleaning action is dispensed and distributed over the surface by the cleaning head, no overdosing occurs." ], [ "1-15.", "(Cancelled) 16.A cleaning device for cleaning surfaces, the device comprising a cleaning head and a handle wherein the handle is adapted to receive at least one single dosage of cleaning material and by delivering means operable to deliver a single dosage of the cleaning material into the cleaning head and to release the cleaning material from the cleaning head.", "17.Cleaning device according to claim 16, adapted to receive a single dosage of cleaning material enclosed in a container made of a water soluble material.", "18.Cleaning device according to claim 16, wherein the handle comprises a hollow shaft extending to the cleaning head forming a passage for insertion and delivery of the single dosage of cleaning material.", "19.Cleaning device according to claim 17, wherein the handle comprises a hollow shaft extending to the cleaning head forming a passage for insertion and delivery of the single dosage of cleaning material.", "20.Cleaning device according to claim 18, wherein the handle further comprises a rod dimensioned to slide within the passage for pushing the single dosage of cleaning material into or next to the cleaning head.", "21.Cleaning device according to claim 20, further comprising a spring acting to push the rod to the bottom of the passage.", "22.Cleaning device according to claim 20, wherein the length of the rod is about half the length of the shaft.", "23.Cleaning device according to claim 21, wherein the length of the rod is about half the length of the shaft.", "24.Cleaning device according to claim 18, further comprising a cutting edge located at a bottom end of the passage for cutting or piercing a container inserted into the passage, the container containing the single dosage of cleaning material.", "25.Cleaning device according to claim 18, wherein the shaft comprises an insertion opening for feeding the single dosage of cleaning material to the cleaning device, the insertion opening located half way between an upper end and a bottom end of the shaft.", "26.Cleaning device according to claim 25, wherein the insertion opening is located at the upper end of the shaft.", "27.Cleaning device according to claim 16, wherein the cleaning head comprises at least one outlet opening through which cleaning material can be released.", "28.Cleaning device according to claim 16, wherein the cleaning head comprises a plurality of bristles and a plurality of outlet openings between the bristles.", "29.A container comprising a single dosage of cleaning material, wherein the container is made of a water soluble foil and contains powder or liquid, non-aqueous material.", "30.Container according to claim 29, wherein the water soluble foil comprises a low-temperature dissolving polyvinylalcohol (PVA).", "31.Container according to claim 29, wherein the container has a cylindrical shape with a circular cross section.", "32.Container according to claim 30, wherein the container has a cylindrical shape with a circular cross section.", "33.A method of cleaning surfaces comprising feeding a container comprising a single dosage of a cleaning material to a cleaning device as claimed in claim 16.34.A method of cleaning surfaces comprising feeding tablet comprising a single dosage of a cleaning material to a cleaning device as claimed in claim 16." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 a - d show a sectional view of an inventive cleaning device and steps of insertion of a container with cleaning material into the device; FIG.", "2 a shows a second inventive device in a first position for the insertion of a cartridge; FIG.", "2 b shows the second inventive device in a second position, e.g.", "for storing the device or for cleaning.", "FIG.", "1 a - d show an inventive cleaning device 1 in a sectional view.", "Four steps of the insertion of a container 15 with cleaning material 17 into the device 1 are depicted.", "detailed-description description=\"Detailed Description\" end=\"lead\"?", "The inventive device 1 comprises a cleaning head 3 in the form of a brush with a plurality of bristles 9 .", "It further comprises a handle 2 having a shaft 2 ′ whose bottom end 2 a comprises the bristles 9 and forms the cleaning head 3 .", "The upper end 2 b of the shaft 2 ′ comprises a grip portion where the user can grip the handle 2 when cleaning.", "The shaft is a hollow tube forming a cylindrical passage 4 .", "The bottom end 5 of the passage 4 is closed.", "A cutting edge 10 is located at the bottom end 5 for piercing a cartridge-like container 15 inserted into the passage 4 and pushed down to the bottom end 5 .", "A cylindrical rod 6 fits tightly into the passage 4 and is able to slide up and down.", "A user can move the rod 6 by moving a slider 13 which is connected to the rod 6 and moves along an axial slit opening 14 in the shaft 2 ′.", "A preferably weak spring 8 contained at the upper end 2 b of the shaft 2 ′ is compressed when the slider 13 respectively the rod 6 is pulled upward, as shown in FIG.", "1 b .", "The spring 14 helps to push the rod downward when moving the slider 13 downward and to keep the rod in the downward position, as shown in FIGS.", "1 a , 1 c and 1 d. FIG.", "1 a shows a cleaning device 1 without a container 15 with cleaning material 17 inserted, the rod 6 being in the downward position.", "When the rod 6 is in the upper position access to the bottom end 5 of the passage 4 can be gained via an introduction opening 12 , which can be additionally closed by a door.", "Through this introduction opening 12 a container 15 with cleaning material 17 is inserted into the passage 4 , as shown in FIG.", "1 b .", "The cartridge 15 is then pushed down by the rod 6 , as shown in FIG.", "1 c .", "As it contacts an is pressed against the cutting edge 10 , here a spike, the foil forming the container walls is pierced and the cleaning material 17 , e.g.", "a powder, is released via outlet openings 16 in the walls of the bottom portion 2 a of the shaft, as shown in FIG.", "1 d .", "The container itself is then dissolved by water entering the bottom end 5 of the passage 4 via the outlet openings 16 .", "The empty cleaning device can then be reused for another cleaning action, i.e.", "inserting a new container, pushing it down, etc.", "FIG.", "2 a, b show a second inventive device 1 ′ in two positions.", "The device 1 ′ in a first “open” position for the insertion of a cartridge (not shown) is shown in FIG.", "2 a ; the device in a second “closed” position, e.g.", "for storing the device or for cleaning, is shown in FIG.", "2 b.", "The inventive device 1 ′, here for the use as toilet brush, comprises a handle 2 ″ and a cleaning head 3 ″ connected to or being an integral part of the handle 2 ″.", "The cleaning head has a plurality of bristles 9 ′ for scrubbing a toilet bowl and the like.", "The handle comprises a first member 19 and an elongate second member 20 which forms the major part of the handle.", "First and second member 19 , 20 are movable with respect to each other by a bar 18 which is connected to the first member 19 an is able to slide within the second member 20 in its axial direction.", "The second member comprises an insertion opening 12 ′ for the cleaning cartridge.", "The opening 12 ′ is open when the two members 19 , 20 are at maximum distance from each other, as shown in FIG.", "2 a .", "At least in the a region 21 extending from the opening 12 ′ to the cleaning head 3 ′ the second member 20 and the cleaning head 3 ′ are hollow, forming a passage 4 ′ extending from the opening to the bottom 5 ′ of the cleaning head 3 ′ for the insertion and delivery of cleaning material.", "In the “open” position as shown in FIG.", "2 a a cartridge can be inserted into the opening.", "It is then pushed down to the bottom 5 ′ of the passage 4 ′ by moving the first member 19 towards the second member 20 .", "Via the bar 18 a rod 6 ′ which is connected to the bar or an integral part of the bar 18 is pushed into the passage, acting to push the cartridge down towards the cleaning head 3 ′.", "The rod 6 ′ closes the opening 12 ′ by fitting tightly into the passage 4 ′.", "In FIG.", "2 b two members 19 , 20 are snapped onto each other to facilitate handling the device.", "In use, water enters the cleaning head 3 ′ through openings 16 ′ which are disposed between the bristles 9 ′.", "Water then dissolves or disintegrates the cartridge with active material or a solid tablet of cleaning material contained inside the head 3 ′.", "Liquid containing active material is then released via the same openings 16 ′ directly to the place of application.", "detailed-description description=\"Detailed Description\" end=\"tail\"?" ], [ "The present invention relates to a cleaning device according to claim 1, especially for cleaning containers with permanent water volume, as toilet bowls and the like.", "The invention further relates to a container containing a single dosage of cleaning material according to claim 11.Cleaning a toilet bowl is typically one of the most undesirable jobs for most persons.", "Nevertheless, toilet bowls must be kept clean in order to prevent sanitary problems, the potential for irritable smells, and the possibility of harmful bacteria buildup.", "Various types of bowl cleaning products are known.", "Such products typically fall within two categories, namely, cleaning by hand with a bowl cleaning device or with automatic “in tank” or “in bowl” cleaners.", "Automatic “in tank” or “in bowl” cleaners comprise cleaning material in the form of a slowly dissolving block or tablet or of a gel or liquid disposed under the rim of the toilet bowl or in the water container.", "These automatic cleaners dispense a dosage of cleaning material upon flushing of the toilet.", "However they are generally not as effective as manual scrubbing.", "Therefore most consumers typically supplement such automatic cleaners with hand scrubbing and cleaning.", "Hand cleaning typically takes the form of a toilet cleaning brush or sponge.", "Most users apply a certain dosage of liquid cleaning material or cleaning powder, stored in a bottle or a container, to the surface of the toilet bowl.", "The right dosage has to be estimated by the user.", "Most people wanting a sufficient cleaning result tend to apply an overdose.", "This may cause foam development that can't be flushed in one go and more burden to the environment than necessary.", "Further, most bottles respectively containers do not allow dosing the cleaning material exactly, as their openings are often wide, dispensing the cleaning material in one splash.", "Some products have angled nozzles to reach under the rim of the toilet but these till result in a lot of the cleaning liquid running into the bowl water and not remaining in the area needed to be cleaned.", "Furthermore, cleaning powder kept in bottles or other containers tends to agglomerate in the humid bathroom atmosphere when the bottle is not closed properly.", "Further, though most containers have a safety cap the material contained is a danger to children who manage to open it.", "Furthermore, regular toilet brushes tend to get dirty and attract germs.", "Soil can be forced into the brush where it remains.", "This means that the user has to periodically clean or replace the brush.", "It is therefore an object of the invention to provide a cleaning device and a container for storing cleaning material, especially for cleaning toilet bowls and the like, which avoids the problems of the cleaning devices mentioned before and which especially facilitates dosing and safe storage of the cleaning material.", "This object is achieved by a cleaning device according to claim 1 and a container containing a single dosage of cleaning material according to claim 11.The object is further achieved by the use of such a container or a material tablet to feed a single dosage of cleaning material to such a cleaning device.", "Beneficial embodiments of the invention are described in the dependent claims.", "An inventive cleaning device comprising a cleaning head, a handle adapted to receive at least one single dosage of cleaning material and delivering means operable, in use, to deliver a single dosage of cleaning material into the cleaning head and to release the cleaning material from the cleaning head has the advantage that a well defined dosage of cleaning material is delivered directly to the cleaning head and is thereby dispensed directly at the surface which is to be cleaned.", "Further, no manual contact with the cleaning head is necessary.", "This ensures that the highest concentration of active material, i.e.", "the cleaning formulation or composition, is within bristles, a sponge, closed-cell phenolic foam or other medium acting as use surface for cleaning or scrubbing, and the material is then distributed from there respectively by them.", "Thereby the active material is used more efficient than when poured directly onto the surface, from where it is at least partially dissolved and flushed without being used for cleaning.", "Another advantage is that a well defined dosage of active material is applied to the surface, ensuring optimum cleaning action at minimum expenses and minimum release of substances burdening the environment.", "The dosage may be adjusted to the application, e.g.", "toilet bowl cleaning, by providing containers with a respective volume and/or concentration of active material.", "A further advantage is that the user does not need to handle the material itself.", "A single material dosage is preferably contained in a container or a tablet which is easy, safe and clean to handle.", "Even with liquid materials no spilling can occur as the active material is contained in a closed container and released from the cleaning head, without the user touching it directly.", "Another advantage is that with the cleaning formula coming from within the brush, it keeps the brush itself clean and prevents germ attraction.", "The inventive device is preferably used for, but not restricted to cleaning surfaces of containers with a permanent water volume.", "Cleaning material is preferably inserted in the form of a material tablet or contained in a cartridge-like container, whose walls consist of water soluble material, preferably Polyvinylalcohol (PVA), which is preferably low-temperature dissolving.", "Water reaching the cleaning material contained in the device through openings in the cleaning head dissolves or disintegrates the active cleaning material and/or its container.", "A liquid, e.g.", "a solution, suspension or dispersion, containing cleaning material is then released through the same openings.", "In case the cleaning material is contained in a container, the container walls dissolve completely during the time normally required for a cleaning action such that, after cleaning the surface, the device is ready for the next use without the need for rinsing or cleaning it.", "In case a material tablet is used the features of the cleaning composition are chosen such that the tablet is completely decomposed under the influence of water during the typical cleaning time, e.g.", "30 seconds.", "The cleaning material itself is not necessarily water soluble, but can for example contain abrasives.", "In a preferred embodiment the handle comprises an at least partially hollow shaft extending to the cleaning head and forming a passage for insertion and delivery of the single dosage of cleaning material.", "Preferably, the handle further comprises a rod dimensioned to slide within the passage for pushing the single dosage of cleaning material into or next to the cleaning head.", "Thereby it is ensured that the single dosage of cleaning material can be inserted at or near the top end of the handle, respectively a portion of the device which is not in direct contact with the surface to clean, at a distance from the cleaning head.", "Thus the insertion area is clean and dry, and the inserted active material is delivered into the cleaning head without the need for manual handling or touching the device near the cleaning head.", "Preferably, the device further comprises a spring acting to push the rod to the bottom of the passage.", "The diameter of rod and passage are designed such that the passage fits tightly around the rod, while the rod is able to slide within the passage.", "Thereby water is prevented from entering an upper portion of the passage, thereby ensuring that the inside of the passage is dry, and a water soluble cartridge inserted into the passage does not get stuck in the passage.", "The rod can comprise a gasket to seal the passage respectively its upper part off from the wet inside of the cleaning head.", "In another preferred embodiment of the invention, the cleaning device comprises a cutting edge, e.g.", "a spike, located in the device, preferably at a bottom end of the passage.", "This edge facilitates cutting or piercing a cartridge-like container inserted into the device, wherein the container contains the single dosage of cleaning material as powder or liquid detergent.", "This has the advantage that the container is pierced when pushed into the device, immediately releasing the cleaning material.", "The inventive container or cartridge contains a single dosage of non-aqueous or anhydrous powder or liquid cleaning material and is made of a water soluble foil.", "Preferably, the water soluble foil consists of a Polyvinylalcohol (PVA) which is preferably low-temperature dissolving.", "PVA films supplied by the following suppliers can be used: Aquafilm ltd. (AQUAFILM), Environmental Polymers ltd. (EP POLY), Cris Craft Inc. (MONO-SOLO●).", "To fit into the inventive cleaning device with a passage having a circular cross section the container preferably has a cylindrical shape, preferably with a circular cross section.", "Cartridges like this are easy to manufacture from a sheet material or a flexible tube.", "Preferably sachets are constructed from a tube that is sealed at the ends, e.g.", "twisted or heat sealed.", "This methods results in no flanges around the edge, i.e.", "the surface in contact with the tube is free of excess PVA.", "The diameter of the cartridge ranges from 10 to 40 mm, preferably around 25 to 35 mm.", "The length of the cartridge ranges from 30 to 80 mm, preferably around 45 to 55 mm.", "The cartridge has an internal volume of approximately 1.5 to 43 cm3, preferably around 18 to 20 cm3.Alternatively, the cartridge is a small pouch containing active material or has spherical shape, as known for single dosage soap containers.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1a-d show a sectional view of an inventive cleaning device and steps of insertion of a container with cleaning material into the device; FIG.", "2a shows a second inventive device in a first position for the insertion of a cartridge; FIG.", "2b shows the second inventive device in a second position, e.g.", "for storing the device or for cleaning.", "FIG.", "1a-d show an inventive cleaning device 1 in a sectional view.", "Four steps of the insertion of a container 15 with cleaning material 17 into the device 1 are depicted.", "The inventive device 1 comprises a cleaning head 3 in the form of a brush with a plurality of bristles 9.It further comprises a handle 2 having a shaft 2′ whose bottom end 2a comprises the bristles 9 and forms the cleaning head 3.The upper end 2b of the shaft 2′ comprises a grip portion where the user can grip the handle 2 when cleaning.", "The shaft is a hollow tube forming a cylindrical passage 4.The bottom end 5 of the passage 4 is closed.", "A cutting edge 10 is located at the bottom end 5 for piercing a cartridge-like container 15 inserted into the passage 4 and pushed down to the bottom end 5.A cylindrical rod 6 fits tightly into the passage 4 and is able to slide up and down.", "A user can move the rod 6 by moving a slider 13 which is connected to the rod 6 and moves along an axial slit opening 14 in the shaft 2′.", "A preferably weak spring 8 contained at the upper end 2b of the shaft 2′ is compressed when the slider 13 respectively the rod 6 is pulled upward, as shown in FIG.", "1b.", "The spring 14 helps to push the rod downward when moving the slider 13 downward and to keep the rod in the downward position, as shown in FIGS.", "1a, 1c and 1d.", "FIG.", "1a shows a cleaning device 1 without a container 15 with cleaning material 17 inserted, the rod 6 being in the downward position.", "When the rod 6 is in the upper position access to the bottom end 5 of the passage 4 can be gained via an introduction opening 12, which can be additionally closed by a door.", "Through this introduction opening 12 a container 15 with cleaning material 17 is inserted into the passage 4, as shown in FIG.", "1b.", "The cartridge 15 is then pushed down by the rod 6, as shown in FIG.", "1c.", "As it contacts an is pressed against the cutting edge 10, here a spike, the foil forming the container walls is pierced and the cleaning material 17, e.g.", "a powder, is released via outlet openings 16 in the walls of the bottom portion 2a of the shaft, as shown in FIG.", "1d.", "The container itself is then dissolved by water entering the bottom end 5 of the passage 4 via the outlet openings 16.The empty cleaning device can then be reused for another cleaning action, i.e.", "inserting a new container, pushing it down, etc.", "FIG.", "2a, b show a second inventive device 1′ in two positions.", "The device 1′ in a first “open” position for the insertion of a cartridge (not shown) is shown in FIG.", "2a; the device in a second “closed” position, e.g.", "for storing the device or for cleaning, is shown in FIG.", "2b.", "The inventive device 1′, here for the use as toilet brush, comprises a handle 2″ and a cleaning head 3″ connected to or being an integral part of the handle 2″.", "The cleaning head has a plurality of bristles 9′ for scrubbing a toilet bowl and the like.", "The handle comprises a first member 19 and an elongate second member 20 which forms the major part of the handle.", "First and second member 19, 20 are movable with respect to each other by a bar 18 which is connected to the first member 19 an is able to slide within the second member 20 in its axial direction.", "The second member comprises an insertion opening 12′ for the cleaning cartridge.", "The opening 12′ is open when the two members 19, 20 are at maximum distance from each other, as shown in FIG.", "2a.", "At least in the a region 21 extending from the opening 12′ to the cleaning head 3′ the second member 20 and the cleaning head 3′ are hollow, forming a passage 4′ extending from the opening to the bottom 5′ of the cleaning head 3′ for the insertion and delivery of cleaning material.", "In the “open” position as shown in FIG.", "2a a cartridge can be inserted into the opening.", "It is then pushed down to the bottom 5′ of the passage 4′ by moving the first member 19 towards the second member 20.Via the bar 18 a rod 6′ which is connected to the bar or an integral part of the bar 18 is pushed into the passage, acting to push the cartridge down towards the cleaning head 3′.", "The rod 6′ closes the opening 12′ by fitting tightly into the passage 4′.", "In FIG.", "2b two members 19, 20 are snapped onto each other to facilitate handling the device.", "In use, water enters the cleaning head 3′ through openings 16′ which are disposed between the bristles 9′.", "Water then dissolves or disintegrates the cartridge with active material or a solid tablet of cleaning material contained inside the head 3′.", "Liquid containing active material is then released via the same openings 16′ directly to the place of application." ] ]

[ [ "Vaccine against Streptococcus pneumoniae", "The present invention relates to the field of bacterial polysaccharide antigen vaccines.", "In particular, the present invention relates to vaccines comprising a pneumococcal polysaccharide antigen, typically a pneumococcal polysaccharide conjugate antigen from Streptococcus pneumoniae selected from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a Th1-inducing adjuvant." ], [ "1.An immunogenic composition comprising at least one Streptococcus pneumoniae polysaccharide antigen and at least one Streptococcus pneumoniae protein antigen selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, or immunologically functional equivalent thereof.", "2.The immunogenic composition of claim 1, wherein the polysaccharide antigen is presented in the form of a polysaccharide-protein carrier conjugate.", "3.The immunogenic composition of claim 2, wherein the carrier protein is selected from the group consisting of: Diphtheria toxoid, Tetanus toxoid, CRM197, Keyhole Limpet Haemocyanin (KLH), protein derivative of Tuberculin (PPD), and protein D from H. influenzae.", "4.An immunogenic composition as claimed in any of claims 1 to 3 wherein the vaccine comprises at least four pneumococcal polysaccharide antigens from different serotypes.", "5.An immunogenic composition as claimed herein additionally comprising an adjuvant.", "6.An immunogenic composition as claimed in claim 5, wherein the adjuvant comprises an aluminium salt.", "7.An immunogenic composition as claimed in claim 5, wherein the adjuvant is a preferential inducer of a TH1 response.", "8.An immunogenic composition as claimed in claim 7, wherein the adjuvant comprises at least one of the following: 3D-MPL, a saponin immunostimulant, or an immunostimulatory CpG oligonucleotide.", "9.An immunogenic composition as claimed in claim 8, wherein the adjuvant comprises a carrier selected from the group comprising: an oil in water emulsion, liposomes, and an aluminium salt.", "10.An immunogenic composition composition as claimed herein for use as a medicament.", "11.A vaccine comprising the immunogenic composition of claims 1-9.12.A method of preventing or ameliorating Streptoccocus pneumoniae infection in a patient over 55 years, comprising administering an effective amount of a vaccine comprising a Streptococcus pneumoniae polysaccharide and at least one Streptococcus pneumoniae protein selected from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a TH1 inducing adjuvant.", "13.Use of a pneumococcal polysaccharide antigen in combination with a Streptoccocus pneumoniae protein antigen selected from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a TH1 inducing adjuvant, in the manufacture of a medicament for the prevention or treatment of pneumonia in patients over 55 years.", "14.Use of a pneumococcal polysaccharide antigen in combination with a Streptoccocus pneumoniae protein antigen selected from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a TH1 inducing adjuvant, in the manufacture of a medicament for the prevention or treatment of otitis media in infants or toddlers.", "15.A method of making an immunogenic composition as claimed herein, comprising the steps of: selecting one or more pneumococcal polysaccharide antigen(s); selecting one or more pneumococcal protein antigen(s) from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133; and mixing said polysaccharide and protein antigens with a suitable excipient.", "16.A method of preventing or ameliorating Otitis media in Infants, comprising administering a safe and effective amount of a vaccine comprising a Streptococcus pneumoniae polysaccharide antigen and a Streptococcus pneumoniae protein antigen selected from the group consisting of PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, optionally with a TH1 adjuvant, to said Infant." ], [ "<SOH> BACKGROUND OF INVENTION <EOH>Streptococcus pneumoniae is a Gram-positive bacteria responsible for considerable morbidity and mortality (particularly in the young and aged), causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonisation, such as acute Otitis media.", "The rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000.In 20% of cases this leads to bacteremia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.", "Pneumococcus is encapsulated with a chemically linked polysaccharide which confers serotype specificity.", "There are 90 known serotypes of pneumococci, and the capsule is the principle virulence determinant for pneumococci, as the capsule not only protects the inner surface of the bacteria from complement, but is itself poorly immunogenic.", "Polysaccharides are T-independent antigens, and can not be processed or presented on MHC molecules to interact with T-cells.", "They can however, stimulate the immune system through an alternate mechanism which involves cross-linking of surface receptors on B cells.", "It was shown in several experiments that protection against invasive pneumococci disease is correlated most strongly with antibody specific for the capsule, and the protection is serotype specific.", "Polysaccharide antigen based vaccines are well known in the art.", "Four that have been licensed for human use include the Vi polysaccharide of Salmonella typhi, the PRP polysaccharide from Haemophilus influenzae, the tetravalent meningococcal vaccine composed of serotypes A, C, W135 and Y, and the 23-Valent pneumococcal vaccine composed of the polysaccharides corresponding to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33 (accounting for at least 90% of pneumococcal blood isolates).", "The latter three vaccines confer protection against bacteria causing respiratory infections resulting in severe morbidity and mortality in infants, yet these vaccines have not been licensed for use in children less than two years of age because they are inadequately immunogenic in this age group [Peltola et al.", "(1984), N. Engl.", "J. Med.", "310:1561-1566 ].", "Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children.", "Likewise, the elderly mount poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol.", "42:265-270], hence the increased incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62:271-285].", "Strategies, which have been designed to overcome this lack of immunogenicity in infants, include the linking of the polysaccharide to large immunogenic proteins, which provide bystander T-cell help and which induce immunological memory against the polysaccharide antigen to which it is conjugated.", "Pneumococcal glycoprotein conjugate vaccines are currently being evaluated for safety, immunogenicity and efficacy in various age groups.", "The 23-valent unconjugated pneumococcal vaccine has shown a wide variation in clinical efficacy, from 0% to 81% (Fedson et al.", "(1994) Arch Intern Med.", "154: 2531-2535).", "The efficacy appears to be related to the risk group that is being immunised, such as the elderly, Hodgkin's disease, splenectomy, sickle cell disease and agammaglobulinemics (Fine et al.", "(1994) Arch Intern Med.", "154:2666-2677), and also to the disease manifestation.", "The 23-valent vaccine does not demonstrate protection against pneumococcal pneumonia (in certain high risk groups such as the elderly) and otitis media diseases.", "There is therefore a need for improved pneumococcal vaccine compositions, particularly ones which will be more effective in the prevention or amelioration of pneumococcal disease (particularly pneumonia) in the elderly and in young children.", "The present invention provides such an improved vaccine." ], [ "<SOH> SUMMARY OF TIE INVENTION <EOH>Accordingly the present invention provides a vaccine composition, comprising at least one Streptococcus pneumoniae polysaccharide antigen (preferably conjugated to a protein carrier) and a Streptococcus pneumoniae protein antigen selected from the group consisting of: Poly Histidine Triad family (Pht; e.g.", "PhtA, PhtB, PhtD, or PhtE), Lyt family (e.g.", "LytA, LytB, or LytC), SpsA, Sp128, Sp130, Sp125, Sp101 and Sp133, or truncate or immunologically functional equivalent thereof, optionally with a Th1 adjuvant (an adjuvant inducing a predominantly Th1 immune response).", "Preferably both a pneumococcal protein and Th1 adjuvant are included.", "Advantageous compositions comprising combinations of the above pneumococcal proteins of the invention with each other and with other pneumococcal proteins are also described.", "The compositions of the invention are particularly suited in the treatment of elderly pneumonia.", "Pneumococcal polysaccharide vaccines (conjugated or not) may not be able to protect against pneumonia in the elderly population for which the incidence of this disease is very high.", "The key defense mechanism against the pneumococcus is opsonophagocytosis (a humoral B-cell/neutrophil mediated event caused by the production of antibodies against the pneumococcal polysaccharide, the bacterium eventually becoming phagocytosed), however parts of the involved opsonic mechanisms are impaired in the elderly, i.e.", "superoxide production by PMN (polymorphonuclear cells), other reactive oxygen species production, mobilization of PMN, apoptosis of PMN, deformability of PMN.", "Antibody responses may also be impaired in the elderly.", "Contrary to the normally accepted dogma, normal levels of anti-capsular polysaccharide antibodies may not be effective in complete clearance of bacteria, as pneumococci may invade host cells to evade this branch of the immune system.", "Surprisingly, the present inventors have found that by simultaneously stimulating the cell mediated branch of the immune system (for instance T-cell meditated immunity) in addition to the humoral brach of the immune system (B-cell mediated), a synergy (or cooperation) may result which is capable of enhancing the clearance of pneumococci from the host.", "This is a discovery which will aid the prevention (or treatment) of pneumococcal infection in general, but will be particularly important for the prevention (or treatment) of pneumonia in the elderly where polysaccharide based vaccines do not show efficacy.", "Without wishing to be bound by any theory, the present inventors have found that both arms of the immune system may synergise in this way if a pneumococcal polysaccharide (preferably conjugated to a protein carrier) is administered with a pneumococcal protein selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133 (proteins which can be processed and presented in the context of Class II and MHC class I on the surface of infected mammalian cells).", "Although one or more of these pneumococcal proteins can trigger cell mediated immunity by itself, the inventors have also found that the presence of a Th1 inducing adjuvant in the vaccine formulation helps this arm of the immune system, and surprisingly further enhances the synergy between both arms of the immune system." ], [ "FIELD OF INVENTION The present invention relates to bacterial polysaccharide antigen vaccines, their manufacture and the use of such polysaccharides in medicines.", "In particular the present invention relates to vaccines comprising a pneumococcal polysaccharide antigen, typically a pneumococcal polysaccharide conjugate antigen, formulated with a protein antigen from Streptococcus pneumoniae and optionally a Th1 inducing adjuvant.", "BACKGROUND OF INVENTION Streptococcus pneumoniae is a Gram-positive bacteria responsible for considerable morbidity and mortality (particularly in the young and aged), causing invasive diseases such as pneumonia, bacteremia and meningitis, and diseases associated with colonisation, such as acute Otitis media.", "The rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000.In 20% of cases this leads to bacteremia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.", "Pneumococcus is encapsulated with a chemically linked polysaccharide which confers serotype specificity.", "There are 90 known serotypes of pneumococci, and the capsule is the principle virulence determinant for pneumococci, as the capsule not only protects the inner surface of the bacteria from complement, but is itself poorly immunogenic.", "Polysaccharides are T-independent antigens, and can not be processed or presented on MHC molecules to interact with T-cells.", "They can however, stimulate the immune system through an alternate mechanism which involves cross-linking of surface receptors on B cells.", "It was shown in several experiments that protection against invasive pneumococci disease is correlated most strongly with antibody specific for the capsule, and the protection is serotype specific.", "Polysaccharide antigen based vaccines are well known in the art.", "Four that have been licensed for human use include the Vi polysaccharide of Salmonella typhi, the PRP polysaccharide from Haemophilus influenzae, the tetravalent meningococcal vaccine composed of serotypes A, C, W135 and Y, and the 23-Valent pneumococcal vaccine composed of the polysaccharides corresponding to serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33 (accounting for at least 90% of pneumococcal blood isolates).", "The latter three vaccines confer protection against bacteria causing respiratory infections resulting in severe morbidity and mortality in infants, yet these vaccines have not been licensed for use in children less than two years of age because they are inadequately immunogenic in this age group [Peltola et al.", "(1984), N. Engl.", "J. Med.", "310:1561-1566].", "Streptococcus pneumoniae is the most common cause of invasive bacterial disease and otitis media in infants and young children.", "Likewise, the elderly mount poor responses to pneumococcal vaccines [Roghmann et al., (1987), J. Gerontol.", "42:265-270], hence the increased incidence of bacterial pneumonia in this population [Verghese and Berk, (1983) Medicine (Baltimore) 62:271-285].", "Strategies, which have been designed to overcome this lack of immunogenicity in infants, include the linking of the polysaccharide to large immunogenic proteins, which provide bystander T-cell help and which induce immunological memory against the polysaccharide antigen to which it is conjugated.", "Pneumococcal glycoprotein conjugate vaccines are currently being evaluated for safety, immunogenicity and efficacy in various age groups.", "The 23-valent unconjugated pneumococcal vaccine has shown a wide variation in clinical efficacy, from 0% to 81% (Fedson et al.", "(1994) Arch Intern Med.", "154: 2531-2535).", "The efficacy appears to be related to the risk group that is being immunised, such as the elderly, Hodgkin's disease, splenectomy, sickle cell disease and agammaglobulinemics (Fine et al.", "(1994) Arch Intern Med.", "154:2666-2677), and also to the disease manifestation.", "The 23-valent vaccine does not demonstrate protection against pneumococcal pneumonia (in certain high risk groups such as the elderly) and otitis media diseases.", "There is therefore a need for improved pneumococcal vaccine compositions, particularly ones which will be more effective in the prevention or amelioration of pneumococcal disease (particularly pneumonia) in the elderly and in young children.", "The present invention provides such an improved vaccine.", "SUMMARY OF TIE INVENTION Accordingly the present invention provides a vaccine composition, comprising at least one Streptococcus pneumoniae polysaccharide antigen (preferably conjugated to a protein carrier) and a Streptococcus pneumoniae protein antigen selected from the group consisting of: Poly Histidine Triad family (Pht; e.g.", "PhtA, PhtB, PhtD, or PhtE), Lyt family (e.g.", "LytA, LytB, or LytC), SpsA, Sp128, Sp130, Sp125, Sp101 and Sp133, or truncate or immunologically functional equivalent thereof, optionally with a Th1 adjuvant (an adjuvant inducing a predominantly Th1 immune response).", "Preferably both a pneumococcal protein and Th1 adjuvant are included.", "Advantageous compositions comprising combinations of the above pneumococcal proteins of the invention with each other and with other pneumococcal proteins are also described.", "The compositions of the invention are particularly suited in the treatment of elderly pneumonia.", "Pneumococcal polysaccharide vaccines (conjugated or not) may not be able to protect against pneumonia in the elderly population for which the incidence of this disease is very high.", "The key defense mechanism against the pneumococcus is opsonophagocytosis (a humoral B-cell/neutrophil mediated event caused by the production of antibodies against the pneumococcal polysaccharide, the bacterium eventually becoming phagocytosed), however parts of the involved opsonic mechanisms are impaired in the elderly, i.e.", "superoxide production by PMN (polymorphonuclear cells), other reactive oxygen species production, mobilization of PMN, apoptosis of PMN, deformability of PMN.", "Antibody responses may also be impaired in the elderly.", "Contrary to the normally accepted dogma, normal levels of anti-capsular polysaccharide antibodies may not be effective in complete clearance of bacteria, as pneumococci may invade host cells to evade this branch of the immune system.", "Surprisingly, the present inventors have found that by simultaneously stimulating the cell mediated branch of the immune system (for instance T-cell meditated immunity) in addition to the humoral brach of the immune system (B-cell mediated), a synergy (or cooperation) may result which is capable of enhancing the clearance of pneumococci from the host.", "This is a discovery which will aid the prevention (or treatment) of pneumococcal infection in general, but will be particularly important for the prevention (or treatment) of pneumonia in the elderly where polysaccharide based vaccines do not show efficacy.", "Without wishing to be bound by any theory, the present inventors have found that both arms of the immune system may synergise in this way if a pneumococcal polysaccharide (preferably conjugated to a protein carrier) is administered with a pneumococcal protein selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133 (proteins which can be processed and presented in the context of Class II and MHC class I on the surface of infected mammalian cells).", "Although one or more of these pneumococcal proteins can trigger cell mediated immunity by itself, the inventors have also found that the presence of a Th1 inducing adjuvant in the vaccine formulation helps this arm of the immune system, and surprisingly further enhances the synergy between both arms of the immune system.", "DESCRIPTION OF THE INVENTION The present invention provides an improved vaccine particularly for the prevention or amelioration of pnemococcal infection of the elderly (and/or infants and toddlers).", "In the context of the invention a patient is considered elderly if they are 55 years or over in age, typically over 60 years and more generally over 65 years.", "Thus in one embodiment of the invention there is provided a vaccine composition, suitable for use in the elderly (and/or Infants and toddlers) comprising at least one Streptococcus pneumoniae polysaccharide antigen and at least one Streptococcus pneumoniae protein antigen(s) selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133.The vaccine may optionally comprise a Th1 adjuvant.", "In a second, preferred, embodiment, the present invention provides a vaccine (suitable for the prevention of pneumonia in the elderly) comprising at least one (2, 3, 4, 5, 6, 7, 8, 9 or 10) Streptococcus pneumoniae polysaccharide antigen(s) and at least one Streptococcus pneumoniae protein antigen selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and, preferably, a Th1 adjuvant.", "In the above embodiments vaccines advantageously comprising combinations of the above pneumococcal proteins of the invention with each other and with other pneumococcal proteins are also envisioned as described below.", "It is envisaged that such a vaccine will be also useful in treating pneumococcal infection (for instance otitis media) in other high risk groups of the population, such as for infants or toddlers.", "STREPTOCOCCUS PNEUMONIAE POLYSACCHARIDE ANTIGENS OF THE INVENTION Typically the Streptococcus pneumoniae vaccine of the present invention will comprise polysaccharide antigens (preferably conjugated to a carrier protein), wherein the polysaccharides are derived from at least four serotypes of pneumococcus.", "Preferably the four serotypes include 6B, 14, 19F and 23F.", "More preferably, at least 7 serotypes are included in the composition, for example those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.", "More preferably still, at least 11 serotypes are included in the composition, for example the composition in one embodiment includes capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated to a carrier protein).", "In a preferred embodiment of the invention at least 13 polysaccharide antigens (preferably conjugated to a carrier protein) are included, although further polysaccharide antigens, for example 23 valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention.", "For elderly vaccination (for instance for the prevention of pneumonia) it is advantageous to include serotypes 8 and 12F (and most preferably 15 and 22 as well) to the 11 valent antigenic composition described above to form a 15 valent vaccine, whereas for infants or toddlers (where otitis media is of more concern) serotypes 6A and 19A are advantageously included to form a 13 valent vaccine.", "Although the above polysaccharides may be used in their full-length, native form, it should be understood that size-reduced polysaccharides may also be used which are still immunogenic (see for example EP 497524 and 497525).", "For the prevention/amelioration of pneumonia in the elderly (+55 years) population and Otitis media in Infants (up to 18 months) and toddlers (typically 18 months to 5 years), it is a preferred embodiment of the invention to combine a multivalent Streptococcus pneumonia polysaccharide as herein described with a Streptococcus pneumoniae protein selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, or immunologically functional equivalent thereof.", "A combination of pneumococcal proteins may also be advantageously utilised as described below.", "PNEUMOCOCCAL PROTEINS OF THE INVENTION For the purposes of this invention, “immunologically functional equivalent” is defined as a peptide of protein comprising at least one protective epitope from the proteins of the invention.", "Such epitopes are characteristically surface-exposed, highly conserved, and can elicit an bactericidal antibody response in a host or prevent toxic effects.", "Preferably, the functional equivalent has at least 15 and preferably 30 or more contiguous amino acids from the protein of the invention.", "Most preferably, fragments, deletions of the protein, such as transmembrane deletion variants thereof (ie the use of the extracellular domain of the proteins), fusions, chemically or genetically detoxified derivatives and the like can be used with the proviso that they are capable of raising substantially the same immune response as the native protein.", "The position of potential B-cell epitopes in a protein sequence may be readily determined by identifying peptides that are both surface-exposed and antigenic using a combination of two methods: 2D-structure prediction and antigenic index prediction.", "The 2D-structure prediction can be made using the PSIPRED program (from David Jones, Brunel Bioinformatics Group, Dept.", "Biological Sciences, Brunel University, Uxbridge UB8 3PH, UK).", "The antigenic index can be calculated on the basis of the method described by Jameson and Wolf (CABIOS 4:181-186 [1988]).", "The proteins of the invention are the following proteins, all of which are exposed on the outer surface of the pneumococcus (capable of being recognised by a host's immune system during at least part of the life cycle of the pneumococcus), or are proteins which are secreted or released by the pneumococcus.", "The Streptococcus pneumoniae protein of the invention is preferably selected from the group consisting of: a protein from the polyhistidine triad family (Pht), a protein from the Lyt family, a choline binding protein, proteins having an LPXTG motif (where X is any amino acid), proteins having a Type II Signal sequence motif of LXXC (where X is any amino acid), and proteins having a Type I Signal sequence motif.", "Preferred examples within these categories (or motifs) are the following proteins (or truncate or immunologically functional equivalent thereof): The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB, PhtD, and PhtE.", "The family is characterised by a lipidation sequence, two domains separated by a proline-rich region and several histidine triads, possibly involved in metal or nucleoside binding or enzymatic activity, (3-5) coiled-coil regions, a conserved N-terminus and a heterogeneous C terminus.", "It is present in all strains of pneumococci tested.", "Homologous proteins have also been found in other Streptococci and Neisseria.", "Preferred members of the family comprise PhtA, PhtB and PhtD.", "More preferably, it comprises PhtA or PhtD.", "It is understood, however, that the terms Pht A, B, D, and E refer to proteins having sequences disclosed in the citations below as well as naturally-occurring (and man-made) variants thereof that have a sequence homology that is at least 90% identical to the referenced proteins.", "Preferably it is at least 95% identical and most preferably it is 97% identical.", "With regards to the Pht proteins, PhtA is disclosed in WO 98/18930, and is also referred to Sp36.As noted above, it is a protein from the polyhistidine triad family and has the type II signal motif of LXXC.", "PhtD is disclosed in WO 00/37105, and is also referred to Sp036D.", "As noted above, it also is a protein from the polyhistidine triad family and has the type II LXXC signal motif.", "PhtB is disclosed in WO 00/37105, and is also referred to Sp036B.", "Another member of the PhtB family is the C3-Degrading Polypeptide, as disclosed in WO 00/17370.This protein also is from the polyhistidine triad family and has the type II LXXC signal motif.", "A preferred immunologically functional equivalent is the protein Sp42 disclosed in WO 98/18930.A PhtB truncate (approximately 79 kD) is disclosed in WO99/15675 which is also considered a member of the PhtX family.", "PhtE is disclosed in WO00/30299 and is referred to as BVH-3.SpsA is a Choline binding protein (Cbp) disclosed in WO 98/39450.The Lyt family is membrane associated proteins associated with cell lysis.", "The N-terminal domain comprises choline binding domain(s), however the Lyt family does not have all the features found in the choline binding protein family (Cbp) family noted below and thus for the present invention, the Lyt family is considered distinct from the Cbp family.", "In contrast with the Cbp family, the C-terminal domain contains the catalytic domain of the Lyt protein family.", "The family comprises LytA, B and C. With regards to the Lyt family, LytA is disclosed in Ronda et al., Eur J Biochem, 164:621-624 (1987).", "LytB is disclosed in WO 98/18930, and is also referred to as Sp46.LytC is also disclosed in WO 98/18930, and is also referred to as Sp91.A preferred member of that family is LytC.", "Another preferred embodiment are Lyt family truncates wherein “Lyt” is defined above and “truncates” refers to proteins lacking 50% or more of the Choline binding region.", "Preferably such proteins lack the entire choline binding region.", "Sp125 is an example of a pneumococcal surface protein with the Cell Wall Anchored motif of LPXTG (where X is any amino acid).", "Any protein within this class of pneumococcal surface protein with this motif has been found to be useful within the context of this invention, and is therefore considered a further protein of the invention.", "Sp125 itself is disclosed in WO 98/18930, and is also known as ZmpB—a zinc metalloproteinase.", "Sp101 is disclosed in WO 98/06734 (where it has the reference # y85993.It is characterised by a Type I signal sequence.", "Sp133 is disclosed in WO 98/06734 (where it has the reference # y85992.It is also characterised by a Type I signal sequence.", "Sp128 and Sp130 are disclosed in WO 00/76540.The proteins used in the present invention are preferably selected from the group PhtD and PhtA, or a combination of both of these proteins.", "ADVANTAGEOUS COMBINATION OF ONE OR MORE PNEUMOCOCCAL PROTEINS OF THE INVENTION WITH OTHER PNEUMOCOCCAL PROTEINS In the vaccine of the invention, each of the above proteins of the invention (preferably either or both of PhtD and PhtA) may also be beneficially combined with one or more pneumococcal proteins from the following list: pneumolysin (also referred to as Ply; preferably detoxified by chemical treatment or mutation) [WO 96/05859, WO 90/06951, WO 99/03884], PsaA and transmembrane deletion variants thereof (Berry & Paton, Infect Immun December 1996; 64(12):5255-62), PspA and transmembrane deletion variants thereof (U.S. Pat.", "No.", "5,804,193, WO 92/14488, WO 99/53940), PspC and transmembrane deletion variants thereof (WO 97/09994, WO 99/53940), a member of the Choline binding protein (Cbp) family [e.g.", "CbpA and transmembrane deletion variants thereof (WO 97/41151; WO 99/51266)], Glyceraldehyde-3-phosphate-dehydrogenase (Infect.", "Immun.", "1996 64:3544), HSP70 (WO 96/40928), PcpA (Sanchez-Beato et al.", "FEMS Microbiol Lett 1998, 164:207-14), M like protein (SB patent application No.", "EP 0837130), and adhesin 18627 (SB Patent application No.", "EP 0834568).", "The present invention also encompasses immunologically functional equivalents or truncates of such proteins (as defined above).", "Concerning the Choline Binding Protein family, members of that family were originally identified as pneumococcal proteins that could be purified by choline-affininty chromatography.", "All of the choline-binding proteins are non-covalently bound to phosphorylcholine moieties of cell wall teichoic acid and membrane-associated lipoteichoic acid.", "Structurally, they have several regions in common over the entire family, although the exact nature of the proteins (amino acid sequence, length, etc.)", "can vary.", "In general, choline binding proteins comprise an N terminal region (N), conserved repeat regions (R1 and/or R2), a proline rich region (P) and a conserved choline binding region (C), made up of multiple repeats, that comprises approximately one half of the protein.", "As used in this application, the term “Choline Binding Protein family (Cbp)” is selected from the group consisting of Choline Binding Proteins as identified in WO 97/41151, PbcA, SpsA, PspC, CbpA, CbpD, and CbpG.", "CbpA is disclosed in WO 97/41151.CbpD and CbpG are disclosed in WO 00/29434.PspC is disclosed in WO 97/09994.PbcA is disclosed in WO 98/21337.Preferably the Choline Binding Proteins are selected from the group consisting of CbpA, PbcA, SpsA and PspC.", "If a Cbp is the further protein utilised it may be a Cbp truncate wherein “Cbp” is defined above and “truncate” refers to proteins lacking 50% or more of the Choline binding region (C).", "Preferably such proteins lack the entire choline binding region.", "More preferably, the such protein truncates lack (i) the choline binding region and (ii) a portion of the N-terminal half of the protein as well, yet retain at least one repeat region (R1 or R2).", "More preferably still, the truncate has 2 repeat regions (R1 and R2).", "Examples of such preferred embodiments are NR1×R2 and R1×R2 as illustrated in WO99/51266 or WO99/51188, however, other choline binding proteins lacking a similar choline binding region are also contemplated within the scope of this invention.", "Cbp truncate-Lyt truncate chimeric proteins (or fusions) may also be used in the vaccine of the invention.", "Preferably this comprises NR1×R2 (or R1×R2) of Cbp and the C-terminal portion (Cterm, i.e., lacking the choline binding domains) of Lyt (e.g., LytCCterm or Sp91Cterm).", "More preferably Cbp is selected from the group consisting of CbpA, PbcA, SpsA and PspC.", "More preferably still, it is CbpA.", "Preferably, Lyt is LytC (also referred to as Sp91).", "A PspA or PsaA truncate lacking the choline binding domain (C) and expressed as a fusion protein with Lyt may also be used.", "Preferably, Lyt is LytC.", "PREFERRED COMBINATIONS OF PNEUMOCOCCAL PROTEINS FOR THE PURPOSES OF THIS INVENTION Preferably the combination of proteins of the invention are selected from 2 or more (3 or 4) different categories such as proteins having a Type II Signal sequence motif of LXXC (where X is any amino acid, e.g., the polyhistidine triad family (Pht)), choline binding proteins (Cbp), proteins having a Type I Signal sequence motif (e.g., Sp101), proteins having a LPXTG motif (where X is any amino acid, e.g., Sp128, Sp130), toxins (e.g., Ply), etc.", "Preferred examples within these categories (or motifs) are the proteins mentioned above, or immunologically functional equivalents thereof.", "Toxin+Pht, toxin+Cbp, Pht+Cbp, and toxin+Pht+Cbp are preferred category combinations.", "Preferred beneficial combinations include, but are not limited to, PhtD+NR1×R2, PhtD+NR1×R2-Sp91Cterm chimeric or fusion proteins, PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+NR1×R2, PhtA+NR1×R2-Sp91Cterm chimeric or fusion proteins, PhtA+Ply, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, NR1×R2+LytC, NR1×R2+PspA, NR1×R2+PsaA, NR1×R2+Sp128, R1×R2+LytC, R1×R2+PspA, R1×R2+PsaA, R1×R2+Sp128, R1×R2+PhtD, R1×R2+PhtA.", "Preferably, NR1×R2 (or R1×R2) is from CbpA or PspC.", "More preferably it is from CbpA.", "A particularly preferred combination of pneumococcal proteins comprises Ply (or a truncate or immunologically functional equivalent thereof)+PhtD (or a truncate or immunologically functional equivalent thereof)+NR1×R2 (or R1×R2).", "Preferably, NR1×R2 (or R1×R2) is from CbpA or PspC.", "More preferably it is from CbpA.", "Without wishing to be bound by any theory, within the composition the pneumococcal protein (or combinations described above) of the invention can help to induce a T-cell mediated response against pneumococcal disease—particularly required for protection against pneumonia—which cooperates with the humoral branch of the immune system to inhibit invasion by pneumococci, and to stimulate opsonophagocytosis.", "A further advantage of including the protein antigen is the presentation of further antigens for the opsonophagocytosis process.", "Accordingly in an embodiment of the invention there is provided a Streptococcus pneumoniae vaccine comprising a pneumococcus polysaccharide conjugate vaccine comprising polysaccharide antigens derived from at least four serotypes, preferably at least seven serotypes, more preferably at least eleven serotypes, and at least one, but preferably 2, 3, or 4, Streptococcus pneumoniae proteins selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133 (or a pneumococcal protein combination as described above).", "Preferably one of the proteins is PhtA (or an immunologically functional equivalent thereof).", "Most preferably one of the proteins is PhtD (or an immunologically functional equivalent thereof).", "As mentioned above, a problem associated with the polysaccharide approach to vaccination, is the fact that polysaccharides per se are poor immunogens.", "To overcome this, polysaccharides may be conjugated to protein carriers, which provide bystander T-cell help.", "It is preferred, therefore, that the polysaccharides utilised in the invention are linked to such a protein carrier.", "Examples of such carriers which are currently commonly used for the production of polysaccharide immunogens include the Diphtheria and Tetanus toxoids (DT, DT CRM197 and TT respectively), Keyhole Limpet Haemocyanin (KLH), OMPC from N. meningitidis, and the purified protein derivative of Tuberculin (PPD).", "A preferred carrier for the pneumococcal polysaccharide based immunogenic compositions (or vaccines) is protein D from Haemophilus influenzae (EP 594610-B), or fragments thereof.", "Fragments suitable for use include fragments encompassing T-helper epitopes.", "In particular a protein D fragment will preferably contain the N-terminal ⅓ of the protein.", "A protein D carrier is surprisingly useful as a carrier in vaccines where multiple pneumococcal polysaccharide antigens are conjugated.", "Epitope suppression is usually likely to occur if the same carrier is used for each polysaccharide.", "Surprisingly, the present inventors have found protein D is particularly suitable for minimising such epitopic suppression effects in combination vaccines.", "One or more pneumococcal polysaccharides in a combination may be advantageously conjugated onto protein D, and preferably all antigens are conjugated onto protein D within such a combination vaccine.", "A further preferred carrier for the pneumococcal polysaccharide is the pneumococcal protein itself (as defined above in section “Pneumococcal Proteins of the invention”).", "The polysaccharide may be linked to the carrier protein by any known method (for example, by Likhite, U.S. Pat.", "No.", "4,372,945 and by Armor et al., U.S. Pat.", "No.", "4,474,757).", "Preferably, CDAP conjugation is carried out (WO 95/08348).", "Preferably the protein:polysaccharide (weight:weight) ratio of the conjugates is 0.3:1 to 1:1, more preferably 0.6:1 to 0.8:1, and most preferably about 0.7:1.The vaccines of the present invention are preferably adjuvanted.", "Suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.", "It is preferred that the adjuvant be selected to be a preferential inducer of a TH1 type of response to aid the cell mediated branch of the immune response.", "TH1 ADJUVANTS OF THE INVENTION High levels of Th1-type cytokines tend to favour the induction of cell mediated immune responses to a given antigen, whilst high levels of Th2-type cytokines tend to favour the induction of humoral immune responses to the antigen.", "It is important to remember that the distinction of Th1 and Th2-type immune response is not absolute.", "In reality an individual will support an immune response which is described as being predominantly Th1 or predominantly Th2.However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties.", "Annual Review of Immunology, 7, p 145-173).", "Traditionally, Th1-type responses are associated with the production of the INF-γ and IL-2 cytokines by T-lymphocytes.", "Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by T-cells, such as IL-12.In contrast, Th2-type responses are associated with the secretion of II-4, IL-5, IL-6, IL-10.Suitable adjuvant systems which promote a predominantly Th1 response include: Monophosphoryl lipid A or a derivative thereof, particularly 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with either an aluminium salt (for instance aluminium phosphate or aluminium hydroxide) or an oil-in-water emulsion.", "In such combinations, antigen and 3D-MPL are contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals.", "Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum-adsorbed antigen [Thoelen et al.", "Vaccine (1998) 16:708-14; EP 689454-B1].", "An enhanced system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210, and is a preferred formulation.", "Preferably the vaccine additionally comprises a saponin, more preferably QS21.The formulation may also comprises an oil in water emulsion and tocopherol (WO 95/17210).", "The present invention also provides a method for producing a vaccine formulation comprising mixing a protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.", "Unmethylated CpG containing oligonucleotides (WO 96/02555) are also preferential inducers of a TH1 response and are suitable for use in the present invention.", "Particularly preferred compositions of the invention comprise one or more conjugated pneumococcal polysaccharides, one or more pneumococcal proteins of the invention and a Th1 adjuvant.", "Without wishing to be bound by any theory, the induction of a cell mediated response by way of a pneumococcal protein (as described above) and the cooperation between both arms of the immune system may be aided using such a Th-1 adjuvant, resulting in a particularly effective vaccine against pneumococcal disease in general, and, importantly, against pneumococcal pneumonia in the elderly.", "In a further aspect of the present invention there is provided an immunogen or vaccine as herein described for use in medicine.", "In one embodiment there is a method of preventing or ameliorating pneumonia in an elderly human (+55 years) comprising administering a safe and effective amount of a vaccine, as described herein, comprising a Streptoccocus pneumoniae polysaccharide antigen and a pneumococcal protein selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a Th1 adjuvant, to said elderly patient.", "In a further embodiment there is provided a method of preventing or ameliorating otitis media in Infants (up to 18 months) or toddlers (typically 18 months to 5 years), comprising administering a safe and effective amount of a vaccine comprising a Streptococcus pneumoniae polysaccharide antigen and a Streptococcus pneumoniae protein antigen selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133, and optionally a Th1 adjuvant, to said Infant or toddler.", "Preferably in the methods of the invention as described above the polysaccharide antigen is present as a polysaccharide protein conjugate.", "VACCINE PREPARATIONS OF THE INVENTION The vaccine preparations of the present invention may be used to protect or treat a mammal susceptible to infection, by means of administering said vaccine via systemic or mucosal route.", "These administrations may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.", "Intranasal administration of vaccines for the treatment of pneumonia or otitis media is preferred (as nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at its earliest stage).", "Although the vaccine of the invention may be administered as a single dose, components thereof may also be co-administered together at the same time or at different times (for instance pneumococcal polysaccharides could be administered separately at the same time or 1-2 weeks after the administration of the bacterial protein component of the vaccine for optimal coordination of the immune responses with respect to each other).", "For co-administration, the optional Th1 adjuvant may be present in any or all of the different administrations, however it is preferred if it is present in combination with the bacterial protein component of the vaccine.", "In addition to a single route of administration, 2 different routes of administration may be used.", "For example, any viral antigens may be administered ID (intradermal), whilst bacterial proteins may be administered IM (intramuscular) or IN (intranasal).", "Polysaccharides may be administered IM (or ID) and bacterial proteins may be administered IN (or ID).", "In addition, the vaccines of the invention may be administered IM for priming doses and IN for booster doses.", "The amount of conjugate antigen in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines.", "Such amount will vary depending upon which specific immunogen is employed and how it is presented.", "Generally, it is expected that each dose will comprise 0.1-100 μg of polysaccharide, preferably 0.1-50 μg, preferably 0.1-10 μg, of which 1 to 5 μg is the most preferable range.", "The content of protein antigens in the vaccine will typically be in the range 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.", "Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects.", "Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced.", "Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M. F. & Newman M.", "J.)", "(1995) Plenum Press New York).", "Encapsulation within liposomes is described by Fullerton, U.S. Pat.", "No.", "4,235,877.Although the vaccines of the present invention may be administered by any route, administration of the described vaccines into the skin (ID) forms one embodiment of the present invention.", "Human skin comprises an outer “horny” cuticle, called the stratum corneum, which overlays the epidermis.", "Underneath this epidermis is a layer called the dermis, which in turn overlays the subcutaneous tissue.", "Researchers have shown that injection of a vaccine into the skin, and in particular the dermis, stimulates an immune response, which may also be associated with a number of additional advantages.", "Intradermal vaccination with the vaccines described herein forms a preferred feature of the present invention.", "The conventional technique of intradermal injection, the “mantoux procedure”, comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow gauge needle (26-31 gauge) facing upwards the needle is inserted at an angle of between 10-15°.", "Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced whilst providing a slight pressure to elevate it under the skin.", "The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.", "More recently, devices that are specifically designed to administer liquid agents into or across the skin have been described, for example the devices described in WO 99/34850 and EP 1092444, also the jet injection devices described for example in WO 01/13977; U.S. Pat.", "No.", "5,480,381, U.S. Pat.", "No.", "5,599,302, U.S. Pat.", "No.", "5,334,144, U.S. Pat.", "No.", "5,993,412, U.S. Pat.", "No.", "5,649,912, U.S. Pat.", "No.", "5,569,189, U.S. Pat.", "No.", "5,704,911, U.S. Pat.", "No.", "5,383,851, U.S. Pat.", "No.", "5,893,397, U.S. Pat.", "No.", "5,466,220, U.S. Pat.", "No.", "5,339,163, U.S. Pat.", "No.", "5,312,335, U.S. Pat.", "No.", "5,503,627, U.S. Pat.", "No.", "5,064,413, U.S. Pat.", "No.", "5,520,639, U.S. Pat.", "No.", "4,596,556, U.S. Pat.", "No.", "4,790,824, U.S. Pat.", "No.", "4,941,880, U.S. Pat.", "No.", "4,940,460, WO 97/37705 and WO 97/13537.Alternative methods of intradermal administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (WO 99/27961), or transdermal patches (WO 97/48440; WO 98/28037); or applied to the surface of the skin (transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).", "When the vaccines of the present invention are to be administered to the skin, or more specifically into the dermis, the vaccine is in a low liquid volume, particularly a volume of between about 0.05 ml and 0.2 ml.", "The content of antigens in the skin or intradermal vaccines of the present invention may be similar to conventional doses as found in intramuscular vaccines (see above).", "However, it is a feature of skin or intradermal vaccines that the formulations may be “low dose”.", "Accordingly the protein antigens in “low dose” vaccines are preferably present in as little as 0.1 to 10 μg, preferably 0.1 to 5 μg per dose; and the polysaccharide (preferably conjugated) antigens may be present in the range of 0.01-1 μg, and preferably between 0.01 to 0.5 μg of polysaccharide per dose.", "As used herein, the term “intradermal delivery” means delivery of the vaccine to the region of the dermis in the skin.", "However, the vaccine will not necessarily be located exclusively in the dermis.", "The dermis is the layer in the skin located between about 1.0 and about 2.0 mm from the surface in human skin, but there is a certain amount of variation between individuals and in different parts of the body.", "In general, it can be expected to reach the dermis by going 1.5 mm below the surface of the skin.", "The dermis is located between the stratum corneum and the epidermis at the surface and the subcutaneous layer below.", "Depending on the mode of delivery, the vaccine may ultimately be located solely or primarily within the dermis, or it may ultimately be distributed within the epidermis and the dermis.", "The present invention also contemplates combination vaccines which provide protection against a range of different pathogens.", "Many Paediatric vaccines are now given as a combination vaccine so as to reduce the number of injections a child has to receive.", "Thus for Paediatric vaccines other antigens from other pathogens may be formulated with the vaccines of the invention.", "For example the vaccines of the invention can be formulated with (or administered separately but at the same time) the well known ‘trivalent’ combination vaccine comprising Diphtheria toxoid (DT), tetanus toxoid (TT), and pertussis components [typically detoxified Pertussis toxoid (PT) and filamentous haemagglutinin (FHA) with optional pertactin (PRN) and/or agglutinin 1+2], for example the marketed vaccine INFANRIX-DTPa™ (SmithKlineBeecham Biologicals) which contains DT, TT, PT, FHA and PRN antigens, or with a whole cell pertussis component for example as marketed by SmithKlineBeecham Biologicals s.a., as Tritanrix™.", "The combined vaccine may also comprise other antigen, such as Hepatitis B surface antigen (HBsAg), Polio virus antigens (for instance inactivated trivalent polio virus—IPV), Moraxella catarrhalis outer membrane proteins, non-typeable Haemophilus influenzae proteins, N. meningitidis B outer membrane proteins.", "Examples of preferred Moraxella catarrhalis protein antigens which can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)]; OMP21; LbpA &/or LbpB [WO 98/55606 (PMC)]; ThpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)]; CopB [Helminen M E, et al.", "(1993) Infect.", "Immun.", "61:2003-2010]; UspA1 and/or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT/EP99/03824); PilQ (PCT/EP99/03823); OMP85 (PCT/EP00/01468); lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11 (GB 9918302.2); lipo18 (GB 9918038.2); P6 (PCT/EP99/03038); D15 (PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and OmpE.", "Examples of non-typeable Haemophilus influenzae antigens which can be included in a combination vaccine (especially for the prevention of otitis media) include: Fimbrin protein [(U.S. Pat.", "No.", "5,766,608—Ohio State Research Foundation)] and fusions comprising peptides therefrom [eg LB1(f) peptide fusions; U.S. Pat.", "No.", "5,843,464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of New York)]; TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); protein D (EP 594610); P2; and P5 (WO 94/26304).", "Other combinations contemplated are the pneumococcal PS & protein of the invention in combination with viral antigens, for example, from influenza (attenuated, split, or subunit [e.g., surface glycoproteins neuraminidase (NA) and haemagglutinin (HA).", "See, e.g., Chaloupka I. et al, Eur.", "Journal Clin.", "Microbiol.", "Infect.", "Dis.", "1996, 15:121-127], RSV (e.g., F and G antigens or F/G fusions, see, eg, Schmidt A. C. et al, J Virol, May 2001, p 4594-4603), PIV3 (e.g., HN and F proteins, see Schmidt et al.", "supra), Varicella (e.g., attenuated, glycoproteins I-V, etc.", "), and any (or all) component(s) of MMR (measles, mumps, rubella).", "A preferred Peadiatric combination vaccine contemplated by the present invention for global treatment or prevention of otitis media comprises: one or more Streptococcus pneumoniae polysaccharide antigen(s) (preferably conjugated to protein D), one or more pneumococcal proteins selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133 (or an immunologically functional equivalent thereof), and one or more surface-exposed antigen from Moraxella catarrhalis and/or non-typeable Haemophilus influenzae.", "Protein D can advantageously be used as a protein carrier for the pneumococcal polysaccharides to overcome epitope suppression problems (as mentioned above), and because it is in itself an immunogen capable of producing B-cell mediated protection against non-typeable H. influenzae (ntHi).", "The Moraxella catarrhalis or non-typeable Haemophilus influenzae antigens can be included in the vaccine in a sub-unit form, or may be added as antigens present on the surface of outer membrane vesicles (blebs) made from the bacteria.", "Preferably the antigenic compositions (and vaccines) hereinbefore described are lyophilised up until they are about to be used, at which point they are extemporaneously reconstituted with diluent.", "More preferably they are lyophilised in the presence of 3D-MPL, and are extemporaneously reconstituted with saline solution.", "Alternatively, the protein and polysaccharide may be stored separately in a vaccination kit (either or both components being lyophilised), the components being reconstituted and either mixed prior to use or administered separately to the patient.", "A Th1 adjuvant (preferably 3D-MPL) may be present with either or both of the components.", "The lyophilisation of vaccines is well known in the art.", "Typically the liquid vaccine is freeze dried in the presence of an anti-caking agent for instance sugars such as sucrose or lactose (present at an initial concentration of 10-200 mg/mL).", "Lyophilisation typically occurs over a series of steps, for instance a cycle starting at −69° C., gradually adjusting to −24° C. over 3 hours, then retaining this temperature for 18 hours, then gradually adjusting to −16° C. over 1 hour, then retaining this temperature for 6 hours, then gradually adjusting to +34° C. over 3 hours, and finally retaining this temperature over 9 hours.", "Lyophilising the compositions results in a more stable composition (for instance it prevents the breakdown of the polysaccharide antigens).", "The process is also surprisingly responsible for a higher antibody titre against the pneumococcal polysaccharides.", "This has been shown to be particularly significant for PS 6B conjugates.", "Another aspect of the invention is thus a lyophilised antigenic composition comprising a PS 6B conjugate adjuvanted with 3D-MPL (preferably devoid of aluminium-based adjuvants) and a pneumococcal protein selected from the group consisting of: PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp125, Sp101, Sp128, Sp130 and Sp133.EXAMPLES The examples illustrate, but do not limit the invention.", "Example 1 S. pneumoniae Capsular Polysaccharide: The 11-valent candidate vaccine includes the capsular polysaccharides serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F which were made essentially as described in EP 72513.Each polysaccharide is activated and derivatised using CDAP chemistry (WO 95/08348) and conjugated to the protein carrier.", "All the polysaccharides are conjugated in their native form, except for the serotype 3 (which was size-reduced to decrease its viscosity).", "Protein Carrier: The protein carrier selected is the recombinant protein D (PD) from Non typeable Haemophilus influenzae, expressed in E. coli.", "Expression of Protein D Haemophilus influenzae Protein D Genetic Construction for Protein D Expression Starting Materials The Protein D Encoding DNA Protein D is highly conserved among H. influenzae of all serotypes and non-typeable strains.", "The vector pHIC348 containing the DNA sequence encoding the entire protein D gene has been obtained from Dr. A. Forsgren, Department of Medical Microbiology, University of Lund, Malmö General Hospital, Malmö, Sweden.", "The DNA sequence of protein D has been published by Janson et al.", "(1991) Infect.", "Immun.", "59:119-125.The Expression Vector pMG1 The expression vector pMG1 is a derivative of pBR322 (Gross et al., 1985) in which bacteriophage λ derived control elements for transcription and translation of foreign inserted genes were introduced (Shatzman et al., 1983).", "In addition, the Ampicillin resistance gene was exchanged with the Kanamycin resistance gene.", "The E. coli Strain AR58 The E. coli strain AR58 was generated by transduction of N99 with a P1 phage stock previously grown on an SA500 derivative (galE::TN10, lambdaKil− cI857 ΔH1).", "N99 and SA500 are E. coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institute of Health.", "The Expression Vector pMG 1 For the production of protein D, the DNA encoding the protein has been cloned into the expression vector pMG 1.This plasmid utilises signals from lambdaphage DNA to drive the transcription and translation of inserted foreign genes.", "The vector contains the promoter PL, operator OL and two utilisation sites (NutL and NutR) to relieve transcriptional polarity effects when N protein is provided (Gross et al., 1985).", "Vectors containing the PL promoter, are introduced into an E. coli lysogenic host to stabilise the plasmid DNA.", "Lysogenic host strains contain replication-defective lambdaphage DNA integrated into the genome (Shatzman et al., 1983).", "The chromosomal lambdaphage DNA directs the synthesis of the cI repressor protein which binds to the OL repressor of the vector and prevents binding of RNA polymerase to the PL promoter and thereby transcription of the inserted gene.", "The cI gene of the expression strain AR58 contains a temperature sensitive mutant so that PL directed transcription can be regulated by temperature shift, i.e.", "an increase in culture temperature inactivates the repressor and synthesis of the foreign protein is initiated.", "This expression system allows controlled synthesis of foreign proteins especially of those that may be toxic to the cell (Shimataka & Rosenberg, 1981).", "The E. coli Strain AR58 The AR58 lysogenic E. coli strain used for the production of the protein D carrier is a derivative of the standard NIH E. coli K12 strain N99 (F− su− galK2, lacZ− thr−).", "It contains a defective lysogenic lambdaphage (galE::TN10, lambdaKil− cI857 ΔH1).", "The Kil− phenotype prevents the shut off of host macromolecular synthesis.", "The cI857 mutation confers a temperature sensitive lesion to the cI repressor.", "The ΔH1 deletion removes the lambdaphage right operon and the hosts bio, uvr3, and chlA loci.", "The AR58 strain was generated by transduction of N99 with a P1 phage stock previously grown on an SA500 derivative (galE::TN10, lambdaKil− cI857 ΔH1).", "The introduction of the defective lysogen into N99 was selected with tetracycline by virtue of the presence of a TN10 transposon coding for tetracyclin resistance in the adjacent galE gene.", "Construction of Vector pMGMDPPrD The pMG 1 vector which contains the gene encoding the non-structural S1 protein of Influenzae virus (pMGNSI) was used to construct pMGMDPPrD.", "The protein D gene was amplified by PCR from the pHIC348 vector (Janson et al.", "1991) with PCR primers containing NcoI and XbaI restriction sites at the 5′ and 3′ ends, respectively.", "The NcoI/XbaI fragment was then introduced into pMGNS1 between NcoI and XbaI thus creating a fusion protein containing the N-terminal 81 amino acids of the NS1 protein followed by the PD protein.", "This vector was labeled pMGNS1PrD.", "Based on the construct described above the final construct for protein D expression was generated.", "A BamHI/BamHI fragment was removed from pMGNS1PrD.", "This DNA hydrolysis removes the NS1 coding region, except for the first three N-terminal residues.", "Upon religation of the vector a gene encoding a fusion protein with the following N-terminal amino acid sequence has been generated: The protein D does not contain a leader peptide or the N-terminal cysteine to which lipid chains are normally attached.", "The protein is therefore neither excreted into the periplasm nor lipidated and remains in the cytoplasm in a soluble form.", "The final construct pMG-MDPPrD was introduced into the AR58 host strain by heat shock at 37° C. Plasmid containing bacteria were selected in the presence of Kanamycin.", "Presence of the protein D encoding DNA insert was demonstrated by digestion of isolated plasmid DNA with selected endonucleases.", "The recombinant E. coli strain is referred to as ECD4.Expression of protein D is under the control of the lambda PL promoter/OL Operator.", "The host strain AR58 contains a temperature-sensitive cI gene in the genome which blocks expression from lambda PL at low temperature by binding to OL.", "Once the temperature is elevated cI is released from OL and protein D is expressed.", "At the end of the fermentation the cells are concentrated and frozen.", "The extraction from harvested cells and the purification of protein D was performed as follows.", "The frozen cell culture pellet is thawed and resuspended in a cell disruption solution (Citrate buffer pH 6.0) to a final OD650=60.The suspension is passed twice through a high pressure homogenizer at P=1000 bar.", "The cell culture homogenate is clarified by centrifugation and cell debris are removed by filtration.", "In the first purification step the filtered lysate is applied to a cation exchange chromatography column (SP Sepharose Fast Flow).", "PD binds to the gel matrix by ionic interaction and is eluted by a step increase of the ionic strength of the elution buffer.", "In a second purification step impurities are retained on an anionic exchange matrix (Q Sepharose Fast Flow).", "PD does not bind onto the gel and can be collected in the flow through.", "In both column chromatography steps fraction collection is monitored by OD.", "The flow through of the anionic exchange column chromatography containing the purified protein D is concentrated by ultrafiltration.", "The protein D containing ultrafiltration retentate is finally passed through a 0.2 μm membrane.", "Chemistry: Activation and Coupling Chemistry: The activation and coupling conditions are specific for each polysaccharide.", "These are given in Table 1.Native polysaccharide (except for PS3) was dissolved in NaCl 2M or in water for injection.", "The optimal polysaccharide concentration was evaluated for all the serotypes.", "From a 100 mg/ml stock solution in acetonitrile, CDAP (CDAP/PS ratio 0.75 mg/mg PS) was added to the polysaccharide solution.", "1.5 minute later, 0.2M triethylamine was added to obtain the specific activation pH.", "The activation of the polysaccharide was performed at this pH during 2 minutes at 25° C. Protein D (the quantity depends on the initial PS/PD ratio) was added to the activated polysaccharide and the coupling reaction was performed at the specific pH for 1 hour.", "The reaction was then quenched with glycine for 30 minutes at 25° C. and overnight at 4° C. The conjugates were purified by gel filtration using a Sephacryl 500HR gel filtration column equilibrated with 0.2M NaCl.", "The carbohydrate and protein content of the eluted fractions was determined.", "The conjugates were pooled and sterile filtered on a 0.22 μm sterilizing membrane.", "The PS/Protein ratios in the conjugate preparations were determined.", "Characterisation: Each conjugate was characterised and met the specifications described in Table 2.The polysaccharide content (μg/ml) was measured by the Resorcinol test and the protein content (μg/ml) by the Lowry test.", "The final PS/PD ratio (w/w) is determined by the ratio of the concentrations.", "Residual DMAP Content (ng/μg PS): The activation of the polysaccharide with CDAP introduces a cyanate group in the polysaccharide and DMAP (4-dimethylamino-pyridin) is liberated.", "The residual DMAP content was determined by a specific assay developed at SB.", "Free Polysaccharide Content (%): The free polysaccharide content of conjugates kept at 4° C. or stored 7 days at 37° C. was determined on the supernatant obtained after incubation with α-PD antibodies and saturated ammonium sulfate, followed by a centrifugation.", "An α-PS/α-PS ELISA was used for the quantification of free polysaccharide in the supernatant.", "The absence of conjugate was also controlled by an α-PD/α-PS ELISA.", "Reducing the quantity of free polysaccharide results in an improved conjugate vaccine.", "Antigenicity: The antigenicity on the same conjugates was analyzed in a sandwich-type ELISA wherein the capture and the detection of antibodies were α-PS and α-PD respectively.", "Free Protein Content (%): The level of “free” residual protein D was determined by using a method with SDS treatment of the sample.", "The conjugate was heated 10 min at 100° C. in presence of SDS 0.1% and injected on a SEC-HPLC gel filtration column (TSK 3000-PWXL).", "As protein D is dimer, there is a risk of overestimating the level of “free” protein D by dissociation the structure with SDS.", "Molecular Size (Kav): The molecular size was performed on a SEC-HPLC gel filtration column (TSK 5000-PWXL).", "Stability: The stability was measured on a HPLC-SEC gel filtration (TSK 6000-PWXL) for conjugates kept at 4° C. and stored for 7 days at 37° C. The 11-valent characterization is given in Table 2 The protein conjugates can be adsorbed onto aluminium phosphate and pooled to form the final vaccine.", "Conclusion: Immunogenic conjugates have been produced, that have since been shown to be components of a promising vaccine.", "The optimised CDAP conditions for the best quality final conjugated pneumococcal polysaccharide product was discovered for each of the 11 valencies.", "TABLE 1 Specific activation/coupling/quenching conditions of PS S. pneumoniae-Protein D conjugates 3 Serotype 1 (μfluid.)", "4 5 6B 7F 9V 14 18C 19F 23F PS conc.", "2.0 3.0 2.0 7.5 5.4 3.0 2.5 2.5 2.0 4.0 3.3 (mg/ml) PS NaCl NaCl H2O H2O NaCl NaCl NaCl NaCl H2O NaCl NaCl dissolution 2 M 2 M 2 M 2 M 2 M 2 M 2 M 2 M PD conc.", "5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 (mg/ml) Initial 1/1 1/1 1/1 1/1 1/1 1/1 1/0.75 1/0.75 1/1 1/0.5 1/1 PS/PD Ratio (w/w) CDAP conc.", "0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pHa = pHc = pHq 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 8.5/8.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 10/9.5/9.0 9.0/9.0/9.0 TABLE 2 Specifications of the 11 valent pneumococcoal PS-PD vaccine (first numbers of the batch code indicates serotype) Criteria D01PDJ227 D03PDJ236 D4PDJ228 D5PDJ235 D6PDJ209 Ratio PS/Prot (w/w) 1/0.66 1/1.09 1/0.86 1/0.86 1/0.69 Free polysac.", "content (%) 1 1 7 9 0 <10% Free protein content (%) 8 <1 19 21 9 <15% DMAP content (ng/μg PS) 0.2 0.6 0.4 1.2 0.3 <0.5 ng/μg PS Molecular size (Kav) 0.18 0.13 0.12 0.11 0.13 Stability no shift no shift no shift low shift no shift D07PDJ225 D09PDJ222 D14PDJ202 D18PDJ221 D19PDJ206 D23PDJ212 Ratio PS/Prot (w/w) 1/0.58 1/0.80 1/0.68 1/0.62 1/0.45 1/0.74 Free polysac.", "content (%) 1 <1 <1 4 4 0 <10% Free protein content (%) 8 0.3 3 21 10 12 <15% DMAP content (ng/μg PS) 0.1 0.6 0.3 0.2 0.1 0.9 <0.5 ng/μg PS Molecular size (Kav) 0.14 0.14 0.17 0.10 0.12 0.12 Stability no shift no shift no shift no shift shift no shift Example 2 Beneficial Impact of the Addition of One or More of the Pneumococcal Proteins of the Invention ±3D-MPL on the Protective Effectiveness of PD-Conjugated 11-valent Polysaccharide Vaccine against Pneumococcal Lung Colonization in Mice Immunological Read-Outs ELISA Dosage of Pneumococcal Protein-Specific Serum IgG Maxisorp Nunc immunoplates are coated for 2 hours at 37° C. with 100 μl/well of 2 μg/ml protein diluted in PBS.", "Plates are washed 3 times with NaCl 0.9% Tween-20 0.05% buffer.", "Then, serial 2-fold dilutions (in PBS/Tween-20 0.05%, 100 μl per well) of an anti-protein serum reference added as a standard curve (starting at 670 ng/ml IgG) and serum samples (starting at a 1/10 dilution) are incubated for 30 minutes at 20° C. under agitation.", "After washing as previously described, peroxydase-conjugated goat anti-mouse IgG (Jackson) diluted 5000× in PBS/Tween-20 0.05% are incubated (100 μl/well) for 30 minutes at 20° C. under agitation.", "After washing, plates are incubated for 15 min at room temperature with 100 μl/well of revelation buffer (OPDA 0.4 mg/ml and H2O2 0.05% in 100 mM pH 4.5 citrate buffer).", "Revelation is stopped by adding 50 μl/well HCl 1N.", "Optical densities are read at 490 and 620 nm by using Emax immunoreader (Molecular Devices).", "Antibody titre is calculated by the 4 parameter mathematical method using SoftMaxPro software.", "Opsonophagocytosis Assay The purpose of this assay is to reproducibly measure the opsonising capacity of test serum samples against Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F or 23F using a method adapted from the published standardized method of the CDC (Steiner et al, Clinical and Diagnostic Laboratory Immunology 4: 415.1997).", "This assay reproduces in vitro what occurs in vivo as the primary mechanism of eliminating invading Streptococcus pneumoniae or pneumococci.", "That is opsonization of the pneumococci followed by phagocytosis and then killing.", "“Phagocytosis” is the process by which cells engulf material and enclose it within a vacuole (phagosome) in the cytoplasm.", "Pneumococci are killed when they are phagocytised by healthy mammalian phagocytes.", "“Opsonization” is the process by which phagocytosis is facilitated by the deposition of opsonins, e.g.", "antibody and complement, on the antigen.", "There have been numerous opsonophagocytic assays reported in the literature.", "The standardized method of the CDC was tested in a multi-lab setting (Steiner et al, ICAAC, Sep. 16-20, 2000, Toronto).", "This latter assay was adapted at SB since it provided a basis for comparison to other laboratories, it used reagents and controls that are generally available, and it expressed the results as the titre (dilution) of serum able to facilitate killing of 50% of viable pneumococci, a unit that is commonly used for this type of assay.", "Indeed, it was shown that the adapted assay could generate results that correponded quite well with 4 other laboratories (Steiner et al, ICAAC, Sep. 16-20, 2000, Toronto).", "The phagocytic cell used in the assay is the HL60 cell line, which originated from an individual with promyelocytic leukemia and was established as a continuous cell line by Collins et al.", "in 1977 (Nature 270: 347-9).", "This cell line is composed of undifferentiated hematopoietic cells, that is 85% blast cells and promyelocytes, 6% myelocytes and 9% differentiated cells.", "Polar compounds can induce the differentiation of the cells into at least two different lineages.", "N,N-dimethylformamide induces granulocytic differentiation which yield polymorphonuclear-like cells (44% myelocytes and metamyelocytes and 53% banded and segmented PMNs).", "In Version A2 of the assay, the sera to be tested are heat-inactivated and 8 two-fold serial dilutions starting at ¼ are made in 96-well microplates in HBSS medium containing 0.3% BSA.", "The final volume of diluted serum in each well is 25 μl.", "Four volumes of HL60 cells at 107 cells/ml (5 or 6 days post differentiation with Dimethyl formamide), 2 volumes of S. pneumoniae bacteria (at the appropriate dilution) and 1 volume of baby rabbit complement are mixed just prior to use, and 25 μl of the mixture is added to each well of the 96-well microplate containing the diluted sera.", "For serotypes 1, and 6B, the amount of complement is increased to 12.5% final concentration, giving Version A3 of the assay.", "After two hours incubation at 37° C. under orbital shaking, the plate is put on ice in order to stop the opsonophagocytosis reaction.", "An estimate is made of the the colony forming units (CFU) in each well by overnight incubation at 37° C. The “opsonic titre” (OT) is defined as the reciprocal dilution of the serum able to reduce by at least 50% the number of S. pneumoniae bacteria in the wells (ie., 50% killing).", "The % killing is calculated by the following fomulae: % killing=(Mean CFU control wells−CFU sample)/Mean CFU control wells×100 Pneumococcal Intranasal Challenge in OF1 Mice Seven week-old OF1 female mice are intranasally inoculated under anesthesia with 5.105 CFU of mouse-adapted S. pneumoniae serotype 2, 4 or 6B.", "Lungs are removed at 6 hours after challenge and homogenized (Ultramax, 24000 rpm, 4° C.) in Todd Hewith Broth (THB, Gibco) medium.", "Serial 10-fold dilutions of lung homogenates are plated overnight at 37° C. onto Petri dishes containing yeast extract-supplemented THB agar.", "Pneumococcal lung infection is determined as the number of CFU/mouse, expressed as logarithmic weighted-average.", "Detection limit is 2.14 log CFU/mouse.", "Example 2A 3D-MPL Adjuvant Effect on Anti-Protein Immune Response In the present example, we can evaluate the impact of 3D-MPL adjuvantation on the immune response to the protein of the invention.", "Groups of 10 female 6 week-old Balb/c mice are intramuscularly immunized at days 0, 14 and 21 with 1 μg protein contained in either A: A1PO4 100 μg; or B: A1PO4 100 μg+5 μg 3D-MPL (3 de-O-acylated monophosphoryl lipid A, supplied by Ribi Immunochem).", "ELISA IgG is measured in post-III sera.", "Whichever the antigen, best immune responses can be shown to be induced in animals vaccinated with 3D-MPL-supplemented formulations.", "Example 2B Beneficial Impact of the Addition of a Protein of the Invention ±3D-MPL Adjuvant on the Protective Effectiveness of PD-Conjugated 11-Valent Polysaccharide Vaccine against Pneumococcal Lung Colonization in OF1 Mice Intranasally Challenged with Serotype 2, 4 or 6B In the present example, we can evaluate the prophylactic efficacy of a vaccine containing the 11-valent polysaccharide-protein D conjugate, a protein of the invention and A1PO4+3D-MPL adjuvants, compared to the classical A1PO4-adsorbed 11-valent polysaccharide-protein D conjugate formulation.", "Groups of 12 female 4 week-old OF1 mice are immunized subcutaneously at days 0 and 14 with formulations containing A: 50 μg A1PO4; B: 0.1 μg PS/serotype of PD-conjugated 11-valent polysaccharide vaccine+50 μg A1PO4; or C: 0.1 μg PS/serotype of PD-conjugated 11-valent polysaccharide vaccine+10 μg protein of the invention+50 μg A1PO4+5 μg 3D-MPL (supplied by Ribi Immunochem).", "Challenge is done at day 21 as described above.", "As can be shown by this method, a significant protection is conferred by the 11-valent polysaccharide conjugate vaccine supplemented with the protein of the invention and adjuvanted with A1PO4+MPL.", "On the contrary, no significant protection is observed in animals immunized with the 11-valent polysaccharide conjugate/A1PO4 formulation.", "This result can prove that the addition of the protein of the invention and 3D-MPL adjuvant enhances the effectiveness of the 11-valent polysaccharide conjugate vaccine against pneumonia.", "Example 2C Immune Correlates of the Protection Showed in Example 2B In order to establish the immune correlates of protection conferred in example 2B, by the 11-valent polysaccharide conjugate vaccine supplemented with a protein of the invention and 3D-MPL, pre-challenge serological antibody responses to polysaccharide 2, 4 or 6B, and the protein of the invention can be measured as described above.", "Antibody titers are then compared to bacteria colony numbers measured in lungs of the corresponding animals collected at 6 hours post-challenge.", "R2 are calculated on Log/Log linear regressions.", "Calculated R2 can show the absence of correlation between humoral immune responses and protection for both antigens.", "Anti-6B (or 2 or 4) antibody titers are not significantly different in the groups immunized with the 11-valent conjugate vaccine or with the same vaccine supplemented with the protein of the invention and 3D-MPL.", "Therefore, the protection improvement seen with formulation C is not solely due to a higher antibody response to polysaccharide 6B (or 2 or 4).", "Taken together, the results can suggest that protection is not mediated by humoral immune responses alone, but rather also by a cell-mediated immunity induced by the protein antigen (preferably in the presence of 3D-MPL).", "This can give additional support to the addition of protein antigen(s) and potent adjuvant(s) in the pneumococcal polysaccharide conjugate vaccine, so as to coordinate both arms of the immune system for optimal protection.", "Example 3 The Cooperation of Both Arms of the Immune System in mice Actively Immunised with a Protein of the Invention and Passively Immunised with Antibodies against Pneumococcal PS Example 3A Find the Concentration of Passively Administered Anti-6B-Polysaccharide (anti-PS) Antibody Protecting Against Pneumonia Method Vaccine Groups: Four groups of 16 mice were passively immunised (i.p.)", "on day-1 with 100 μl of undiluted rat anti-polysaccharide antisera according to the groups detailed below.", "(total 64 mice) Group Specificity IgG Concentration in Antisera G1 α-PS -6B 5 μg/ml.", "G2 α-PS -6B 2 μg/ml.", "G3 α-PS -6B 0.75 μg/ml.", "G4 Control 0 μg/ml.", "Animals: 64 male CD-1 mice from Charles River, Canada, weighing approx 35 g (approx 10 weeks old).", "Anesthesia.", "Mice were anesthetized with isoflurane (3%) plus O2(1 L/min).", "Organism: S. pneumoniae N1387 (serotype 6) was harvested from trypticase soy agar plates (TSA) supplemented with 5% horse blood and suspended in 6 ml of PBS.", "Immediately prior to infection, 1 ml bacterial suspension was diluted into 9 ml of cooled molten nutrient agar (BBL) and kept at 41° C. Mice received approx 6.0 log10 cfu/mouse in a volume 50 ul.", "Infection: On day 0 mice were anesthetized as described above and infected with S. pneumoniae N1387 (50 μl cooled bacterial suspension) by intra-bronchial instillation via non-surgical intra-tracheal intubation.", "This method was described by Woodnut and Berry (Antimicrob.", "Ag.", "Chemotherap.", "43: 29 (1999)).", "Samples: On day 3 post infection, 8 mice/group were sacrificed by CO2 overdose and lungs were excised and homogenized in 1 ml PBS.", "Tenfold serial dilutions were prepared in PBS to enumerate viable bacterial numbers.", "Samples were inoculated (20 μl) in triplicate onto TSA plates supplemented with 5% horse blood and incubated overnight at 37° C. prior to evaluation.", "Further sets of mice were sacrificed on day 7 and sampled as above.", "Results: IgG conc Bacterial numbers (ug/ml) (log 10 cfu/lungs) at days post infection in rat sera 3 8 5 6.7 ± 0.7 (1/7) 7.2 ± 0.7 (5/8) 2 6.5 ± 0.7 (1/7) 6.9 ± 1.8 (4/7) 0.75 7.7 ± 0.5 (5/8) 4.8 ± 1.4 (2/8) 0 6.7 ± 1.5 (3/6) 6.3 ± 1.5 (3/9) Figures in parenthesis are numbers of animals that died prior to sample time.", "Conclusion: In general, there was no significant difference in bacterial numbers isolated from any of the treatment groups.", "This indicates that no measurable protection was afforded by the anti-polysaccharide antibody at concentrations up to and including 5 μg/ml.", "This is similar to what is observed in some human clinical trials, that is, anti-polysaccharide antibody is insufficient to protect against pneumococcal pneumonia in some populations.", "Example 3B Determine the Protection from Pneumonia Afforded by Active Administration of a Protein of the Invention with or without Adjuvant, and Synergy with Sub-Optimal Anti-PS Antibody Method Animals: 128 male CD-1 mice (6 weeks old at old at immunisation, 10 weeks old at infection) from Charles River, St.", "Constant, Quebec, Canada Animals weighed approx 20 gm at 6 weeks and 38 g at 10 weeks.", "Immunisations: Six groups of 16 mice are immunised by subcutaneous injection on days-22 and -14 with 100 ul of vaccine as detailed below.", "(Total 128 mice).", "3D-MPL is obtained from Ribi/Corixa.", "On day-1, specific groups (see Table below) are immunised (i.p.", "100 μl) passively with a concentration of 4.26 μg/ml (4 ml of 5 μg/ml+1.3 ml of 2 μg/ml) mouse anti-polysaccharide antibody.", "Injection Vaccine given Injection Volume days −22, −14 Volume Passive IgG Group Active (Dosage μg) Passive (day −1) 1-1 100 μl s.c. Protein/AlPO4 (10/50) None 1-2 100 μl s.c. Protein/MPL/AlPO4 None (10/5/50) 1-3 100 μl s.c. Protein/AlPO4 (10/50) 100 μl i.p.", "α-PS 1-4 100 μl s.c. Protein/MPL/AlPO4 100 μl i.p.", "α-PS (10/5/50) 1-5 100 μl s.c. MPL/AlPO4 (5/50) 100 μl i.p.", "α-PS 1-6 100 μl s.c. MPL/AlPO4 (5/50) None Infection: On day 0, mice are anesthetized (3% isoflurane plus 1 L/min O2).", "Bacterial inocula are prepared by harvesting growth of S. pneumoniae N1387 (serotype 6) from trypticase soy agar plates (TSA) supplemented with 5% horse blood and suspending in 6 ml of PBS.", "A ten-fold dilution (1 ml plus 9 ml) is prepared in cooled molten nutrient agar (kept at 4° C.) immediately prior to infection.", "Mice are infected by intra-bronchial instillation via intra-tracheal intubation and receive approximately 6.0 log10 cfu/mouse in a volume of 50 μl.", "This method was described by Woodnut and Berry (Antimicrob.", "Ag.", "Chemotherap.", "43: 29 (1999)).", "Samples: At 72 post infection, 8 mice/group are sacrificed by CO2 overdose and the lungs are excised and homogenized in 1 ml PBS.", "Tenfold serial dilutions are prepared in PBS to enumerate viable bacterial numbers.", "Samples are inoculated (20 μl) in triplicate onto TSA plates supplemented with 5% horse blood and incubated overnight 37° C. prior to evaluation.", "Further sets of mice are sacrificed on day 8 post-infection and samples as above.", "Analysis of Data The outcome measure for comparison of treatment is the number of bacteria in the lungs at 3 and 7 day post infection.", "Results can be presented as group means with standard deviations.", "Statistical analysis should be performed using the Students t-test where a P value of <0.05 is considered significant.", "As demonstrated above, anti-polysaccharide antibody alone (Group 1-5) can be shown not to afford protection against growth of pneumococci in the lung.", "Pneumococcal protein adjuvanted with A1PO4 (Group 1-1) may not confer protection either, but will do to a better extent when Protein is combined with 3D-MPL (Group 1-2).", "Most significant protection can be seen in groups with both anti-polysaccharide antibody and protein, particularly in the group having all three elements, Protein, 3D-MPL and passively administered anti-polysaccharide antibody (Group 1-4).", "This conclusion can also be supported by the mortality rate.", "Groups 1-3 and, particularly, 1-4 will have fewer deaths compared to the other groups.", "Conclusion: As the experiment is done with passively immunised animals, the synergistic effect of also actively immunising with protein (±MPL) cannot be due to an increase in the level of antibodies against the polysaccharide antigen.", "Significant protection against pneumococcal pneumonia can be seen in groups immunised with both protein plus passively administered anti-polysaccharide antibody, particularly if 3D-MPL is also present, indicating the synergy of this combination.", "If the anti-polysaccharide immunisation is carried out actively (preferably with conjugated polysaccharide), this effect will be even more marked, as the effect of B-cell memory, and constant levels of anti-PS antibody throughout the experiment will contribute to the immune response cooperation.", "Example 4 Method to Determine Synergy by Correlate of Protection The principle mechanism of protection that the human body uses to eliminate infecting pneumococcus is antibody mediated opsonophagocytosis (Bruyn et al.", "Clin.", "Infect.", "Dis.", "14: 251 (1992)).", "Whereas several ELISA methods have been developed to measure the antibody concentration to capsular polysaccharide as a correlate of protection, it has become apparent that the in vitro opsonophagocytosis assay is a better correlate of protection (Musher et al.", "J. Infect.", "Dis.", "182: 158 (2000)).", "The Pneumococcal proteins of the invention provide protection against pneumococcal infection by mechanisms that are different from antibody mediated opsonophagocytosis.", "In example 2, active immunisation with both conjugate and protein can show a synergic effect, which can not be explained by the antibody concentration differences since they are the same in both groups.", "Thus the residual protection that can be observed must come from a synergistic effect.", "Similarly, since the antibody is added passively, the same conclusion can be reached in example 3.In many cases, the pneumococcal proteins of the invention are surface associated, and are expected to provide some opsonic activity themselves.", "In this case it is possible to distinguish the mechanism of protection via a quantitative measure of the opsonising capacity of the the anti-pneumococcal protein, which can be used to estimate the relative contribution of opsonic activity to other synergeic mechanisms of protection.", "In the mouse lung colonisation model, the relative protection of each vaccine can be estimated from the clearance of bacteria from the lungs.", "Or alternatively, the vaccine efficacy can be estimated from case rates, as normally determined for vaccines.", "% Protection=(CFU/lung Control−CFU/lung Vaccine)/(CFU/lung Control) % Efficacy=(Cases Control−Cases Vaccine)/(Cases Control) To determine the portion of the protection or efficacy that originates from the synergistic effect, it is a matter of determining which portion of the efficacy would be expected based on the ratio of the opsonic titres.", "In Example 3 above, the % protection by the combination is due to synergy between the protein/antibody components as neither the protein nor the anti-polysacharide antibody alone can provide much protection themselves.", "It is possible to estimate the amount of protection of the synergeic effect on the basis of the relative opsonic activity.", "If the opsonic activity afforded by a anti-capsular polysacharide antibody is X, and the opsonic activity afforded by anti-pneumococcal protein antibody is Y, then the total opsonic activity can be shown to be X+Y, and the relative portion of the opsonic activity of the protein would be Y/X+Y.", "This is compared to the relative protective efficacy of a vaccine, where the anti-polysaccharide portion of the vaccine provides a protective efficacy of A %, and the protective efficacy of the vaccine of polysaccharide plus protein is B %.", "The additional efficacy that can not be accounted by opsonic acitivity is then estimated as Residual Protective Activity (Synergy)=B %−A %−B %*(Y/X+Y) This example is not intended to limit the ways to estimate the effect of synergy.", "Once other correlates of protection have been identified, they could be used to estimate this synergisic effect.", "All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth." ] ]

[ [ "Island network and method for operation of an island network", "The invention relates to an island network with at least one energy generator, using regenerative energy sources, whereby the energy generator is preferably a wind energy plant with a first synchronous generator, a DC link, at least one first power rectifier and a power inverter, a second synchronous generator and an internal combustion engine which may be coupled with the second synchronous generator.", "A fully controllable wind energy unit (10) and an electromagnetic coupling (34) between the second synchronous generator (32) and the internal combustion engine (30) are provided in order to establish an island network in which the internal combustion engine can be switched off completely, so long as the wind energy unit is generating enough power for all connected users with an efficiency which is as high as possible." ], [ "1.An isolated electrical network with at least one first energy producer that uses a regenerative energy source, wherein the energy producer is preferably a wind energy system with a generator, wherein a second generator that can be coupled to an internal combustion engine is provided, characterized in that a) the wind energy system is controllable in regard to its rotational speed and blade adjustment; b) the wind energy system can be controlled such that it always produces only the required electric power, the required power being composed of the consumption of electric power in the network and the power needed to charge an interim electricity storage unit; and c) when the power produced by the wind energy system falls below power R, network power is initially not provided by the internal combustion engine, but instead interim electricity storage units are called upon to release energy to the network.", "2.The isolated electrical network according to claim 1, characterized in that the first energy producer has a synchronous generator which contains an inverter with a dc link with at least one rectifier and a dc-ac converter.", "3.The isolated electrical network according to claim 1, characterized by at least one electrical element connected to the dc link for feeding in dc electrical energy.", "4.The isolated electrical network according to claim 3, characterized in that the electrical element is a photovoltaic element and/or a mechanical energy accumulator and/or an electrochemical storage unit and/or a capacitor and/or a chemical storage unit as electrical interim storage unit.", "5.The isolated electrical network according to claim 1, characterized by a flywheel that can be coupled to the second or a third generator.", "6.The isolated electrical network according to claim 1, characterized by several internal combustion engines, each of which can be coupled to a generator.", "7.The isolated electrical network according to claim 1, characterized by a controller for controlling the isolated network.", "8.The isolated electrical network according to claim 1, characterized by a step-up or step-down converter between the electrical element and the dc link.", "9.The isolated electrical network according to claim 1, characterized by charge/discharge circuits between the electrical element and the dc link.", "10.The isolated electrical network according to claim 1, characterized by a flywheel with a generator and a downstream rectifier for feeding electrical energy into dc links.", "11.The isolated electrical network according to claim 1, characterized in that all energy producers using regenerative energy sources and interim storage units feed a shared dc link.", "12.The isolated electrical network according to claim 1, characterized by a line-commuted dc-ac converter.", "13.The isolated electrical network according to claim 1, characterized in that the energy for operating the electromagnetic clutch is provided by an electricity storage unit and/or by the primary energy producer.", "14.The isolated electrical network according to claim 1, characterized in that a seawater desalination/usable water production plant is connected to the isolated network and produces usable water whenever the power supply from the primary energy producer is greater than the power consumption of the other electric loads connected to the isolated network.", "15.The isolated electrical network according to claim 1, characterized in that a pump storage plant which receives its electrical energy from the primary energy producer is provided.", "16.An isolated electrical network comprising: at least one first primary energy producer for producing electrical energy for an isolated electrical network; a synchronous generator that has the function of a pulse-former, wherein the synchronous generator can operate in motor mode and the energy required operation in motor mode is provided by the primary energy producer.", "17.The isolated electrical network according to claim 16, characterized in that the generator can be connected via a clutch to an internal combustion engine that is turned off whenever the electric power from the primary energy producer is greater than or roughly as large as the consumed electric power in the isolated network.", "18.The method according to claim 1, characterized in that internal combustion engines are provided to drive at least one second generator and the internal combustion engines are switched on only if the energy emitted by energy producers using regenerative energy sources and/or by interim electricity storage units falls below a specificiable threshold value for a specificiable time span.", "19.The method according to claim 18, characterized in that more energy than is required for the loads connected to the network is produced from regenerative sources in order to charge the interim storage units.", "20.An electrical supply network comprising: a synchronous generator as a pulse-former for a line-commutated dc-ac converter for feeding an alternating current into the electricity supply network, wherein the generator operates in motor mode and the driving of the generator to operate in motor mode is accomplished by a flywheel or by providing electrical energy from a regenerative energy producer." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention pertains to an isolated electrical network with at least one energy producer that is coupled to a first generator.", "A second generator, which may be coupled to an internal combustion engine, is also provided.", "In such an isolated network, the energy producer connected to the first generator is frequently a regenerative energy producer such as a wind energy system, a hydroelectric power plant, etc.", "2.Description of the Related Art Such isolated networks are generally known and serve particularly to provide power to areas that are not connected to a central power supply network but in which regenerative energy sources such as wind and/or solar and/or water power are available.", "These areas may be islands or remote and/or inaccessible areas with peculiarities with regard to size, location and/or climatic conditions.", "Even in such areas, however, a supply of electricity, water and heat is necessary.", "The energy required for this, at least the electrical energy, is provided and distributed by the isolated network.", "Modern electrically operated equipment also requires compliance with relatively narrow limit values for voltage and frequency fluctuations in the isolated network for proper functioning.", "Among other ways to comply with these limit values, wind/diesel systems are used, in which a wind energy system is used as the primary energy source.", "The alternating current produced by the wind energy system is rectified and subsequently converted via an inverter into alternating current at the required network frequency.", "In this way, a network frequency is generated that is independent of the rotational speed of the generator in the wind energy system and thus of the frequency of the latter.", "The network frequency is thus determined by the inverter.", "Two different variants are available in this regard.", "The first variant is a so-called self-commutated inverter, which is capable itself of generating a stable network frequency.", "Such self-commutated inverters, however, require a high degree of technical effort and are correspondingly expensive.", "An alternative to self-commutated inverters are line-commutated inverters, which synchronize the frequency of their output voltage: to an existing network.", "Such inverters are considerably more economical than self-commutated inverters, but always require a network to which they can synchronize themselves.", "Therefore, a pulse-former that supplies the control parameters necessary for line commutation must always be provided for a line-commutated inverter.", "For known isolated networks, such a pulse-former is, for instance, a synchronous generator that is driven by an internal combustion engine, such as a diesel engine.", "That implies that the internal combustion engine must run continuously to drive the synchronous generator as a pulse-former.", "This too is disadvantageous for reasons of maintenance requirements, fuel consumption and pollution of the environment with exhaust because, even if the internal combustion engine need provide only a fraction of its available power for driving the generator as a pulse-former—the power often amounts to only 3-5 kW—the fuel consumption is not inconsiderable and amounts to several liters of fuel per hour.", "An additional problem for known isolated networks consists in the fact that reactive loads referred to as “dump loads,” which consume the excess energy produced by the primary energy producer, must be present so that, when loads are disconnected, the primary energy producer does not go into idle operation, which could in turn lead to mechanical damage in the primary energy producer due to an excessive rotational speed.", "This is very problematic particularly for wind energy systems as the primary energy producer." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention is based on avoiding the aforementioned disadvantages to solve the problem of the prior art and improving the efficiency of an isolated network.", "The problem is solved according to the invention with an isolated electrical network according to claims 1 and 16 and a method of controlling the operation of an isolated network according to claim 18 .", "Advantageous refinements are described in the subordinate claims.", "The invention is based on the recognition that the second generator, which has the function of a pulse-former, can also be driven by the electrical energy of the first generator, which is usually the primary energy producer, such as a wind energy system, so that the internal combustion engine can be shut off completely and decoupled from the second generator.", "In this case the second generator is not in generator mode but rather in motor mode, the required electrical energy being supplied by the primary electrical energy producer or the first generator.", "If the clutch between the second generator and the internal combustion engine is an electromagnetic clutch, then this clutch can be actuated by the application of electrical energy from the primary energy producer or its generator.", "If the electrical energy is shut off at the clutch, the clutch is disengaged.", "When the internal combustion engine is not operating, electrical energy is then applied to the second generator, as described above, and it is driven in motor mode so that the pulse-former remains in operation, despite the shut-down internal combustion engine.", "Whenever it is necessary to start the engine and go into generator mode, the internal combustion engine can be started and coupled to the second generator by means of the electrically operated clutch so that, in generator mode, this second generator can provide additional energy for the isolated electrical network.", "The use of a fully controllable wind energy system makes it possible to do without “dump loads,” since the wind energy system is capable by virtue of its complete controllability, i.e., its variable speed and variable blade adjustment, of producing precisely the required amount of power so that “disposal” is not necessary, since the wind energy system produces precisely the required power.", "Because the wind power system produces only as much energy as is needed in the network or for further charging of interim storage, no excess energy need be eliminated uselessly and the overall efficiency of the wind energy system, but also that of the isolated network, is considerably better than when “dump loads” are used.", "In a preferred embodiment of the invention, the wind energy system contains a synchronous generator with a downstream dc-ac converter.", "This dc-ac converter consists of a rectifier, a dc link and a variable-frequency inverter.", "If another source providing a dc voltage or direct current such as a photovoltaic element is installed in the network, then it is expedient for such additional primary energy producers such as photovoltaic elements to be connected to the dc link of the dc-ac converter, so that the energy of the additional regenerative energy source can be fed into the dc link.", "In that way, the energy supply available from the first primary energy producer can be increased.", "In order to compensate for fluctuations in the available power and/or an increased power demand spontaneously as well as to make use of available energy that is non-instantaneously in demand, it is preferable to provide interim storage units that can store electrical energy and release it quickly when needed.", "Such storage units can be electrochemical storage devices such as rechargeable batteries, but also capacitors (caps) or chemical storage units such as hydrogen accumulators, in which hydrogen produced by electrolysis from the excess electrical energy is stored.", "In order to release their electrical energy, such storage units are also connected, directly or via appropriate charge/discharge circuitry, to the dc link of the dc-ac converter.", "An additional form of energy storage that may be used is conversion into energy of rotation, which is stored in a flywheel.", "This flywheel is connected in a preferred refinement of the invention to the second synchronous generator and thus likewise makes it possible to utilize the stored energy to drive the pulse-former.", "Electrical energy can be supplied to all storage units whenever the consumption of energy in the isolated network is less than the power capacity of the primary energy producer, for instance, the wind energy system.", "If, for example, the primary energy producer is a wind energy system with 1.5 MW nominal power or a 10 MW nominal power wind park with several wind energy systems and wind conditions are such that the primary energy producer can be run at nominal operation, but the power consumption in the isolated network is clearly less than the nominal power of the primary energy producers, it is possible in such an operation (especially at night and during times of low consumption in the isolated network) for the primary energy producer to be run such that all energy storage units are charged (filled), so that in those times when the power consumption of the isolated network is greater than power supply of the primary energy producer the energy storage units can be turned on first, sometimes only for a short time.", "In a preferred refinement of the invention all energy producers and interim storage units except the energy component, for example, the internal combustion engine, or flywheel, connected to the second generator can be connected to a shared dc link configured like a bus and terminated by a single line-commutated inverter (dc-ac converter).", "By using a single line-commutated dc-ac converter on a dc link, a very economical arrangement is created.", "It is also advantageous if additional or redundant internal combustion engines and third generators (e.g., synchronous generators) are provided so that, in case of a greater demand for power than is available from the regenerative energy producers and stored energy, it can be produced by operating the additional or redundant production systems." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention pertains to an isolated electrical network with at least one energy producer that is coupled to a first generator.", "A second generator, which may be coupled to an internal combustion engine, is also provided.", "In such an isolated network, the energy producer connected to the first generator is frequently a regenerative energy producer such as a wind energy system, a hydroelectric power plant, etc.", "2.Description of the Related Art Such isolated networks are generally known and serve particularly to provide power to areas that are not connected to a central power supply network but in which regenerative energy sources such as wind and/or solar and/or water power are available.", "These areas may be islands or remote and/or inaccessible areas with peculiarities with regard to size, location and/or climatic conditions.", "Even in such areas, however, a supply of electricity, water and heat is necessary.", "The energy required for this, at least the electrical energy, is provided and distributed by the isolated network.", "Modern electrically operated equipment also requires compliance with relatively narrow limit values for voltage and frequency fluctuations in the isolated network for proper functioning.", "Among other ways to comply with these limit values, wind/diesel systems are used, in which a wind energy system is used as the primary energy source.", "The alternating current produced by the wind energy system is rectified and subsequently converted via an inverter into alternating current at the required network frequency.", "In this way, a network frequency is generated that is independent of the rotational speed of the generator in the wind energy system and thus of the frequency of the latter.", "The network frequency is thus determined by the inverter.", "Two different variants are available in this regard.", "The first variant is a so-called self-commutated inverter, which is capable itself of generating a stable network frequency.", "Such self-commutated inverters, however, require a high degree of technical effort and are correspondingly expensive.", "An alternative to self-commutated inverters are line-commutated inverters, which synchronize the frequency of their output voltage: to an existing network.", "Such inverters are considerably more economical than self-commutated inverters, but always require a network to which they can synchronize themselves.", "Therefore, a pulse-former that supplies the control parameters necessary for line commutation must always be provided for a line-commutated inverter.", "For known isolated networks, such a pulse-former is, for instance, a synchronous generator that is driven by an internal combustion engine, such as a diesel engine.", "That implies that the internal combustion engine must run continuously to drive the synchronous generator as a pulse-former.", "This too is disadvantageous for reasons of maintenance requirements, fuel consumption and pollution of the environment with exhaust because, even if the internal combustion engine need provide only a fraction of its available power for driving the generator as a pulse-former—the power often amounts to only 3-5 kW—the fuel consumption is not inconsiderable and amounts to several liters of fuel per hour.", "An additional problem for known isolated networks consists in the fact that reactive loads referred to as “dump loads,” which consume the excess energy produced by the primary energy producer, must be present so that, when loads are disconnected, the primary energy producer does not go into idle operation, which could in turn lead to mechanical damage in the primary energy producer due to an excessive rotational speed.", "This is very problematic particularly for wind energy systems as the primary energy producer.", "SUMMARY OF THE INVENTION The invention is based on avoiding the aforementioned disadvantages to solve the problem of the prior art and improving the efficiency of an isolated network.", "The problem is solved according to the invention with an isolated electrical network according to claims 1 and 16 and a method of controlling the operation of an isolated network according to claim 18.Advantageous refinements are described in the subordinate claims.", "The invention is based on the recognition that the second generator, which has the function of a pulse-former, can also be driven by the electrical energy of the first generator, which is usually the primary energy producer, such as a wind energy system, so that the internal combustion engine can be shut off completely and decoupled from the second generator.", "In this case the second generator is not in generator mode but rather in motor mode, the required electrical energy being supplied by the primary electrical energy producer or the first generator.", "If the clutch between the second generator and the internal combustion engine is an electromagnetic clutch, then this clutch can be actuated by the application of electrical energy from the primary energy producer or its generator.", "If the electrical energy is shut off at the clutch, the clutch is disengaged.", "When the internal combustion engine is not operating, electrical energy is then applied to the second generator, as described above, and it is driven in motor mode so that the pulse-former remains in operation, despite the shut-down internal combustion engine.", "Whenever it is necessary to start the engine and go into generator mode, the internal combustion engine can be started and coupled to the second generator by means of the electrically operated clutch so that, in generator mode, this second generator can provide additional energy for the isolated electrical network.", "The use of a fully controllable wind energy system makes it possible to do without “dump loads,” since the wind energy system is capable by virtue of its complete controllability, i.e., its variable speed and variable blade adjustment, of producing precisely the required amount of power so that “disposal” is not necessary, since the wind energy system produces precisely the required power.", "Because the wind power system produces only as much energy as is needed in the network or for further charging of interim storage, no excess energy need be eliminated uselessly and the overall efficiency of the wind energy system, but also that of the isolated network, is considerably better than when “dump loads” are used.", "In a preferred embodiment of the invention, the wind energy system contains a synchronous generator with a downstream dc-ac converter.", "This dc-ac converter consists of a rectifier, a dc link and a variable-frequency inverter.", "If another source providing a dc voltage or direct current such as a photovoltaic element is installed in the network, then it is expedient for such additional primary energy producers such as photovoltaic elements to be connected to the dc link of the dc-ac converter, so that the energy of the additional regenerative energy source can be fed into the dc link.", "In that way, the energy supply available from the first primary energy producer can be increased.", "In order to compensate for fluctuations in the available power and/or an increased power demand spontaneously as well as to make use of available energy that is non-instantaneously in demand, it is preferable to provide interim storage units that can store electrical energy and release it quickly when needed.", "Such storage units can be electrochemical storage devices such as rechargeable batteries, but also capacitors (caps) or chemical storage units such as hydrogen accumulators, in which hydrogen produced by electrolysis from the excess electrical energy is stored.", "In order to release their electrical energy, such storage units are also connected, directly or via appropriate charge/discharge circuitry, to the dc link of the dc-ac converter.", "An additional form of energy storage that may be used is conversion into energy of rotation, which is stored in a flywheel.", "This flywheel is connected in a preferred refinement of the invention to the second synchronous generator and thus likewise makes it possible to utilize the stored energy to drive the pulse-former.", "Electrical energy can be supplied to all storage units whenever the consumption of energy in the isolated network is less than the power capacity of the primary energy producer, for instance, the wind energy system.", "If, for example, the primary energy producer is a wind energy system with 1.5 MW nominal power or a 10 MW nominal power wind park with several wind energy systems and wind conditions are such that the primary energy producer can be run at nominal operation, but the power consumption in the isolated network is clearly less than the nominal power of the primary energy producers, it is possible in such an operation (especially at night and during times of low consumption in the isolated network) for the primary energy producer to be run such that all energy storage units are charged (filled), so that in those times when the power consumption of the isolated network is greater than power supply of the primary energy producer the energy storage units can be turned on first, sometimes only for a short time.", "In a preferred refinement of the invention all energy producers and interim storage units except the energy component, for example, the internal combustion engine, or flywheel, connected to the second generator can be connected to a shared dc link configured like a bus and terminated by a single line-commutated inverter (dc-ac converter).", "By using a single line-commutated dc-ac converter on a dc link, a very economical arrangement is created.", "It is also advantageous if additional or redundant internal combustion engines and third generators (e.g., synchronous generators) are provided so that, in case of a greater demand for power than is available from the regenerative energy producers and stored energy, it can be produced by operating the additional or redundant production systems.", "BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention are described in greater detail below for the sake of example.", "Shown are: FIG.", "1, a schematic circuit diagram of an isolated network according to the invention; FIG.", "2, a variant of the schematic shown in FIG.", "1 and FIG.", "3, a preferred embodiment of an isolated network according to the invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG.", "1 shows a wind energy system 10 having a first generator therein with a downstream inverter consisting of a rectifier 20, via which the wind energy system is connected to a dc link 28, as well as a dc-ac converter 24 connected to the output of dc link 28.A second synchronous generator 32, connected in turn via an electromagnetic clutch 34 to an internal combustion engine 30, is connected in parallel to the output of dc-ac converter 24.The output lines of dc-ac converter 24 and second synchronous generator 32 supply the loads (not shown) with the required energy.", "Wind energy system 10 produces the power for supplying the loads.", "The energy produced by wind energy system 10 is rectified by rectifier 20 and fed into dc link 28.The dc-ac converter 24 produces alternating current from the direct current applied to it and feeds it into the isolated network.", "Since dc-ac converter 24 is designed as a line-commutated dc-ac converter 24 for reasons of cost, a pulse-former is present, to which the dc-ac converter can synchronize itself.", "This pulse-former is the second synchronous generator 32.This synchronous generator 32 operates in motor mode with internal combustion engine 30 turned off and acts as a pulse-former.", "In this mode the driving energy is the electrical energy from the wind energy system 10.This energy for driving synchronous generator 32, just like the losses of rectifier 20 and dc-ac converter 24, must be additionally produced by wind energy system 10.In addition to its function as a pulse-former, second synchronous generator 32 fulfills other tasks such as producing reactive energy in the network, supplying short-circuit current, acting as a flicker filter and regulating voltage.", "If loads are switched off and the energy requirements therefore decrease, then wind energy system 10 is controlled in a known manner such that it produces correspondingly less energy, so that the use of dump loads can be dispensed with.", "If the energy demands of the loads increase to the point that they can no longer be covered by the wind energy system alone, internal combustion engine 28 can start up and voltage is applied to electromagnetic clutch 34.Clutch 34 thereby creates a mechanical connection between internal combustion engine 30 and second synchronous generator 32.The generator 32 is now in generator mode, and it continues to operate as a pulse-former, and it also supplies the additional required energy.", "By appropriate dimensioning of wind energy system 10 it is possible on average for enough energy to supply the loads to be provided from wind energy.", "The usage of internal combustion engine 30 and the associated fuel consumption can thereby be reduced to a minimum.", "FIG.", "2 shows a variant of the isolated network shown in FIG.", "1.The structure essentially corresponds to the solution shown in FIG.", "1.The difference is that here no internal combustion engine 30 is associated with second generator 32, which acts as a pulse-former.", "Internal combustion engine 30 is instead connected to an additional, third (synchronous) generator 36 which can be turned on as needed.", "Second synchronous generator 32 thus constantly operates in motor mode as pulse-former, reactive power producer, short-circuit current source, flicker filter and voltage regulator.", "FIG.", "3 shows an additional preferred embodiment of an isolated network.", "In this figure, three wind energy systems 10, forming a wind park as an example, are shown with (synchronous) generators, each connected to a rectifier 20.The rectifiers 20 are connected in parallel on the output side and feed the energy produced by wind energy systems 10 into a dc link 28.Also shown are three photovoltaic elements 12, each connected to a step-up converter 22.The output sides of the step-up converters 22 are likewise connected in parallel to dc link 28.Also shown is a storage battery block 14 which symbolically stands for an interim storage unit.", "In addition to being an electrochemical storage unit such as storage battery 14, this interim storage unit can also be a chemical one such as a hydrogen accumulator (not shown).", "The hydrogen accumulator can be filled, for instance, with hydrogen obtained by electrolysis.", "Illustrated next to it is a capacitor block 18 which shows the possibility of using appropriate capacitors as interim storage.", "These capacitors could, for instance, be so-called Ultra-Caps made by the Siemens company, which are distinguished by low losses as well as high storage capacity.", "Accumulator block 14 and capacitor block 18 (each block can also be formed from more than one unit) are connected via charge/discharge circuits 26 to dc link 28.The dc link 28 is terminated by a single dc-ac converter 24 (or a plurality of dc-ac converters in parallel), dc-ac converter 24 preferably being constructed to be line-commutated.", "A distributor 40 (possibly with a transformer) that is supplied with the line voltage by dc-ac converter 24 is connected to the output side of dc-ac converter 24.Likewise connected to the output side of dc-ac converter 24 is a second synchronous generator 32.This synchronous generator 32 is the pulse-former, reactive power and short-circuit current producer, flicker filter and voltage regulator of the isolated network.", "A flywheel 16 is coupled to second synchronous generator 32.This flywheel 16 is likewise an interim storage unit and can store energy, for instance, during motor-mode operation of the pulse-former.", "An internal combustion engine 30 and an electromagnetic clutch 34, which drive generator 32 in generator mode in case of insufficient power from regenerative sources, can likewise be associated with second synchronous generator 32.In this way, needed energy can be fed into the isolated network.", "Internal combustion engine 30 associated with second synchronous generator 32 and electromagnetic clutch 34 are shown in dashed lines to clarify that second synchronous generator (if desired, with a flywheel as interim storage unit) can alternatively be operated only in motor mode as pulse-former; reactive power and short-circuit current producer, flicker filter and voltage regulator.", "Particularly if second synchronous generator 32 is provided without internal combustion engine 30, a third synchronous generator 36 can be provided with an internal combustion engine to compensate for a lengthier power deficit.", "In the idle state, this third synchronous generator 36 can be separated by a switching unit 44 from the isolated network so as not to burden the isolated network as an additional load.", "Finally, a microprocessor or computer controller 42 is provided, which controls the individual components of the isolated network and thus allows a largely automated operation of the isolated network.", "By appropriate design of the individual components of the isolated network, it is possible for wind energy systems 10 on average to produce sufficient energy for the loads.", "This supply of energy is augmented by the photovoltaic elements, if needed.", "If the supply of power available from wind energy systems 10 and/or photovoltaic elements 12 is smaller/larger than the needs of the loads, interim storage units 14, 16, 18 can be called upon (discharged/charged), either to provide the missing power (discharging) or to store the surplus power (charging).", "Interim storage units 14, 16, 18 thus smooth out the always-fluctuating supply of regenerative energy.", "What power fluctuation can be compensated for what span of time is largely a function of the storage capacity of interim storage units 14, 16, 18.For a generous dimensioning of the interim storage units, time spans of a few hours to a few days are possible.", "Starting up internal combustion engines 30 and second or third synchronous generators 32, 36 is necessary only for power deficits that exceed the capacity of interim storage units 14, 16, 18.In the above description of embodiments, the primary energy producer was always one that uses a regenerative energy source, such as wind or solar (light).", "The primary energy producer can also make use of another regenerative energy source, for instance, hydropower, or be a producer that consumes fossil fuels.", "It is also possible for a seawater desalination plant (not shown) to be connected to the isolated network so that in times when the loads on the isolated network require considerably less energy than the primary energy producers can provide, the seawater desalination plant will consume the “surplus” electric power, i.e., the additional amount that could be provided, to produce usable water/drinking water, which can then be stored in catch basins.", "Should the energy consumption of the isolated network be so great that all energy producers are just barely able to provide this power, then the seawater desalination plant will be reduced to a minimal operation, or possibly turned off entirely.", "The control of the seawater desalination plant can also be accomplished via controller 42.In times when only part of the electric power from the primary energy producers is required by the isolated network, it is also possible to operate a pump storage plant, also not shown, by means of which water (or other fluid media) is brought from a lower to a higher potential, so that the electric power from the pump storage plant can be used if needed.", "Control of the pump storage plant can also be accomplished via controller 42.It is also possible for the seawater desalination plant and a pump storage plant to be combined by pumping the usable water (drinking water) produced by the seawater desalination plant to a higher potential, which can then be used to drive the generators of the pump storage plant.", "Of course, various combinations of the components of the systems shown in FIGS.", "1-3 can also be constructed and these fall within the scope of the present invention.", "From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention.", "Accordingly, the invention is not limited except as by the appended claims." ] ]

[ [ "4-Amino-quinazolines", "Quinazolines of the formula (I) in which R, R1, R2, R3, R4 and Y have the meaning indicated in Patent claim 1, and their salts or solvates as glycoprotein IbIX antagonists." ], [ "1.Compounds of the formula I in which R and R1 are independently of each other H, A, OH, OA, Hal, N(R5)2, NO2, CN, CHO, COA, CON(R5)2, COOR5, allyl, CH═CH—COOR5, CH═CHCON(R5)2, SO2A or phenyl, which is unsubstituted or mono-, di- or trisubstituted by A, R2 and R3 are independently of each other H, A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)o—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p-(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, provided that R2 and R3 together are not H, or NR2R3 together form a saturated monocyclic heterocyclic radical having 5 to 6 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by OH, Ar, OAr or arylalkyl, R4 is Ar or Het1, R5 is H or A, R5 is benzo[1,3]dioxol-5-yl, R7 is H, A, cycloalkyl or (CH2)q—OR5, Q is O or S, Y is a direct bond, (CH2)n or —NR5—(CH2)m—, Z is phenylene, cyclohexylene, —NR5—, O, —CH(OH)—, —CA2- or A is unbranched or branched alkyl having 1 to 6 carbon atoms, Ar is phenyl, naphthyl or biphenyl, which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, cycloalkyloxy, O—(CH2)p-Ph, CF3, OCF3, Hal, CN, CHO, COA, COOR5, —(CH2)p—N(R7)2, NR5—COA, NO2, SO2N(R5)2, mor, SO2-mor, 5-methyl-3-oxo-2,4-dihydropyrazol-2-yl, naphthyl or Het2, Het is a saturated, partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, carbonyl oxygen, COOR5, benzyl, Het2 or phenyl which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, CF3, OCF3, Hal, CN, COOR5, N(R5)2, NO2, SO2N(R5)2, Het1 is thiophen-2-yl substituted with Ar or Het2, Het2 is a unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, —(CH2)p—(CHA)p-N(R5)—(CH2)q—COR5, CHO, COA or COOR5, Het3 is a partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, SO2A or COOR5 provided that the heterocyclic radical is not bondend via an N atom, Hal is F, Cl, Br or I, mor is morpholin-4-yl, Ph is phenyl, n is 1, 2, 3, 4, 5 or 6, m is 0, 1, 2, 3, 4, 5 or 6, o is 1, 2, 3, 4, 5, 6 or 7, p is 0, 1, 2, 3 or 4, q is 1, 2, 3 or 4, and their pharmaceutically tolerable salts and solvates.", "2.A compound of claim 1, wherein R is H or OA.", "3.A compound according to claims 1 or 2, wherein R1 is H, A, OA or Hal.", "4.A compound according to at least one of claims 1 to 3, wherein Y is (CH2)n and n is 1, 2, 3, 4, 5 or 6.5.A compound according to at least one of claims 1 to 3, wherein Y is a direct bond.", "6.A compound according to at least one of claims 1 to 3, wherein Y is —N(R5)—(CH2)m— and R5 is H or A.", "7.Compounds of the formula I according to at least one of claims 1 to 6 a) [2-(benzo[1,3]dioxol-5-yloxy)-ethyl]-[2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-amine, b) [2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine, and their physiologically acceptable salts and solvates.", "8.Process for the preparation of the compounds of the formula I in which R and R1 are independently of each other H, A, OH, OA, Hal, N(R5)2, NO2, CN, CHO, COA, CON(R5)2, COOR5, allyl, CH═CH—COOR5, CH═CHCON(R5)2, SO2A or phenyl, which is unsubstituted or mono-, di- or trisubstituted by A, R2 and R3 are independently of each other H, A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)o—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p—(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, provided that R2 and R3 together are not H, or NR2R3 together form a saturated monocyclic heterocyclic radical having 5 to 6 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by OH, Ar, OAr or arylalkyl, R4 is Ar or Het1, R5 is H or A, R6 is benzo[1,3]dioxol-5-yl, R7 is H, A, cycloalkyl or —(CH2)q—OR5, Q is O or S, Y is a direct bond, (CH2)n or —NR5—(CH2)m—, Z is phenylene, cyclohexylene, —NR5—, O, —CH(OH)—, —CA2- or A is unbranched or branched alkyl having 1 to 6 carbon atoms, Ar is phenyl, naphthyl or biphenyl, which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, cycloalkyloxy, O—(CH2)p-Ph, CF3, OCF3, Hal, CN, CHO, COA, COOR5, —(CH2)p—N(R7)2, NR5—COA, NO2, SO2N(R5)2, mor, SO2-mor, 5-methyl-3-oxo-2,4-dihydropyrazol-2-yl, naphthyl or Het2, Het is a saturated, partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, carbonyl oxygen, COOR5, benzyl, Het2 or phenyl which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, CF3, OCF3, Hal, CN, COOR5, N(R5)2, NO2, SO2N(R5)2, Het1 is thiophen-2-yl substituted with Ar or Het2, Het2 is a unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, —(CH2)p—(CHA)p-N(R5)—(CH2)q—COR5, CHO, COA or COOR5, Het3 is a-partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, SO2A or COOR5 provided that the heterocyclic radical is not bondend via an N atom, Hal is F, Cl, Br or I, mor is morpholin-4-yl, Ph is phenyl, n is 1, 2, 3, 4, 5 or 6, m is 0, 1, 2, 3, 4, 5 or 6, o is 1, 2, 3, 4, 5, 6 or 7, p is 0, 1, 2, 3 or 4, q is 1, 2, 3 or 4, and their pharmaceutically tolerable salts and solvates according to at least one of claims 1 to 7, characterized in that a) a compound of the formula I is liberated from one of its functional derivatives by treating with a solvolysing or hydrogenolysing agent, or b) for compounds in which Y is a direct bond or (CH2)n in stage 1) a compound of the formula II in which R and R1 have a meaning indicated in claim 1, is reacted with a compound of the formula III in which R4 has a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, and X is Cl, Br, OH or a reactive esterified OH group followed by converting the cyano group to an amide group by conventional means to give a compound of formula IV in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, in stage 2) a compound of formula IV as indicated above is reacted with a base to give a compound of formula V in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, in stage 3) a compound of formula V as indicated above is reacted with a chlorinating agent to give a compound of formula VI in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, and in stage 4) a compound of formula VI as indicated above is reacted with a compound of formula VII in which R2 and R3 or NR2R3 have a meaning indicated in claim 1, or b) for compounds in which Y is NR5—(CH2)m— in stage 1) a compound of the formula VIII in which R and R1 have a meaning indicated in claim 1, is reacted with a compound of formula VII in which R2 and R3 or NR2R3 have a meaning indicated in claim 1, to give a compound of formula IX in which R, R1, R2, R3 and NR2R3 have a meaning indicated in claim 1, and in stage 2) a compound of formula IX as indicated above is reacted with a compound of formula X in which R4, R5 and m have a meaning indicated in claim 1 or d) a radical R, R1,R2, R3 and/or R4 is converted into another radical R, R1, R2, R3 and/or R4 by, for example reducing a nitro group, sulfonyl group or sulfoxyl group, etherifying an OH group or subjecting an OA group to ether cleavage, alkylating a primary or secondary amino group, partially or completely hydrolysing a CN group, cleaving an ester group or esterifying a carboxylic acid radical, reacting an aryl bromide, aryl iodide, heteroaryl bromide or heteroaryliodide to give the corresponding coupling products by means of a Suzuki coupling with boronic acids, reacting a iodoquinazoline or bromoquinazoline to give the corresponding coupling products by means of a Stille coupling with allyltributyltin, reacting a iodoquinazoline or bromoquinazoline to give the corresponding coupling products by means of a Heck coupling with acrylates, or carrying out a nucleophilic or electrophilic substitution, or a base or acid of the formula I is converted into one of its salts or solvates.", "9.Compounds of the formula I according to at least one of claims 1 to 7 and their physiologically acceptable salts or solvates as pharmaceutical active compounds.", "10.Compounds of the formula I according to claim 9 and their physiologically acceptable salts or solvates as glycoprotein IbIX antagonists.", "11.Compounds of the formula I according to claim 9 and their physiologically acceptable salts or solvates as glycoprotein IbIX antagonists for the control of thrombotic disorders and sequelae deriving therefrom.", "12.Pharmaceutical preparation characterized in that it contains at least one compound of the formula I according to at least one of claims 1 to 7 or one of its physiologically acceptable salts or solvates.", "13.Use of compounds of the formula I according to at least one of claims 1 to 7 and/or their physiologically acceptable salts or solvates for the production of a pharmaceutical preparation for the control of thrombotic disorders and sequelae deriving therefrom or for use as anti-adhesive substances.", "14.Use of compounds of the formula I according to at least one of claims 1 to 7 and/or their physiologically acceptable salts or solvates for the production of a pharmaceutical preparation for the treatment of illnesses, such as for the prophylaxis and/or therapy of thrombotic disorders, as well as sequelae such as, for example, myocardial infarct, arteriosclerosis, angina pectoris, acute coronary syndromes, peripheral circulatory disorders, stroke, transient ischaemic attacks, reocclusion/restenosis after angioplasty/stent implantations or as anti-adhesive substances for implants, catheters or heart pacemakers." ], [ "The invention relates to substituted 4-amino-quinazolines of the formula I in which R and R1 are independently of each other H, A, OH, OA, Hal, N(R5)2, NO2, CN, CHO, COA, CON(R5)2, COOR5, allyl, CH═CH—COOR5, CH═CHCON(R5)2, SO2A or phenyl, which is unsubstituted or mono-, di- or trisubstituted by A, R2 and R3 are independently of each other H, A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)o—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p-(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, provided that R2 and R3 together are not H, or NR2R3 together form a saturated monocyclic heterocyclic radical having 5 to 6 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by OH, Ar, OAr or arylalkyl, R4 is Ar or Het1, R5 is H or A, R6 is benzo[1,3]dioxol-5-yl, R7 is H, A, cycloalkyl or —(CH2)q—OR5, Q is O or S, Y is a direct bond, (CH2)n or —NR5—(CH2)m—, Z is phenylene, cyclohexylene, —NR5—, O, —CH(OH)—, —CA2- or A is unbranched or branched alkyl having 1 to 6 carbon atoms, Ar is phenyl, naphthyl or biphenyl, which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, cycloalkyloxy, O—(CH2)p-Ph, CF3, OCF3, Hal, CN, CHO, COA, COOR5, —(CH2)p—N(R7)2, NR5—COA, NO2, SO2N(R5)2, mor, SO2-mor, 5-methyl-3-oxo-2,4-dihydropyrazol-2-yl, naphthyl or Het2, Het is a saturated, partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, carbonyl oxygen, COOR5, benzyl, Het2 or phenyl which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, CF3, OCF3, Hal, CN, COOR5, N(R5)2, NO2, SO2N(R5)2, Het1 is thiophen-2-yl which is substituted by Ar or Het2, Het2 is a unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, —(CH2)p—(CHA)p-N(R5)—(CH2)q—COR5, CHO, COA or COOR5, Het3 is a partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, SO2A or COOR5 provided that the heterocyclic radical is not bondend via an N atom, Hal is F, Cl, Br or I, mor is morpholin-4-yl, Ph is phenyl, n is 1, 2, 3, 4, 5 or 6, m is 0, 1, 2, 3, 4, 5 or 6, o is 1, 2, 3, 4, 5, 6 or 7, p is 0, 1, 2, 3 or 4, q is 1, 2, 3 or 4, and their pharmaceutically tolerable salts and solvates.", "Similar 4-amino substituted quinazolines are disclosed in WO 99/09986, Mastafanova, Li et al, Khim.-Farm.Zh.", "1982, 16, 938-42, U.S. Pat.", "No.", "5,436,233 or DE 2135172.The invention is based on the object of finding novel compounds having valuable properties, in particular those which can be used for the production of medicaments.", "It has been found that the compounds of the formula I and their salts or solvates have very valuable pharmacological properties together with good tolerability.", "They act especially as GPIbIX inhibitors, in particular inhibiting the interaction of this receptor with the ligand von Willebrand factor (vWF).", "This action can be demonstrated, for example, by a method which is described by S. Meyer et al.", "in J. Biol.", "Chem.", "1993, 268, 20555-20562.The property as GPIbIX alpha-thrombin receptor (N. J. Greco, Biochemistry 1996, 35, 915-921) can also be blocked by the compounds mentioned.", "The significance of GPIbIX as an adhesion receptor on platelets, which mediates the primary interaction of platelets with an arteriosclerotically modified vascular wall via binding to the vWF expressed there, has been described by many authors (e.g.", "Z. M. Ruggeri in Thromb.", "Hemost.", "1997, 78, 611-616).", "The activation of another platelet adhesion receptor, GPIIbIIIa, following the GPIbIX-vWF interaction, leads to platelet aggregation and thus to thrombotic vascular occlusion.", "A GPIbIX antagonist can thus prevent the start of thrombus formation and thus also release of active substances from the platelets which, for example, promote thrombus growth and have an additional trophic action on the vascular wall.", "This has been shown with inhibitory peptides or antibodies in various experimental models (e.g.", "H Yamamoto et al., Thromb.", "Hemost.", "1998, 79, 202-210).", "In the case of higher shear forces, the blocking action of GPIbIX inhibitors exerts its maximum effect, as described by J. J. Sixma et al.", "in Arteriosclerosis, Thrombosis, and Vascular Biology 1996, 16, 64-71.According to the flow chamber method used there, the compounds of the formula I can be characterized as GPIbIX inhibitors in whole blood.", "The inhibition of thrombus formation of the GPIbIX inhibitors can be measured by a modified Born method (Nature 1962, 4832, 927-929) using botrocetin or ristocetin as an aggregation stimulant.", "The compounds of the formula I according to the invention can therefore be employed as pharmaceutical active compounds in human and veterinary medicine.", "They act as adhesion receptor antagonists, in particular as glycoprotein IbIX antagonists, and are suitable for the prophylaxis and/or therapy of thrombotic disorders and sequelae deriving therefrom.", "The preferentially best action is to be expected in the case of thrombotic disorders in the arterial vascular system, but GPIbIX inhibitors also have an effect in the case of thrombotic disorders in the venous vascular bed.", "The disorders are acute coronary syndromes, angina pectoris, myocardial infarct, peripheral circulatory disorders, stroke, transient ischaemic attacks, arteriosclerosis, reocclusion/restenosis after angioplasty/stent implantation.", "The compounds can furthermore be employed as anti-adhesive substances where the body comes into contact with foreign surfaces such as implants, catheters or cardiac pacemakers.", "Therefore, the invention relates further to compounds of the formula I according to claim 1 and their physiologically acceptable salts or solvates as pharmaceutical active compounds.", "The invention relates to compounds of the formula I according to claim 1 and their physiologically acceptable salts or solvates as glycoprotein IbIX antagonists.", "Comparison medication introduced onto the market which may be mentioned are aspirin and GPIIbIIIa antagonists.", "The invention relates to the compounds of the formula I and their salts or solvates, and to a process for the preparation of these compounds and their salts or solvates, characterized in that a) a compound of the formula I is liberated from one of its functional derivatives by treating with a solvolysing or hydrogenolysing agent, or b) for compounds in which Y is a direct bond or (CH2)n in stage 1) a compound of the formula II in which R and R1 have a meaning indicated in claim 1, is reacted with a compound of the formula III in which R4 has a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, and X is Cl, Br, OH or a reactive esterified OH group followed by converting the cyano group to an amide group by conventional means to give a compound of formula IV in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, in stage 2) a compound of formula IV as indicated above is reacted with a base to give a compound of formula V in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, in stage 3) a compound of formula V as indicated above is reacted with a chlorinating agent to give a compound of formula VI in which R, R1 and R4 have a meaning indicated in claim 1 and Y is a direct bond or (CH2)n, and in stage 4) a compound of formula VI as indicated above is reacted with a compound of formula VII in which R2 and R3 or NR2R3 have a meaning indicated in claim 1, or b) for compounds in which Y is NR5—(CH2)m—, in stage 1) a compound of the formula VIII in which R and R1 have a meaning indicated in claim 1, is reacted with a compound of formula VII in which R2 and R3 or NR2R3 have a meaning indicated in claim 1, to give a compound of formula IX in which R, R1, R2, R3 and NR2R3 have a meaning indicated in claim 1, and in stage 2) a compound of formula IX as indicated above is reacted with a compound of formula X in which R4, R5 and m have a meaning indicated in claim 1 or d) a radical R, R1, R2, R3 and or R4 is converted into another radical R, R1, R2, R3 and/or R4 by, for example reducing a nitro group, sulfonyl group or sulfoxyl group, etherifying an OH group or subjecting an OA group to ether cleavage, alkylating a primary or secondary amino group, partially or completely hydrolysing a CN group, cleaving an ester group or esterifying a carboxylic acid radical, reacting an aryl bromide, aryl iodide, heteroaryl bromide or heteroaryliodide to give the corresponding coupling products by means of a Suzuki coupling with boronic acids, reacting a iodoquinazoline or bromoquinazoline to give the corresponding coupling products by means of a Stille coupling with allyltributyltin, reacting a iodoquinazoline or bromoquinazoline to give the corresponding coupling products by means of a Heck coupling with acrylates, or carrying out a nucleophilic or electrophilic substitution, or a base or acid of the formula I is converted into one of its salts or solvates.", "The compounds of the formula I can have a chiral center and therefore occur in a number of stereoisomeric forms.", "All these forms (e.g.", "R and S forms) and their mixtures (e.g.", "the RS forms) are included in the formula I.", "The compounds according to the invention also include so-called prodrug derivatives, i.e.", "compounds of the formula I modified with, for example, alkyl or acyl groups, sugars or oligopeptides and which are rapidly cleaved in the body to give the active compounds according to the invention.", "Furthermore, free amino groups as substituents of compounds of the formula I can be provided with appropriate conventional protective groups.", "Solvates of the compounds of the formula I are understood as meaning adducts of inert solvent molecules to the compounds of the formula I which are formed on account of their mutual power of attraction.", "Solvates are, for example, mono- or dihydrates or alcoholates.", "The abbreviations used have the following meanings: Ac acetyl, Bu n-butyl, DBU 1,8-diazabicyclo[5.4.0]undec-7-ene, DMA dimethylacetamide, DMF dimethylformamide, dppf 1,1′-bis(diphenylphosphino)ferrocene, Et ethyl, iPr isopropyl, Me methyl, Ph phenyl, TEA triethylamine, TFA trifluoroacetic acid.", "In the above formulae, A is alkyl and has 1 to 6, preferably 1, 2, 3 or 4 C atoms.", "Alkyl is preferably methyl, furthermore ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl, additionally also pentyl, 1-, 2- or 3-methylbutyl, 1,1-, 1,2- or 2,2-dimethylpropyl, 1 -ethylpropyl, hexyl, 1-, 2-, 3- or 4-methylpentyl, 1,1-, 1,2-, 1,3-, 2,2-, 2,3- or 3,3-dimethylbutyl, 1 - or 2-ethylbutyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, 1,1,2- or 1,2,2-trimethylpropyl.", "A is preferentially methyl, ethyl, propyl, isopropyl, butyl or pentyl.", "Ar is phenyl, naphthyl or biphenyl, which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, cycloalkyloxy, O—(CH2)P-Ph, CF3, OCF3, Hal, CN, CHO, COA, COOR5, —(CH2)p—N(R7)2, NR5—COA, NO2, SO2N(R5)2, mor, SO2-mor, 5-methyl-3-oxo-2,4-dihydropyrazol-2-yl, naphthyl or Het2.Ar is preferentially phenyl, preferably—as indicated—mono- di- or trisubstituted phenyl, specifically preferentially phenyl, 2-, 3- or 4-methylphenyl, 2-, 3- or 4-ethylphenyl, 2-, 3- or 4-propylphenyl, 2-, 3- or 4-isopropylphenyl, 2-, 3- or 4-butylphenyl, 2-, 3- or 4-tert-butylphenyl, 2-, 3- or 4-aminophenyl, 2-, 3- or 4-N,N-dimethylaminophenyl, 2-, 3- or 4-sulfamoylphenyl, 2-, 3- or 4-nitrophenyl, 2-, 3- or 4-hydroxyphenyl, 2-, 3- or 4-methoxyphenyl, 2-, 3- or 4-ethoxyphenyl, 2-, 3- or 4-pentoxyphenyl, 2-, 3- or 4-phenoxyphenyl, 2-, 3- or 4-phenylmethoxyphenyl, 2-, 3- or 4-trifluoromethylphenyl, 2-, 3- or 4-trifluoromethoxyphenyl, 2-, 3- or 4-cyclopentyloxyphenyl, 2-, 3- or 4-carboxyphenyl, 2-, 3- or 4-(N,N-diethyl)sulfamoylphenyl, 4-(3-methyl-butyramido)-phenyl, 2-, 3- or 4-cyanophenyl, 2-, 3- or 4-fluorophenyl, 2-, 3- or 4-chlorophenyl, 2-, 3- or 4-bromophenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-dichlorophenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or3,5-dibromophenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-dimethoxyphenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-di(trifluoromethyl)phenyl, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- or 3,5-di(phenylmethoxy)phenyl, 2-chloro-6-methylphenyl, 2-chloro-4-fluorophenyl, 3-bromo-6-fluorophenyl, 3,4,5-trimethoxyphenyl, 4-(morpholin-4-yl)phenyl, 4-(morpholin-4-yl-sulfonyl)phenyl, 4-(5-methyl-3-oxo-2,4-dihydropyrazol-2-yl)phenyl, 4-(4,6-dimethoxy-pyrimidin-2-yl)phenyl, 3-(4,6-dimethoxy-pyrimidin-2-yl)phenyl, 4-(pyrid-3-yl)phenyl, 3-(pyrid-3-yl)phenyl, 4-(thiophen-2-yl)phenyl, 4-(thiophen-3-yl)phenyl, 4-(2-formyl-thiophen-3-yl)phenyl, 3-(thiophen-2-yl)phenyl, 4-(benzo[c]thiophen-2-yl)phenyl, 4-(naphthalen-1-yl)phenyl or Furthermore, however, Ar is also preferentially unsubstituted naphthyl or biphenyl—as indicated—or alternatively mono-, di- or trisubstituted biphenyl, specifically preferentially biphenyl-4-yl or biphenyl-3-yl, 2′-methylbiphenyl-4-yl, 3′-methylbiphenyl-4-yl, 4′-methylbiphenyl-4-yl, 2′-methylbiphenyl-3-yl, 3′-methylbiphenyl-3-yl, 4′-methylbiphenyl-3-yl, 2-methylbiphenyl-4-yl, 3-methylbiphenyl-4-yl, 2-methylbiphenyl-3-yl, 4-methylbiphenyl-3-yl, 2′-tert-butylbiphenyl-4-yl, 3′-tert-butylbiphenyl-4-yl, 4′-tert-butylbiphenyl-4-yl, 2′-tert-butylbiphenyl-3-yl, 3′-tert-butylbiphenyl-3-yl, 4′-tert-butylbiphenyl-3-yl, 2-tert-butylbiphenyl-4-yl, 3-tert-butylbiphenyl-4-yl, 2-tertbutylbiphenyl-3-yl, 4-tert-butylbiphenyl-3-yl, 2′-isopropylbiphenyl-4-yl, 3′-isopropylbiphenyl-4-yl, 4′-isopropylbiphenyl-4-yl, 2′-isopropylbiphenyl-3-yl, 3′-isopropylbiphenyl-3-yl, 4′-isopropylbiphenyl-3-yl, 2-isopropylbiphenyl-4-yl, 3-isopropylbiphenyl-4-yl, 2-isopropylbiphenyl), 4-isopropylbiphenyl-3-yl, 2′-fluorobiphenyl-4-yphenyl, 4′-fluorobiphenyl-4-yl, 2′-fluorobiphenyl-3-yl, 3′-fluorobiphenyl-3-yl, 4′-fluorobiphenyl-3-yl, 2-fluorobiphenyl-4-yl, 3-fluorobiphenyl-4-yl, 2-fluorobiphenyl-3-yl, 4-fluorobiphenyl-3-yl, 2′-chlorobiphenyl-4-yl, 3-chlorobiphenyl-4-yl, 4′-chlorobiphenyl-4-yl, 2′-chlorobiphenyl-3-yl, 3′-chlorobiphenyl-3-yl, 4′-chlorobiphenyl-3-yl, 2-chlorobiphenyl-4-yl, 3-chlorobiphenyl-4-yl, 2-chlorobiphenyl-3-yl, 4-chlorobiphenyl-3-yl, 2′-methoxybiphenyl-4-yl, 3′-methoxybiphenyl-4-yl, 4′-methoxybiphenyl-4-yl, 2′-methoxybiphenyl-3-yl, 3′-methoxybiphenyl-3-yl, 4′-methoxybiphenyl-3-yl, 2-methoxybiphenyl-4-yl, 3-methoxybiphenyl-4-yl, 2-methoxybiphenyl-3-yl, 4-methoxybiphenyl-3-yl, 2′-nitrobiphenyl-4-yl, 3′-nitrobiphenyl-4-yl, 4′-nitrobiphenyl-4-yl, 2′-nitrobiphenyl-3-yl, 3′-nitrobiphenyl-3-yl, 4′-nitrobiphenyl-3-yl, 2-nitrobiphenyl-4-yl, 3-nitrobiphenyl-4-yl, 2-nitrobiphenyl-3-yl, 4-nitrobiphenyl-3-yl, 2′-trifluoromethylbiphenyl-4-yl, 3′-trifluoromethylbiphenyl-4-yl, 4′-trifluoromethyl-biphenyl-4-yl, 2′-trifluoromethylbiphenyl-3-yl, 3′-trifluoromethylbiphenyl-3-yl, 4′-trifluoromethylbiphenyl-3-yl, 2-trifluoromethylbiphenyl-4-yl, 3-trifluoromethylbiphenyl-4-yl, 2-trifluoromethylbiphenyl-3-yl, 4-trifluoromethylbiphenyl-3-yl, 2′-trifluoromethoxybiphenyl-4-yl, 3′-trifluoromethoxybiphenyl-4-yl, 4′-trifluoromethoxybiphenyl-4-yl, 2′-trifluoromethoxybiphenyl-3-yl, 3′-trifluoromethoxybiphenyl-3-yl, 4′-trifluoromethoxybiphenyl-3-yl, 2-trifluoromethoxybiphenyl-4-yl, 3-trifluoromethoxybiphenyl-4-yl, 2-trifluoromethoxybiphenyl-3-yl, 4-trifluoromethoxybiphenyl-3-yl, 3′-acetylbiphenyl-4-yl, 3′-acetylaminobiphenyl-4-yl, 3′-aminobiphenyl-4-yl, 3′-formylbiphenyl-4-yl, 4′-formylbiphenyl-4-yl, 4′-propylaminomethylbiphenyl-4-yl, 3′-methoxyethylaminomethylbiphenyl-4-yl, 4′-cyclohexylmethylaminomethylbiphenyl-4-yl or 3′-hydroxypropylaminomethylbiphenyl-4-yl, furthermore preferentially disubstituted biphenyls, such as 2′-methyl-3′-nitrobiphenyl-4-yl, 2′-methyl-4′-nitrobiphenyl-4-yl, 2′--methyl-5′-nitrobiphenyl-4-yl, 2′-methyl-6′-nitrobiphenyl-4-yl, 3′-methyl-2′-nitrobiphenyl-4-yl, 3′-methyl-4′-nitrobiphenyl-4-yl, 3′-methyl-5′-nitrobiphenyl-4-yl, 3′-methyl-6′- nitrobiphenyl-4-yl, 4′-methyl-2′-nitrobiphenyl-4-yl, 4′-methyl-3′-nitrobiphenyl-4-yl, 2′-methyl-3′-nitrobiphenyl-3-yl, 2′-methyl-4′-nitrobiphenyl-3-yl, 2′-methyl-5′-nitrobiphenyl-3-yl, 2′-methyl-6′-nitrobiphenyl-3-yl, 3′-methyl-2′-nitrobiphenyl-3-yl, 3′-methyl-4′-nitrobiphenyl-3-yl, 3′-methyl-5′-nitrobiphenyl-3-yl, 3′-methyl-6′-nitrobiphenyl-3-yl, 4′-methyl-2′-nitrobiphenyl-3-yl, 4′-methyl-3′-nitrobiphenyl-3-yl, 2′-methoxy-2-methylbiphenyl-4-yl, 3′-methoxy-2-methylbiphenyl-4-yl, 4′-methoxy-2-methylbiphenyl-4-yl, 4′-methoxy-3-nitrobiphenyl-4-yl, 2′-chloro-3′-fluorobiphenyl-4-yl, 2′-chloro-4′- fluorobiphenyl-4-yl, 2′-chloro-5′-fluorobiphenyl-4-yl, 2′-chloro-6′-fluorobiphenyl-4-yl, 3′-chloro-2′-fluorobiphenyl-4-yl, 3′-chloro-4′-fluorobiphenyl-4-yl, 3′-chloro-5′-fluorobiphenyl-4-yl, 3′-chloro-6′-fluorobiphenyl-4-yl, 4′-chloro-2′-fluorobiphenyl-4-yl, 4′-chloro-3′-fluorobiphenyl-4-yl, 2′-chloro-3′-fluorobiphenyl-3-yl, 2′-chloro-4′-fluorobiphenyl-3-yl, 2′-chloro-5′-fluorobiphenyl-3-yl, 2′-chloro-6′-fluorobiphenyl-3-yl, 3′-chloro-2′-fluorobiphenyl-3-yl, 3′-chloro-4′-fluorobiphenyl-3-yl, 3′-chloro-5′-fluorobiphenyl-3-yl, 3′-chloro-6′-fluorobiphenyl-3-yl, 4′-chloro-2′-fluorobiphenyl-3-yl, 4′-chloro-3′- fluorobiphenyl-3-yl, (2,3′-diethyl)biphenyl-4-yl, (3,3′-diethyl)biphenyl-4-yl), (2,2′-diethyl)biphenyl-4-yl, (2,4′-diethyl)biphenyl-4-yl, (2′,3′-dimethoxy)biphenyl-4-yl, (2′,4′-dimethoxy)biphenyl-4-yl, (2′,5′-dimethoxy)biphenyl-4-yl, (2′,6′-dimethoxy)-biphenyl-4-yl, (3′,4′-dimethoxy)biphenyl-4-yl, (3′,5′-dimethoxy)biphenyl-4-yl, (2′,3′-dimethoxy)-biphenyl-3-yl, (2′,4′-dimethoxy)biphenyl-3-yl, (2′,5′-dimethoxy)biphenyl-3-yl, (2′,6′-dimethoxy)-biphenyl-3-yl, (3′,4′-dimethoxy)biphenyl)-3-yl, (3′,5′-dimethoxy)biphenyl-3-yl, (3′,5′-dichloro)biphenyl-4-yl, (3′,5′-dichloro)biphenyl-3-yl, (2′,4′-dichloro)biphenyl-4-yl, (3′,4′,5′-trimethoxy)biphenyl-4-yl, (2′,3′-di(trifluoromethyl))biphenyl-4-yl, (2′,4′-di(trifluoromethyl))biphenyl-4-yl, (2′,5′-di(trifluoromethyl))biphenyl-4-yl, (2′,6′-di(trifluoromethyl))biphenyl-4-yl, (3′,4′-di(trifluoromethyl))biphenyl-4-yl, (3′,5′-di(trifluoromethyl))biphenyl-4-yl, (2′,3′-di(trifluoromethyl))biphenyl-3-yl, (2′,4′-di(trifluoromethyl))biphenyl-3-yl, (2′,5′-di(trifluoromethyl))biphenyl-3-yl, (2′,6′-di(trifluoromethyl))biphenyl-3-yl, (3′,4′-di(trifluoromethyl))biphenyl-3-yl, (3′,5′-di(trifluoromethyl)biphenyl-3-yl, (2,2′-dimethyl)biphenyl-4-yl, (2,′3-dimethyl)biphenyl-4-yl, (2,4′-dimethyl)biphenyl-4-yl, (2,2′-dimethyl)biphenyl-3-yl, (2,3′-dimethyl)biphenyl-3-yl or (2,4′-dimethyl)biphenyl-3-yl.", "Phenyl, 2-, 3- or 4-fluorophenyl, 2-, 3- or 4-chlorophenyl, 4-bromophenyl, 2,4- or 3,4-dichlorophenyl, 2,5- or 3,4-dimethoxyphenyl, 3,5-bis(trifluoromethyl)phenyl, 4-aminophenyl, 4-dimethylaminophenyl, 2-, 3- or 4-methylphenyl, 2-, 3- or 4-methoxyphenyl, 4-propyiphenyl, 4-isopropylphenyl, 4-butylphenyl, 4-tert.-butylphenyl, 2-, 3- or 4-nitrophenyl, 2-cyanophenyl, 2-, 3- or 4-pentoxyphenyl, 3- or 4-phenoxyphenyl, 2- or 4-benzyioxyphenyl, 2-, 3- or 4-trifluoromethylphenyl, 2-, 3- or 4-trifluoromethoxyphenyl, 2- or 4-cyclopentyloxyphenyl, 3- or 4-carboxyphenyl, 2-, 3- or 4-(N,N-diethyl)sulfamoylphenyl, 3,4-di(benzyloxy)phenyl, 4-(3-methyl-butyramido)-phenyl, 2-chloro-6-methylphenyl, 2-chloro-4-fluorophenyl, 3-bromo-6-fluorophenyl, 3,4,5-trimethoxyphenyl, 4-(morpholin-4-yl)phenyl, 4-(morpholin-4-yl-sulfonyl)phenyl, 4-(5-methyl-3-oxo-2,4-dihydropyrazol-2-yl)phenyl, 4-(4,6-dimethoxy-pyrimidin-2-yl)phenyl, 3-(4,6-dimethoxy-pyrimidin-2-yl)phenyl, 4-(pyridin-3-yl)phenyl, 3-(pyridin-3-yl)phenyl, 4-(thiophen-2-yl)phenyl, 3-(thiophen-2-yl)phenyl, 4-(benzo[c]thiophen-2-yl)phenyl, 4-(naphthalen-1-yl)phenyl, 4-(thiophen-3-yl)phenyl, 4-(2-formyl-thiophen-3-yl)phenyl, naphthyl, biphenyl-4-yl, 2′-fluorobiphenyl-4-yl, 4′-fluorobiphenyl-4-yl, 4′-fluorobiphenyl-3-yl, 4′-chlorobiphenyl-4-yl, 4′-chlorobiphenyl-3-yl, 4′-methoxybiphenyl-4-yl, 4′-methoxybiphenyl-3-yl, 3′-nitrobiphenyl-4-yl, 3′-acetylbiphenyl-4-yl, 3′-acetylaminobiphenyl-4-yl, 3′-aminobiphenyl-4-yl, 3′-formylbiphenyl-4-yl, 4′-formylbiphenyl-4-yl, 4′-propylaminomethylbiphenyl-4-yl, 3′-methoxyethyl-aminomethylbiphenyl-4-yl, 4′-cyclohexylmethylaminomethylbiphenyl-4-yl, 3′-hydroxypropylaminomethylbiphenyl-4-yl, (2,3′-diethyl)biphenyl-4-yl, (2,4′-diethyl)biphenyl-4-yl, (2,2′-diethyl)biphenyl-4-yl, (3′,5′-dichloro)biphenyl-3-yl, (3′,4′-dimethoxy)biphenyl-4-yl, (2′,4′-dichloro)biphenyl-4-yl, (3′,4′,5′-trimethoxy)biphenyl-4-yl, (3′,5′-di(trifluoromethyl))biphenyl-4-yl or is particularly preferred for Ar.", "Arylalkyl is preferentially benzyl.", "O—(CH2)P-Ph is phenylalkyloxy, in which p can be 0, 1, 2, 3 or 4.Benzyloxy or phenyloxy is particularly preferred.", "Cycloalkyl preferably has 3-7 C atoms and is preferably cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and further also cyclopentylmethyl, cyclopentylethyl or cyclohexylmethyl; cyclopentyl, cyclohexylmethyl or cyclohexyl are particularly preferred.", "Hal is preferably F, Cl, Br or I. Het is a saturated, partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, carbonyl oxygen, COOR5, Het2, benzyl or phenyl which is unsubstituted or mono-, di- or trisubstituted by A, OH, OA, CF3, OCF3, Hal, CN, COOR5, N(R5)2, NO2, SO2N(R5)2.Het is preferably unsubstituted 2- or 3-furyl, 2- or 3-thiophenyl, 1-,-2-.or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 2-, 3- or 4-pyridyl, 2-, 4-, 5- or 6-pyrimidinyl, furthermore preferably 1,2,3-triazol-1-, -4- or -5-yl, 1,2,4-triazol-1-, -4- or -5-yl, 1- or 5-tetrazolyl, 1,2,3-oxadiazol-4- or -5-yl, 1,2,4-oxadiazol-3- or -5-yl, 1,3,4-thiadiazol-2- or -5-yl, 1,2,4-thiadiazol-3- or -5-yl, 1,2,3-thiadiazol-4- or -5-yl, 2-, 3-, 4-, 5- or 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 3- or 4-pyridazinyl, pyrazinyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothiophenyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-1H-indolyl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 2-, 4-, 5-, 6- or 7-benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisoxazolyl, 2-, 4-, 5-, 6- or 7-benzothiazolyl, 2-, 4-, 5-, 6- or 7-benzisothiazolyl, 4-, 5-, 6- or 7-benz-2,1,3-oxadiazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolinyl, 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-acridinyl, 3-, 4-, 5-, 6-, 7- or 8-cinnolinyl, 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl.", "The heterocyclic radicals can also be partially or completely hydrogenated.", "Het can thus also be 2,3-dihydro-2-, -3-, -4- or -5-furyl, 2,5-dihydro-2-, -3-, -4- or -5-furyl, tetrahydro-2- or -3-furyl, 1,3-dioxolan-4-yl, tetrahydro-2- or -3-thiophenyl, 2,3-dihydro-1-, -2-, -3-, -4- or -5-pyrrolyl, 2,5-dihydro-1-, -2-, -3-, -4- or -5-pyrrolyl, 1-, 2- or 3-pyrrolidinyl, tetrahydro-1-, -2- or -3-pyrrolyl, tetrahydro-1-, -2- or 4-imidazolyl, 2,3-dihydro-1-2-, -3-, -4-, -5-, -6-, -7-1H-indolyl, 2,3-dihydro-1-, -2-, -3-, -4- or -5-pyrazolyl, tetrahydro-1-3- or -4-pyrazolyl, 1,4-dihydro-1-, -2-, -3- or -4-pyridyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5- or -6-pyridyl, 1,2,3,6-tetrahydro-1-, -2-, -3-, -4-, -5- or -6-pyridyl, 1-, 2-, 3- or 4-piperidinyl, 1-, 2-, 3- or 4-azepanyl, 2-, 3- or 4-morpholinyl, tetrahydro-2-, -3- or -4-pyranyl, 1,4-dioxanyl, 1,3-dioxan-2-, -4- or -5-yl, hexahydro-1-, -3- or -4-pyridazinyl, hexahydro-1-, -2-, -4- or -5-pyrimidinyl, 1-, 2- or 3-piperazinyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5-, -6-, -7- or -8-quinolinyl, 1,2,3,4-tetrahydro-1-, -2-, -3-, -4-, -5-, -6-, -7- or -8-isoquinolinyl which can be substituted as indicated above or particularly substituted by A, OA, carbonyl oxygen, NO2, Het2 or phenyl which is substituted by Hal, CN or OA.", "Thiophen-2-yl, tetrahydro-furan-2-yl, 1-methyl-octahydro-indol-3-yl, benzo[1,3]dioxol-5-yl, piperazin-1-yl, 4-methyl-piperazin-1-yl, piperidin-1-yl, piperidin-4-yl, 1-methyl-piperidin-3-yl, 4-benzyl-piperidin-1-yl, 2-methyl-piperidin-1-yl, 1-ethyl-pyrrolidin-2-yl, 1-methyl-pyrrolidin-2-yl, 2-oxo-pyrrolidin-1-yl, pyridin-2-yl, pyridin-4-yl, 5-nitro-pyridin-2-yl, imidazol-1-yl, morpholin-4-yl, 5-methoxy-1H-indol-2-yl is particularly preferred for Het.", "Het1 is thiophen-2-yl which is substituted by Ar or Het2, in which Ar and Het2 have one of the above or below mentioned meanings.", "5-(4-Fluorophenyl)-thiophen-2-yl, 5-(2-methoxyphenyl)-thiophen-2-yl, 5-(2-cyanophenyl)-thiophen-2-yl, 5-(2,5-dimethoxyphenyl)-thiophen-2-yl, 2-[2,2′]bithiophenyl-5-yl, 5-(pyridin-4-yl)-thiophen-2-yl, 5-(1H-indol-5-yl)-thiophen-2-yl, 5-quinolin-8-yl-thiophen-2-yl or 5-(benzo[b]thiophen-2-yl)-thiophen-2-yl is particularly preferred for Het1.Het2 is a unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N and/or 1 or 2 S or O atoms can be present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, —(CH2)p—(CHA)p-N(R5)—(CH2)q—COR5, CHO, COA or COOR5.Thiophen-2-yl, 2-formyl-thiophen-3-yl, pyridin-3-yl, pyridin-2-yl, pyridin-4-yl, indol-5-yl, quinolin-8-yl, 4,6-dimethoxy-pyrimidin-2-yl, benzo[b]thiophen-2-yl or is particularly preferred for Het2.Het3 is a partially or completely unsaturated mono- or bicyclic heterocyclic radical having 5 to 10 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by A, Hal, OH, OA, CF3, OCF3, N(R5)2, SO2A or COOR5 provided that the heterocyclic radical is not bondend via an N atom.", "Quinolin-5-yl and 1-methanesulfonyl-2,3-dihydro-1H-indol-5-yl is particularly preferred for Het3.", "(CH2)o-Het is preferentially thiophen-2-yl-ethyl, tetrahydro-furan-2-yl-methyl, 1-methyl-octahydro-indol-3-yl-methyl, 1-methyl-octahydro-indol-3-yl-ethyl benzo[1,3]dioxol-5-yl-methyl, benzo[1,3]dioxol-5-yl-ethyl, piperazin-1-yl-ethyl, 4-methyl-piperazin-1-yl-propyl, piperidin-1-yl-ethyl, piperidin-4-yl-methyl, 1-methyl-piperidin-3-yl-ethyl, 4-benzyl-piperidin-1-yl-ethyl, 2-methyl-piperidin-1-yl-propyl, 1-ethyl-pyrrolidin-2-yl-methyl, 1-methyl-pyrrolidin-2-yl-methyl, 2-oxo-pyrrolidin-1-yl-propyl, pyridin-2-yl-ethyl, pyridin-4-yl-methyl, pyridin-4-yl-ethyl, imidazol-1-yl-propyl, morpholin-4-yl-propyl or morpholin-4-yl-ethyl.", "(CH2)o—NR5-Het is preferentially (5-nitro-pyridin-2-yl)-amino-ethyl.", "(CH2)o—OR5 is preferentially (CH2)2—OCH3, (CH2)3—OCH3 or (CH2)3—O(iPr).", "(CH2)o—OR6 is preferentially (CH2)o-Z-(CH2)q—N(R5)2 is preferentially (CH2)p—(CHA)p-(CH2)m—Ar is preferentially phenyl, R and R1 are independently of each other H, A, OH, OA, Hal, N(R5)2, NO2, CN, CHO, COA, CON(R5)2, COOR5, allyl, CH═CH—COOR5, CH═CHCON(R5)2, SO2A or phenyl, which is unsubstituted or mono-, di- or trisubstituted by A, where A and Hal have a preferred meaning indicated beforehand and R5 have a preferred meaning indicated in the following.", "R is preferentially H or OA.", "R1 is preferentially H, A, OA, Hal, allyl, CH═CH—COOR5, CH═CHCON(R5)2 or phenyl, which is unsubstituted or monosubstituted by A. H, A, OA or Cl is particularly preferred for R1.The preferred position of R1 is the 6- or 7-position of the quinazoline ring system.", "R2 and R3 are independently of each other H, A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p-(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, provided that R2 and R3 together are not H, where A, Ar, cycloalkyl, Het or Het3 have a preferred meaning indicated beforehand and R5, R6, Q, Z, m, o, p and q have a preferred meaning indicated in the following.", "R2 is preferentially H or A. R3 is preferentially A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)o—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p-(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, Furthermore NR2R3 together form a saturated monocyclic heterocyclic radical having 5 to 6 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by OH, Ar, OAr or arylalkyl, where Ar or arylalkyl have a preferred meaning indicated beforehand.", "Preferred saturated monocyclic heterocyclic radicals can be piperidine or piperazine.", "are particularly preferred for NR2R3.R4 is Ar or Het1, where Ar or Het1 have a preferred meaning indicated beforehand.", "Phenyl, 4-bromophenyl, 3,5-bis-(trifluoromethyl)phenyl, 4-tert.-butylphenyl, 3-bromo-6-fluorophenyl, 4-(pyridin-3-yl)phenyl, 3-(pyridin-3-yl)phenyl, 4-(thiophen-2-yl)phenyl, 3-(thiophen-2-yl)phenyl, naphthyl, biphenyl-4-yl, 4′-fluorobiphenyl-4-yl, 4′-fluorobiphenyl-3-yl, 4′-chlorobiphenyl-4-yl, 4′-chlorobiphenyl-3-yl, 4′-methoxybiphenyl-4-yl, 4′-methoxybiphenyl-3-yl, (3′,5′-dichloro)biphenyl-3-yl is particularly preferred as Ar in R4.Phenyl, 4-bromophenyl, 3,5-bis-(trifluoromethyl)phenyl, 4-tert.-butylphenyl, 3-bromo-6-fluorophenyl, 4-(pyridin-3-yl)phenyl, 3-(pyridin-3-yl)phenyl, 4-(thiophen-2-yl)phenyl, 3-(thiophen-2-yl)phenyl, naphthyl, biphenyl-4-yl, 4′-fluorobiphenyl-4-yl, 4′-fluorobiphenyl-3-yl, 4′-chlorobiphenyl4-yl, 4′-chlorobiphenyl-3-yl, 4′-methoxybiphenyl4-yl, 4′-methoxybiphenyl-3-yl, (3′,5′-dichloro)biphenyl-3-yl, 5-(4-fluorophenyl)-thiophen-2-yl, 5-(2-methoxyphenyl)-thiophen-2-yl, 5-(2-cyanophenyl)-thiophen-2-yl, 5-(2,5-dimethoxyphenyl)-thiophen-2-yl, 2-[2,2′]bithiophenyl-5-yl, 5-(pyridin4-yl)-thiophen-2-yl, 5-(1H-indol-5-yl)-thiophen-2-yl, 5-quinolin-8-yl-thiophen-2-yl or 5-(benzo[b]thiophen-2-yl)-thiophen-2-yl is particularly preferred for R4.R5 is H or A, where A has a preferred meaning indicated beforehand.", "R6 is benzo[1,3]dioxol-5-yl.", "R7 is H, A, cycloalkyl or (CH2)q—OR5, where A, cycloalkyl and R5 have a preferred meaning indicated beforehand and q is preferrably 2 or 3.Q is O or S, preferentially O. Y is a direct bond, (CH2)n or —NR5—(CH2)m—, where R5 has a preferred meaning indicated beforehand and n and mr have a preferred meaning indicated in the following.", "Y is preferentially a direct bond or (CH2)n, very particularly preferably a direct bond.", "Z is phenylene, cyclohexylene, —NR5—, O, —CH(OH)—, —CA2- or where R5 and A have a preferred meaning indicated beforehand.", "Phenylene and/or cyclohexylene are particularly bonded in 1,4- or 1,3-position.", "n is 1, 2, 3, 4, 5 or 6, preferentially 4.m is 0, 1, 2, 3, 4, 5 or 6, preferentially 0, 1, 2 or 3.o is 1, 2, 3, 4, 5, 6 or 7, preferentially 1, 2, 3 or 7.p is 0, 1, 2, 3 or 4, preferentially 0, 1 or 2.q is 1, 2, 3 or 4, preferentially 1, 2 or 3.Some preferred groups of compounds can be expressed by the following subformulae Ia to In, which correspond to the formula I and in which the radicals not designated in greater detail have the meanings indicated in formula I, but in which in Ia R is H or OA and R1 is H, A, OA or Hal; in Ib R is H or OA, R1 is H, A, OA or Hal and Y is (CH2)n; in Ic R is H or OA, R1 is H, A, OA or Hal, R4 is Ar, Ar is unsubstituted phenyl and Y is (CH2)n; in Id R is H or OA, R1 is H, A, OA or Hal and Y is a direct bond; in Ie R is H or OA, R1 is H, A, OA or Hal and Y is —N(R5)—(CH2)m—; in If R is H or OA, R1 is H. A, OA or Hal, R4 is Ar and Y is a direct bond; in Ig R is H or OA, R1 is H, A, OA or Hal, R4 Ar, Ar is phenyl or biphenyl, which is unsubstituted or substituted by Hal, Het2, OA or —(CH2)p—N(R7)2, R7 is H, A, cycloalkyl, (CH2)3—OR5 or (CH2)2—OR5 and Y is a direct bond; in Ih R is H, R1 is Hal, R2 is H, R3 is —(CH2)o-Het, —(CHA)p-(CH2)o—N(R5)2 or —(CH2)p—(CHA)p-(CH2)m—Ar, R4 is Ar Ar is unsubstituted phenyl and Y is a direct bond; in Ii R is H, R1 is Hal, R2 is H, R3 is —(CH2)o-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)o—NR5-Het, —(CH2)o-Z-(CH2)q—N(R5)2 or —(CH2)p—(CHA)p-(CH2)m—Ar, R4 is Ar, Ar is biphenyl-4-yl and Y is a direct bond; in Ij R is H, R1 is Hal, R2 is H or A, R3 is A, cycloalkyl, -Het3, —(CH2)o—OR5, —(CH2)o—OR6, —(CH2)o-Het, —(CH2)o—NR5-Het, —(CHA)p-(CH2)o—N(R5)2, —(CH2)p—(CHA)p-(CH2)m—Ar, —(CH2)o-Z-(CH2)q—N(R5)2, or NR2R3 together form a saturated monocyclic heterocyclic radical having 5 to 6 ring members, where 1 or 2 N atoms are present and the heterocyclic radical can be mono- or disubstituted by OH, Ar, OAr or arylalkyl, R4 is Ar, Ar is phenyl which is substituted by Br and Y is a direct bond; in Ik R is H, R1 is Hal, R2 is H, R3 is —(CH2)o-Het or —(CHA)p-(CH2)o—N(R5)2, R4 is Ar, Ar is phenyl or biphenyl, which is unsubstituted or substituted by Hal, Het2, OA or —(CH2)p—N(R7)2, R7 is H, A, cycloalkyl, (CH2)3—OR5 or (CH2)2—OR5 and Y is a direct bond; in Im R is H, R1 is Hal, R2 is H, R3 is —(CH2)o-Het or —(CHA)p-(CH2)o—N(R5)2, R4 is Het1, Het1 is thiophen-2-yl, which is substituted by Ar or Het2, R7 is H, A, cycloalkyl, (CH2)3—OR5 or (CH2)2—OR5 and Y is a direct bond; in In R is H, R1 is Hal, R2 is H, R3 is —(CH2)o-Het or —(CHA)p-(CH2)o—N(R5)2, R4 is Het1, Het1 is 5-(4-fluoro-phenyl)-thiophen-2-yl, 5-(2-methoxy-phenyl)-thiophen-2-yl, 5-(2-cyano-phenyl)-thiophen-2-yl, 5-(2,5-dimethoxy-phenyl)-thiophen-2-yl, 2-[2,2′]bithiophenyl-5-yl, 5-(1H-indol-5-yl)-thiophen-2-yl, 5-pyridine-4-yl-thiophen-2-yl, 5-quinolin-8-yl-thiophen-2-yl or 5-benzo[b]thiophen-2-yl-thiophen-2-yl R7 is H, A, cycloalkyl, (CH2)3—OR5 or (CH2)2—OR5 and Y is a direct bond.", "The compounds of the formula I and also the starting substances for their preparation are otherwise prepared by methods known per se, such as are described in the literature (e.g.", "in the standard works such as Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg-Thieme-Verlag, Stuttgart), namely under reaction conditions which are known and suitable for the reactions mentioned.", "In this case, use can also be made of variants which are known per se, but not mentioned here in greater detail.", "The starting substances, if desired, can also be formed in situ such that they are not isolated from the reaction mixture, but immediately reacted further to give the compounds of the formula I.", "The compounds of the formula I can be obtained by liberating them from their functional derivatives by solvolysis, in particular hydrolysis or by hydrogenolysis.", "Preferred starting substances for the solvolysis or hydrogenolysis are those which otherwise correspond to the formula I, but instead of one or more free amino and/or hydroxyl groups contain corresponding protected amino and/or hydroxyl groups, in particular those which instead of an H—N— group carry an R′—N— group, in which R′ is an amino protective group and/or those which instead of the H atom of a hydroxyl group carry a hydroxyl protective group, e.g.", "those which correspond to the formula I, but instead of a group —COOH carry a group —COOR″, in which R″ is a hydroxyl protective group.", "A number of—identical or different—protected amino and/or hydroxyl groups can also be present in the molecule of the starting substance.", "If the protective groups present are different from one another, in many cases they can be removed selectively (lit.", ": T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Chemistry, 2nd ed., Wiley, New York 1991 or P. J. Kocienski, Protecting Groups, 1st ed., Georg Thieme Verlag, Stuttgart-New-York, 1994).", "The expression “amino protective group” is generally known and relates to groups which are suitable for protecting (for blocking) an amino group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out at other positions in the molecule.", "Typical groups of this type are, in particular, unsubstituted or substituted acyl, aryl, aralkoxymethyl or aralkyl groups.", "Since the amino protective groups are removed after the desired reaction (or reaction sequence), their nature and size is otherwise not critical; however, those having 1-20, in particular 1-8, C atoms are preferred.", "The expression “acyl group” is to be interpreted in the widest sense in connection with the present process.", "It includes acyl groups derived from aliphatic, araliphatic, aromatic or heterocyclic carboxylic acids or sulfonic acids and, in particular, alkoxycarbonyl groups, aryloxycarbonyl groups and especially aralkoxycarbonyl groups.", "Examples of acyl groups of this type are alkanoyl such as acetyl, propionyl, butyryl; aralkanoyl such as phenylacetyl; aroyl such as benzoyl or toluyl; aryloxyalkanoyl such as POA; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, BOC, 2-iodoethoxycarbonyl; aralkyloxycarbonyl such as CBZ (“carbobenzoxy”), 4-methoxybenzyloxycarbonyl (MOZ), 4-Nitro-benzyloxycarbonyl oder 9-fluorenylmethoxycarbonyl (Fmoc); 2-(phenylsulfonyl)ethoxycarbonyl; trimethylsilylethoxycarbonyl (Teoc) or arylsulfonyl such as 4-methoxy-2,3,6-trimethylphenyl-sulfonyl (Mtr).", "Preferred amino protective groups are BOC, furthermore CBZ, Fmoc, benzyl and acetyl; particularly preferred Fmoc.", "The expression “hydroxyl protective group” is also generally known and relates to groups which are suitable for protecting a hydroxyl group against chemical reactions, but which are easily removable after the desired chemical reaction has been carried out at other positions in the molecule.", "Typical groups of this type are the above mentioned unsubstituted or substituted aryl, aralkyl, aroyl or acyl groups, furthermore also alkylgroups, alkyl-, aryl- or aralkylsilylgroups or O,O- or O,S-acetals.", "The nature and size of the hydroxyl protective groups is not critical, since they are removed again after the desired chemical reaction or reaction sequence; groups having 1-20, in particular 1-10 C atoms, are preferred.", "Examples of hydroxyl protective groups are, inter alia, benzyl, 4-methoxybenzyl or 2,4-dimethoxybenzyl, aroyl groups such as benzoyl or p-nitrobenzoyl, acyl groups such as acetyl or pivaloyl, p-toluolsulfonyl, alkyl groups such as methyl or tert-butyl, but also allyl, alkylsilyl groups such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), tert-butyidimethylsilyl (TBS) or triethylsilyl, trimethylsilylethyl, aralkylsilyl groups such as tert-butyidiphenylsilyl (TBDPS), cyclic acetals such as isopropylidene-, cyclopentylidene-, cyclohexylidene-, benzylidene-, p-methoxybenzylidene- or o,p-dimethoxybenzylideneacetal, acyclic acetales such as tetrahydropyranyl (Thp), methoxymethyl (MOM), methoxyethoxymethyl (MEM), benzyloxymethyl (BOM) or methylthiomethyl (MTM).", "Acetyl, benzyl, tert-butyl or TBS being particularly preferred.", "The liberation of the compounds of the formula I from their functional derivatives depending on the protective group used is known in the present literature such as T. W. Greene, P. G. M.. Wuts, Protective Groups in Organic Chemistry, 2nd ed., Wiley, New York 1991, P. J. Kocienski, Protecting Groups, 1st ed., Georg Thieme Verlag, Stuttgart-New-York, 1994.In this case, use can also be made of variants which are known per se, but not mentioned here in greater detail.", "The groups BOC and O-tert-butyl can preferably be removed, for example, using TFA in dichloromethane or using approximately 3 to 5N HCl in dioxane at 15-30° C., the Fmoc group using an approximately 5 to 50% solution of dimethylamine, diethylamine or piperidine in DMF at 15-30° C. Preferred starting substances for the solvolysis or hydrogenolysis includes also those which otherwise correspond to the formula I, but are attached to a solid phase.", "The liberation of the compounds of the formula I from the solid phase is known in the present literature such as Novabiochem—The Combinatorial Chemistry Catalog, March 99 and cited literature.", "The solid phase with a carbonate moiety as terminal functional group can preferably be removed, for example, using TFA (50%) in dichloromethane.", "The quinazolines of formula I can also preferably be prepared, using either solution or solid-phase techniques.", "The term solid phase indicates a resin for solid-phase chemistry, especially for combinatorial chemistry, i.e.", "by robot- and computer-assisted syntheses, and subjected to mass screening as indicated in U.S. Pat.", "No.", "5,463,564; M. A. Gallop et al., J. Med.", "Chem.", "1994, 37,1233-1251 and 1385-1401 and M. J. Sofia, Drug Discovery Today 1996,1, 27-34).", "The polymeric material of the solid phase is generally chosen from the group consisting of cross-linked polystyrene, cross-linked polyacrylamide or other resins, natural polymers or silicagels.", "The group of cross-linked polystyrene, cross-linked polyacrylamide or other resins includes e.g.", "polyacrylamide, polymethacrylamide, polyhydroxyethylmethacrylate, polyamide, polystyrene, (meth)acrylate copolymers, for instance from (methy)acrylic acid, esters of (meth)acrylic acid and/or 2-methylene-succinic acid, but-2-enoic acid or maleic acid, polyurethanes or other copolymers.", "Suitable terminal functional groups or linkers on the surface of the resin have to be chosen to attach the compounds to the resin.", "There exists a variety of commercially available resins, e.g.", "in Novabiochem—The Combinatorial Chemistry-Catalog, March 99.Examples for suitable resins are carbonate resins with a modified carbonate group as terminal functional group like p-nitrophenylcarbonate resin, halogenated resins like Merrifield resin (chloromethylpolystyrene) or carboxy resins like carboxy polystyrene resin or NovaSyn® TG Carboxy Resin.", "p-Nitrophenylcarbonate resin is particularly preferred.", "These and other types of resins well known in the art can be used in the subject invention.", "The quinazolines of formula I, in which Y is a direct bond or (CH2)n, can preferably be prepared by combining and reacting a 2-amino-benzonitrile of formula II with an aldehyde of formula III followed by converting the cyano group to an amide group, reacting the given formula IV with a base, chlorinating the given quinazolin-4-one of formula V and reacting the given formula VI with an amine of formula VII.", "The conversion of the cyano group to the amide group occurs by conventional means which are known to a skilled artisan.", "Particularly, the conversion occurs via oxidation within the presence of a base.", "The quinazolines of formula I, in which Y is —N(R5)—(CH2)m—, can be prepared by reacting a 2,4-dichloro-quinazoline of formula VIII with an amine of formula VII and reacting the given formula IX with an amine of formula X.", "As a rule, the starting compounds of the formulae II, III, VII, VIII and X are known or commercially available.", "The unknown compounds, however, can be prepared by methods known per se.", "The 2,4-dichloro-quinazolines of formula II in which R and R1 have a meaning indicated in claim 1 can be prepared by reacting a substituted anthranilic acid with KOCN/acetic acid in the presence of a base and chlorinating the given 1H-quinazoline-2,4-dione.", "The aldehydes of formula III, as a rule, are also commercially available.", "Furthermore, syntheses for the preparation of aldehydes of formula III, such as, for example, the oxidation of an alcohol, can be used.", "The amines of formula VII or X in which R2, R3, NR2R3, R5, R4 and m have a meaning indicated in claim 1, as a rule, are also commercially available and can be attached to the suitable resin or to a compound of formula VI, VIII or IX by coupling procedures well known in the art and as described in the ensuing Examples.", "Furthermore, syntheses for the preparation of amines of formula VII or X, such as, for example, the Gabriel synthesis, can be used.", "For the preparation of compounds of the formula I in which R4 is unsubstituted or substituted biphenyl, heteroarylsubstituted phenyl or aryl- or heteroaryl-substituted thiophenyl, an appropriate compound of the formula I in which R4 is phenyl chloride, phenyl bromide, phenyl iodide, thiophenyl chloride, thiophenyl bromide or thiophenyl iodide can be reacted with the appropriate boronic acid derivatives in a Suzuki type coupling reaction.", "This reaction is expediently carried out under Palladium catalysis with different phosphines as coordination ligands, e.g.", "Pd(P(Ph)3)2, Pd(II)Cl2dppf, PdOAc2+P(R*)3(R*=phenyl, cyclohexyl, tert-butyl) etc.", "in the presence of a base such as potassium carbonate, cesium carbonate, DBU, NaOH, in an inert solvent or solvent mixture, e.g.", "DMF or 1,4-dioxane at temperatures between 0° and 150°, preferably between 60° and 120°.", "Depending on the conditions used, the reaction time is between a few minutes and a number of days.", "The boronic acid derivatives can be prepared by conventional methods or are commercially available.", "The reactions can be carried out in analogy to the methods indicated in Suzuki et al., J.", "Am.", "Chem.", "Soc.", "1989,111, 314ff., Suzuki et al., Chem.", "Rev.", "1995, 95, 2457ff and G. C. Fu et al.", "Angew.", "Chem 1998, 110, 3586.The Suzuki type coupling reaction can be furthermore used to convert radicals R and R1 into other radicals R and R1, for e.g.", "to convert a halogen substituted quinazolines to a quinazoline substituted by substituted or unsubstituted phenyl.", "For the preparation of compounds of the formula I in which R or R1 is allyl, an appropriate compound of the formula I in which R4 is quinazoline chloride, quinazoline bromide or quinazoline iodide can be reacted with allyltributyltin in a Stille type coupling reaction.", "This reaction is expediently carried out under Palladium catalysis with different phosphines as coordination ligands, e.g.", "Pd(P(Ph)3)2, Pd(II)Cl2dppf, PdOAC2+P(R*)3(R*=phenyl, cyclohexyl, tert-butyl) etc.", "in an inert solvent or solvent mixture, e.g.", "DMF or 1,4-dioxane at temperatures between 0° and 150°, preferably between 60° and 120°.", "Depending on the conditions used, the reaction time is between a few minutes and a number of days.", "For the preparation of compounds of the formula I in which R or R1 is CH═CH—COOR5 or CH═CH—CON(R5)2, an appropriate compound of the formula I in which R4 is quinazoline chloride, quinazoline bromide or quinazoline iodide can be reacted with substituted acrylate in a Heck type coupling reaction.", "This reaction is expediently carried out under Palladium catalysis with different phosphines as coordination ligands, e.g.", "Pd(P(Ph)3)2, Pd(II)Cl2dppf, PdOAc2+P(R*)3(R*=phenyl, cyclohexyl, tert-butyl) etc.", "in the presence of a base such as triethyl amine or a catalyst tetrabutylammonium iodide, in an inert solvent or-solvent mixture, e.g.", "DMF or 1,4-dioxane at temperatures between 0° and 150°, preferably between 60° and 120°.", "Depending on the conditions used, the reaction time is between a few minutes and a number of days.", "A base of the formula I can be converted into the associated acid addition salt using an acid, for example by reaction of equivalent amounts of the base and of the acid in an inert solvent such as ethanol and subsequent evaporation.", "Acids which give physiologically acceptable salts are particularly suitable for this reaction.", "Thus inorganic acids can be used, e.g.", "sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric acid or hydrobromic acid, phosphoric acids such as orthophosphoric acid, sulfamic acid, furthermore organic acids, in particular aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono- or polybasic carboxylic, sulfonic or sulfuric acids, e.g.", "formic acid, acetic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, malic acid, citric acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methane- or ethanesulfonic acid, p-toluenesulfonic acid, naphthalenemono- and disulfonic acids or laurylsulfuric acid.", "Salts with physiologically unacceptable acids, e.g.", "picrates, can be used for the isolation and/or purification of the compounds of the formula I.", "On the other hand, compounds of the formula I with bases (e.g sodium or potassium hydroxide or carbonate) can be converted into the corresponding metal salts, in particular alkali metal or alkaline earth metal salts, or into the corresponding ammonium salts.", "The invention furthermore relates to pharmaceutical preparations comprising at least one compound of the formula I and/or one of its physiologically acceptable salts, which are prepared, in particular, in an non-chemical way.", "In this case, the compounds of the formula I according to the invention can be brought into a suitable dose form together with at least one solid, liquid and/or semi-liquid excipient or auxiliary and, if appropriate, in combination with one or more other active compounds.", "These preparations can be used as medicaments in human or veterinary medicine.", "Possible excipients are organic or inorganic substances which are suitable for enteral (e.g.", "oral) or parenteral administration or topical application and do not react with the novel compounds, for example water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glyceryl triacetate, gelatin, carbohydrates such as lactose or starch, magnesium stearate, talc and petroleum jelly.", "Tablets, pills, coated tablets, capsules, powders, granules, syrups, juices or drops are used, in particular, for oral administration, suppositories are used for rectal administration, solutions, preferably oily or aqueous solutions, furthermore suspensions, emulsions or implants, are used for parenteral administration, and ointments, creams or powders are used for topical application.", "The novel compounds can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection preparations.", "The preparations indicated can be sterilized and/or can contain auxiliaries such as lubricants, preservatives, stabilizers and/or wetting agents, emulsifiers, salts for affecting the osmotic pressure, buffer substances, colorants, flavorings and/or one or more other active compounds, e.g.", "one or more vitamins.", "The compounds of the formula I and their physiologically acceptable salts act as adhesion receptor antagonists, in particular glycoprotein IbIX antagonists, and can be employed for the prophylaxis and/or therapy of thrombotic disorders and sequelae deriving therefrom.", "The disorders are acute coronary syndromes, angina pectoris, myocardial infarct, peripheral circulatory disorders, stroke, transient ischaemic attacks, arteriosclerosis and reocclusion/restenosis after angioplasty/stent implantation.", "In this case, the substances according to the invention are as a rule administered in the dose of the glycoprotein IIbIIIa antagonist ReoPro® of preferably between approximately 1 and 500 mg, in particular between 5 and 100 mg, per dose unit.", "The daily dose is preferably between approximately 0.02 and 10 mg/kg of body weight.", "The specific dose for each patient depends, however, on all sorts of factors, for example on the efficacy of the specific compound employed, on the age, body weight, general state of health and sex, on the diet, on the time and route of administration, and on the excretion rate, pharmaceutical combination and severity of the particular disorder to which the therapy applies.", "Oral administration is preferred.", "Above and below, all temperatures are indicated in ° C. In the following examples, “customary working-up” for solution reactions means: if necessary, water is added, if necessary, depending on the constitution of the final product, the mixture is adjusted to pHs between 2 and 10 and extracted with ethyl acetate or dichloromethane, the organic phase is separated off, dried over sodium sulfate and evaporated, and the residue is purified by chromatography on silica gel and/or by crystallization.", "“Customary working-up” for solid-phase reactions means: the crude reaction is filtered and washed with DMF twice, then sucessively with methanol and methylene chloride three times, and finally once with methyl tert-butyl ether.", "The resin is then dried in vacuo.", "Mass spectrometry (MS) apparatuses Kratos OMIT and Finnigan LCQ.", "(M+H)+ values or M+ values are determined.", "EXAMPLES Example 1 1.Pyridine (0.144 mole) is given to a solution of 2-amino-4-chlorobenzonitrile (0,131 mole) in 100 ml tetrahydrofuran (THF) and a solution of 4-bromobenzoylchloride (31.6 g, 0.144 mole) in 50 ml THF is added under nitrogen.", "After addition of 100 ml THF, the mixture is stirred for 6 h at room temperature (rt).", "The crude reaction is then customary worked up for solution reactions affording 4-bromo-N-(5-chloro-2-cyano-phenyl)-benzamide as a solid; m.p.", "151-152°.", "MS calc.", ": 335,6; found: 336, 338.2.A suspension of 4-bromo-N-(5-chloro-2-cyano-phenyl)-benzamide (30 g, 89.4 mmol) in 500 ml methanol is mixed with 130 ml NaOH (2N) and perhydrite tablets [(H2O2), 50 g].", "The mixture is heated to boiling for 2 hrs.", "After cooling to rt and customary working up 4-bromo-N-(5-chloro-2-aminocarbonyl-phenyl)-benzamide is obtained as a solid; m.p.", "172-173°.", "MS calc.", ": 354; found: 354.3.4-Bromo-N-(5-chloro-2-aminocarbonyl-phenyl)-benzamide (18.25 g, 51.6 mmol) is solved in 250 ml dioxane and 250 ml NaOH (1 N) are added.", "The mixture is heated to boiling for 5 days.", "After cooling to rt and customary working up 2-(4-bromo-phenyl)-7-chloro-3H-quinazolin-4-one is obtained as a solid; m.p.", ">300°.", "MS calc.", ": 336; found: 336.4.2-(4-Bromo-phenyl)-7-chloro-3H-quinazolin-4-one (38,7 mmol) is added to 50 ml thionylchloride and is heated at 40°.", "The mixture is treated with 6 ml dimethylformamide (DMF).", "After cooling to rt the mixture is stirred for 3 hrs.", "Customary working up gives 2-(4-bromo-phenyl)-4,7-dichloroquinazoline as a solid; m.p.", "189-190°.", "MS calc.", ": 354.0; found: 355.5.A solution of 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline (0.085 mmol, 30 mg) in 2 ml THF is treated with aniline (0.01 ml, 0.11 mmol).", "The suspension is heated to 60° and stirred for 18 hrs.", "The reaction mixture is filtered and the crystals are washed with THF and dried.", "[2-(4-Bromo-phenyl)-7-chloro-quinazolin-4-yl]-phenyl-amine is obtained.", "MS calc.", ": 410,7; found: 411.Example 2 Analogously to example 1,1-(4-bromo-phenyl)-4,7-dichloro-quinazoline is reacted with HNR2R3 to obtain compounds of formula II according to table 1.TABLE 1 4-bromophenyl-quinazolines of formula I1 R2 in HNR2R3 R3 in HNR2R3 MS and in I1 and in I1 calc.", "found H 506.8 507 H 494.8 495 H 494.8 495 H 559.9 560 H 545.9 546 H 548.8 549 H 548.8 550 H 461.8 463 H 495.8 496 H 438.8 439 H 438.8 439 H 438.8 439 H 452.8 453 H 452.8 453 H 466.8 467 H 502.8 503 H 502.8 503 H 529.9 530 Example 3 A solution of 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline (0.085 mmol, 30 mg) [prepared analogously to example 1] in 2 ml THF is treated with benzylamine (0.174 mmol, 0.019 ml).", "The mixture is heated at 60° for 6 hrs.", "After cooling to rt, the reaction mixture is filtered through a tentacle ion exchanger (LiChrolut® SCX: Merck ChromBook, 2nd ed.", "page 31).", "Evaporation of the solvent afforded benzyl-[2-(4-bromo-phenyl)-7-chloroquinazolin-4-yl]-amine.", "MS calc.", ": 424,7; found: 425.Example 4 Analogously to example 3, 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline is reacted with HNR2R3 to obtain compounds of formula II according to table 2.TABLE 2 4-bromophenyl-quinazolines of formula I1 R2 in HNR2R3 R3 in HNR2R3 and in I1 and in I1 MS calc.", "found H 438.8 440 H 459.2 460 H 442.7 444 H 442.7 444 H 442.7 444 H 454.8 456 H 473.2 474 H 493.6 494 H 493.6 494 H 459.2 460 H 454.8 456 H 484.8 486 H 468.7 470 H 438.8 439 H 456.8 457 H 498.8 499 H 518.8 519 H 560.9 561 H 569.9 561 H 542.9 543 H 442.8 444 H 482.8 483 H 468.8 469 H 444.8 445 H 439.8 440 H 439.8 440 H 459.8 460 H 447.8 449 H 445.8 447 H —(CH2)2—OCH3 392.7 393 H —(CH2)2—N(CH3)2 405.7 405 H —(CH2)3—N(CH3)2 419.8 420 H —(CH2)2—N(Et)2 433.8 434 H —(CH2)3—OCH3 406.7 407 H 448.8 449 H 418.7 419 H 461.8 462 H 499.9 500 H Bu 390.7 391 H Pentyl 404.7 406 H 474.8 475 Example 5 Analogously to example 3, 2-(4-bromo-phenyl)-4-chloro-6-methylquinazoline is reacted with 1-propyl-pyrrolidin-2-one to obtain 1-{3-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-ylamino]-propyl}-pyrrolidin-2-one; MS calc.", ": 439.4; found: 439.6; with [2-(2-aminomethyl-phenylsulfanyl)-phenyl]-methanol to obtain [2-(2-{[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-ylamino]-methyl}-phenylsulfanyl)-phenyl]-methanol; MS calc.", ": 542.5 ; found: 544.2; with 3-(2-methyl-piperidin-1-yl)-propylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-[3-(2-methyl-piperidin-1-yl)-propyl]-amine; MS calc.", ": 453.4 ; found: 453.3; with 2-pyridin-2-yl-ethylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-(2-pyridin-2-yl-ethyl)-amine; MS calc.", ": 419.3; found: 419.3; with 1-(3-trifluoromethyl-phenyl)-piperazine to obtain 2-(4-bromo-phenyl)-6-methyl-4-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-quinazoline; MS calc.", ": 419.3; found: 419.3; with C-(1-ethyl-pyrrolidin-2-yl)-methylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-(1-ethyl-pyrrolidin-2-ylmethyl)-amine; MS calc.", ": 425.4; found: 425.6; with 4-benzyl-piperidin-4-ol to obtain 4-benzyl-1-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-piperidin-4-ol; MS calc.", ": 488.4 ; found: 490.3; with 1-phenyl-piperazine to obtain 2-(4-bromo-phenyl)-6-methyl-4-(4-phenyl-piperazin-1-yl)-quinazoline; MS calc.", ": 459.4 ; found: 459.4; with 3-morpholin-4-yl-propylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-(3-morpholin-4-yl-propyl)-amine; MS calc.", ": 441.4; found: 441.3; with 2-(1-methyl-pyrrolidin-2-yl)-ethylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amine; MS calc.", ": 425.4; found: 425.4; with 3-(4-methyl-piperazin-1-yl)-propylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-[3-(4-methyl-piperazin-1-yl)-propyl]-amine; MS calc.", ": 454.4 ; found: 454.4; with 3,4,5-trimethoxy-benzylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-(3,4,5-trimethoxy-benzyl)-amine; MS calc.", ": 494.4 ; found: 496.1; with 2-fluoro-benzylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-(2-fluoro-benzyl)-amine; MS calc.", ": 422.3; found: 422.4; with benzyl-methyl-amine to obtain benzyl-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-methyl-amine; MS calc.", ": 418.2; found: 418.4; with methyl-(1-methyl-piperidin-4-ylmethyl)-amine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-methyl-(1-methyl-piperidin-4-ylmethyl)-amine; MS calc.", ": 425.3 ; found: 425.2; with cyclohexyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin4-yl]-cyclohexyl-methyl-amine; MS calc.", ": 410.4 ; found: 412.1; with N1,N1-dimethyl-ethane-1,2-diamine to obtain N′-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-N,N-dimethyl-ethane-1,2-diamine; MS calc.", ": 385.3 ; found: 386.3; with butyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-butyl-methyl-amine; MS calc.", ": 384.3 ; found: 384.4; with N,N,N′-trimethyl-propane-1,3-diamine to obtain N-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-N,N′,N′-trimethyl-propane-1,3-diamine; MS calc.", ": 413.4; found: 415.2; with 4-benzyl-piperidine to obtain 4-(4 benzyl-piperidin-1-yl)-2-(4-bromo-phenyl)-6-methyl-quinazoline; MS calc.", ": 472.4 ; found: 474.3; with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine; MS calc.", ": 455.4; found: 455.3; with butylamine to obtain [2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-butyl-amine; with N,N-diethyl-propane-1,3-diamine to obtain N′-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-N, N-diethyl-propane-1,3-diamine; with benzylamine to obtain benzyl-[2-(4-bromo-phenyl)-6-methyl-quinazolin-4-yl]-amine.", "Example 6 Analogously to example 3, 2-(4-bromo-phenyl)-4,6-dichloro-quinazoline is reacted with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine; MS calc.", ": 475.86 ; found: 476; with 4-benzyl-piperidine to obtain 4-(4-benzyl-piperidin-1-yl)-2-(4-bromo-phenyl)-6-chloro-quinazoline; MS calc.", ": 492.84; found: 493; with N,N,N′-trimethyl-propane-1,3-diamine to obtain N-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-N,N′,N′-trimethyl-propane-1,3-diamine; MS calc.", ": 433.78; found: 434; with butyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-butyl-methyl-amine; MS calc.", ": 404.74; found: 405; with N1,N1-dimethyl-ethane-1,2-diamine to obtain N′-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-N,N-dimethyl-ethane-1,2-diamine; with N1,N1-diethyl-ethane-1,2-diamine to obtain N′-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-N,N-diethyl-ethane-1,2-diamine; with cyclohexyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-cyclohexyl-methyl-amine; MS calc.", ": 430.77; found: 431; with methyl-(1-methyl-piperidin-4-ylmethyl)-amine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-methyl-(1-methyl-piperidin-4-ylmethyl)-amine; MS calc.", ": 445.79; found: 446; with benzyl-methyl-amine to obtain benzyl-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-methyl-amine; MS calc.", ": 438.75; found: 439; with 2-fluoro-benzylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-(2-fluoro-benzyl)-amine; MS calc.", ": 442.72; found: 443; with 3,4,5-trimethoxy-benzylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-(3,4,5-trimethoxy-benzyl)-amine; MS calc.", ": 514.80; found: 515; with 3-(4-methyl-piperazin-1-yl)-propylamine to obtain (2-(4-!bromo-phenyl)-6-chloro-quinazolin-4-yl]-[3-(4-methyl-piperazin-1-yl)-propyl]-amine; MS calc.", ": 474.8 ; found: 474; with 2-(1-methyl-pyrrolidin-2-yl)-ethylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amine; MS calc.", ": 445.79; found: 446; with 3-morpholin-4-yl-propylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-(3-morpholin-4-yl-propyl)-amine; MS calc.", ": 461.78; found: 462; with 1-phenyl-piperazine to obtain 2-(4-bromo-phenyl)-6-chloro-4-(4-phenyl-piperazin-1-yl)-quinazoline; MS calc.", ": 479.81; found: 480; with 4-benzyl-piperidin-4-ol to obtain 4-benzyl-1-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-piperidin-4-ol; MS calc.", ": 508.84; found: 509; with C-(1-ethyl-pyrrolidin-2-yl)-methylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-(1-ethyl-pyrrolidin-2-ylmethyl)-amine; MS calc.", ": 445.78; found: 446; with 1-(3-trifluoromethyl-phenyl)-piperazine to obtain 2-(4-bromo-phenyl)-6-chloro-4-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-quinazoline; MS calc.", ": 547.80; found: 549; with 2-pyridin-2-yl-ethylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-(2-pyridin-2-yl-ethyl)-amine; MS calc.", ": 439.74; found: 440; with 3-(2-methyl-piperidin-1-yl)-propylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-[3-(2-methyl-piperidin-1-yl)-propyl]-amine; MS calc.", ": 472.83; found: 473; with [2-(2-aminomethyl-phenylsulfanyl)-phenyl]-methanol to obtain [2-(2-{[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-ylamino]-methyl}-phenylsulfanyl)-phenyl]-methanol; MS calc.", ": 562.92; found: 563; with 1-propyl-pyrrolidin-2-one to obtain 1-{3-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-ylamino]-propyl}-pyrrolidin-2-one; MS calc.", ": 459.77; found: 460; with butylamine to obtain [2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-butyl-amine; with N,N-diethyl-propane-1,3-diamine to obtain N′-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine; with benzylamine to obtain benzyl-[2-(4-bromo-phenyl)-6-chloro-quinazolin-4-yl]-amine.", "Example 7 Analogously to example 3, 2-(4-bromo-phenyl)-4-chloro-6,7-dimethoxy-quinazoline is reacted with 1-propyl-pyrrolidin-2-one to obtain 1-{3-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-ylamino]-propyl}-pyrrolidin-2-one; with [2-(2-aminomethyl-phenylsulfanyl)-phenyl]-methanol to obtain [2-(2-{[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-ylamino]-methyl}-phenylsulfanyl)-phenyl]-methanol; MS calc.", ": 588.52; found: 589; with 3-(2-methyl-piperidin-1-yl)-propylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-[3-(2-methyl-piperidin-1-yl)-propyl]-amine; MS calc.", ": 499.45; found: 500; with 2-pyridin-2-yl-ethylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-(2-pyridin-2-yl-ethyl)-amine; MS calc.", ": 465.35; found: 466; with 1-(3-trifluoromethyl-phenyl)-piperazine to obtain 2-(4-bromo-phenyl)-6,7-dimethoxy-4-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-quinazoline; MS calc.", ": 573.41; found: 574; with C-(1-ethyl-pyrrolidin-2-yl)-methylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-(1-ethyl-pyrrolidin-2-ylmethyl)-amine; MS calc.", ": 483.41; found: 484; with 4-benzyl-piperidin-4-ol to obtain 4-benzyl-1-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-piperidin-4-ol; MS calc.", ": 534.45; found: 535; with 1-phenyl-piperazine to obtain 2-(4-bromo-phenyl)-6,7-dimethoxy-4-(4-phenyl-piperazin-1-yl)-quinazoline; MS calc.", ": 505.41; found: 506; with 3-morpholin-4-yl-propylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-(3-morpholin-4-yl-propyl)-amine; MS calc.", ": 487.39; found: 488; with 2-(1-methyl-pyrrolidin-2-yl)-ethylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amine; MS calc.", ": 471.39; found: 472; with 3-(4-methyl-piperazin-1-yl)-propylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-[3-(4-methyl-piperazin-1-yl)-propyl]-amine; MS calc.", ": 486.43; found: 487; with 3,4,5-trimethoxy-benzylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-(3,4,5-trimethoxy-benzyl)-amine; MS calc.", ": 540.41; found: 541; with 2-fluoro-benzylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-(2-fluoro-benzyl)-amine; MS calc.", ": 468.32; found: 469; with benzyl-methyl-amine to obtain benzyl-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-methyl-amine; MS calc.", ": 464.36; found: 465; with methyl-(1-methyl-piperidin-4-ylmethyl)-amine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-methyl-(1-methyl-piperidin-4-ylmethyl)-amine; MS calc.", ": 471.40; found: 472; with cyclohexyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-cyclohexyl-methyl-amine; MS calc.", ": 456.38; found: 457; with N1,N1-dimethyl-ethane-1,2-diamine to obtain N′-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-N,N-dimethyl-ethane-1,2-diamine; MS calc.", ": 431.33; found: 432; with N1,N1-diethyl-ethane-1,2-diamine to obtain N′-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-N,N-diethyl-ethane-1,2-diamine; with butyl-methyl-amine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-butyl-methyl-amine; MS calc.", ": 431.35; found: 432; with N,N,N′-trimethyl-propane-1,3-diamine to obtain N-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-N,N′,N′-trimethyl-propane-1,3-diamine; MS calc.", ": 459.38; found: 460; with 4-benzyl-piperidine to obtain 4-(4-benzyl-piperidin-1-yl)-2-(4-bromo-phenyl)-6,7-dimethoxy-quinazoline; MS calc.", ": 518.45; found: 519; with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine; MS calc.", ": 501.47; found: 502; with butylamine to obtain [2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-butyl-amine; with N,N-diethyl-propane-1,3-diamine to obtain N′-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine; with benzylamine to obtain benzyl-[2-(4-bromo-phenyl)-6,7-dimethoxy-quinazolin-4-yl]-amine.", "Example 8 Analogously to example 3, 2-phenyl-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and benzoylchloride] is reacted with 3-imidazol-1-yl-propylamine to obtain (7-chloro-2-phenyl-quinazolin-4-yl)-(3-imidazol-1-yl-propyl)-amine; with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-(7-chloro-2-phenyl-quinazolin-4-yl)-N1, N1-diethyl-pentane-1,4-diamine; with 3-morpholin-4-yl-propylamine to obtain (7-chloro-2-phenyl-quinazolin-4-yl)-(3-morpholin-4-yl-propyl)-amine; with phenyl-amine to obtain (7-chloro-2-phenyl-quinazolin-4-yl)-phenyl-amine.", "Analogously to example 3, N-[4-(4,7-dichloro-quinazolin-2-yl)-phenyl]-3-methyl-butyramide [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and 4-(3-methyl-butyrylamino)-benzoyl chloride] is reacted with N1,N1-diethyl-propane-1,3-diamine to obtain N-{4-[7-chloro-4-(3-diethylamino-propylamino)-quinazolin-2-yl)-phenyl}-3-methyl-butyramide.", "Example 9 Analogously to example 3, 2-biphenyl-4-yl-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and biphenyl-4-carbonyl chloride] is reacted with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-N,N1-diethyl-pentane-1,4-diamine; with 3-imidazol-1-yl-propylamine to obtain (2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-(3-imidazol-1-yl-propyl)-amine; with N1,N1-diethyl-ethane-1,2-diamine to obtain N′-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-N, N-diethyl-ethane-1,2-diamine; with N1,N1-diethyl-propane-1,3-diamine to obtain N′-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-N, N-diethyl-propane-1,3-diamine; with 3-morpholin-4-yl-propylamine to obtain (2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-(3-morpholin-4-yl-propyl)-amine; with 1-(3-amino-propyl)-pyrrolidin-2-one to obtain 1-[3-(2-biphenyl-4-yl-7chloro-quinazolin-4-ylamino)-propyl]-pyrrolidin-2-one; with 4-(2-amino-ethyl-phenylamine to obtain [2-(4-amino-phenyl)-ethyl]-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-amine; with N1-(5-nitro-pyridin-2-yl)-ethane-1,2-diamine to obtain N-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-N′-(5-nitro-pyridin-2-yl)-ethane-1,2-diamine; with 2-piperazin-1-yl-ethylamine to obtain (2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-(2-piperazin-1-yl-ethyl)-amine; with (4-aminomethyl-phenyl)-dimethyl-amine to obtain (2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-(4-dimethylamino-benzyl)-amine; with 2-pyridin-2-yl-ethylamine to obtain (2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-(2-pyridin-2-yl-ethyl)-amine; with C-(3-aminomethyl-cyclohexyl)-methylamine to obtain (3-aminomethyl-cyclohexylmethyl)-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-amine; with heptane-1,7-diamine to obtain N1-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-heptane-1,7-diamine; with propane-1,3-diamine to obtain N1-(2-biphenyl-4-yl-7-chloro-quinazolin-4-yl)-propane-1,3-diamine.", "Example 10 Analogously to example 3, 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and 4-bromobenzoylchloride] is reacted with N1,N1-diethyl-propane-1,3-diamine to obtain N′-[2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine.", "1,2 Equivalents of K2CO3, 1,2 equivalents of 4-chlorophenylboronic acid and 10 mol % of Pd((PPh3)4 are added to a solution of N′-[2-(4-bromo-phenyl)-7-chloro-quinazolin4-yl]-N,N-diethyl-propane-1,3-diamine (16,5 mmol) in 80 ml of DMF and it is heated at 800 until conversion is complete.", "After filtering off the catalyst and customary working up for solution reactions, N′-[7-chloro-2-(4′-chloro-biphenyl-4-yl)-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine is obtained.", "Example 11 Analogously to example 10, N′-[2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine is reacted with 4-methoxyphenylboronic acid to obtain N′-[7-chloro-2-(4′-methoxy-biphenyl-4-yl)-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine; with 3-acetylaminophenylboronic acid to obtain N-{4′-[7-chloro-4-(3-diethylamino-propylamino)-quinazolin-2-yl]-biphenyl-3-yl}-acetamide; with 3-formylphenylboronic acid to obtain 4′-[7-chloro-4-(3-diethylamino-propylamino)-quinazolin-2-yl]-biphenyl-3-carbaldehyde; with 4-[(cyclohexylmethyl-amino)-methyl]-phenylboronic acid to obtain N-(7-chloro-2-{4′-[(cyclohexylmethyl-amino)-methyl]-biphenyl-4-yl}-quinazolin-4-yl)-N′, N′-diethyl-propane-1,3-diamine.", "Analogously to example 10, N′-[2-(3-bromo-phenyl)-7-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine is reacted with 3,5-dichlorophenylboronic acid to obtain N′-[7-chloro-2-(3′,5′-dichloro-biphenyl-3-yl)-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine; with 4-fluorophenylboronic acid to obtain N′-[7-chloro-2-(4′-fluoro-biphenyl-3-yl)-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine.", "Example 12 Analogously to example 10, N4-[2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine (prepared according to example 3) is reacted with 4-chlorophenylboronic acid to obtain N4-[7-chloro-2-(4′-chloro-biphenyl-4-yl)-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine; with 3-formylphenylboronic acid to obtain 4′-[7-chloro-4-(4-diethylamino-1-methyl-butylamino)-quinazolin-2-yl]-biphenyl-3-carbaldehyde; with 3-[(hydroxypropyl-amino)-methyl]-phenylboronic acid to obtain 3-({4′-[7-chloro-4-(4-diethylamino-1-methyl-butylamino)-quinazolin-2-yl]-biphenyl-3-ylmethyl}-amino)-propan-1-ol.", "Analogously to example 10, N4-[2-(3-bromo-phenyl)-7-chloro-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine is reacted with 4-methoxyphenylboronic acid to obtain N4-[7-chloro-2-(4′-methoxy-biphenyl-3-yl)-quinazolin-4-yl]-N1 ,N1-diethyl-pentane-1,4-diamine; with (pyridin-3-yl)boronic acid to obtain N4-[7-chloro-2-(3-pyridin-3-yl-phenyl)-quinazolin-4-yl]-N1 ,N1-diethyl-pentane-1,4-diamine.", "Example 13 Analogously to example 3, 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and 4-bromobenzoylchloride] is reacted with 2-(1-methyl-octahydro-indol-3-yl)-ethylamine to obtain [2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine.", "Analogously to example 10, [2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine is reacted with 4-fluorophenylboronic acid to obtain [7-chloro-2-(4′-fluoro-biphenyl-4-yl)-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with (thiophen-2-yl)boronic acid to obtain [7-chloro-2-(4-thiophen-2-yl-phenyl)-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with (pyridin-3-yl)boronic acid to obtain [7-chloro-2-(4-pyridin-3-yl-phenyl)-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine.", "Analogously to example 10, [2-(3-bromo-phenyl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine is reacted with (thiophen-2-yl)boronic acid to obtain [7-chloro-2-(3-thiophen-2-yl-phenyl)-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with 3-(acetylamino)-phenylboronic acid to obtain N-(3′-{7-chloro-4-[2-(1-methyl-octahydro-indol-3-yl)-ethylamino]-quinazolin-2-yl}-biphenyl-3-yl)-formamide.", "Example 14 Analogously to example 3, 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and 4-bromobenzoylchloride] is reacted with 2-(1-methyl-piperidin-3-yl)-ethylamine to obtain [7-chloro-2-(4-bromo-phenyl)-quinazolin-4-yl]-[2-(1-methyl-piperidin-3-yl)-ethyl]-amine.", "Analogously to example 10, [7-chloro-2-(4-bromo-phenyl)-quinazolin-4-yl]-[2-(1-methyl-piperidin-3-yl)-ethyl]-amine is reacted with 3-formylphenylboronic acid to obtain 4′-{7-chloro-4-[2-(1-methyl-piperidin-3-yl)-ethylamino]-quinazolin-2-yl}-biphenyl-3-carbaldehyde; with (thiophen-3-yl)boronic acid to obtain [7-chloro-2-(4-thiophen-3-yl-phenyl)-quinazolin-4-yl]-[2-(1-methyl-piperidin-3-yl)-ethyl]-amine; with 2-(formyl-thiophen-3-yl)boronic acid to obtain 3-(4-{7-chloro-4-[2-( 1-methyl-piperidin-3-yl)-ethylamino]-quinazolin-2-yl}-phenyl)-thiophene-2-carbaldehyde; with 3-[(2-methoxy-ethylamino)-methyl]-phenylboronic acid to obtain (7-chloro-2-{3′-[(2-methoxy-ethylamino)-methyl)-biphenyl-4-yl}-quinazolin-4-yl}-[2-(1-methyl-piperidin-3-yl)-ethyl]-amine; with {2-[1-(methoxycarbonylmethyl-amino)-ethyl]-thiophen-3-yl}-boronic acid to obtain {1-[3-(4-{7-chloro-4-[2-(1-methyl-piperidin-3-yl)-ethylamino]-quinazolin-2-yl}-phenyl)-thiophen-2-yl]-ethylamino}-acetic acid methyl ester Analogously to example 3, 2-(4-bromo-phenyl)-4,7-dichloro-quinazoline [prepared analogously to example 1] is reacted with 2-morpholin-4-yl-ethylamine to obtain [2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-(2-morpholin-4-yl-ethyl)-amine.", "Analogously to example 10, [2-(4-bromo-phenyl)-7-chloro-quinazolin-4-yl]-(2-morpholin-4-yl-ethyl)-amine is reacted with 3,4-dimethoxyphenylboronic acid to obtain [7-chloro-2-(3′,4′-dimethoxy-biphenyl-4-yl)-quinazolin-4-yl]-(2-morpholin-4-yl-ethyl)-amine; with 4-formylphenylboronic acid to obtain 4′-[7-chloro-4-(2-morpholin-4-yl-ethylamino)-quinazolin-2-yl]-biphenyl-4-carbaldehyde; with 2-(formyl-thiophen-3-yl)boronic acid to obtain 3-{4-[7-chloro-4-(2-morpholin-4-yl-ethylamino)-quinazolin-2-yl]-phenyl}-thiophene-2-carbaldehyde; with 4-(propylaminomethyl)-phenylboronic acid to obtain [7-chloro-2-(4′-propylaminomethyl-biphenyl-4-yl)-quinazolin-4-yl]-(2-morpholin-4-yl-ethyl)-amine.", "Example 15 Analogously to example 3, 2-(5-bromo-thiophen-2-yl)-4,7-dichloro-quinazoline [prepared analogously to example 1, starting compounds 2-amino-4-chlorobenzonitrile and 5-bromo-thiophene-2-carbonyl chloride] is reacted with 2-(1-methyl-octahydro-indol-3-yl)-ethylamine to obtain [2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine.", "Analogously to example 10, [2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine is reacted with 4-fluorophenylboronic acid to obtain {7-chloro-2-[5-(4-fluoro-phenyl)-thiophen-2-yl]-quinazolin-4-yl}-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with 2-methoxyphenylboronic acid to obtain {7-chloro-2-[5-(2-methoxy-phenyl)-thiophen-2-yl]-quinazolin-4-yl}-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with (1H-indol-5-yl)boronic acid to obtain {7-chloro-2-[5-(1H-indol-5-yl)-thiophen-2-yl]-quinazolin-4-yl}-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine; with (quinolin-8-yl)boronic acid to obtain [7-chloro-2-(5-quinolin-8-yl-thiophen-2-yl)-quinazolin-4-yl]-[2-(1-methyl-octahydro-indol-3-yl)-ethyl]-amine.", "Analogously to example 3, 2-(5-bromo-thiophen-2-yl)-4,7-dichloro-quinazoline [prepared analogously to example 1] is reacted with N1,N1-diethyl-propane-1,3-diamine to obtain N′-[2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine.", "Analogously to example 10, N′-[2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine is reacted with (thiophen-2-yl)boronic acid to obtain N′-(2-[2,2′]bithiophenyl-5-yl-7-chloro-quinazolin-4-yl)-N,N-diethyl-propane-1,3-diamine; with (pyridin-4-yl)boronic acid to obtain N′-[7-chloro-2-(5-pyridin-4-yl-thiophen-2-yl)-quinazolin-4-yl]-N,N-diethyl-propane-1,3-diamine; with (2,5-dimethoxy)phenylboronic acid to obtain N′-{7-chloro-2-[5-(2,5-dimethoxy-phenyl)-thiophen-2-yl]-quinazolin-4-yl}-N,N-diethyl-propane-1,3-diamine.", "Analogously to example 3, 2-(5-bromo-thiophen-2-yl)-4,7-dichloro-quinazoline [prepared analogously to example 1] is reacted with N1,N1-diethyl-pentane-1,4-diamine to obtain N4-[2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine.", "Analogously to example 10, N4-[2-(5-bromo-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-N′,N′-diethyl-pentane-1,4-diamine is reacted with 2-cyanophenylboronic acid to obtain 2-{5-[7-chloro-4-(4-diethylamino-1-methyl-butylamino)-quinazolin-2-yl]-thiophen-2-yl}-benzonitrile; with (benzo[b]thiophen-2-yl)boronic acid to obtain N4-[2-(5-benzo[b]thiophen-2-yl-thiophen-2-yl)-7-chloro-quinazolin-4-yl]-N1,N1-diethyl-pentane-1,4-diamine.", "Example 16 Analogously to example 3, 4-chloro-2-(4-phenyl-butyl)-quinazoline [prepared analogously to example 1, starting compounds 2-amino-benzonitrile and 5-phenyl-pentanoyl chloride] is reacted with N1,N1-diethyl-pentane-1,4-diamine to obtain N1 ,N1-diethyl-N4-[2-(4-phenyl-butyl)-quinazolin-4-yl]-pentane-1,4-diamine; with C-(3-aminomethyl-cyclohexyl)-methylamine to obtain (3-aminomethyl-cyclohexylmethyl)-[2-(4-phenyl-butyl)-quinazolin-4-yl]-amine; MS calc.", ": 402.6 ; found: 403.6.Example 17 1.A solution of 2,4,7-trichloro-quinazoline (38.7 mmol) in 50 ml DMF is treated with N1,N1-diethyl-pentane-1,4-diamine (50 mmol).", "The mixture is stirred at rt for 4 hrs.", "A solution of C-(5-methoxy-1H-indol-3-yl)-methylamine (50 mmol) in 10 ml DMF is added and the mixture is heated to 80-100° for 3 days.", "Customary working up gives 7-chloro-N4-(4-diethylamino-1-methyl-butyl)-N2-(5-methoxy-1H-indol-3-ylmethyl)-quinazoline-2,4-diamine Analogously to example 17, 2,4,7-trichloro-quinazoline is reacted with N1,N1-diethyl-pentane-1,4-diamine to give N4-(2,7-dichloro-quinazolin-4-yl)-N1,N1-diethyl-pentane-1,4-diamine and is further reacted with 5-bromo-2-fluoro-benzylamine to obtain N2-(5-bromo-2-fluoro-benzyl)-7-chloro-N4-(4-diethylamino-1-methyl-butyl)-quinazoline-2,4-diamine; with 3,5-bis-trifluoromethyl-benzylamine to obtain N2-(3,5-bis-trifluoromethyl-benzyl)-7-chloro-N4-(4-diethylamino-1-methyl-butyl)-quinazoline-2,4-diamine; with 4-tert-butyl-benzylamine to obtain N2-(4-tert-butyl-benzyl)-7-chloro-N4-(4-diethylamino-1-methyl-butyl)-quinazoline-2,4-diamine.", "Analogously to example 17, 2,4,7-trichloro-quinazoline is reacted with 2-pyridin-2-yl-ethylamine to give (7-chloro-quinazolin-4-yl)-(2-pyridin-2-yl-ethyl)-amine and is further reacted with phenethylamine to obtain 7-chloro-N2-phenethyl-N4-(2-pyridin-2-yl-ethyl)-quinazoline-2,4-diamine; with 3-morpholin-4-yl-propylamine to obtain 7-chloro-N2-(3-morpholin-4-yl-propyl)-N4-(2-pyridin-2-yl-ethyl)-quinazoline-2,4-diamine.", "Analogously to example 17, 2,4,7-trichloro-quinazoline is reacted with diethylamine to give (2,7-dichloro-quinazolin-4-yl)-diethyl-amine and is further reacted with biphenyl-4-ylamine to obtain N2-biphenyl-4-yl-7-chloro-N4,N4-diethyl-quinazoline-2,4-diamine.", "The following examples relate to pharmaceutical preparations: Example A Injection Vials A solution of 100 g of an active compound of the formula I and 5 g of disodium hydrogenphosphate is adjusted to pH 6.5 in 3 l of double-distilled water using 2N hydrochloric acid, sterile-filtered, dispensed into injection vials, lyophilized under sterile conditions and aseptically sealed.", "Each injection vial contains 5 mg of active compound.", "Example B Suppositories A mixture of 20 g of an active compound of the formula I is melted with 100 g of soya lecithin and 1400 g of cocoa butter, poured into moulds and allowed to cool.", "Each suppository contains 20 mg of active compound.", "Example C Solution A solution is prepared from 1 g of an active compound of the formula I, 9.38 g of NaH2PO4.2H2O, 28.48 g of Na2HPO4.12H2O and 0.1 g of benzalkonium chloride in 940 ml of double-distilled water.", "The mixture is adjusted to pH 6.8, made up to 1 l and sterilized by irradiation.", "This solution can be used in the form of eye drops.", "Example D Ointment 500 mg of an active compound of the formula I is mixed with 99.5 g of petroleum jelly under aseptic conditions.", "Example E Tablets A mixture of 1 kg of active compound of the formula I, 4 kg of lactose, 1.2 kg of potato starch, 0.2 g of talc and 0.1 kg of magnesium stearate is compressed in a customary manner to give tablets such that each tablet contains 10 mg of active compound.", "Example F Coated Tablets Analogously to Example E, tablets are pressed which are then coated with a coating of sucrose, potato starch, talc, tragacanth and colorant in a customary manner.", "Example G Capsules 2 kg of active compound of the formula I are dispensed into hard gelatin capsules in a customary manner such that each capsule contains 20 mg of the active compound.", "Example H Ampules A solution of 1 kg of active compound of the formula I in 60 ml of double-distilled water is sterile-filtered, dispensed into ampoules, lyophilized under sterile conditions and aseptically sealed.", "Each ampule contains 10 mg of active compound." ] ]

[ [ "Method and system for performing electronic commerce", "The present invention relates generally to electronic commerce, and more particularly, to a method and system for performing electronic commerce over the Internet." ], [ "1.A method for performing electronic commerce, comprising the steps of: receiving a query including at least one code-specific and/or predetermined value over a network; and querying a database connected to said network to find at least one result related to said code-specific and/or predetermined value.", "2.A method for performing electronic commerce as in claim 1, further comprising the step of returning said at least one found result to a client associated with said query.", "3.A method for performing electronic commerce as in claim 1, wherein the at least one pre-determined value is a latitude and longitude location value.", "4.A method for performing electronic commerce as in claim 1, wherein the at least one code-specific value is indicative of a at least one code selected from the group consisting of a sequence identifier, a URL type identifier, a URL format identifier, and a language translation identifier.", "5.A method for performing electronic commerce as in claim 2, wherein the at least one found result includes information regarding a particular vendor.", "6.A method for performing electronic commerce as in claim 5, wherein the at least one pre-determined value is a value indicative of a maximum distance between said vendor and said client.", "7.A method for performing electronic commerce as in claim 1, wherein said step of returning said at least one found result further includes the step of returning a Uniform Resource Locator (URL) corresponding to a network location associated with a particular vendor.", "8.A method for performing electronic commerce as in claim 1, wherein said step of returning said at least one found result further includes the step of returning a global business identifier identifying a business category corresponding to a particular vendor.", "9.A method for performing electronic commerce as in claim 1, wherein said step of returning said at least one found result further includes the step of returning a global location identifier identifying the location of a particular vendor.", "10.A method for performing electronic commerce as in claim 1, wherein said step of returning said at least one found result further includes the step of returning a distance between a latitude and longitude value and a particular vendor.", "11.A method for performing electronic commerce as in claim 1, further comprising the steps of: receiving information from a trading vendor relating to said vendor's products and/or services; formatting said information into a format including at least one data segment; tagging said at least one data segment using at least one code; and storing said at least one tagged data segment in said database.", "12.A method for performing electronic commerce as in claim 11, wherein said at least one code is selected from the group consisting of the Universal Standard Products and Services Classification (UNSPSC) code, a global business identifier, and a global location identifier.", "13.A system for performing electronic commerce comprising: a server coupled to a network for receiving a query and searching vendor information, wherein the query includes at least one code-specific and/or predetermined value; and a database accessible by said server and storing said vendor information, wherein said vendor information is tagged with at least one identifier.", "14.A system for performing electronic commerce as in claim 13, wherein said at least one identifier is a global business identifier.", "15.A system for performing electronic commerce as in claim 13, wherein said at least one identifier is a global location identifier.", "16.A system for performing electronic commerce as in claim 13, wherein said vendor information includes at least a Uniform Resource Locator (URL), a URL type identifier, a URL format identifier, and a language translation identifier.", "17.A system for performing electronic commerce, comprising: means for receiving a query including at least one code-specific and/or predetermined value over a network; and means for querying a database connected to said network to find at least one result related to said code-specific and/or predetermined value.", "18.A system for performing electronic commerce as in claim 17, further comprising means for returning said at least one found result to a client associated with said query.", "19.A system for performing electronic commerce as in claim 17, wherein the at least one pre-determined value is a latitude and longitude location value.", "20.A system for performing electronic commerce as in claim 17, wherein the at least one code-specific value is indicative of at least one code selected from the group consisting of a sequence identifier, a URL type identifier, a URL format identifier and a language translation identifier.", "21.A system for performing electronic commerce as in claim 18, wherein the at least one found result includes information regarding a particular vendor.", "22.A system for performing electronic commerce as in claim 21, wherein the at least one pre-determined value is a value indicative of a maximum distance between said vendor and said client.", "23.A system for performing electronic commerce as in claim 17, wherein said at least one found result includes a Uniform Resource Locator (URL) corresponding to a network location associated with a particular vendor.", "24.A system for performing electronic commerce as in claim 17, wherein said at least one found result includes a global business identifier identifying a business category corresponding to a particular vendor.", "25.A system for performing electronic commerce as in claim 17, wherein said at least one found result includes a global location identifier identifying the location of a particular vendor.", "26.A system for performing electronic commerce as in claim 17, wherein said at least one found result includes a distance between a latitude and longitude value and a particular vendor.", "27.A system for performing electronic commerce as in claim 17, further comprising: means for receiving information from a trading vendor relating to said vendor's products and/or services; means for formatting said information into a format including at least one data segment; means for tagging said at least one data segment using at least one code; and means for storing said at least one tagged data segment in said database.", "28.A system for performing electronic commerce as in claim 27, wherein said at least one code is selected from the group consisting of the Universal Standard Products and Services Classification (UNSPSC) code, a global business identifier, and a global location identifier.", "29.A method for performing electronic commerce, comprising the steps of: receiving information from a trading vendor relating to said vendor's products and/or services; formatting said information into a format including at least one data segment; tagging said at least one data segment using at least one code; and storing said at least one tagged data segment in a database.", "30.A method for performing electronic commerce as in claim 29, wherein said at least one code is selected from the group consisting of the Universal Standard Products and Services Classification (UNSPSC) code, a global business identifier, and a global location identifier." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates generally to electronic commerce, and more particularly, to a method and system for performing electronic commerce over the Internet.", "2.Description of the Related Art Since HTTP's (Hypertext Transfer Protocol) introduction, the Internet has opened many avenues for electronic commerce.", "For example, vendors around the world can now offer product catalogs on www (World Wide Web) pages, thus reaching not just their local potential customers but also potential customers at remote locations around the world.", "However, such vendors have no guarantee that maintaining such a www page will increase business.", "Plainly, before business can increase, potential customers must be able to find a vendor's www page, e.g., potential customers must first know that a vendor exists.", "Conventional methods of finding vendors on the Internet invariably involve use of search engines.", "Search engines are web applications that search for web pages matching a certain criterion.", "For example, users use search engines daily to find web pages that store information on subjects like sports, art, technology and science.", "A search engine is actually a set of programs accessible at a network site within a network, for example a local area network (LAN) at a company or the Internet and World Wide Web.", "One program, called a “robot” or “spider,” pre-traverses a network in search of documents and builds large index files of keywords found in the documents.", "A user of the search engine formulates a query comprising one or more keywords and submits the query to another program of the search engine.", "In response, the search engine inspects its own index files and displays a list of documents that match the search query, typically as hyperlinks.", "When a user activates one of the hyperlinks to see the information contained in the document, the user exits the site of the search engine and terminates the search process.", "In the www's infancy, few search engines existed.", "Today, they number in the tens of thousands with most free for public use, such as Alta Vista, Excite, Google, HotBot, Lycos and Yahoo!.", "Of these, some—like those listed above—purport to search the entire www, while others are more specialized and cover specific industries only.", "Finally, the majority search through just a single web site, usually of a large corporate enterprise, such as ibm.com.", "Search engines, like most database applications, apply traditional search-and-retrieve mechanisms.", "However, search engines are at a disadvantage when compared to desktop or mainframe applications, chiefly due to the www's structure.", "To appreciate the difference, consider this: most corporate databases stringently adhere to a specific structure, a structure that database designers and administrators establish prior to entering data.", "This structure serves as a roadmap “written in stone”; all data must conform to it without exception.", "Consequently, developers can easily search and retrieve because they structure their queries based on the database architecture.", "The web works differently and is effectively the “wild west” of databases.", "Every web site is organized differently and these deviations, no matter how slight, can make transactional search and retrieval approaches more difficult.", "This is partially due to the web's underlying technologies.", "Web developers basically cannot perform logical web indexing.", "Hypertext Markup Language (HTML)—what web pages consist of—offers no way to differentiate one data element from another data element.", "For example, while one can use HTML to visually label a list item as an address element, one cannot use it to logically or programmatically label it.", "Hence, while a user can visually recognize an address element as such, a machine cannot comprehend it in this way.", "This limits automated web indexing to regular expression searches and similar procedures.", "Here, lexical scanners examine thousands of lines of HTML looking for a matching text pattern.", "Unfortunately, the indexing procedures of such search engines are insufficient for the purpose of finding web sites associated with potential vendors, rather than, e.g., web sites associated with a particular manufacturer who is not a vendor.", "Although users adept in Boolean expressions (logical operators OR, AND or NOT) can reap a more incisive result, even when one uses such measures, a search for “tractors” or “office desks” can return tens of thousands of links.", "Thus, generating a viable list of vendors for tractors or other products/services is impractical.", "Moreover, other problems exist, even if the query search returns only a limited number of web site links, i.e., URLs, etc.", "One problem is that a user must venture forward to investigate these links, one at a time.", "The user cannot guarantee that the limited number of links will be valid.", "Some studies suggest that as many as three out of every ten links will return a 404 error (i.e., Page Not Found error).", "Further, even if the links lead to valid pages, the user cannot anticipate the purpose, content, or structure of those web site pages.", "Thus, the user has no guarantee that such links will be product specifications, price catalogs, or pages designed to be read by automated procurement systems.", "With the increasing complexity of web syntax, even if the links are valid and the content is meaningful, the user has no guarantee that his clients will be able to interact with, read, or otherwise use the information in any meaningful way.", "Therefore, before Internet-based global (and automated) electronic commerce can fully bloom, we must first offer buyers and suppliers, i.e., vendors, the ability to ascertain six things: 1.Where (on the network) a vendor's information is stored.", "2.What type of network resource (or page) the vendor maintains.", "3.In what format the vendor stores its information.", "4.In what language the vendor creates its content.", "5.The vendor's geographical location.", "6.The vendor's purported online trading identity." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>Accordingly, it is an aspect of the present invention to provide a method and system for performing electronic commerce which overcome the disadvantages of the prior art methods and systems.", "It is another aspect of the present invention to provide a protocol that will simplify and automate many electronic commerce tasks, including procurement, purchasing, and the mining and qualifying of potential online trading partners.", "According to the present invention, a system for performing electronic commerce is provided.", "The system includes a query client, such as a terminal, coupled to a network, e.g., the Internet, for searching online trading vendor information; and a global business registry including trading vendor information tagged with at least one unique identifier, e.g., a global business identifier and/or a global location identifier.", "Additionally, a particular trading vendor's information is associated with at least a Uniform Resource Locator (URL), a URL type identifier, a URL format identifier, and a language translation identifier to ensure that the vendor's information is compatible with the query client.", "The global business registry is essentially a database for storing and receiving high-quality, low-level infrastructure data foundational to all electronic commerce transactions.", "According to another aspect of the present invention, a method for performing electronic commerce is also provided.", "The method includes the steps of receiving a query including at least one code-specific or predetermined value over a network, e.g., the Internet; querying a database which includes formatted information and is connected to the network to find at least one result related to the code-specific or pre-determined value; and returning at least one found result to a client associated with the query.", "The at least one code-specific or pre-determined value can be one or more of the following: a sequence identifier code, a latitude and longitude location value, a URL type identifier, a URL format identifier, and a language translation identifier.", "Additionally, the method provides for creating and appending the database, i.e., the global business registry by receiving information from an online trading vendor relating to the vendor's products and/or services; formatting the information into a standard file format including at least one data segment; tagging the at least one data segment with a code; and storing the tagged data segment in the database.", "Preferably, the code used to tag the vendor information is the Universal Standard Products and Services Classification (UNSPSC) code." ], [ "PRIORITY This application claims priority to an application entitled “METHOD AND SYSTEM FOR PERFORMING ELECTRONIC COMMERCE” filed in the United States Patent and Trademark Office on Sept. 25, 2000 and assigned Ser.", "No.", "60/235,031, the contents of which are hereby incorporated by reference.", "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates generally to electronic commerce, and more particularly, to a method and system for performing electronic commerce over the Internet.", "2.Description of the Related Art Since HTTP's (Hypertext Transfer Protocol) introduction, the Internet has opened many avenues for electronic commerce.", "For example, vendors around the world can now offer product catalogs on www (World Wide Web) pages, thus reaching not just their local potential customers but also potential customers at remote locations around the world.", "However, such vendors have no guarantee that maintaining such a www page will increase business.", "Plainly, before business can increase, potential customers must be able to find a vendor's www page, e.g., potential customers must first know that a vendor exists.", "Conventional methods of finding vendors on the Internet invariably involve use of search engines.", "Search engines are web applications that search for web pages matching a certain criterion.", "For example, users use search engines daily to find web pages that store information on subjects like sports, art, technology and science.", "A search engine is actually a set of programs accessible at a network site within a network, for example a local area network (LAN) at a company or the Internet and World Wide Web.", "One program, called a “robot” or “spider,” pre-traverses a network in search of documents and builds large index files of keywords found in the documents.", "A user of the search engine formulates a query comprising one or more keywords and submits the query to another program of the search engine.", "In response, the search engine inspects its own index files and displays a list of documents that match the search query, typically as hyperlinks.", "When a user activates one of the hyperlinks to see the information contained in the document, the user exits the site of the search engine and terminates the search process.", "In the www's infancy, few search engines existed.", "Today, they number in the tens of thousands with most free for public use, such as Alta Vista, Excite, Google, HotBot, Lycos and Yahoo!.", "Of these, some—like those listed above—purport to search the entire www, while others are more specialized and cover specific industries only.", "Finally, the majority search through just a single web site, usually of a large corporate enterprise, such as ibm.com.", "Search engines, like most database applications, apply traditional search-and-retrieve mechanisms.", "However, search engines are at a disadvantage when compared to desktop or mainframe applications, chiefly due to the www's structure.", "To appreciate the difference, consider this: most corporate databases stringently adhere to a specific structure, a structure that database designers and administrators establish prior to entering data.", "This structure serves as a roadmap “written in stone”; all data must conform to it without exception.", "Consequently, developers can easily search and retrieve because they structure their queries based on the database architecture.", "The web works differently and is effectively the “wild west” of databases.", "Every web site is organized differently and these deviations, no matter how slight, can make transactional search and retrieval approaches more difficult.", "This is partially due to the web's underlying technologies.", "Web developers basically cannot perform logical web indexing.", "Hypertext Markup Language (HTML)—what web pages consist of—offers no way to differentiate one data element from another data element.", "For example, while one can use HTML to visually label a list item as an address element, one cannot use it to logically or programmatically label it.", "Hence, while a user can visually recognize an address element as such, a machine cannot comprehend it in this way.", "This limits automated web indexing to regular expression searches and similar procedures.", "Here, lexical scanners examine thousands of lines of HTML looking for a matching text pattern.", "Unfortunately, the indexing procedures of such search engines are insufficient for the purpose of finding web sites associated with potential vendors, rather than, e.g., web sites associated with a particular manufacturer who is not a vendor.", "Although users adept in Boolean expressions (logical operators OR, AND or NOT) can reap a more incisive result, even when one uses such measures, a search for “tractors” or “office desks” can return tens of thousands of links.", "Thus, generating a viable list of vendors for tractors or other products/services is impractical.", "Moreover, other problems exist, even if the query search returns only a limited number of web site links, i.e., URLs, etc.", "One problem is that a user must venture forward to investigate these links, one at a time.", "The user cannot guarantee that the limited number of links will be valid.", "Some studies suggest that as many as three out of every ten links will return a 404 error (i.e., Page Not Found error).", "Further, even if the links lead to valid pages, the user cannot anticipate the purpose, content, or structure of those web site pages.", "Thus, the user has no guarantee that such links will be product specifications, price catalogs, or pages designed to be read by automated procurement systems.", "With the increasing complexity of web syntax, even if the links are valid and the content is meaningful, the user has no guarantee that his clients will be able to interact with, read, or otherwise use the information in any meaningful way.", "Therefore, before Internet-based global (and automated) electronic commerce can fully bloom, we must first offer buyers and suppliers, i.e., vendors, the ability to ascertain six things: 1.Where (on the network) a vendor's information is stored.", "2.What type of network resource (or page) the vendor maintains.", "3.In what format the vendor stores its information.", "4.In what language the vendor creates its content.", "5.The vendor's geographical location.", "6.The vendor's purported online trading identity.", "SUMMARY OF THE INVENTION Accordingly, it is an aspect of the present invention to provide a method and system for performing electronic commerce which overcome the disadvantages of the prior art methods and systems.", "It is another aspect of the present invention to provide a protocol that will simplify and automate many electronic commerce tasks, including procurement, purchasing, and the mining and qualifying of potential online trading partners.", "According to the present invention, a system for performing electronic commerce is provided.", "The system includes a query client, such as a terminal, coupled to a network, e.g., the Internet, for searching online trading vendor information; and a global business registry including trading vendor information tagged with at least one unique identifier, e.g., a global business identifier and/or a global location identifier.", "Additionally, a particular trading vendor's information is associated with at least a Uniform Resource Locator (URL), a URL type identifier, a URL format identifier, and a language translation identifier to ensure that the vendor's information is compatible with the query client.", "The global business registry is essentially a database for storing and receiving high-quality, low-level infrastructure data foundational to all electronic commerce transactions.", "According to another aspect of the present invention, a method for performing electronic commerce is also provided.", "The method includes the steps of receiving a query including at least one code-specific or predetermined value over a network, e.g., the Internet; querying a database which includes formatted information and is connected to the network to find at least one result related to the code-specific or pre-determined value; and returning at least one found result to a client associated with the query.", "The at least one code-specific or pre-determined value can be one or more of the following: a sequence identifier code, a latitude and longitude location value, a URL type identifier, a URL format identifier, and a language translation identifier.", "Additionally, the method provides for creating and appending the database, i.e., the global business registry by receiving information from an online trading vendor relating to the vendor's products and/or services; formatting the information into a standard file format including at least one data segment; tagging the at least one data segment with a code; and storing the tagged data segment in the database.", "Preferably, the code used to tag the vendor information is the Universal Standard Products and Services Classification (UNSPSC) code.", "BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which: FIG.", "1 is a block diagram illustrating a method for performing electronic commerce according to the present invention; FIG.", "2 is a flowchart illustrating a method for tagging supplier information according to the present invention; FIG.", "3 is a block diagram illustrating a single database record according to the present invention; FIG.", "4 is a flowchart illustrating a method for finding vendor information according to the present invention; and FIG.", "5 is a block diagram illustrating a system for performing electronic commerce according to the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A preferred embodiment of the present invention will be described herein below with reference to the accompanying drawings.", "In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail.", "Referring to FIG.", "1, the method and system of the present invention allow a buyer 106 to search a network for potential vendors and create Buy-Side catalogs and/or place orders by reading Sell-Side catalogs from a vendor or a supplier's web site 104.The buyer 106 evaluates the supplier's web site 104 and controls what is incorporated in the Buy-Side catalog.", "The supplier 102 can control access to the data on its website 104.From a technology point of view it is efficient for the buyer 106 to pull data when it is needed (i.e., update Buy-Side catalog: Office supplies on Monday, Office furniture on Tuesday, Fasteners on Wednesday, etc.)", "rather than have to receive input from the supplier 102 when the supplier 102 wants to send it, e.g., via e-mail.", "From a supplier implementation perspective, it is irrelevant whether the supplier 102 sends data to the buyer 106 or the buyer 106 pulls it from the suppliers web site 104.With continued reference to FIG.", "1, the inventive method is implemented by creating a global business registry (GBR) 100.The global business registry 100 is a central location for the supplier 102 to post, and for the buyer 106 to search, high-quality, low-level infrastructure data.", "The global business registry 100 operates under a whosells protocol developed by ResolveNet (IOM) Limited and which is a protocol for validating, cataloging, searching, and retrieving updated information in real-time.", "Data is validated and cataloged the moment it is entered and immediately following any administrative editing on the supplier's part.", "Cataloging actually involves breaking down all of the supplier's low-level data, validating it, and assigning each element the appropriate standardized tag, or code, that identifies each data type.", "The tagged data elements are then stored in the global business registry 100.That is, to build the database of the global business registry 100, information supplied by online trading vendors, or suppliers, is tagged.", "The tagging process adds standard code tags and globally unique identifiers to the supplier's catalogs, address files, transactional and legacy data to improve the data quality and usability in performing automated electronic commerce.", "By utilizing the whosells protocol, the buyer 106 then searches the global business registry 100, and retrieves results.", "The results retrieved are updated real-time and accurate, as only the supplier 102 himself has control over the content displayed on his web site 104.Because the global business registry 100 is built on open standards, and specifically to support “Open Procurement” (the process of analyzing all possible suppliers before selecting the most appropriate), it is inherently a central registry for accessing by a multitude of e-marketplaces, procurement portals, and information exchanges.", "The open source code of the global business registry 100 affords anyone the ability to incorporate the search protocols on their website or within software applications.", "FIG.", "2 is a flowchart illustrating a tagging procedure in accordance with the present invention.", "Specifically, FIG.", "2 illustrates a tagging procedure for tagging a supplier's information file with the UNSPSC code.", "After receiving an information file from a supplier or vendor at step 200, the file is reviewed at step 202 to determine whether more information is needed to satisfactorily tag the file.", "After the file is reviewed, the information is formatted into a standardize file format to be received into a Sell-Side and a Buy-Side catalog file at steps 204 and 206, respectively.", "The catalog files are then sent to a computer assisted classification system for automated tagging with the UNSPSC code at step 208.If the file is satisfactorily tagged as determined by step 210, the file undergoes a quality review at step 218 and is returned at step 220 to be used at the supplier's web site 104.If the file was not satisfactorily tagged as determined by step 210, a domain expert will attempt to manually tag the file at step 212.If the file is satisfactorily tagged as determined by step 214, it is passed onto a quality review at step 218 as described above.", "Otherwise, if a satisfactory code cannot be found, a change request can be submitted to the code's governing body at step 222 to initiate a code more suitable for the particular vendor's product or service.", "The change request process is described in PCT Patent Application No.", "PCT/US01/22496 entitled “METHOD AND SYSTEM FOR MANAGING A CODE” filed on Jul.", "18, 2001 by Peter R. Benson, the contents of which are incorporated by reference.", "Further, when the file is received at step 220 and reviewed at step 202, it may be processed in a spend analysis file in steps 230 to 238 in a way similar to the process described above for the Sell-Side catalog file.", "By coding the file, the buyer can see how much or how little they spend on a commodity and from which particular vendor or supplier the commodity comes from.", "The tagging process of FIG.", "2 is repeated to tag the file using additional codes besides the UNSPSC code to further tag the supplier's files with unique code/parameter identifiers.", "An example of a single tagged record 300 is shown in FIG.", "3.Briefly, the record 300 includes the following data segments: a global business identifier (GBI) 302, the UNSPSC code 304, a global location identifier (GLI) 306, a URL type identifier (EUTI) 308, a URL format identifier (EUFI) 310, a URL language translation identifier (ELTI) 312 and a Uniform Resource Locator (URL) 314.Each data segment will be described in more detail below by the way of an example.", "The global business registry 100 is then assembled with a multitude of records 300 to assist buyers in finding potential suppliers.", "Referring to FIG.", "4, a search for potential suppliers is conducted by interacting with the global business registry 100.To begin the search, a client, i.e., a buyer, enters a query relating to a specific product or service desired at step 400.According to the method of the present invention, the data segments of the tagged records 300 are searched for matching results.", "First, the method ensures that returned vendors sell the desired product or service the buyer seeks at step 402.This alone is a tremendous improvement in relevance over standard web searches.", "Next, the method determines if the desired information is located at a given URL at step 406.This does not merely specify the vendor's online location, but gives clues as to whether the buyer can generally interact with the vendor's system.", "For instance, a URL could be merely an e-mail address or File Transfer Protocol (FTP) site.", "If so, the chances that the specified vendor supports automated procurement is slim: an e-mail address is merely a contact point, and an FTP site is archival.", "Next, the method determines the supplier's URL type identifier (EUTI) at step 410.This specifies the URL's purpose.", "If a buyer is seeking price information, he naturally wants a price catalog type EUTI.", "If a returned supplier's EUTI is simply a home page, the buyer might well disqualify that vendor for the purposes of the instant search.", "If, on the other hand, the EUTI type reported is a price catalog, the search can continue.", "Then, the method of the present invention checks the URL's format type identifier (EUFI) at step 414.From this, one will learn what format types the vendor's site supports.", "For example, suppose that one is seeking financial data and their system reads XBRL (the Extensible Business Reporting Language).", "The method described allows one to filter out all potential trading partners that do not return a EUFI XBRL identifier.", "Next, the method verifies the language translation identifier (ELTI) at step 418.From this, one can learn what languages the vendor's site supports.", "For example, suppose that a buyer is seeking only English content.", "The buyer is enabled to filter out all potential trading partners whose content is not in English.", "It is to be understood that if any of the above-mentioned steps incurs an incompatible data segment, the current record 300 being searched will be disregarded at steps 404, 408, 412, 416 or 420 and other available records 300 will be analyzed by the inventive method.", "Referring back to FIG.", "4, if a vendor meets all of the buyers needs (the correct URL type, page type, format type, and language), the buyer will be provided with more information, starting with whom the specified vendor is and where they are located.", "The method provides this information through a global business identifier (GBI) and a global location identifier (GLI) at step 422.The GBI can be used with an identify protocol to access all the identifiers of a business (Legal name, main telephone number, DUNS number, TIN, etc.)", "and the GLI can be used with a geolocate protocol to identify the exact or approximate longitude and latitude as well physical address information of the business entity.", "The method then terminates at step 424.By way of example as shown in FIG.", "5, the following is a description of a system 500 embodying the method described above designed to implement the open standards established by the Electronic Commerce Code Management Association (ECCMA).", "The system 500 employs the whosells protocol designed to facilitate electronic commerce in general and specifically open procurement.", "It includes an interactive, distributed database 504, based upon the client-server model.", "Users can retrieve information in one or more ways, such as over the network 506.For the Internet community at large, such information can be retrieved by conventional means with any HTTP client.", "The information can also be reached from a CLI (Command Line Interface) on both the UNIX and Windows NT platforms.", "Herein below, only the CLI server and client specification are described.", "whosells Server The WHOSELLS server 502 is an HTTP based query/response server running on http://whosells.resolvenet.net.", "The server 502 provides directory services to Internet users via a URL-based query and response.", "The server 502 reports data that online trading entities wish to publish to facilitate and engage in open procurement.", "Terminologies and Notation In this document, the following acronyms appear: ECCMA—Electronic Commerce Code Management Association EGCI—ECCMA Global Commodity Identifier BTI—ECCMA Business Type Identifier EUTI—ECCMA URL Type Identifier EUFI—ECCMA URL Format Identifier ELTI—ECCMA Language Translation Identifier GBI—Global Business Identifier GLI—Global Location Identifier LAT—Latitude LONG—Longitude N\\L—Newline SQL—Structured Query Language UNSPSC—Universal Standard Products and Services Classification Sequence Identifier—A numeric value that identifies a specific code record in any one of the ECCMA Schemas (i.e., EGCI, EUTI, EUFI, ELTI are all valid Sequence Identifiers in their respective schemas).", "WHOSELLS Service Description The server 502 is designed to facilitate open procurement and electronic commerce.", "It is a URL based web page with input parameters specified as part of the URL.", "The server 502 accepts thirteen arguments in its query string from any client 508 or web browser: [egci.bti] [lat] [long] [city] [state] [country] [euti] [eufi] [elti] [distunit] [maxdist] [maxrec] [skip] The following are descriptions of each input argument: [egci.bti]—The EGCI is The ECCMA Global Commodity Identifier, a valid Sequence Identifier from The Universal Standard Products and Services Classification (UNSPSC).", "The UNSPSC coding system is an open, hierarchical, global electronic commerce standard that provides a logical framework for classifying goods and services.", "The BTI is the ECCMA Business Type Identifier, a Valid Sequence Identifier from the UNSPSC that identifies the type of business (i.e.", "wholesale, retail, manufacturer).", "The BTI is appended to the EGCI and the EGCI/BTI combination consists of two six-digit numbers separated by a dot (i.e.", "123456.123456).", "The egci.bti argument thus identifies the product or service and the type of business you seek.", "It is a REQUIRED argument.", "[lat]—The user's latitude, which is a point that identities the angular distance north or south of the earth's equator, measured in degrees along a meridian.", "This, coupled with the user's longitude, identifies the user's location.", "The user expresses latitude in decimal notation (e.g., 33.58).", "If the user desires a search based on latitude and longitude, then [lat] is a REQUIRED argument together with [long] when a [city], [state] and [country] combination is not passed.", "[long]—The user's longitude, a point that identifies the angular distance on the earth's surface, measured east or west from the prime meridian at Greenwich, England, to the meridian passing through a position, expressed in degrees (or hours), minutes, and seconds.", "This, coupled with the user's latitude, identifies the user's location.", "The user expresses longitude in decimal notation (e.g., 85.85).", "If the user desires a search based on latitude and longitude, then [long] is a REQUIRED argument together with [lat] when a [city], [state] and country combination is not passed.", "[city]—The user's city.", "If the user desires a geographical search, then [city] is a REQUIRED argument together with [country] (and [state] if [country] is US) when a [lat] and [long] pair is not passed.", "[state]—The user's state.", "If the user is doing a geographical search, and the [country] value passed is US, then [state] is a REQUIRED argument along with [city] when a [lat] and [long] pair is not passed.", "[country]—The user's country.", "If the user is doing a geographical search, then [country] is a REQUIRED argument together with [city] (and [state] if [country] is US]) when a [lat] and [long] pair is not passed.", "If [city] and [state] are passed, without a value for [country] then [country] defaults to US.", "Note: The arguments listed as REQUIRED with respect to location are only truly REQUIRED if the user is concerned with the location.", "If not, then all of the location arguments (lat, long, city, state, country, distunit and maxdist) MAY be omitted.", "[euti]—The ECCMA URL Type Identifier, a valid Sequence Identifier from the ECCMA URL Type Schema (EUTS) that identifies the trading partner's URL type, e.g., home page, product catalog, price catalog, and is a OPTIONAL argument.", "[eufi]—The ECCMA URL Format Identifier, a valid Sequence Identifier from the ECCMA URL Format Schema (EUFS) that identifies your desired data types, or, what data types that your procurement client supports.", "EUFIs consist of both high-level, generic data types (HTML, XML, EDI, etc.)", "as well as industry-specific types e.g., Ariba Catalog, or Commerce One's XML Common Business Library xCBL 2.0.The EUFI thus identifies the data format you seek and can manipulate or read, and is an OPTIONAL argument [elti]—The ECCMA Language Translation Identifier, a valid Sequence Identifier from the ECCMA Language Translation Schema (ELTS) that identifies the desired language for the web pages that are returned.", "The ELTI is an OPTIONAL argument.", "[distunit]—The units of measurement that should be returned as the distance value.", "Either “MI” for miles or “KM” for kilometers.", "This argument is only used on conjunction with a geographical search (i.e.", "lat/long or city/state/country).", "It is OPTIONAL and will default to miles for a geographical search if it is not passed.", "[maxdist]—The maximum distance from the user.", "This specifies the maximum allowable distance that a trading partner can be from you.", "This argument is only used on conjunction with a geographical search (i.e.", "lat/long or city/state/country).", "The maximum distance argument is OPTIONAL.", "[maxrec]—The maximum number of records that WHOSELLS SHOULD return.", "The maximum record argument is OPTIONAL and if not specified, its default maximum is 1,000.", "[skip]—The skip argument is OPTIONAL and controls which records (out of your maxrec return) you receive.", "The skip argument lets you specify several methods of traversing those records.", "One method is to randomize results.", "That is, instead of returning the specified number of records sorted from closest to farthest, the server will return those records as a random sample of vendors within the specified distance range.", "Still another method is to skip a specified range of records.", "For example, suppose you ask for a maxrec of 100 within a specified distance range but the server 502 reports that 1000 records exist.", "Rather than pull a new query that returns records you've already seen, you can instead specify that you want to skip the first 100, or 200, or 300.This argument is only used on conjunction with a geographical search (i.e.", "lat/long or city/state/country).", "In response, the server returns an XML document in the following format: <?xml version=“1.0” encoding=“UTF-8”?> <ResolvenetWhosellsResponse xmlns:xsi=“http://www.w3.org/2000/10/XMLSchema-instance” xsi:noNamespaceSchemaLocation=“ http://www.ResolveNet.net/ XMLSchemas/WhosellsSchema.xsd”> <Results> <TotalRecordsFound>2</TotalRecordsFound> <RecordsSkipped>0</RecordsSkipped> <Result> <URL ELTI=“000001” EUFI=“900041” EUTI=“000001”> http://www.myCompany.com</URL> <GBI>1234-2345-6789</GBI> <GLI>3456-2345-1234</GLI> <Distance Units=“MI”>35</Distance> </Result> <Result> <URL ELTI=“000001” EUFI=“900041” EUTI=“000001”> http://www.yourCompany.com</URL> <GBI>1234-2345-6789</GBI> <GLI>3456-2345-1234</GLI> <Distance Units=“MI”>56</Distance> </Result> </Results> </ResolvenetWhosellsResponse> For Each <Result> element, the sub elements and attributes are described as follows: XML Element <URL>—The Uniform Resource Locator of the vendor's page or server from which it transacts business.", "XML Attribute [EUTI]—The ECCMA URL Type Identifier, the Sequence Identifier from the ECCMA URL Type Schema (EUTS) that identifies the trading partner's URL type, e.g., home page, product catalog, price catalog, etc.", "XML Attribute [EUFI]—The ECCMA URL Format Identifier, the Sequence Identifier from the ECCMA URL Format Schema (EUFS) that identifies the data type of the web page identified by a URL.", "XML Attribute [ELTI]—The ECCMA Language Translation Identifier, the Sequence Identifier from the ECCMA Language Translation Schema (ELTS) that identifies the language of the web page identified by a URL.", "XML Element <GBI>—The Global Business Identifier.", "The Global Business Identifier (GBI) is a twelve-digit, unique identifier that both uniquely identifies a trading entity and (can) point to all other business identifiers assigned to the same.", "In global electronic commerce systems (such as those maintained by ResolveNet), the GBI serves as a pointer to all business identifiers associated with a specified trading partner.", "To learn more about GBI, please visit ResolveNet at http://www.ResolveNet.net.", "XML Element <GLI>—The Global Location Identifier(GLI).", "The GLI is a twelve-digit, unique identifier that identifies the trading partner's mailing address and location (i.e.", "long, lat).", "For more information on the GLI, please visit the ResolveNet GLI page at http://www.ResolveNet.net.", "XML Element <Distance>—The distance between the returned vendor and the user, which WHOSELLS will return either in kilometers or miles based on the user's preference.", "WHOSELLS Query and Response Examples Users query the Whosells server 502 database using a standard URL with a query string.", "Syntax: http.//whosells.resolvenet.net?egci=######.######&lat=+###.## &long=−###.##&city=$$$$$$$$$&state=$$&euti=######&eufi=######& elti=######&distunit=MI&maxdist=######&maxrec=####&skip=#### A typical query looks like this: http://whosells.resolvenet.net?egci=009286.010644&lat=+34.03 &long=−118.19&euti=000001&eufi=000001&elti=000001&distance=500 &max=10&skip=0 (Note: the “lat” and “long”0 name/value pair MAY be replaced with the “city”, “state”, “country” name/value combination, or all geographical information MAY be omitted if location is of no concern.)", "The above query consist of the following values: egci 009286.010644 (009286 is the ECCMA Global Commodity Identifier for Computer Services, 010644 is the Business Type Identifier (BTI) for Retail) lat +34.03 long −118.19 euti 000001 (Home pages) eufi 000001 (HTML) elti 000001 (English) distunit Miles maxdist 500 (500 miles) maxrec 10 (only 10 records) skip Start with the first record As such, the aforementioned query makes the follow definitive statement: “I want all vendors within 500 miles of Los Angeles that deal in Computer Services who have a home page formatted in English and HTML, and I only want the first ten records.” The server 502 would then respond with one or more <Result> records in XML format as follows: <?xml version=“1.0” encoding=“UTF-8”?> <ResolvenetWhosellsResponse xmlns:xsi=“http://www.w3.org/2000/10/XMLSchema-instance” xsi:noNamespaceSchemaLocation=“http://www.ResolveNet.net/ XMLSchemas/whosellsSchema.xsd”> <Results> <TotalRecordsFound>1</TotalRecordsFound> <RecordsSkipped>0</RecordsSkipped> <Result> <URL EUTI=“000001” EUFI=“000001” ELTI=“000001”>http://www.Company.com</URL> <GBI>1234-1345-6789</GBI> <GLI>3456-2345-1234</GLI> <Distance Units=“Miles”>35</Distance> </Result> </Results> </ResolvenetWhosellsResponse> This response reports the following information: URL http://www.Company.com EUTI 000001 (Home Page) EUFI 000001 (HTML) ELTI 000001 (English) GBI 1234-1345-6789 GLI 3456-2345-1234 Distance 35 (35 miles from Los Angeles) A similar query and response based on the city/state/country combination would appear as follows.", "Query: http://whosells.resolvenet.net?egci=009286.010644&city=Boston &state=MA&country=us&euti=000001&eufi=000001&elti=000001& distance=500&max=10&skip=0 Response: <?xml version=“1.0” encoding=“UTF-8”?> <ResolvenetWhosellsResponse xmlns:xsi=“http://www.w3.org/2000/10/XMLSchema-instance” xsi:noNamespaceSchemaLocation=“http://www.ResolveNet.net/ XMLSchemas/whosellsSchema.xsd”> <Results> <TotalRecordsFound>1</TotalRecordsFound> <RecordsSkipped>0</RecordsSkipped> <Result> <URL ELTI=“000001” EUFI=“900041” EUTI=“000001”> http://www.myCompany.com</URL> <GBI>1234-2345-6789</GBI> <GLI>3456-2345-1234</GLI> <Distance Units=“MI”>35</Distance> <City>Cambridge</City> <State>MA</State> <Country>US</Country> </Result> </Results> </ResolvenetWhosellsResponse> In many cases, output may well be more extensive.", "Many businesses will register more than one EUTI or EUFI and a resulting vendor record would contain multiple results.", "It can be appreciate that various suppliers 510 can communicate with the server 502 utilizing the whosells protocol to supply and validate their records 300.It is also to be understood that upon returning favorable results to a client 508, the client 508 can immediately access the supplier 510 and their information through the network 506, such as the Internet.", "While the invention has been shown and described with reference to certain preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims." ] ]

[ [ "Methods for inducing apolipoprotein e secretion", "The invention provides a method for increasing apolipoprotein E (ApoE) in plasma and in tissues of a mammal by using a combination of an ApoE increasing amount of an activator of the orphan nuclear receptor FXR and an ApoE increasing amount of an activator of the orphan nuclear receptor LXRα.", "Also provided is the use of a combination of an ApoE increasing amount of a FXR activator and of an ApoE increasing amount of a LXRα activator, for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal." ], [ "1.A method for increasing apolipoprotein E (ApoE) in plasma and in tissues of a mammal comprising administering to said mammal an ApoE increasing amount of an activator of the orphan nuclear receptor FXR and an ApoE increasing amount of an activator of the orphan nuclear receptor LXRα.", "2.A method for increasing ApoE in plasma and in tissues of a mammal comprising admininstering to said mammal an ApoE increasing amount of a FXR activator.", "3.A method for increasing ApoE in plasma and in tissues of a mammal comprising administering to said mammal an ApoE increasing amount of a LXRα activator.", "7.A method of regulating regulating cholesterol homeostasis in a mammal comprising administering to said mammal at least one of a FXR activator or a LXRα activator.", "8.The method of claim 1, wherein the FXR activator is an isoprenoid or a bile acid.", "9.The method of claim 8, wherein the activator of the orphan nuclear receptor FXR is selected from: farrnesol (trans,trans-farnesol;-3,7,11-trimethyl-2,6,10dodecatrien-1-ol), methyl farnesyl ether, (methyl-3,7,11-trimethyl-2,6,10dodecatrien-1-yl ether), ethyl farnesyl ether, ethyl-3,7,11-trimethyl-2,6,10dodecatrien-1-yl ether), methyl farnesoate, (-3,7,11-tnmethyl-2,6,10-dodecatrienoic acid methyl ester), ethyl farnesoate, (-3,7,11-trimethyl-2,6,10-dodecatrienoic acid ethyl ester), juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl2,6-nonadienoic acid methyl ester), 7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid ethyl ester and chenodeoxycholic acid (3a,7a-dihydroxy-5(3-cholanic acid).", "10.The method of claim 9, wherein the activator of the orphan nuclear receptor FXR is selected from: farnesol (trans,trans-fanesol;-3,7,11-trimethyl-2,6,10dodecatrien-1-ol), juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl2,6-nonadienoic acid methyl ester) and chenodeoxycholic acid (3a,7(x-dihydroxy-5(3-cholanic acid).", "11.The method of claim 1, wherein the activator of the orphan nuclear receptor LXRα is a cholesterol derivative.", "12.The method of claim 11, wherein the cholesterol derivative is selected from: 22(R)-hydroxycholesterol (5-cholestene-3(3,22-diol) 24(S)-hydroxycholesterol, 24(5),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol.", "13.The method of claim 12, wherein the activator of the orphan nuclear receptor LXRα is 22(R)-hydroxycholesterol (5-cholestene-3β,22-diol).", "14.A pharmaceutical composition having apoE increasing activity and comprising both an FXR activator and a LXRα activator in a pharmaceutically acceptable excipient.", "15.The pharmaceutical composition of claim 14, wherein the FXR activator is an isoprenoid or a bile acid.", "16.The pharmaceutical composition of claim 15, wherein the activator of the orphan nuclear receptor FXR is selected from: farrnesol (trans,trans-farnesol;-3,7,11-trimethyl-2,6,10dodecatrien-1-ol), methyl farnesyl ether, (methyl-3,7,11-trimethyl-2,6,10dodecatrien-1-yl ether), ethyl farnesyl ether, ethyl -3,7,11-trimethyl-2,6,10dodecatrien-1-yl ether), methyl farnesoate, (-3,7,11-tnmethyl-2,6,10-dodecatrienoic acid methyl ester), ethyl farnesoate, (-3,7,11-trimethyl-2,6,10-dodecatrienoic acid ethyl ester), juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl2,6-nonadienoic acid methyl ester), 7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid ethyl ester and chenodeoxycholic acid (3a,7a-dihydroxy-5(3-cholanic acid).", "17.The method of claim 1, wherein said mammal has atherosclerosis.", "18.The pharmaceutical composition of claim 14, wherein the activator of the orphan nuclear receptor LXRα is a cholesterol derivative.", "19.The method of claim 1, wherein said mammal has macular degeneration and retinitis pigmentosa.", "20.The method of claim 1, wherein said mammal has suffered a stroke.", "21.The method of claim 1, wherein said mammal has a degenerative neuropathy, such as diabetic neuropathies or multiple sclerosis.", "22.The method of claim 1, wherein said mammal has Alzheimer's disease or dementia.", "23.The method of claim 25, wherein said method comprises the combination of an in vitro assay and an in vivo animal model assay, said in vitro assay comprising testing said candidate compound for activity in inhibiting or inducing the secretion of ApoE in the THP-1 cell line, and said in vivo animal model assay comprising testing said candidate compound in a rat for a modulating effect on plasma ApoE.", "24.A method of screening a candidate compound for therapeutic use as an ApoE modulator comprising selecting a compound and screening said candidate compound for FXR or LXRα activator activity wherein FXR or LXRα activator activity identifies the candidate compound as an ApoE modulator.", "25.The method of claim 24, further comprising the step of assessing the ApoE modulatory activity of said candidate compound.", "26.The method of claim 24, further comprising the step of formulating said compound to form a pharmaceutical composition.", "27.The pharmaceutical composition of claim 18, wherein the cholesterol derivative is selected from: 22(R)-hydroxycholesterol (5-cholestene-3(3,22-diol) 24(S)-hydroxycholesterol, 24(5),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Apolipoprotein E (ApoE) is a polymorphic, multifunctional protein synthesized by several cell types and tissues, including liver, kidney, skin, adipose tissue, macrophages and brain.", "The wide distribution of ApoE is associated with the maintenance of key cellular functions such as intracellular cholesterol trafficking, cholesterol distribution between cells, and tissue reparation.", "The amino acid sequence of the ApoE protein is well conserved throughout species.", "ApoE can be viewed as a regulator of cholesterol homeostasis in tissues such as the Central Nervous System (CNS) and Peripheral Nervous System (PNS) and the arterial wall (cell-cell) or between tissues via the circulating plasma lipoproteins (tissue-tissue).", "The major role of plasma ApoE containing lipoproteins is to transfer lipids (cholesterol) from peripheral tissues to the liver and to remove excess cholesterol from peripheral tissues via the reverse cholesterol transport system.", "Dysregulation of this mechanism leads to excess cholesterol deposition in peripheral tissues such as arteries (arterosclerosis) and skin (xanthomas and xanthelasmas).", "ApoE has also been shown to have a direct effect on lymphocyte proliferation and thus has an immunomodulatory role.", "After the liver, the brain is the second major site of ApoE synthesis.", "ApoE is the only lipoprotein synthesized by brain tissue where the key role of ApoE is cholesterol transport between cells of the central nervous system (CNS).", "Local secretion of ApoE by cells such as macrophages or macrophage-derived cells is essential for the uptake of excess tissue cholesterol or provides cholesterol for specific needs such as nerve repair and remyelinisation.", "Up to the present time, the compounds affecting Apo E production in vitro and in vivo have not been investigated thoroughly.", "Only hormone-like estrogens and corticoids have been shown to change Apo E levels under various experimental conditions; see for instance: Srivastava R A K, Srivastava N, Averna M, Lin R C, Korach K S, Lubahn D B, Schonfeld G “Estrogen up-regulates apolipoprotein E (ApoE) gene expression by increasing ApoE mRNA in the translating pool via the estrogen receptor alpha-mediated pathway” J Biol Chem, 1997, 272:33360-33366 and Stone D J, Rozovsky I, Morgan T E, Anderson C P, Hajian H, Finch C E “Astrocytes and microglia respond to estrogen with increased ApoE mRNA.", "in vivo and in vitro” Experimental Neurology, 1997, 143:313-318.Orphan Nuclear Receptors FXR and LXR Steroid hormones (glucocorticoids, mineralocorticoids, estrogens, progestins, androgens, and vitamin D) bind to their nuclear receptors which are transcription factors and by this means regulate expression of gene coding for specific proteins and control critical cellular activities; see for instance Meier, C., A.", "Journal of Receptor & Signal Transduction Research 1997, 17, 319-335.In the last ten years more than 100 mammalian genes have extended the family of steroid nuclear receptors and have been classified as orphan nuclear receptors for which ligands are unknown; see for instance Enmark, E. and Gustafsson, J.", "A. Molecular Endocrinology 1996, 10, 1293-1307.The farnesoid X activated receptor (FXR; NR1H4) was identified in 1995 by Forman et al.", "Cell 1995, 81, 687-693.FXR functions as a heterodimer with the Retinoid X Receptor (RXR) and binds to the DNA via an inverted repeat element IR-1.FXR was originally described as activated by isoprenoids such as farnesol and juvenile hormone III.", "More recently, several investigators came to the conclusion that bile acids are the physiological ligands and activators of FXR; see for instance Makishima, M. et al Science 1999, 284, 1362-1365.Parks, D. J. et al Science 1999, 284, 1365-1368.Wang, H. B. et al Molecular Cell 1999, 3, 543-553.The Liver X Receptor was identified as an orphan nuclear receptor by Willy et al Genes & Development 1995, 9, 1033-1045.and its ligand-binding domain shares 37% identity with FXR.", "The DNA binding sequence of LXR is a direct repeat separated by 4 nucleotides (DR-4) and differs from the DNA binding sequence of FXR (IR-1).", "Two genes encode for LXR proteins (LXRα;NR1H3 and LXRβ;NR1H2) and both receptors are activated by various oxysterols, the most potent being 22(R)-hydroxycholesterol, 24 (S)-hydroxycholesterol, 24(S),25-epoxycholesterol and 7-hydroxycholesterol; see for instance: Janowsky B.", "A. et al Proc Natl Acad Sci USA 1999, 96, 266-271.Thus LXRα appears to be a sensor of both isoprenoids and oxysterols but the exact physiological and pharmacological applications of the activators and ligands of any these receptors (FXR and LXR) are yet to be established (Niesor E et al Current Pharmaceutical Drug Design 2000 in press)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The applicants have discovered that representative ligands and activators of FXR (e.g.", "the isoprenoid farnesol, the bile acid derivative chenodeoxycholic acid) and of LXRα (e.g.", "the hydroxysterol 22(R)-hydroxycholesterol) are potent inducers of ApoE secretion and might be useful in the treatment of diseases requiring an increased production of plasma and/or tissue ApoE.", "Compounds which modulate ApoE synthesis and secretion should have application in the treatment of diseases such as atherosclerosis, excess lipid deposition in peripheral tissues such as skin (xanthomas), stroke, memory loss, optic nerve and retinal pathologies (i.e.", "macular degeneration, retinitis pigmentosa), repair of traumatic damage of the central nervous system (brain tissue), repair of traumatic damage of the peripheral nervous system (i.e.", "nerve section compression or crush), prevention of the degenerative process due to aging (i.e.", "Alzheimer's disease), prevention of degenerative neuropathies occurring in diseases such as diabetic neuropathies and multiple sclerosis, autoimmune diseases and activation of the innate immune system.", "ApoE in Atherosclerosis As a component of all lipoprotein fractions, ApoE plays a important role in cholesterol homeostasis, by mediating their interaction with receptors such as the apoB, low-density lipoprotein (LDL) and other specific receptors.", "The important role of ApoE in cardiovascular diseases is demonstrated by the ApoE knock-out mouse model where the animals rapidly develop hypercholesterolemia and atherosclerosis with pathological features similar to human atherosclerosis; see for instance: Plump A “Atherosclerosis and the mouse—a decade of experience [review]” Annals of Medicine 1997, 29:193-198.In man, in the absence of a functional ApoE protein (human variants; see for instance: Richard P, Dezulueta M P, Beucler I, Degennes J L, Cassaigne A, Iron A “Identification of a new apolipoprotein E variant (E(2) Arg(142)- ]Leu) in type-III hyperlipidemia” Atherosclerosis.", "1995, 112:19-28) and in the ApoE knock out mouse, plasma levels of cholesterol and triglycerides are abnormally high (even on a low fat diet) and atherosclerosis develops rapidly.", "In the animal model, these changes are prevented by infusion of ApoE, transplantation of macrophage producing ApoE or gene therapy by introducing the human ApoE gene into ApoE knock out mice, demonstrating the direct beneficial role of ApoE; see for instance: Linton M F, Atkinson J B, Fazio S “Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation” Science 1995, 267:1034-1037.A recent study has examined the response of ApoE knock out mice to diets with increasing cholesterol content and has concluded that ApoE plays a critical role in decreasing the absorption of dietary cholesterol and in increasing biliary secretion, see for instance Sehayek E, Shefer S, Nguyen L B, Ono J G, Merkel M and Breslow J L,“Apolipoprotein E regulates dietary absorption and biliary cholesterol excretion: Studies in C57BL/6 ApoE knock out mice” Proc Natl Acad Sci USA 2000, 97, 3433-3437.The results of this study further suggest that increasing ApoE should decrease the absorption of dietary cholesterol and prevent the formation of biliary cholesterol stones.", "ApoE in the Central Nervous System (CNS) ApoE also plays a critical role in the central nervous system.", "In the brain ApoE is synthesized and secreted by astrocytes, its principal role being cholesterol transport between cells.", "ApoE is considered to redistribute lipids and to participate in the cholesterol homeostasis of the brain.", "ApoE is linked to the neuropathological lesions characteristic of Alzheimer's disease with one isoform, ApoE4, strongly associated with the age of onset of the disease; (see for instance: Poirier J “Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease” [review]Trends in Neurosciences 1994, 17:525-530 and Rubinsztein D C “Apolipoprotein E—a review of its roles in lipoprotein metabolism, neuronal growth and repair and as a risk factor for Alzheimer's disease” Psychological Medicine 1995, 25:223-229), while another isoform, ApoE3, is believed to help maintain healthy microtubules.", "In the brains of patients having Alzheimer's disease the observed increase in both ApoE mRNA and the number of astrocytes suggests that the ApoE increase represents an attempt of the astrocytes to repair the damage within the nervous cells.", "In the absence of the ApoE gene (ApoE knock out mice) memory deficit, defect in the repair of brain injury and deposition of the Alzheimer's associated β-amyloid variant APP V717F were demonstrated; see for instance: Oitzl M S, Mulder M, Lucassen P J, Havekes L M, Grootendorst J, Dekloet E R “Severe learaing deficits in apolipoprotein E knockout mice in a water maze task” Brain Research 1997, 752:189-196 and Laskowitz D T, Sheng H X, Bart R D, Joyner K A, Roses A D and Warner D S “Apolipoprotein E-deficient mice have increased susceptibility.", "to focal cerebral ischemia” Journal of Cerebral Blood Flow and Metabolism 1997, 17: 753-758 and Walker L C, Parker C A, Lipinski W J, Callahan M J, Carroll R T, Gandy S E, Smith J D, Jucker M and Bisgaier C L “Cerebral lipid deposition in aged Apolipoprotein E-deficient mice”, American Journal of Pathology 1997, 151:1371-1377.Thus, increasing ApoE production in patients bearing the E2 and E3 isoforms of ApoE might have a beneficial effect on the occurrence of Alzheimer's or other spontaneous or traumatic neurological diseases.", "ApoE in the Peripheral Nervous System (PNS) The important role of ApoE in nerve regeneration in the peripheral nervous system is demonstrated by the observation that ApoE synthesis is dramatically induced when nerves are injured, see for intance: Poirier J 1994 “Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease” [review], Trends in Neurosciences 1994, 17:525-530.The maintenance and/or repair of the myelin sheets involves the participation of ApoE secreted by support cells such as glial and Schwann cells.", "The ApoE synthesis and concentration were found to be abnormally low in degenerative diseases of nervous tissues such as in Multiple Sclerosis, see for instance: Gaillard O, Gervais A, Meillet D, Delattre J, Lyoncaen O, Schuller E “Apolipoprotein E intrathecal synthesis is decreased in multiple sclerosis patients” Annals of Clinical Biochemistry 1996, 33:2:148-150.ApoE is also considered to stabilize the cytoskeleton apparatus and support neurite elongation thus having a major effect on the development and remodelling following injury of the nervous system occurring late in life.", "The above evidence has establislied the beneficial role of ApoE in lipid (cholesterol and triglyceride) homeostasis and also in nervous tissue homeostasis and recovery from injury.", "There is thus a rationale for the development of agents which effectively increase ApoE in plasma and tissues (liver, brain, central or peripheral nervous system) in order to treat lipid disorders such as atherosclerosis; or neurodegenerative disorders such as Alzheimer's disease or dementia and multiple sclerosis.", "ApoE as Modulators of the Immune System ApoE affects the immune system by acting on lymphocyte proliferation.", "Furthermore ApoE knock out mice are highly sensitive to bacterial infection due to a defect in their innate immune systems suggesting that increasing ApoE production should ameliorate the immune response; see for instance: Roselaar S E, Daugherty A “Apolipoprotein E-deficient mice have impaired innate immune responses to listeria monocytogenes in vivo” J Lipid Res 1998, 39:1740-1743.Potential Uses of ApoE Inducers in the Treatment of Human Diseases (i) In Atherosclerosis (Role of Plasma HDL ApoE) Increasing ApoE plasma levels will decrease plasma atherogenic lipoproteins (VLDL, IDL and LDL) by increasing their uptake by the liver.", "Increasing ApoE in HDL will increase the removal of cholesterol from loaded tissues (atherosclerotic arteries) by the reverse cholesterol transport mechanism.", "(ii) In the Central Nervous System (CNS) Increasing ApoE in the brain will prevent the deposition of plaques associated with Alzheimer's disease and increase the repair mechanism of brain injuries due to mechanical traumas or strokes.", "Through the increase of neurite extension synaptic sprouting the overall brain activity (i.e.", "memory) should improve.", "(iii) In the Peripheral Nervous System (PNS) ApoE plays an important role in nerve regeneration and increasing ApoE in traumatised nerves (nerve section, crush etc) or degenerative nerves (multiple sclerosis) will increase the speed of the healing process or prevent degeneration.", "(iv) In skin Diseases Due to Disturbances in Skin Lipid Metabolism The skin constitutes a lipophilic barrier and lipid homeostasis is well controlled in cells such as keratinocytes.", "Cholesterol deposition (xanthelasma and xanthomas) will be prevented by increasing the level of ApoE in skin tissue.", "(v) As Modulators of the Immune System ApoE affects the immune system by acting on lymphocyte proliferation.", "Furthermore ApoE knock out mice are highly sensitive to bacterial infection due to a defect in innate immune system suggesting that increasing ApoE production should ameliorate the immune response.", "For the above reasons the development of compounds which increase ApoE prodution might be useful to treat numerous disease states.", "Until now only hormones have been shown to affect ApoE synthesis and secretion.", "The discovery of new small molecules with ApoE modulating activities should lead to the discovery of drugs having application in the treatment of diseases such as: atherosclerosis, excess lipid deposition in peripheral tissues such as skin (xanthomas), stroke, memory loss, optic nerve and retinal pathologies (i.e.", "macular degeneration, retinitis pigmentosa), repair of traumatic damage of the central nervous system (brain tissue), repair of traumatic damage of the peripheral nervous system (i.e.", "nerve section compression or crush), prevention of the degenerative process due to aging (i.e.", "Alzheimer's disease), prevention of degenerative neuropathies occuring in diseases such as diabetic neuropathies and multiple sclerosis, autoimmune diseases and activation of the innate immune system.", "The applicants have now found that certain FXR and LXRα activators increase ApoE production.", "Furthermore, the applicants have found that a screening system comprising an in vitro assay allows the rapid identification of ApoE modulators.", "This in vitro assay involves testing the effects of FXR and LXRα activators on the secretion of ApoE in the THP-1 cell line.", "Activators of FXR can be identified by means of the assays described by Forman et al.", "Cell 1995, 81, 687-693, Makishima, M. et al Science 1999, 284, 1362-1365, Parks, D. J. et al Science 1999, 284, 1365-1368 and Wang, H. B. et al Molecular Cell 1999, 3, 543-553.Activators of LXRα can be identified by means of the assays described by Willy et al Genes & Development 1995, 9, 1033-1045 and Janowsky B.", "A. et al.", "Proc Natl.", "Acad Sci USA 1999, 96, 266-271.The present invention is-based on the finding by the inventors that activators of the orphan nuclear receptors FXR such as farnesol, chenodeoxycholic acid and activators of the orphan nuclear receptor LXRα such as 22(R)-hydroxycholesterol, are ApoE modulators and more specifically are ApoE inducers and thus are potentially useful as agents for the treatment of atherosclerotic or neurological diseases such as those previously discussed.", "Accordingly, in one aspect, the invention provides a method for increasing Apolipoprotein E (ApoE) in plasma and in tissues of a mammal by using a combination of an ApoE increasing amount of an activator of the orphan nuclear receptor FXR and an ApoE increasing amount of an activator of the orphan nuclear receptor LXRα.", "In another aspect, the invention provides a method for increasing ApoE in plasma and in tissues of a mammal by using an ApoE increasing amount of a FXR activator.", "In a further aspect, the invention provides a method for increasing ApoE in plasma and in tissues of a mammal by using an ApoE increasing amount of a LXRα activator.", "The invention also provides the use of a combination of an ApoE increasing amount of a FXR activator and of an ApoE increasing amount of a LXRα activator, for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "In another aspect, the invention provides the use of a FXR activator for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "In yet another aspect, the invention provides the use of a LXRα activator for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "Moreover the invention also provides the above-mentioned activators, farnesol, chenodeoxycholic acid and 22(R)-hydroxycholesterol for use in medicine, for example for use in therapy, e.g.", "for use in the treatment or prophylaxis of a disease or condition as hereinbefore defined, and as set out in the claims appended hereto.", "The invention also provides the use of FXR and LXRα activators for the manufacture of a medicament for the treatment or prophylaxis of a disease or condition as hereinbefore defined or as set out in the claims appended hereto.", "The invention further provides a method of treatment or prophylaxis of a disease state or condition as hereinbefore defined, or as set out in the claims appended hereto, which method comprises administering to a subject (e.g.", "a mammal such as a human) in need thereof, a therapeutically or prophylactically effective (and preferably non-toxic) amount of the FXR activator or the LXRα activator.", "A further method of treatment of the above-mentioned disease state or condition comprises administering to said subject a combination of an effective amount of the FXR activator and an effective amount of the LXRα activator, by taking advantage of the synergistic effect of the FXR and LXRα activators.", "In a further aspect, the invention provides a pharmaceutical composition having apoE increasing activity and comprising both an FXR activator and a LXRα activator.", "The composition can contain, for example, a mixture of the FXR activator and LXRα activator.", "The apoE increasing activity of a composition containing both activators may usefully exhibit an apoE increasing activity which is greater than the sum of the apoE increasing activities of the individual activators, i.e.", "may exhibit synergy.", "Such synergistic mixtures, compositions containing them and their uses form a further aspect of the invention.", "The farnesoid X activated receptor (FXR; NR1H4) is activated by natural compounds from two different chemical classes which comprise certain isoprenoids and certain bile acids.", "The isoprenoids that are FXR activators include the following: farnesol, farnesal, farnesyl acetate, farnesoic acid and its esters and juvenile hormone III.", "It is important to note that not all farnesoids are FXR activators: for instance geranylgeraniol does not activate FXR.", "The majority of bile acids are FXR, activators, chenodeoxycholic acid being one of the most potent activators.", "Examples of bile acid compounds that are not FXR activators are: cholic acid and ursodeoxycholic acid.", "Whether or not a compound is a FXR activator can be determined by the assays described by Forman et al.", "Cell 1995, 81, 687-693, Makishima, M. et al Science 1999, 284, 1362-1365, Parks, D. J. et al Science 1999, 284, 1365-1368 and Wang, H. B. et al Molecular Cell 1999, 3, 543-553.The currently preferred FXR activators are: farnesol (trans,trans-farnesol; [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) methyl farnesyl ether, (methyl [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl ether) ethyl farnesyl ether, ethyl [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl ether) methyl farnesoate, ([2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrienoic acid methyl ester), ethyl farnesoate, ([2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrienoic acid ethyl ester), juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methyl ester), 7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid ethyl ester and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid).", "The currently most preferred FXR activators are: farnesol (trans,trans-farnesol; [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-ol) juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methyl ester) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid).", "The Liver X alpha receptor (LXRα, NR1H3) is activated by certain oxysterols which comprise the following: 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 24(S),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol.", "It is again important to note that oxysterols are not all potent activators of LXRα, for instance 22(S)-hydroxycholesterol (the enantiomer of the activator 22(R)-hydroxycholesterol) is a less potent activator of LXRα.", "Whether or not a compound is a LXRα activator can be determined by the assay described by Willy et al Genes & Development 1995, 9, 1033-1045 and Janowsky B.", "A. et al Proc Natl Acad Sci USA 1999, 96, 266-271.The currently preferred LXRα activators are: 22(R)-hydroxycholesterol (5-cholestene-3β,22[R]-diol) 24(S)-hydroxycholesterol, 24(S),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol The currently most preferred LXRα activators are: 22(R)-hydroxycholesterol (5-cholestene-3β,22[R]-diol) 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol.", "This invention arises from the finding by the inventors that ligands and activators of FXR and LXRα are potent inducers of ApoE secretion and might be useful in the treatment of diseases requiring an increased production of plasma and/or tissue ApoE described above.", "The ApoE inducing activity of these compounds was demonstrated in an in vitro model, the ApoE-producing cell line THP-1, where a representative member of each chemical class of activators was tested in parallel with a closely related compound which is not an activator of the specific nuclear receptor.", "The test results show that the activators are several-fold more potent in inducing ApoE than their non-inducer analogs, thereby establishing the potency and specificity of the activity of the FXR and LXRα activators.", "In a further aspect, the invention provides a method for identifying a candidate compound for use in increasing apolipoprotein E (ApoE) levels and tissues and plasma of a mammal; which method comprises selecting a test compound having FXR nuclear receptor activator activity and thereafter subjecting the test compound to an assay to determine its apoE inducing effect.", "In another aspect the invention provides a method for identifying a candidate compound for use in increasing apolipoprotein E (ApoE) levels and tissues and plasma of a mammal; which method comprises selecting a test compound having LXRα nuclear receptor activator activity and thereafter subjecting the test compound to an assay to determine its ApoE inducing effect.", "Each of the above methods may further comprise a preliminary step of subjecting a test compound to an assay to determine whether the compound is a FXR or LXRα activator respectively.", "The aforementioned methods may optionally comprise formulating a candidate compound identified by the method as a pharmaceutical formulation as a medicament.", "The medicaments prepared according to the foregoing methods may be used for any of the therapeutic purposes disclosed herein for apoE modulator compounds.", "The FXR or the LXRα activator of the invention can be administered by any of a variety of routes.", "Thus, for example, they can be administered orally, or by delivery across another mucosal surface (for example across the nasal, buccal, bronchial or rectal mucosa), transdermally, or by injection (for example intradermal, intraperitoneal, intravenous or intramuscular injection).", "When the compounds are intended for oral administration, they can be formulated, for example, as tablets, capsules, granules, pills, lozenges, powders, solutions, emulsions, syrups, suspensions, or any other pharmaceutical form suitable for oral administration.", "Oral dosage forms can, if desired, be coated with one or more release delaying coatings to allow the release of the active compound to be controlled or targeted at a particular part of the enteric tract.", "Tablets and other solid or liquid oral dosage forms can be prepared (e.g.", "in standard fashion) from the FXR or the LXRα activator and a pharmaceutically acceptable solubilizer, diluent or carrier.", "Examples of solubilizers, diluents or carriers include sugars such as lactose, starches, cellulose and its derivatives, powdered tracaganth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols such as glycerol, propyleneglycol and polyethyleneglycols, alginic acids and alginates, agar, pyrogen free water, isotonic.", "saline, phosphate buffered solutions, and optionally other pharmaceutical excipients such as disintegrants, lubricants, wetting agents such as sodium lauryl sulfate, coloring agents, flavoring agents and preservatives, etc.", "Capsules can be of the hard or soft variety and can contain the active compound in solid, liquid or semisolid form.", "Typically such capsules are formed from gelatine or an equivalent substance and can be coated or uncoated.", "If it is desired to delay the release of the active compound until the capsule has passed through the stomach and into the intestine, the capsule can be provided with a pH sensitive coating adapted to dissolve at the pH found in the duodenum or ileum.", "Examples of such coatings include the Eudragits, the uses of which are well known.", "Formulations for injection will usually be made up of the appropriate, solubilizers such as detergents which may also include compounds and excipients such as buffering agents to provide an isotonic solution having the correct physiological pH.", "The injectable solutions are typically pyrogen-free and can be provided in sealed vials or ampoules containing a unit dose of compound.", "A unit dosage form of the compounds of the invention typically will contain from 0.1% to 99% by weight of the active substance, more usually from 5% to 75% of the active substance.", "By way of example, a unit dosage form can contain from 1 mg to 1 g of the compound, more usually from 10 mg to 500 mg, for example between 50 mg and 400 mg, and typically in doses of 100 mg to 200 mg.", "The compounds of the invention will be administered in amounts which are effective to provide the desired therapeutic effect.", "The concentrations necessary to provide the desired therapeutic effect will vary according to among other things the precise nature of the disease, the size, weight and age of the patient and the severity of the disease.", "The doses.", "administered will preferably be non-toxic to the patient, although in certain circumstances the severity of the disease under treatment may necessitate administering an amount of compound which causes some signs of toxicity.", "Typically, the compounds of the invention will be administered in amounts in the range 0.01 mg/kg to 100 mg/kg body weight, more preferably 0.1 mg/kg to 10 mg/kg body weight and particularly 1 mg/kg to 5 mg/kg body weight.", "For an average human of 70 kg weight, a typical daily dosage of the compounds of the invention would be in the range of 70 mg to 700 mg.", "Such a dosage can be administered, for example from two to four times daily.", "Ultimately however, the size of the doses administered and the frequency of administration will be at the discretion and judgement of the physician treating the patient.", "For therapeutic use the.", "compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.", "For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.", "They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.", "For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.", "The choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated.", "The choice of mode of administration and dosage is within the skill of the art.", "The FXR or LXRα activators and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions or as solids for example, tablets, capsules and lozenges.", "A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agents.", "A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.", "Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.", "A composition in the form of a capsule can be prepared using routine encapsulation procedures.", "For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.", "Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.", "Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.", "A typical suppository formulation comprises the FXR or LXRα activator or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.", "Preferably the composition is in unit dose form such as a tablet or capsule.", "Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of the FXR or LXRα activator or a pharmaceutically acceptable salt thereof calculated as the free base.", "The pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.", "For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the FXR or LXRα activator or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.", "Disease states which could benefit from increasing plasma and tissue ApoE levels include, but are not limited to: atherosclerosis, neurodegenerative disorders such as Alzheimer's disease or dementia.", "The compounds of this invention modulate ApoE and are therefore of value in the treatment of any of these conditions.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "The present invention relates to respectively activators of the orphan nuclear receptor FXR such as farnesol, chenodeoxycholic acid and activators of the orphan nuclear receptor LXRα such as 22(R)-hydroxycholesterol, pharmaceutical compositions containing them and their use in therapy for modulating and in particular for increasing apolipoprotein E in plasma and in tissues.", "BACKGROUND OF THE INVENTION Apolipoprotein E (ApoE) is a polymorphic, multifunctional protein synthesized by several cell types and tissues, including liver, kidney, skin, adipose tissue, macrophages and brain.", "The wide distribution of ApoE is associated with the maintenance of key cellular functions such as intracellular cholesterol trafficking, cholesterol distribution between cells, and tissue reparation.", "The amino acid sequence of the ApoE protein is well conserved throughout species.", "ApoE can be viewed as a regulator of cholesterol homeostasis in tissues such as the Central Nervous System (CNS) and Peripheral Nervous System (PNS) and the arterial wall (cell-cell) or between tissues via the circulating plasma lipoproteins (tissue-tissue).", "The major role of plasma ApoE containing lipoproteins is to transfer lipids (cholesterol) from peripheral tissues to the liver and to remove excess cholesterol from peripheral tissues via the reverse cholesterol transport system.", "Dysregulation of this mechanism leads to excess cholesterol deposition in peripheral tissues such as arteries (arterosclerosis) and skin (xanthomas and xanthelasmas).", "ApoE has also been shown to have a direct effect on lymphocyte proliferation and thus has an immunomodulatory role.", "After the liver, the brain is the second major site of ApoE synthesis.", "ApoE is the only lipoprotein synthesized by brain tissue where the key role of ApoE is cholesterol transport between cells of the central nervous system (CNS).", "Local secretion of ApoE by cells such as macrophages or macrophage-derived cells is essential for the uptake of excess tissue cholesterol or provides cholesterol for specific needs such as nerve repair and remyelinisation.", "Up to the present time, the compounds affecting Apo E production in vitro and in vivo have not been investigated thoroughly.", "Only hormone-like estrogens and corticoids have been shown to change Apo E levels under various experimental conditions; see for instance: Srivastava R A K, Srivastava N, Averna M, Lin R C, Korach K S, Lubahn D B, Schonfeld G “Estrogen up-regulates apolipoprotein E (ApoE) gene expression by increasing ApoE mRNA in the translating pool via the estrogen receptor alpha-mediated pathway” J Biol Chem, 1997, 272:33360-33366 and Stone D J, Rozovsky I, Morgan T E, Anderson C P, Hajian H, Finch C E “Astrocytes and microglia respond to estrogen with increased ApoE mRNA.", "in vivo and in vitro” Experimental Neurology, 1997, 143:313-318.Orphan Nuclear Receptors FXR and LXR Steroid hormones (glucocorticoids, mineralocorticoids, estrogens, progestins, androgens, and vitamin D) bind to their nuclear receptors which are transcription factors and by this means regulate expression of gene coding for specific proteins and control critical cellular activities; see for instance Meier, C., A.", "Journal of Receptor & Signal Transduction Research 1997, 17, 319-335.In the last ten years more than 100 mammalian genes have extended the family of steroid nuclear receptors and have been classified as orphan nuclear receptors for which ligands are unknown; see for instance Enmark, E. and Gustafsson, J.", "A. Molecular Endocrinology 1996, 10, 1293-1307.The farnesoid X activated receptor (FXR; NR1H4) was identified in 1995 by Forman et al.", "Cell 1995, 81, 687-693.FXR functions as a heterodimer with the Retinoid X Receptor (RXR) and binds to the DNA via an inverted repeat element IR-1.FXR was originally described as activated by isoprenoids such as farnesol and juvenile hormone III.", "More recently, several investigators came to the conclusion that bile acids are the physiological ligands and activators of FXR; see for instance Makishima, M. et al Science 1999, 284, 1362-1365.Parks, D. J. et al Science 1999, 284, 1365-1368.Wang, H. B. et al Molecular Cell 1999, 3, 543-553.The Liver X Receptor was identified as an orphan nuclear receptor by Willy et al Genes & Development 1995, 9, 1033-1045.and its ligand-binding domain shares 37% identity with FXR.", "The DNA binding sequence of LXR is a direct repeat separated by 4 nucleotides (DR-4) and differs from the DNA binding sequence of FXR (IR-1).", "Two genes encode for LXR proteins (LXRα;NR1H3 and LXRβ;NR1H2) and both receptors are activated by various oxysterols, the most potent being 22(R)-hydroxycholesterol, 24 (S)-hydroxycholesterol, 24(S),25-epoxycholesterol and 7-hydroxycholesterol; see for instance: Janowsky B.", "A. et al Proc Natl Acad Sci USA 1999, 96, 266-271.Thus LXRα appears to be a sensor of both isoprenoids and oxysterols but the exact physiological and pharmacological applications of the activators and ligands of any these receptors (FXR and LXR) are yet to be established (Niesor E et al Current Pharmaceutical Drug Design 2000 in press).", "SUMMARY OF THE INVENTION The applicants have discovered that representative ligands and activators of FXR (e.g.", "the isoprenoid farnesol, the bile acid derivative chenodeoxycholic acid) and of LXRα (e.g.", "the hydroxysterol 22(R)-hydroxycholesterol) are potent inducers of ApoE secretion and might be useful in the treatment of diseases requiring an increased production of plasma and/or tissue ApoE.", "Compounds which modulate ApoE synthesis and secretion should have application in the treatment of diseases such as atherosclerosis, excess lipid deposition in peripheral tissues such as skin (xanthomas), stroke, memory loss, optic nerve and retinal pathologies (i.e.", "macular degeneration, retinitis pigmentosa), repair of traumatic damage of the central nervous system (brain tissue), repair of traumatic damage of the peripheral nervous system (i.e.", "nerve section compression or crush), prevention of the degenerative process due to aging (i.e.", "Alzheimer's disease), prevention of degenerative neuropathies occurring in diseases such as diabetic neuropathies and multiple sclerosis, autoimmune diseases and activation of the innate immune system.", "ApoE in Atherosclerosis As a component of all lipoprotein fractions, ApoE plays a important role in cholesterol homeostasis, by mediating their interaction with receptors such as the apoB, low-density lipoprotein (LDL) and other specific receptors.", "The important role of ApoE in cardiovascular diseases is demonstrated by the ApoE knock-out mouse model where the animals rapidly develop hypercholesterolemia and atherosclerosis with pathological features similar to human atherosclerosis; see for instance: Plump A “Atherosclerosis and the mouse—a decade of experience [review]” Annals of Medicine 1997, 29:193-198.In man, in the absence of a functional ApoE protein (human variants; see for instance: Richard P, Dezulueta M P, Beucler I, Degennes J L, Cassaigne A, Iron A “Identification of a new apolipoprotein E variant (E(2) Arg(142)- ]Leu) in type-III hyperlipidemia” Atherosclerosis.", "1995, 112:19-28) and in the ApoE knock out mouse, plasma levels of cholesterol and triglycerides are abnormally high (even on a low fat diet) and atherosclerosis develops rapidly.", "In the animal model, these changes are prevented by infusion of ApoE, transplantation of macrophage producing ApoE or gene therapy by introducing the human ApoE gene into ApoE knock out mice, demonstrating the direct beneficial role of ApoE; see for instance: Linton M F, Atkinson J B, Fazio S “Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation” Science 1995, 267:1034-1037.A recent study has examined the response of ApoE knock out mice to diets with increasing cholesterol content and has concluded that ApoE plays a critical role in decreasing the absorption of dietary cholesterol and in increasing biliary secretion, see for instance Sehayek E, Shefer S, Nguyen L B, Ono J G, Merkel M and Breslow J L,“Apolipoprotein E regulates dietary absorption and biliary cholesterol excretion: Studies in C57BL/6 ApoE knock out mice” Proc Natl Acad Sci USA 2000, 97, 3433-3437.The results of this study further suggest that increasing ApoE should decrease the absorption of dietary cholesterol and prevent the formation of biliary cholesterol stones.", "ApoE in the Central Nervous System (CNS) ApoE also plays a critical role in the central nervous system.", "In the brain ApoE is synthesized and secreted by astrocytes, its principal role being cholesterol transport between cells.", "ApoE is considered to redistribute lipids and to participate in the cholesterol homeostasis of the brain.", "ApoE is linked to the neuropathological lesions characteristic of Alzheimer's disease with one isoform, ApoE4, strongly associated with the age of onset of the disease; (see for instance: Poirier J “Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease” [review]Trends in Neurosciences 1994, 17:525-530 and Rubinsztein D C “Apolipoprotein E—a review of its roles in lipoprotein metabolism, neuronal growth and repair and as a risk factor for Alzheimer's disease” Psychological Medicine 1995, 25:223-229), while another isoform, ApoE3, is believed to help maintain healthy microtubules.", "In the brains of patients having Alzheimer's disease the observed increase in both ApoE mRNA and the number of astrocytes suggests that the ApoE increase represents an attempt of the astrocytes to repair the damage within the nervous cells.", "In the absence of the ApoE gene (ApoE knock out mice) memory deficit, defect in the repair of brain injury and deposition of the Alzheimer's associated β-amyloid variant APPV717F were demonstrated; see for instance: Oitzl M S, Mulder M, Lucassen P J, Havekes L M, Grootendorst J, Dekloet E R “Severe learaing deficits in apolipoprotein E knockout mice in a water maze task” Brain Research 1997, 752:189-196 and Laskowitz D T, Sheng H X, Bart R D, Joyner K A, Roses A D and Warner D S “Apolipoprotein E-deficient mice have increased susceptibility.", "to focal cerebral ischemia” Journal of Cerebral Blood Flow and Metabolism 1997, 17: 753-758 and Walker L C, Parker C A, Lipinski W J, Callahan M J, Carroll R T, Gandy S E, Smith J D, Jucker M and Bisgaier C L “Cerebral lipid deposition in aged Apolipoprotein E-deficient mice”, American Journal of Pathology 1997, 151:1371-1377.Thus, increasing ApoE production in patients bearing the E2 and E3 isoforms of ApoE might have a beneficial effect on the occurrence of Alzheimer's or other spontaneous or traumatic neurological diseases.", "ApoE in the Peripheral Nervous System (PNS) The important role of ApoE in nerve regeneration in the peripheral nervous system is demonstrated by the observation that ApoE synthesis is dramatically induced when nerves are injured, see for intance: Poirier J 1994 “Apolipoprotein E in animal models of CNS injury and in Alzheimer's disease” [review], Trends in Neurosciences 1994, 17:525-530.The maintenance and/or repair of the myelin sheets involves the participation of ApoE secreted by support cells such as glial and Schwann cells.", "The ApoE synthesis and concentration were found to be abnormally low in degenerative diseases of nervous tissues such as in Multiple Sclerosis, see for instance: Gaillard O, Gervais A, Meillet D, Delattre J, Lyoncaen O, Schuller E “Apolipoprotein E intrathecal synthesis is decreased in multiple sclerosis patients” Annals of Clinical Biochemistry 1996, 33:2:148-150.ApoE is also considered to stabilize the cytoskeleton apparatus and support neurite elongation thus having a major effect on the development and remodelling following injury of the nervous system occurring late in life.", "The above evidence has establislied the beneficial role of ApoE in lipid (cholesterol and triglyceride) homeostasis and also in nervous tissue homeostasis and recovery from injury.", "There is thus a rationale for the development of agents which effectively increase ApoE in plasma and tissues (liver, brain, central or peripheral nervous system) in order to treat lipid disorders such as atherosclerosis; or neurodegenerative disorders such as Alzheimer's disease or dementia and multiple sclerosis.", "ApoE as Modulators of the Immune System ApoE affects the immune system by acting on lymphocyte proliferation.", "Furthermore ApoE knock out mice are highly sensitive to bacterial infection due to a defect in their innate immune systems suggesting that increasing ApoE production should ameliorate the immune response; see for instance: Roselaar S E, Daugherty A “Apolipoprotein E-deficient mice have impaired innate immune responses to listeria monocytogenes in vivo” J Lipid Res 1998, 39:1740-1743.Potential Uses of ApoE Inducers in the Treatment of Human Diseases (i) In Atherosclerosis (Role of Plasma HDL ApoE) Increasing ApoE plasma levels will decrease plasma atherogenic lipoproteins (VLDL, IDL and LDL) by increasing their uptake by the liver.", "Increasing ApoE in HDL will increase the removal of cholesterol from loaded tissues (atherosclerotic arteries) by the reverse cholesterol transport mechanism.", "(ii) In the Central Nervous System (CNS) Increasing ApoE in the brain will prevent the deposition of plaques associated with Alzheimer's disease and increase the repair mechanism of brain injuries due to mechanical traumas or strokes.", "Through the increase of neurite extension synaptic sprouting the overall brain activity (i.e.", "memory) should improve.", "(iii) In the Peripheral Nervous System (PNS) ApoE plays an important role in nerve regeneration and increasing ApoE in traumatised nerves (nerve section, crush etc) or degenerative nerves (multiple sclerosis) will increase the speed of the healing process or prevent degeneration.", "(iv) In skin Diseases Due to Disturbances in Skin Lipid Metabolism The skin constitutes a lipophilic barrier and lipid homeostasis is well controlled in cells such as keratinocytes.", "Cholesterol deposition (xanthelasma and xanthomas) will be prevented by increasing the level of ApoE in skin tissue.", "(v) As Modulators of the Immune System ApoE affects the immune system by acting on lymphocyte proliferation.", "Furthermore ApoE knock out mice are highly sensitive to bacterial infection due to a defect in innate immune system suggesting that increasing ApoE production should ameliorate the immune response.", "For the above reasons the development of compounds which increase ApoE prodution might be useful to treat numerous disease states.", "Until now only hormones have been shown to affect ApoE synthesis and secretion.", "The discovery of new small molecules with ApoE modulating activities should lead to the discovery of drugs having application in the treatment of diseases such as: atherosclerosis, excess lipid deposition in peripheral tissues such as skin (xanthomas), stroke, memory loss, optic nerve and retinal pathologies (i.e.", "macular degeneration, retinitis pigmentosa), repair of traumatic damage of the central nervous system (brain tissue), repair of traumatic damage of the peripheral nervous system (i.e.", "nerve section compression or crush), prevention of the degenerative process due to aging (i.e.", "Alzheimer's disease), prevention of degenerative neuropathies occuring in diseases such as diabetic neuropathies and multiple sclerosis, autoimmune diseases and activation of the innate immune system.", "The applicants have now found that certain FXR and LXRα activators increase ApoE production.", "Furthermore, the applicants have found that a screening system comprising an in vitro assay allows the rapid identification of ApoE modulators.", "This in vitro assay involves testing the effects of FXR and LXRα activators on the secretion of ApoE in the THP-1 cell line.", "Activators of FXR can be identified by means of the assays described by Forman et al.", "Cell 1995, 81, 687-693, Makishima, M. et al Science 1999, 284, 1362-1365, Parks, D. J. et al Science 1999, 284, 1365-1368 and Wang, H. B. et al Molecular Cell 1999, 3, 543-553.Activators of LXRα can be identified by means of the assays described by Willy et al Genes & Development 1995, 9, 1033-1045 and Janowsky B.", "A. et al.", "Proc Natl.", "Acad Sci USA 1999, 96, 266-271.The present invention is-based on the finding by the inventors that activators of the orphan nuclear receptors FXR such as farnesol, chenodeoxycholic acid and activators of the orphan nuclear receptor LXRα such as 22(R)-hydroxycholesterol, are ApoE modulators and more specifically are ApoE inducers and thus are potentially useful as agents for the treatment of atherosclerotic or neurological diseases such as those previously discussed.", "Accordingly, in one aspect, the invention provides a method for increasing Apolipoprotein E (ApoE) in plasma and in tissues of a mammal by using a combination of an ApoE increasing amount of an activator of the orphan nuclear receptor FXR and an ApoE increasing amount of an activator of the orphan nuclear receptor LXRα.", "In another aspect, the invention provides a method for increasing ApoE in plasma and in tissues of a mammal by using an ApoE increasing amount of a FXR activator.", "In a further aspect, the invention provides a method for increasing ApoE in plasma and in tissues of a mammal by using an ApoE increasing amount of a LXRα activator.", "The invention also provides the use of a combination of an ApoE increasing amount of a FXR activator and of an ApoE increasing amount of a LXRα activator, for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "In another aspect, the invention provides the use of a FXR activator for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "In yet another aspect, the invention provides the use of a LXRα activator for the manufacture of a medicament for increasing ApoE in plasma and in tissues of a mammal.", "Moreover the invention also provides the above-mentioned activators, farnesol, chenodeoxycholic acid and 22(R)-hydroxycholesterol for use in medicine, for example for use in therapy, e.g.", "for use in the treatment or prophylaxis of a disease or condition as hereinbefore defined, and as set out in the claims appended hereto.", "The invention also provides the use of FXR and LXRα activators for the manufacture of a medicament for the treatment or prophylaxis of a disease or condition as hereinbefore defined or as set out in the claims appended hereto.", "The invention further provides a method of treatment or prophylaxis of a disease state or condition as hereinbefore defined, or as set out in the claims appended hereto, which method comprises administering to a subject (e.g.", "a mammal such as a human) in need thereof, a therapeutically or prophylactically effective (and preferably non-toxic) amount of the FXR activator or the LXRα activator.", "A further method of treatment of the above-mentioned disease state or condition comprises administering to said subject a combination of an effective amount of the FXR activator and an effective amount of the LXRα activator, by taking advantage of the synergistic effect of the FXR and LXRα activators.", "In a further aspect, the invention provides a pharmaceutical composition having apoE increasing activity and comprising both an FXR activator and a LXRα activator.", "The composition can contain, for example, a mixture of the FXR activator and LXRα activator.", "The apoE increasing activity of a composition containing both activators may usefully exhibit an apoE increasing activity which is greater than the sum of the apoE increasing activities of the individual activators, i.e.", "may exhibit synergy.", "Such synergistic mixtures, compositions containing them and their uses form a further aspect of the invention.", "The farnesoid X activated receptor (FXR; NR1H4) is activated by natural compounds from two different chemical classes which comprise certain isoprenoids and certain bile acids.", "The isoprenoids that are FXR activators include the following: farnesol, farnesal, farnesyl acetate, farnesoic acid and its esters and juvenile hormone III.", "It is important to note that not all farnesoids are FXR activators: for instance geranylgeraniol does not activate FXR.", "The majority of bile acids are FXR, activators, chenodeoxycholic acid being one of the most potent activators.", "Examples of bile acid compounds that are not FXR activators are: cholic acid and ursodeoxycholic acid.", "Whether or not a compound is a FXR activator can be determined by the assays described by Forman et al.", "Cell 1995, 81, 687-693, Makishima, M. et al Science 1999, 284, 1362-1365, Parks, D. J. et al Science 1999, 284, 1365-1368 and Wang, H. B. et al Molecular Cell 1999, 3, 543-553.The currently preferred FXR activators are: farnesol (trans,trans-farnesol; [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) methyl farnesyl ether, (methyl [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl ether) ethyl farnesyl ether, ethyl [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl ether) methyl farnesoate, ([2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrienoic acid methyl ester), ethyl farnesoate, ([2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrienoic acid ethyl ester), juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methyl ester), 7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid ethyl ester and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid).", "The currently most preferred FXR activators are: farnesol (trans,trans-farnesol; [2E,6E]-3,7,11-trimethyl-2,6,10-dodecatrien-ol) juvenile hormone III (7-methyl-9-(3,3-dimethyloxiranyl)-3-methyl-2,6-nonadienoic acid methyl ester) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid).", "The Liver X alpha receptor (LXRα, NR1H3) is activated by certain oxysterols which comprise the following: 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 24(S),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol.", "It is again important to note that oxysterols are not all potent activators of LXRα, for instance 22(S)-hydroxycholesterol (the enantiomer of the activator 22(R)-hydroxycholesterol) is a less potent activator of LXRα.", "Whether or not a compound is a LXRα activator can be determined by the assay described by Willy et al Genes & Development 1995, 9, 1033-1045 and Janowsky B.", "A. et al Proc Natl Acad Sci USA 1999, 96, 266-271.The currently preferred LXRα activators are: 22(R)-hydroxycholesterol (5-cholestene-3β,22[R]-diol) 24(S)-hydroxycholesterol, 24(S),25-epoxycholesterol, 5,6-24-(S)diepoxycholesterol and 7-hydroxycholesterol The currently most preferred LXRα activators are: 22(R)-hydroxycholesterol (5-cholestene-3β,22[R]-diol) 24(S)-hydroxycholesterol and 24(S),25-epoxycholesterol.", "This invention arises from the finding by the inventors that ligands and activators of FXR and LXRα are potent inducers of ApoE secretion and might be useful in the treatment of diseases requiring an increased production of plasma and/or tissue ApoE described above.", "The ApoE inducing activity of these compounds was demonstrated in an in vitro model, the ApoE-producing cell line THP-1, where a representative member of each chemical class of activators was tested in parallel with a closely related compound which is not an activator of the specific nuclear receptor.", "The test results show that the activators are several-fold more potent in inducing ApoE than their non-inducer analogs, thereby establishing the potency and specificity of the activity of the FXR and LXRα activators.", "In a further aspect, the invention provides a method for identifying a candidate compound for use in increasing apolipoprotein E (ApoE) levels and tissues and plasma of a mammal; which method comprises selecting a test compound having FXR nuclear receptor activator activity and thereafter subjecting the test compound to an assay to determine its apoE inducing effect.", "In another aspect the invention provides a method for identifying a candidate compound for use in increasing apolipoprotein E (ApoE) levels and tissues and plasma of a mammal; which method comprises selecting a test compound having LXRα nuclear receptor activator activity and thereafter subjecting the test compound to an assay to determine its ApoE inducing effect.", "Each of the above methods may further comprise a preliminary step of subjecting a test compound to an assay to determine whether the compound is a FXR or LXRα activator respectively.", "The aforementioned methods may optionally comprise formulating a candidate compound identified by the method as a pharmaceutical formulation as a medicament.", "The medicaments prepared according to the foregoing methods may be used for any of the therapeutic purposes disclosed herein for apoE modulator compounds.", "The FXR or the LXRα activator of the invention can be administered by any of a variety of routes.", "Thus, for example, they can be administered orally, or by delivery across another mucosal surface (for example across the nasal, buccal, bronchial or rectal mucosa), transdermally, or by injection (for example intradermal, intraperitoneal, intravenous or intramuscular injection).", "When the compounds are intended for oral administration, they can be formulated, for example, as tablets, capsules, granules, pills, lozenges, powders, solutions, emulsions, syrups, suspensions, or any other pharmaceutical form suitable for oral administration.", "Oral dosage forms can, if desired, be coated with one or more release delaying coatings to allow the release of the active compound to be controlled or targeted at a particular part of the enteric tract.", "Tablets and other solid or liquid oral dosage forms can be prepared (e.g.", "in standard fashion) from the FXR or the LXRα activator and a pharmaceutically acceptable solubilizer, diluent or carrier.", "Examples of solubilizers, diluents or carriers include sugars such as lactose, starches, cellulose and its derivatives, powdered tracaganth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols such as glycerol, propyleneglycol and polyethyleneglycols, alginic acids and alginates, agar, pyrogen free water, isotonic.", "saline, phosphate buffered solutions, and optionally other pharmaceutical excipients such as disintegrants, lubricants, wetting agents such as sodium lauryl sulfate, coloring agents, flavoring agents and preservatives, etc.", "Capsules can be of the hard or soft variety and can contain the active compound in solid, liquid or semisolid form.", "Typically such capsules are formed from gelatine or an equivalent substance and can be coated or uncoated.", "If it is desired to delay the release of the active compound until the capsule has passed through the stomach and into the intestine, the capsule can be provided with a pH sensitive coating adapted to dissolve at the pH found in the duodenum or ileum.", "Examples of such coatings include the Eudragits, the uses of which are well known.", "Formulations for injection will usually be made up of the appropriate, solubilizers such as detergents which may also include compounds and excipients such as buffering agents to provide an isotonic solution having the correct physiological pH.", "The injectable solutions are typically pyrogen-free and can be provided in sealed vials or ampoules containing a unit dose of compound.", "A unit dosage form of the compounds of the invention typically will contain from 0.1% to 99% by weight of the active substance, more usually from 5% to 75% of the active substance.", "By way of example, a unit dosage form can contain from 1 mg to 1 g of the compound, more usually from 10 mg to 500 mg, for example between 50 mg and 400 mg, and typically in doses of 100 mg to 200 mg.", "The compounds of the invention will be administered in amounts which are effective to provide the desired therapeutic effect.", "The concentrations necessary to provide the desired therapeutic effect will vary according to among other things the precise nature of the disease, the size, weight and age of the patient and the severity of the disease.", "The doses.", "administered will preferably be non-toxic to the patient, although in certain circumstances the severity of the disease under treatment may necessitate administering an amount of compound which causes some signs of toxicity.", "Typically, the compounds of the invention will be administered in amounts in the range 0.01 mg/kg to 100 mg/kg body weight, more preferably 0.1 mg/kg to 10 mg/kg body weight and particularly 1 mg/kg to 5 mg/kg body weight.", "For an average human of 70 kg weight, a typical daily dosage of the compounds of the invention would be in the range of 70 mg to 700 mg.", "Such a dosage can be administered, for example from two to four times daily.", "Ultimately however, the size of the doses administered and the frequency of administration will be at the discretion and judgement of the physician treating the patient.", "For therapeutic use the.", "compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.", "For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents.", "They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.", "For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.", "The choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated.", "The choice of mode of administration and dosage is within the skill of the art.", "The FXR or LXRα activators and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions or as solids for example, tablets, capsules and lozenges.", "A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agents.", "A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.", "Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.", "A composition in the form of a capsule can be prepared using routine encapsulation procedures.", "For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.", "Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.", "Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.", "A typical suppository formulation comprises the FXR or LXRα activator or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.", "Preferably the composition is in unit dose form such as a tablet or capsule.", "Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of the FXR or LXRα activator or a pharmaceutically acceptable salt thereof calculated as the free base.", "The pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.", "For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the FXR or LXRα activator or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day.", "Disease states which could benefit from increasing plasma and tissue ApoE levels include, but are not limited to: atherosclerosis, neurodegenerative disorders such as Alzheimer's disease or dementia.", "The compounds of this invention modulate ApoE and are therefore of value in the treatment of any of these conditions.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention will be now illustrated, but not limited, by reference to the following examples.", "EXAMPLE 1 A.", "Method of Determining Biological Activity: The FXR and LXRα activators of the invention increase the ApoE production in vitro.", "The activities of the compounds can be determined an in vitro assay which comprises testing the effects of FXR and LXRα activators on the secretion of ApoE in the THP-1 cell line.", "Previous studies performed by the applicants had shown that test compounds which induce the secretion of ApoE by the ApoE-secreting cell line (THP-1 cells), are active in vivo for increasing plasma and tissue ApoE when given to animals from a wide range of species.", "B.", "Cell Culture The THP1 cell line was derived from the peripheral blood of a 1-year-old boy with acute monocytic leukemia.", "These cells did not express surface and cytoplasmic immunoglobulins; they were phagocytic and differentiated into macrophage-like cells.", "These cells were obtained from the European Collection of Animal Cell Cultures (ECACC, #88081201).", "The cells were grown as non-adherent cells in RPMI 1640 culture medium, 2 mM glutamine, 20 μM 2-mercaptoethanol.", "Fresh medium was added to maintain cell density between 2 and 9×105 cells/ml.", "Once a week, new cultures were initiated by inoculating 10 ml of medium with 2×106 cells in a 75 cm2 plate.", "The plates were kept at 37° C. in a 5% CO2 atmosphere.", "For screening, cells were seeded in 24-well plates at the density of 2×105 cells per well.", "Phorbol-12-myristat-13-acetate (PMA) was added at 0 and 2.5 nM to initiate THP1 differentiation into adherent macrophage-like cells.", "Vehicles, reference compounds and test compounds were added simultaneously at concentrations varying from 1 to 50 μM and incubated for 72 hours.", "The culture medium was then recovered, centrifuged at 300 g for 5 min to remove any unattached cell and stored at −20° C. before analysis.", "C. Apo E Determination by ELISA 96 well-microtiter plates were coated by incubating with a 5% gelatine solution from porcine skin, in 50 mM carbonate-bicarbonate buffer, pH9.6 at the concentration of 200 μl/well, for 2 hours at 37° C. The coating solution was carefully removed and the test compound was added (100 μl/well) at the appropriate dilution.", "Dilutions of a human ApoE standard (ICN BiomedicalsInc, USA) were simultaneously assayed.", "Test compounds and antibodies were diluted in the following buffer solution: PBS, 1% BSA, 0.1% Tween 20, pH 7.4.Test compounds were incubated for 1 hour at 37° C., and the wells were washed 3 times with 200 μl of buffer solution.", "One hundred microliters per well of the primary antibody (goat anti-human ApoE IgG) diluted 10000 fold was incubated for 1 hour at 37° C. under continuous shaking.", "After the third wash, 100 μl/well of the secondary antibody (anti-goat-IgG peroxidase conjugate) diluted 5000 fold was incubated for 1 hour at 37° C. with continuous shaking.", "Wells were washed 5 times and 100 μl/well of substrate (ortho-phenylenediamine dihydrochloride) was incubated for the appropriate time at room temperature in the dark with continuous shaking.", "The reaction was stopped by adding 50 μl/well of 3M sulfuric acid and incubating for 1 min at room temperature.", "The absorbance at 492 run versus 620 nm was read on a microplate photometer and the results were then converted into human ApoE ng equivalent.", "D. ApoE Inducing Activity of Farnesol, Chenoxycholic Acid and 22(R)-Hydroxycholesterol Representative examples of FXR and LXRα activators were tested for their activity in modulating the secretion of ApoE by THP-1 cells in the conditions described above and the results obtained are reported in Tables 1, 2 and 3.In each instance the FXR or the LXRα activator, was tested at the same concentration as that of a close analog from the same chemical class which is not an activator, or is a much weaker activator, of the nuclear receptor.", "This experimental design was devised to demonstrate the potency and selectivity of the FXR and LXRα activators.", "Thus, the ApoE inducing effect of two different chemical classes of FXR activator is demonstrated in Table 1 and 2.Farnesol, a representative example of FXR activator selected from the isoprenoid class, increases ApoE secretion by 125% whereas the inactive isoprenoid geranylgeraniol increases ApoE by only 36% (Table 1).", "Likewise, the bile acid derivative chenodeoxycholic acid, a well characterised ligand and activator of FXR, increased ApoE production by 138% while the close analog, cholic acid which does not activate FXR, did not change ApoE secretion by THP-1 cells (Table 2).", "The direct involvement of the orphan nuclear receptor LXRα in the induction of ApoE is clearly demonstrated by the fact that the activator enantiomer, 22(R)-hydroxycholesterol increases ApoE secretion by 250% whereas the much weaker enantiomer, 22(S)-hydroxycholesterol, was inactive: +23% at the same concentration (Table 3).", "In particular, the potency of 22(R)-hydroxycholesterol should be noted since significant increases in ApoE secretion could already be measured at submicromolar concentrations.", "All the compounds tested were commercially available and were purchased from Aldrich (Farnesol, # 27,754-1) or Sigma (Geranylgeraniol, # G3278, Chenodeoxycholic acid, # C9377, Cholic acid, # C1129, 22(R)-hydroxycholesterol, # H9384 and 22(S)-hydroxycholesterol, # H5884).", "TABLE 1 Chemical Class: Isoprenoid Compound Interaction with Change in Apo E (20 μM) nuclear receptor production Farnesol FXR activator 1) +125% (Trans,trans-Farnesol; [2E,6E]-3,7,11-trimethyl- 2,6,10-dodecatrien-ol) Geranylgeraniol Does not activate +36% (All trans-3,7,11,15-tetra- FXR 1) methyl-2,6,10,14-hexadeca- tetraen-1-ol) 1) See for instance: Forman et al.", "Cell 1995, 81, 687-693 TABLE 2 Chemical Class: Bile Acid Compound Interaction with Change in Apo E (50 μM) Nuclear receptor production Chenodeoxycholic acid FXR activator 2) +138% (3α,7α-dihydroxy-5β- cholanic acid) Cholic acid Does not activate +6% (3α,7α,12α-trihydroxy- FXR 2) 5β-cholan-24-oic acid) 2) See for instance: Makishima, M. et al Science 1999, 284, 1362-1365.Parks, D. J. et al Science 1999, 284, 1365-1368.Wang, H. B. et al Molecular Cell 1999, 3, 543-553.TABLE 3 Chemical Class: Hydroxy Sterol Compound Interaction with Change in Apo E (1 μM) Nuclear receptor production 22(R)-hydroxycholesterol LXRα activator 3) +249% (5-cholestene-3β,22[R]-diol) 22(S)-hydroxycholesterol Less potent activator +23% (5-cholestene-3β,22[S]-diol) of LXRα 3) 3) See for instance: Janowsky B.", "A. et al Proc Natl Acad Sci USA 1999, 96, 266-271 The foregoing examples are intended merely to be illustrative of the invention and are not intended to limit the scope of the invention in any way.", "It will readily be apparent that numerous modifications and alterations may be made to the examples without departing from the principles underlying the invention and all such modifications and alterations are intended to be embraced by this application." ] ]

[ [ "Clothing piece", "A garment of a stretch fabric knit in a fine-meshed manner, particularly a pair of cycling pants, comprises a front part (10) and a rear part (12).", "The front part (10) and the rear part (12) are knit simultaneously.", "Along at least one connecting edge (14,18), the front part is knit together with the rear part in a seamless manner.", "Thus, a seamless garment is manufactured.", "Since the garment does not have any bead-like seams, the occurrence of pressure marks and chafes caused by the seams is avoided." ], [ "1.A garment of a stretch fabric knit in a fine-meshed manner, particularly a pair of cycling pants, comprising a front part (10) and a rear part (12) knit independently of each other and simultaneously, the front part (10) and the rear part (12) being knit together in a seamless manner along at least one connecting edge (14,18).", "2.The garment according to claim 1, characterized in that the stretch fabric is smooth and free of beads along the connecting edge (14,18) at both sides thereof.", "3.The garment according to claim 1 or 2, characterized in that the entire garment is manufactured of the same yarn.", "4.The garment according to one of claims 1-3, characterized in that each row of the front part (10) is knit together with a row of the rear part (12) so that an uninterrupted connecting edge (14,18) is produced.", "5.The garment according to one of claims 1-4, characterized in that the front part (10) is knit together with the rear part (12) during the knitting of the front (10) and rear parts (12).", "6.The garment according to one of claims 1-5, characterized in that knitting together the front part (10) and the rear part (12) is effected by connecting the yarn with which the front part (10) is knit and the yarn with which the rear part (12) is knit.", "7.The garment according to one of claims 1-6, characterized in that the mesh width at the connecting edge (14,18) substantially corresponds to the mesh width of the front (10) and rear parts (12).", "8.The garment according to one of claims 1-7, characterized in that the front (10) and/or the rear part (12) comprise portions knit in a wide-meshed manner to increase the air permeability.", "9.Cycling pants according to one of claims 1-8, characterized in that knit straps (24) are connected with the front part (10) and a substantially closed back part (26) is connected with the rear part (12)." ], [ "The invention relates to a garment made of a stretch fabric knit in a fine-meshed manner.", "Particularly, the invention relates to sportswear such as sport shorts and cycling pants.", "Known garments of stretch fabric are mostly made of two fine-meshed knit parts with a front part and a rear part.", "The front part is sewn together with the rear part.", "Upon sewing together, bead-like seams are produced which are particularly disagreeable in garments of stretch fabric which generally have a close fit at the body.", "If the seams are located at positions where pressure is exerted, e.g., by a cycle saddle, painful pressure marks may be produced at the body.", "Further, the seams may cause chafes at the body when the garment moves in the region of the seam and thus chafes at the skin.", "Further, it is known to produce sport shorts such as cycling pants made of stretch fabric such that a tube is knit.", "From one side, the tube is then cut in in longitudinal direction and sewn along this edge of cut such that two trouser legs are formed.", "In doing so, another bead-like seam is produced at the inside of the thighs as well as in the seat region of the cycling pants.", "Pressure marks and chafes are particularly disagreeable at these body spots.", "From DE 79 22 268 U1, a pair of cycling pants is known which comprises elastically stretchable portions in the form of longitudinal strips of knitwear.", "It cannot be gathered how these portions of the pants are fastened to the remaining portions of the pants.", "From U.S. Pat.", "No.", "4,961,233, a pair of pants for cyclists is further known which is made of synthetic nonwoven and heat-deformable leather.", "The pants are made of a single piece.", "It is the object of the invention to increase the wearing comfort of garments of stretch fabric knit in a fine-meshed manner, particularly of cycling pants.", "Especially, it is the object of the invention to avoid the occurrence of pressure marks, chafes and the like.", "This object is solved, according to the invention, with the features of claim 1.According to the invention, the garment is seamless.", "Thus, the entire garment does not have any bead-like seams.", "Particularly at those spots especially critical in cycling pants, for example, no bead-like seams are provided.", "Hence, the pants according to the invention do not comprise any bead-like seams at the thighs or in the seat region.", "Since the pants according to the invention do not have any seams, a good elasticity of the pants is ensured.", "In the region of seams, a so-called blocking occurs since cloth is not extensible or only very restrictedly extensible in the region of seams.", "This blocking also occurs directly next to the seam.", "Particularly in the crotch and seat region of the pants, respectively, an especially high stress as to the stretching ability of the pants, e.g.", "cycling pants or other sportswear, occurs.", "Since the pants according to the invention have no seam in the crotch and seat region, respectively, damage to the pants such as a tearing-up of seams, for example, cannot happen.", "According to the invention, for manufacturing a seamless garment of stretch fabric knit in a fine-meshed manner, a front part and a rear part are knit independently of each other.", "The knitting is done simultaneously so that the two parts are manufactured in parallel with each other.", "During the manufacture, the two parts are arranged such with respect to each other that the outer edges are adjacent to each other.", "During the knitting procedure, the front part and the rear part are knit together at a connecting edge without a seam.", "Thereby, it is possible to manufacture a garment which is completely seamless.", "Such a knitting is also effected along the inner seam of the legs of the pants so that no bead-like seam is produced in this region, either.", "Thus, the stretch fabric is smooth and free of beads along the connecting edges at both sides thereof.", "Since no seam is produced along the connecting edge due to the knitting, both the inside and the outside of the garment are free of beads and smooth.", "Particularly with tight-fitting garments such as cycling pants, this has the advantage that no pressure marks and chafes can be caused by seams.", "Because of the knitting along the connecting edge, this region is also free of grooves and steps.", "Preferably, the garment is manufactured from the same yarn.", "This means that apart from additional elements such as pockets and the like which are attached later on, the entire garment consists of one and the same material and no material with other properties is used in the production of seams.", "To produce an uninterrupted connecting edge that does not have any holes and the like either, each row of the front part is knit together with a row of the rear part.", "This means that upon knitting the front part row by row, the yarn of both parts is guided such when an outer edge is respectively reached that they are connected with each other, i.e., knit together.", "Hence, at each connecting edge, the number of connection points between the front and the rear part corresponds to the number of rows of the front and rear parts, respectively.", "To shape the garment, the number of rows of the front and rear parts may vary by a few rows.", "Preferably, the knitting of the front part to the rear part is effected during the knitting procedure of the front and rear parts.", "Thus, the front and rear parts are not knit separately from each other and joined subsequently but rather already connected with each other during the knitting procedure.", "This has the advantage that the additional operational step of connecting is eliminated and the connection is further effected with the yarn used for the front and rear parts, respectively.", "An additional thread for connecting the two parts is not required.", "Even in garments that are knit together according to the invention and do not have a seam, there is, in any case, an edge at least being visible against the light along the connecting edge.", "This is an edge merely visually visible which cannot be felt upon wearing.", "To reduce the visibility of this edge, the mesh width at the connecting edge is substantially as large as the mesh width of the front and rear parts.", "In the ideal case, it is possible to connect the front and rear parts with each other such that even along the connecting edge, no line is visible any more.", "In the front and/or rear part, the garment may comprise portions which are knit in a more wide-meshed manner.", "Such portions serve to increase the air permeability.", "This is particularly advantageous with cycling pants.", "Since the entire pants are knit, it is possible to produce the wide-meshed portions by controlling the knitting machine correspondingly.", "Therefore, it is not required to sew in portions with a more wide-meshed structure.", "Thus, portions with a more wide-meshed structure can be provided in the pants according to the invention without producing seams thereby which, in turn, cause pressure marks and the like.", "The manufacture of a pair of cycling pants or another garment is effected by a knitting machine with a front and a back needle bed.", "Through the needle bed, so-called double-needle-bar meshes are knit.", "These are extremely fine-meshed meshes.", "At the end of each row, the back needle bed, for example, produces a loop.", "The loop is taken up in front of a needle of the front needle bed so that the yarn of the front needle bed is threaded through the loop.", "Thereby, the border portions are knit together along a connecting edge.", "The same connection was made on the other outside of the two needle beds as well.", "In order to manufacture trouser legs, two further connecting edges have to be manufactured in the middle, extending at the inner surface of the thighs.", "Thus, the two needle beds are configured such that one of the needle beds throws out a loop in this region as well, which is taken up by the respectively other needle bed.", "By controlling the individual needles of the needle beds correspondingly, the length of the rows can be varied so that trouser legs can be knit in one piece, which, for example, become tighter downwards to have an as even contact with the thigh as possible.", "Due to the fact that, according to the invention, it is possible to manufacture the garments without seams it is possible to manufacture the cycling pants illustrated in the drawing.", "The cycling pants illustrated in the drawing is a schematic view of a pair of pants with straps.", "The cycling pants comprise a front part and a rear part 12 lying behind the front part 10 in the drawing.", "The front part 10 is knit together with the rear part 12 along outer connecting edges 14.Further, the front part 10 is knit together with the rear part 12 along an inner connecting edge 18 extending at the inside of the thighs in order to form trouser legs 16.The connecting edge 18 is configured such that the legs 16 of the pants taper towards the ends 20 of the legs of the pants.", "Thus, the lengths of the individual rows of the front part 10 and the rear part 12 vary.", "At an edge 22 of the front part 10 opposite to the trouser legs 16, two straps 24 are connected with the front part 10.At the same level, i.e., at the level of the waistband, the rear part 12 is connected with a back part 26.When the pants are worn in a conventional manner, the back part 26 fits the back of the cyclist.", "The straps 24 extend over the breast of the cyclist.", "If the weather is warm, the cyclist wears the pants such that the straps extend over the breast.", "Thus, a good cooling is effected in this region by the airstream.", "Then, the back part 26 serves to absorb sweat.", "If the weather is cold, the pants can be worn the other way round so that the back part 26 fits the cyclist's breast in front.", "Then, the back part serves to keep off the cold airstream.", "Turning the pants in such a manner, with the wearing comfort remaining the same, is only possible because of the seamless configuration of the cycling pants." ] ]

[ [ "Deflector devices", "A deflector device (22) for use with a tow lie between a towing vessel (10) and a tow (16) in water behind the vessel, the device comprising a principal wing-shaped body (28) shaped to produce in use a sideways force which urges the tow line (16) laterally with respect to the direction of movement of the towing vessel, a boom (32) extending rearwardly from the principal wing-shaped body (28), and an auxiliary wing-shaped body (52), smaller than the principal wing-shaped body (28), secured to the end of the boom (32) remote from the principal wing-shaped body (28) and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body (28)." ], [ "1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the principal wing-shaped body, and an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.", "2.A deflector device as claimed in claim 1, further comprising remotely-operable means for adjusting the angle between the boom and the principal wing-shaped body to vary the sideways force produced by the principal wing-shaped body.", "3.A deflector device as claimed in claim 2, wherein the auxiliary wing-shaped body is secured to the boom at or near its trailing edge, with its chord extending outwardly from the boom, on the side thereof opposite to the principal wing-shaped body, at an angle to the boom substantially equal to half the angle through which the boom is adjustable.", "4.A deflector device as claimed in claim 2 or claim 3, wherein the remotely-operable means includes a hydraulically-actuated telescopic strut.", "5.A deflector device as claimed in claim 1, wherein the angle between the boom and the principal wing-shaped body is substantially fixed, and further comprising remotely operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.", "6.A deflector device as claimed in claim 5, wherein the remotely-operable means comprises an electric motor.", "7.A deflector device as claimed in claim 5, wherein the remotely-operable means comprises a hydraulic actuator.", "8.A deflector device as claimed in any preceding claim, wherein the auxiliary, wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.", "9.A deflector device as claimed in any preceding claim, wherein the end of the boom remote from the principal wing-shaped body is adapted to be connected to the tow.", "10.A method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic streamers over an area to be surveyed, wherein the lateral position of at least one of the streamers is controlled by a deflector device in accordance with any one of the preceding claims." ], [ "This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.", "A deflector device of this kind is described in detail in our U.S. Pat.", "No.", "5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.", "In use, the wing-shaped deflector body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.", "As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.", "This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.", "The deflector device of U.S. Pat.", "No.", "5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.", "In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.", "However, under certain circumstances, eg in bad weather, at low towing speeds and when towing relatively short streamers, the drag produced by the tow can vary and/or become too low, to an extent greater than is desirable for stability purposes.", "It is an object of the present invention to alleviate this problem.", "As already mentioned, the lift produced by the deflector device of U.S. Pat.", "No.", "5,357,892 is varied by varying the angle of the boom attached to the wing-shaped body.", "This is achieved in current versions of the MONOWING deflector device by means of a hydraulic system which is disposed within the wing-shaped body, and which operates a telescopic actuator strut connected between the wing-shaped body and the boom to pivot the boom towards or away from the wing-shaped body.", "It is another object of the present invention, in one of its preferred embodiments, to provide a simpler and less power-consuming mechanism for varying the lift produced by the deflector device.", "According to the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a principal wing-shaped body shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the principal wing-shaped body, and an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body.", "In a first implementation of the invention, the device further comprises remotely-operable means for adjusting the angle between the boom and the principal wing-shaped body to vary the sideways force produced by the principal wing-shaped body.", "In this case, the auxiliary wing-shaped body is preferably secured to the boom at or near its trailing edge, with its chord extending outwardly from the boom, on the side thereof opposite to the principal wing-shaped body, at an angle to the boom substantially equal to half the angle through which the boom is adjustable.", "Also, the remotely-operable means preferably includes a hydraulically-actuated telescopic strut.", "In a second and preferred implementation of the invention, the angle between the boom and the principal wing-shaped body is substantially fixed, and the device further comprises remotely-operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.", "In this case, the remotely-operable means may comprise an electric motor or a hydraulic actuator.", "Advantageously, the auxiliary wing-shaped body is provided with a trailing edge flap angled away from the boom, typically at about 35°.", "The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic streamers over an area to be surveyed, wherein the lateral position of at least one of the streamers is controlled by a deflector device in accordance with any one of the preceding statements of invention.", "The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.", "1 is a partial schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.", "2 is a somewhat schematic part-sectional view of a first embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.", "1; FIGS.", "3 and 4 show different operating positions of part of the deflector device of FIG.", "2; FIG.", "5 is a somewhat schematic part-sectional view of a second embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.", "1; and FIG.", "6 is a front view of part of the deflector device of FIG.", "5.The seismic survey vessel shown in FIG.", "1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.", "PCT/GB98/01832 (WO 99/00295).", "The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.", "No.", "4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.", "PCT/IB98/01435 (WO 99/15913).", "The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.", "The deflector devices 22 are suspended from respective floats (not shown), and act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.", "One of the deflector devices 22 is shown in more detail in FIG.", "2.The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.", "No.", "5,357,892, but is a much improved version of it.", "In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.", "No.", "5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 29, both of which features enhance lift.", "Also, the main wing-shaped body 28 is provided with vortex controlling end plates (not shown) of the kind described in our PCT Patent Application No.", "PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight.", "Additionally, the angle lever 10 of U.S. Pat.", "No.", "5,357,892 is replaced by a rearwardly extending boom 32, which is pivotally connected at one end 34 to the low pressure side 36 of the body 28 near the midpoint of that side of the body, at a mounting bracket 38.The other end 40 of the boom 32 has a towing eye (not shown) which is coupled to the forward end 24 of a respective one of the two outermost streamers 18.Pivotal movement of the boom 32 is controlled by a mechanism comprising first and second struts 41, 42, which are pivotally connected to each other at 44 and to each end of the boom at 34 and 46, forming with the boom a triangle, and an extending hydraulic actuator strut 48 pivotally connected between the apex of the triangle, ie the pivotal connection point 44 of the struts 41, 42, and a pivotal connection point 50 positioned on the low pressure side 36 of the body 28 between its midpoint and its trailing edge.", "The actuator strut 48 is connected to be operated by a remotely-operable hydraulic control system (not shown) disposed within the body 28.It will be appreciated that extension of the hydraulic actuator strut 48, from its unextended position of FIG.", "2, will move the boom 32 outwardly from the low pressure side 36 of the body 28, from its closest position shown in FIG.", "2.The extent of the outward movement is preferably about 20°, as shown in FIGS.", "3 and 4.In accordance with the present invention, an auxiliary wing-shaped body 52, which is much smaller than the body 28 in length, thickness and chord, is secured to the end 40 of the boom 32 with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.", "2) extending parallel to the longitudinal axis of the body 28.The body 52 is fixedly secured to the boom 32 at or near the midpoint of its trailing edge 54, and its leading edge 56 is inclined away from the body 28 such that the chord of the body 52 (ie the line connecting its leading edge 56 and its trailing edge 54) is at an angle of about 10° to the boom 32.This angle is chosen because it is about half the angular extent of the movement of the boom 32.The shape of the body 52 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because it will be appreciated that the direction of the force varies in use as the boom 32 moves).", "This sideways force is increased by providing the body 52 with a fixed trailing edge flap 58, angled away from the boom 32 at an angle of about 35°.", "As the boom 32 is pivoted away from the body 28, the sideways, force produced by the body 52 acts as a restoring force, and thus varies the angle of the body 28 with respect to the direction of tow, so changing the lift produced by the body 28.This restoring force augments the restoring force produced by the drag of the towed streamers 18 (and in particular, reduces the effect of any stability-reducing variations or reductions in that drag).", "Indeed, the deflector device 22 will remain stable with no streamer attached, eg if its streamer 18 breaks or is severed at its forward end 24.FIGS.", "5 and 6 show at 60 an alternative embodiment of the deflector device 22 of FIGS.", "2 to 4, with corresponding parts having the same reference numbers as were used in FIGS.", "2 to 4.The principal difference between this alternative embodiment and the embodiment of FIGS.", "2 to 4 is that in the deflector device 60, the boom 32 is secured at a fixed angle, typically about 10°, to the low pressure side 36 of the main wing-shaped body 28, while the angle of the chord of the auxiliary wing-shaped body 52 with respect to the boom 32 is variable by means of a remotely operable electric stepper motor 62.As best seen in FIG.", "6, the electric stepper motor 62 is secured to the boom 32 with its axis extending laterally of the boom, and is disposed in a-cut-out or aperture 64 in the auxiliary wing-shaped body 52.Coaxial drive shafts 66 protrude from each axial end of the motor 62 and are secured to the body 52 to rotate it about the common axis of the drive shafts.", "A slot 65 is provided in the body 52 between the aperture 64 and the trailing edge of the body, to accommodate the boom 32 as the body 52 is rotated by the motor 62.As an alternative to the apertured and slotted implementation of the body 52, the body 52 can be implemented in two separate but symmetrical halves disposed on respective sides of the boom 32 and each attached to a respective one of the drive shafts 66 of the motor 62.The boom 32 is of sandwich construction: it is made of two similarly shaped plates 68 which are bolted together at intervals along their length and which sandwich between them both a mounting flange 70 of the motor 62 and the boom mounting bracket 38 secured to the low pressure side of the main wing-shaped body 28.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 60 is on the vessel 10, for ease of stowage.", "As in the embodiment of FIGS.", "2 to 4, the end 40 of the boom is provided with a towing eye, indicated at 74 in FIG.", "5, for connection to a streamer 18.However, as mentioned earlier, since stability is no longer dependent upon a streamer 18 being connected to the end 40 of the boom 32, the towing eye 74 can be omitted, and the streamer 18 can be towed from the lead-in 20 at a point near the attachment point of the deflector device 60.The same is true for the deflector device 22.It will be appreciated that varying the angle of the auxiliary wing-shaped body 52 of the deflector device 60 has the same effect as varying the angle of the boom 32 of the deflector device 22, ie i changes the angle of the main wing-shaped body 28 with respect to the direction of tow and so changes the lift produced by the main wing-shaped body.", "However, for the deflector device 60, less power is required to produce a given change in angle of the main wing-shaped body 28, because of the increased leverage provided by the position of the auxiliary wing-shaped body 52 towards the end 40 of the boom 32 (as opposed to the position of the hydraulically-operated actuator strut 48 of the deflector device 22).", "It is this which permits the use of the relatively low-powered electric stepper motor 62 in the deflector device 60, in place of the relatively more powerful hydraulic system which operates the mechanism based on the strut 48 in the deflector device 22.However, if desired, the electric stepper motor 62 can be replaced by a simple hydraulic actuator secured to the boom 32, since this also would not need to be as powerful as the hydraulic system which operates the mechanism based on the strut 48.An additional advantage of replacing the hydraulic system and the mechanism based on the strut 48 with the electric stepper motor 62 or a simple hydraulic actuator is the considerable weight saving which can be achieved.", "It will be appreciated that many other modifications can be made to the described embodiments of the invention.", "In particular, the deflector devices 22 and 60 can be used with tows other than streamers, for example seismic sources.", "Although the deflector devices 22 and 60 are adapted for use with separate floats, this is not an essential feature of the invention, since for example they can incorporate a float, as described in our co-pending United Kingdom Patent Applications Nos.", "0023775.0 and 0025719.6.And although the invention has been described in relation to deflector devices whose lift can be varied by varying the angle of the device with respect to the direction of tow, it is also applicable in its broadest aspect to a fixed angle deflector device, eg of the kind referred to as a “door”." ] ]

[ [ "Servo control system and its control method", "After a controller #1 has started giving an interpolating instruction, the length on an interpolation line is calculated by a first calculation means in synchronism with a clock signal from a clock synchronous circuit 45, and after having executed a first step for generating a synthetic locus-use frame 100 based upon the calculated value, the synthetic locus-use frame 100 is transmitted to controller #2 so that, after controller #2 has executed a second step for receiving a synthetic locus calculation-use frame from a receiving means, controllers #1, #2 execute a third step for calculating the position on the interpolation line based upon the synthetic locus-use frame 100 by using a second calculation means." ], [ "1.A servo-control system, comprising: a first controller which controls a first and third shafts; a second controller which controls a second and fourth shafts; wherein said first controller includes a first storing means in which information used for calculating the length on a first interpolation line is written, a first calculation means for reading the information from said first storing means to calculate the length on said first interpolation line, a transmitting means, and a receiving means, said second controller includes a second storing means in which information used for calculating the length on a second interpolation line is written, a second calculation means for reading the information from said second storing means to calculate the length on said second interpolation line, a transmitting means, and a receiving means, and said first and second controllers transmit or receive mutually the lengths on said first interpolation line and second interpolation line through said transmitting means or receiving means, whereby calculating the positions of said first and second axes based upon the length on the first interpolation line, and calculating the positions of said third and fourth axes based upon the length on the second interpolation line.", "2.A servo-control system according to claim 1, wherein the calculation of the length on the first interpolation line by said first calculation means is simultaneously executed with the calculation of the length on the second interpolation line by said second calculation means.", "3.A servo-control system according to claim 1, wherein said first and second controllers further comprise a fourth storing means respectively, the fourth storing means in said first controller stores information needed for interpolation instruction for said first and second axes, which includes a current position, a target position, a maximum speed value, an acceleration time, and a deceleration time, the fourth storing means in said second controller stores information needed for interpolation instruction to said third and fourth axes, which includes a current position, a target position, a maximum speed value, an acceleration time, and a deceleration time, the first and second controllers generate respectively respective-axis calculation frame every target position based upon the information from said fourth storing means to transmit or receive mutually the respective axis calculation frame before said target position control starts.", "4.A servo-control system according to claim 1, wherein said first and second controllers transmit or receive mutually an in-position instruction and an instruction type, along with the length on the first or second interpolation line, wherein the in-position instruction serves as a reaching instruction that is generated by reaching a predetermined range in the previous positioning instruction position and wherein the instruction type shows a controlled state including a control completion instruction.", "5.A servo-control system according to claim 1, wherein said first and second controllers further comprise an upper controller, the upper controller containing information needed for interpolation instruction to the first and second axes, including a current position, a target position, a maximum speed value, an acceleration time, and deceleration time; and information needed for interpolation instruction to the third and fourth axes, including a current position, a target position, a maximum speed value, an acceleration time, and deceleration time; the respective-axis calculation frame being generated based upon said information to transmit to said first and second controllers.", "6.A control method of a servo-control system using a first controller which controls a first and third shafts and a second controller which controls a second and fourth shafts, and mutually controlling between said first and second controllers, comprising the steps of: executing simultaneously a synthetic locus calculation needed for respective axes calculation for said first and second axes in said first controller and a synthetic locus calculation needed for respective axes calculation for said third and fourth axes in said second controller, transmitting the result of said synthetic locus calculation in the first controller to the second controller, transmitting the result of said synthetic locus calculation in the second controller to the first controller, and executing respective axes calculation for said first to fourth axes based upon the result of said synthetic locus calculations." ], [ "<SOH> BACKGROUND ART <EOH>Referring to FIG.", "8 , a conventional servo control system which is disclosed in Japanese Laid-Open Patent Publication No.", "9-269811, will be explained.", "In FIG.", "8 , the servo control system is constituted by a host CPU 6 which outputs controlling instructions to the entire system as an upper controlling unit, a plurality of servo-CPUs 8 , 9 which executes the same calculations as the host CPU 6 and serve as lower controlling unit, drivers D 1 to D 3 which are connected to the servo-CPU 8 and also connected to servo motors M 1 to M 3 of an orthogonal-type robot (Cartesian type robot) 15 , and drivers D 4 , D 5 which are connected to the servo-CPU 9 and also connected to servo motors M 4 ,M 5 of a joint-type robot 17 .", "To the host CPU 6 are connected a key board 10 through which positional data for positioning points, for example, are inputted, an instruction device 11 for giving instructions about positional data as to positioning target points, and an external input-output circuit 13 that allows transmitting and receiving operations to or from an external device.", "The following description will discuss an operation of the servo-control system having the above-mentioned arrangement shown in FIG.", "8 .", "Upon starting an interpolation controlling process, the host CPU 6 transmits operating instructions for the next target point etc., to the servo-CPUs 8 and 9 , and then the servo-CPUs 8 , 9 execute the same calculations respectively for the synchronous control.", "Based on the result of the above calculations, the servo-CPUs 8 , 9 execute the position feed-back controlling calculations of the respective servo-motors M 1 to M 3 , M 4 , M 5 to transmit positioning completion instructions to the host CPU 6 .", "In the above-mentioned servo control system, the respective servo-CPus 8 , 9 to which operating instructions are transmitted from the host CPU 6 can execute the same calculations respectively to carry out the synchronous control among the robots 15 and 17 .", "Therefore, the above-mentioned system provides an effective control system for synchronous control when the number of controlled axes is comparatively small.", "However, in a servo control system in which a number of motors are synchronously controlled among the respective servo-CPUs 8 , 9 for interpolation controls, for example, in a servo control system for driving a tire molding machine with a number of control axes, as will be described later, the calculation time required in the respective servo-CPUs 8 , 9 increases as the number of motors to be synchronously controlled increases, when each servo CPUs 8 and 9 need to execute the same calculations.", "This results in a longer interpolation controlling time.", "In order to solve this problem, utilization of those servo-CPUs 8 , 9 with a higher processing speed is recommendable, however, there is an inevitable limitation to the processing speed.", "Here, in the case when respective motors are mutually subjected to an interpolation controlling process, with respect to multiple axes M 1 -M 5 as shown in FIG.", "8 , these are simply shown by two axes form, that is, X-axis and Y-axis as shown in FIG.", "9 .", "In FIG.", "9 , the interpolation controlling calculations consist of synthetic locus calculations for calculating a length Lt 1 on an interpolation line and respective-axis calculations for calculating positions Xt 1 and Yt 1 of the respective axes based on the length Lt 1 on the interpolation line.", "Since the synthetic locus calculations are expected to be common to the respective motors, there is no need for the respective servo-CPUs 8 , 9 to execute the same synthetic locus calculations, respectively.", "Therefore, it is possible that either one of the servo CPU 8 ( 9 ) executes the synthetic locus calculations, and the other respective servo CPUs 8 , 9 execute respective-axis calculations using the resultant value of the synthetic locus calculations.", "However, the respective servo CPUs 8 , 9 can not execute the respective axis calculations while one of the servo CPU 8 ( 9 ) is executing the synthetic locus calculations, therefore, the total interpolation-control processing time which is the sum of the synthetic locus calculation time and respective axis calculation time is not substantially reduced.", "The present invention has been made to solve the above-mentioned problem in the servo-control system which carries out an interpolation control of respective axes of motors, and the object of the invention is to provide a servo control system and a control method thereof which can decrease the interpolation-control processing time of this entire system among two or more controllers without using CPU with a higher processing speed." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG.", "1 is a schematic front view of the tire molding equipment which is driven and controlled by a servo control system in accordance with one embodiment of this invention; FIG.", "2 is a block diagram of the entire servo control system showed in FIG.", "1 ; FIG.", "3 ( a ) is a block diagram showing the synthetic locus calculation frame which is used in the system shown in FIG.", "2 ; FIG.", "3 ( b ) is a block diagram of a frame for respective axes calculation; FIG.", "4 is an explanatory curve showing an interpolation controlling process which is executed by the servo control system shown in FIG.", "1 , in which positioning points P 0 , P 1 , P 2 , P 11 , P 12 are indicated on a plane defined by the X-Y axes; FIG.", "5 is a flow chart showing a process in which a target is shifted to positioning points shown in FIG.", "4 by using the servo control system of FIG.", "1 ; FIG.", "6 is a time chart showing a process in which a target is shifted to positioning points, P 0 , P 1 , P 2 , Pl 1 , P 12 shown in FIG.", "4 , by using the servo control system of FIG.", "1 ; FIG.", "7 is a block diagram showing the entire constitution of a servo control system according to another embodiment of this invention; FIG.", "8 is a schematic diagram showing the entire constitution of the conventional servo control system; FIG.", "9 is a curve showing the positioning points P 0 , P 1 , and P 2 .", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to a servo control system having a plurality of servo-controllers, and more specifically relates to interpolation controlling of respective axes of servo-motors.", "BACKGROUND ART Referring to FIG.", "8, a conventional servo control system which is disclosed in Japanese Laid-Open Patent Publication No.", "9-269811, will be explained.", "In FIG.", "8, the servo control system is constituted by a host CPU 6 which outputs controlling instructions to the entire system as an upper controlling unit, a plurality of servo-CPUs 8, 9 which executes the same calculations as the host CPU 6 and serve as lower controlling unit, drivers D1 to D3 which are connected to the servo-CPU 8 and also connected to servo motors M1 to M3 of an orthogonal-type robot (Cartesian type robot) 15, and drivers D4, D5 which are connected to the servo-CPU 9 and also connected to servo motors M4,M5 of a joint-type robot 17.To the host CPU 6 are connected a key board 10 through which positional data for positioning points, for example, are inputted, an instruction device 11 for giving instructions about positional data as to positioning target points, and an external input-output circuit 13 that allows transmitting and receiving operations to or from an external device.", "The following description will discuss an operation of the servo-control system having the above-mentioned arrangement shown in FIG.", "8.Upon starting an interpolation controlling process, the host CPU 6 transmits operating instructions for the next target point etc., to the servo-CPUs 8 and 9, and then the servo-CPUs 8, 9 execute the same calculations respectively for the synchronous control.", "Based on the result of the above calculations, the servo-CPUs 8, 9 execute the position feed-back controlling calculations of the respective servo-motors M1 to M3, M4, M5 to transmit positioning completion instructions to the host CPU 6.In the above-mentioned servo control system, the respective servo-CPus 8, 9 to which operating instructions are transmitted from the host CPU 6 can execute the same calculations respectively to carry out the synchronous control among the robots 15 and 17.Therefore, the above-mentioned system provides an effective control system for synchronous control when the number of controlled axes is comparatively small.", "However, in a servo control system in which a number of motors are synchronously controlled among the respective servo-CPUs 8, 9 for interpolation controls, for example, in a servo control system for driving a tire molding machine with a number of control axes, as will be described later, the calculation time required in the respective servo-CPUs 8, 9 increases as the number of motors to be synchronously controlled increases, when each servo CPUs 8 and 9 need to execute the same calculations.", "This results in a longer interpolation controlling time.", "In order to solve this problem, utilization of those servo-CPUs 8, 9 with a higher processing speed is recommendable, however, there is an inevitable limitation to the processing speed.", "Here, in the case when respective motors are mutually subjected to an interpolation controlling process, with respect to multiple axes M1-M5 as shown in FIG.", "8, these are simply shown by two axes form, that is, X-axis and Y-axis as shown in FIG.", "9.In FIG.", "9, the interpolation controlling calculations consist of synthetic locus calculations for calculating a length Lt1 on an interpolation line and respective-axis calculations for calculating positions Xt1 and Yt1 of the respective axes based on the length Lt1 on the interpolation line.", "Since the synthetic locus calculations are expected to be common to the respective motors, there is no need for the respective servo-CPUs 8, 9 to execute the same synthetic locus calculations, respectively.", "Therefore, it is possible that either one of the servo CPU 8 (9) executes the synthetic locus calculations, and the other respective servo CPUs 8, 9 execute respective-axis calculations using the resultant value of the synthetic locus calculations.", "However, the respective servo CPUs 8, 9 can not execute the respective axis calculations while one of the servo CPU 8 (9) is executing the synthetic locus calculations, therefore, the total interpolation-control processing time which is the sum of the synthetic locus calculation time and respective axis calculation time is not substantially reduced.", "The present invention has been made to solve the above-mentioned problem in the servo-control system which carries out an interpolation control of respective axes of motors, and the object of the invention is to provide a servo control system and a control method thereof which can decrease the interpolation-control processing time of this entire system among two or more controllers without using CPU with a higher processing speed.", "DISCLOSURE OF INVENTION A servo control system according to a first invention is provided with: a first controller which carries out an interpolation-controlling process by calculating the length on a first interpolation line based on respective first axes of the first and second motors, and a second controller which carries out an interpolation-controlling process by calculating the length on a second interpolation line based on respective second axes of the third and fourth motors, wherein the first controller includes a first storing means for storing information used for calculating the length on the first interpolation line, wherein the second controller includes a second storing means for storing information used for calculating the length on the second interpolation line, wherein the first and second controllers include a third storing means for storing information used for calculating the positions of the first and second respective axes based on the lengths on the first and second interpolation lines, a first calculating means which, after initiation of the interpolation instruction, reads out information stored in the first and second storing means to calculate the lengths on the first and second interpolation lines, and which produces a first frame based on the resultant calculated values, a first transmitting means for transmitting the first frame to the other of the controllers, a receiving means for receiving the first frame, and a second calculating means which reads out information stored in the second storing means to calculate the positions of the respective first and second axes based on the lengths on the first and second interpolation lines.", "According to such a servo control system, the first and second controllers allow the first calculating means to calculate the lengths on the first and second interpolation lines, and the first frame, formed based on the calculated value, is transmitted from the first controller to the second controller as well from the second controller to the first controller by the first transmitting means, and after the first and second controllers have received the lengths on the interpolation lines through the receiving means, the positions of the respective first and second axes are calculated by the second calculation means based on the lengths on the first and second interpolation lines.", "Therefore, since after the first and second controllers have simultaneously calculated the lengths on the first and second interpolation lines by the first calculation means, the positions of the respective first and second axes are calculated by the second calculation means, it is possible to shorten the interpolation controlling time in comparison with a servo control system in which, after the first controller has calculated the length on the first interpolation line, the second controller successively calculates the length on the second interpolation line to calculate the positions of the respective first and second axes.", "Here, the interpolation controlling time refers to the sum of a time for calculating the length on the interpolation line and a time for calculating the positions of the respective axes.", "A servo control system according to the second invention is characterized in that the first and second controllers in the first invention further include: the fourth storing means for storing an interpolation instruction, a respective-axes instruction generating means which reads out the interpolation instruction from the fourth storing means based on initiation of the interpolation instruction to generate instruction values for the respective first and second axes and a second frame based on the instruction values, the second transmitting means for transmitting the second frame to the other controller, and the second receiving means for receiving the second frame.", "According to the servo control system, the respective-axes instruction generating means generates the respective-axes instructions for calculating the positions of the respective axes, and the second frame is transmitted to the other controller so that the first and second controllers are allowed to receive the respective-axes instructions.", "Therefore, since the positions of the respective axes are calculated by the respective axes instruction generating means for each of the interpolation instructions, it becomes possible to construct a servo-control system by using only the controllers without receiving the respective-axes instructions from upper controllers.", "A servo control system according to the third invention is characterized in that the servo control system according to the first invention further include an upper controller for transmitting an interpolation instruction to the first and second controllers, wherein the upper controller is provided with a respective-axes instruction generating means which generates first and second respective-axes instructions based on the interpolation instruction, and transmits the first and second respective-axes instructions to the first and second controllers, and the first and second controllers have the third receiving means for receiving the first and second respective-axes instructions.", "According to the third invention, the upper controller allows the respective-axes instruction generating means to generate the respective-axes instructions used for calculating the positions of the respective first and second axes, and transmits these to the first and second controllers so that the second controller receives the respective-axes instructions.", "Therefore, since the first and second controllers need not to generate the first and second respective-axes instructions, it is possible to reduce the operation load on the first and second controllers, and the operation load on the upper controller is also mitigated since it only needs to generate the respective-axes instructions.", "According to the fourth invention, a control method for the servo control system comprising a first controller which carries out an interpolation controlling process by calculating the length on the first interpolation line based on the respective first axis of the first and second motors, and a second controller which carries out an interpolation controlling process by calculating the length on the second interpolation line based on the respective second axes of the third and fourth motors, includes: a first step, after the first and second controllers have started the interpolation instructions, calculating the lengths on the first and second interpolation lines by using the first calculation means, to generate a first frame based on the calculated value, a second step of transmitting the first frame from either of the first and second controllers to the other controller by the transmission means, so that the first frame is received by the other controller through the transmission means, and a third step of calculating the positions of the respective first and second axes based on the first frame by using the second calculation means in the first and second controllers.", "According to the control method for the servo control system, since the first and second controllers simultaneously calculate the lengths on the first and second interpolation lines by using the first calculation means, and calculate the respective first and second axes positions based on the calculated lengths on the first and second interpolation lines, it is possible to shorten interpolation controlling time in comparison with the conventional system in which the first and second controllers calculate sequentially the length on the first interpolation line and the length on the second interpolation line.", "The control method of the servo control system according to the fifth invention is characterized in that the first to third steps according to the third invention are executed after the first and second controllers allowed the respective-axes instruction generating means to generate the instruction values of the respective first and second axes based on the start of the interpolation instruction, and after having transmitted the second frame formed based upon the instruction values of the respective axes to the other controller.", "According to the control method, a servo control system can be built only by controllers, without receiving respective axial instructions from an upper controller, since the respective axial instruction generating means calculates the respective axial position for every interpolation instruction.", "The control method of the servo control system according to the sixth invention is characterized in that the first to third steps according to the fourth invention are executed after an upper controller generated the instructions for the respective first and second axes based on the start of the interpolation instruction, and after having transmitted the instruction for the respective first and second axes to the first and second controllers.", "According to the control method of the servo control system, there is no need for the first and the second controllers to generate the respective axes instructions.", "Therefore, the calculations load of the first and second controllers is mitigated and the calculations load of the upper controller is also effectively reduced, since it only generates respective axes instructions.", "A servo control system according to the seventh invention is characterized in that the first frame according to the first or second invention includes a reaching instruction that is generated when a predetermined range of the previous positioning instruction position has been reached based on the calculated values of the respective first and second axes.", "According to the servo control system, the next positioning instruction can be effectively generated in accordance with the reaching instruction based on the calculated values of the respective first and second axes.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic front view of the tire molding equipment which is driven and controlled by a servo control system in accordance with one embodiment of this invention; FIG.", "2 is a block diagram of the entire servo control system showed in FIG.", "1; FIG.", "3 (a) is a block diagram showing the synthetic locus calculation frame which is used in the system shown in FIG.", "2; FIG.", "3 (b) is a block diagram of a frame for respective axes calculation; FIG.", "4 is an explanatory curve showing an interpolation controlling process which is executed by the servo control system shown in FIG.", "1, in which positioning points P0, P1, P2, P11, P12 are indicated on a plane defined by the X-Y axes; FIG.", "5 is a flow chart showing a process in which a target is shifted to positioning points shown in FIG.", "4 by using the servo control system of FIG.", "1; FIG.", "6 is a time chart showing a process in which a target is shifted to positioning points, P0, P1, P2, Pl1, P12 shown in FIG.", "4, by using the servo control system of FIG.", "1; FIG.", "7 is a block diagram showing the entire constitution of a servo control system according to another embodiment of this invention; FIG.", "8 is a schematic diagram showing the entire constitution of the conventional servo control system; FIG.", "9 is a curve showing the positioning points P0, P1, and P2.BEST MODE FOR CARRYING OUT THE INVENTION Embodiment 1 Referring to FIG.", "1 to 4, a servo control system according to an embodiment of the present invention will be explained.", "FIG.", "1 is a schematic front view of the tire molding equipment in which drive control is carried out by a servo control system according to this invention.", "FIG.", "2 is a block diagram showing the entire servo-control system for driving the tire molding equipment showed in FIG.", "1.FIG.", "3(a) is a block diagram showing the synthetic locus calculation frame used for the system of FIG.", "1.FIG.", "3(b) is a block diagram of the respective-axes calculation frame.", "FIG.", "4 is a curve that shows an interpolation controlling process executed by the servo control system of FIG.", "1, which includes positioning points P0, P1, P2, P11, P12 on the X-Y axis plane.", "In FIG.", "1, a tire molding apparatus 20 for molding a tire using a servo control system is provided with a servo motor M1 for rotating a drum 21 which forms a tire and which is placed in the center; an A material supplying device 22 for supplying a tire forming material at the left-hand side of the drum 21 from a viewpoint facing to the drawing; and a B material supplying device 24 for supplying a tire forming material at the right-hand side of the drum 21.The A material supplying device 22 in the left-hand side comprises a first material supplying unit and a second material supplying unit.", "The first material supplying unit is provided with belt-conveyers B2, B3 by which the material a is supplied and which is driven by servo motors M2, M3, and a guiding belt-conveyer B6 which is located in the neighborhood of the dram 21 and having the servo motor M6.The second material supplying unit is provided with belt-conveyers B4, B5 by which the material b is supplied and which is driven by servo motors M4, M5.The B material supplying device 24 in the right-hand side comprises a third material supplying unit and a fourth material supplying unit.", "The third material supplying unit is provided with belt-conveyers B8, B9 by which the material c is supplied and which is driven by servo motors M8, M9, and a guiding belt-conveyer B7 which is located in the neighborhood of the dram 21 and having a servo motor M7.The fourth material supplying unit is provided with belt-conveyers B10, B11 by which the material d is supplied and which is driven by servo motors M10, M11.In order to supply the tire materials a to d to the dram 21, the tire molding apparatus 20 is designed in such a manner that the servo motors M2 through M6 and M8 through M11 which constitutes the above-mentioned material supplying units, the servo motor M1 of the dram 21, and the servo-motors M6 (M7) of the guiding belt-conveyers B6 (B7) are synchronously controlled properly each other (interpolation-controlled).", "Here, in general, the tire molding apparatus 20 uses approximately 20 kind of materials, and about 100 axes in most cases, but FIG.", "1 shows only a simplified structure.", "In FIG.", "2, the servo control system 25 is provided with controllers #1 to #3 which is connected to a bus 30 and drive servo motors M1-M11.The controller #1 serving as the first controller is connected to servo motors M1 to M4 through the servo drivers 71a to 71d, the controller #2 serving as the second controller is connected to servo motors M5 to M8 through the servo drivers 72a to 72d, and the controller #3 is connected to servo motors M9 to M11 through the servo drivers 73a to 73c.", "Thus, in order to carry out the synchronous control (interpolation control), the respective axes of the servo motors M2 to M5 and M8 to M11 which form the respective material supplying units, the servo motor M1 of the drum 21, and the servo motor M6 (M7) of guide-use belt-conveyor B6 (B7) are constituted so that predetermined controlling operation are carried out between controller #1 and controller #2 (#3).", "Respective controllers #1 to #3 are provided with bus-control-use I/Fs 31 to 33 that are connected to a bus 30, CPUs 41-43 which are connected to the I/F 31-33 through interrupt control circuits 35 to 37, clock synchronous circuits 45 to 47 which are connected to bus-control-use I/Fs 31 to 33 and serving as a clock generating means for generating clock signals with a constant cycle, memory 51 to 53 connected to CPUs 41-43, and communication-use I/F 61-63.The respective servo drivers 71a to 71d, 72a to 72d, and 73a to 73d are connected to the communication-use I/F 61 to 63.Memory 51 to 53 are respectively provided with synthetic locus calculation unit 51b serving as the first storing means, synthetic locus calculation units 52b, 53b serving as the second storing means, respective axes calculation units 51c to 53c serving as the third storing means, and control instruction units 51a to 53a serving as the fourth storing means.", "In FIG.", "4, the interpolation control on respective axes consists of the synthetic locus calculations for calculating the length Lt1 and Lt11 on the interpolation line, and the respective axes calculations for calculating positions Xt1, Yt1 on the first respective axes (on interpolation line) based on the length Lt1 on the interpolation line and for calculating positions Xt1 and Yt11 on the second respective axes (on interpolation line) based on the length Lt11 on the interpolation line.", "The control instruction unit 51a stores a starting position (current value) P0 serving as an interpolation instruction, target positions P1, P2, a maximum speed value, an acceleration time, and a deceleration time.", "The control instruction unit 52a stores a starting position (current value) P0 serving as an interpolation instruction, target positions P11, P12, a maximum speed value, an acceleration time, and a deceleration time.", "Furthermore, the control instruction units 52a stores a starting position (current value) P0 serving as an interpolation instruction, target positions P21 and P22 (not shown) a maximum speed value, an acceleration time, and a deceleration time.", "The synthetic locus calculation unit 51b stores parameters such as a total shift distance, a speed, an acceleration time, and a deceleration time, so as to carry out the synthetic locus calculations used as information on the interpolation line between a starting position P0 and target positions P1, P2.The unit 51b also stores a predetermined interpolation calculation expression and a calculated synthetic locus calculation values.", "The synthetic locus calculation unit 52b (53b) stores parameters such as a total shift distance, a speed, an acceleration time, and a deceleration time, so as to carry out the synthetic locus calculations used as information on the interpolation line between a starting position P0 and target positions P11 (P12), P12 (P22), and the unit 52b (53b) also stores a predetermined interpolation calculation expression and a calculated synthetic locus calculations values.", "Each of the respective axes calculation units 51c to 53c stores information such as a current position, a total shift distance and a total shift amount of each axis.", "Moreover, each of the units 51c to 53c also stores a predetermined interpolation calculation expression and calculated respective axial calculation values.", "FIG.", "3(a) shows a format of the synthetic locus frame 100 serving as the first frame which is transmitted between controllers #1 to #3, for example, synthetic locus calculation value calculated by a controller #1 is transmitted to the other controllers #2 and #3.A synthetic locus frame 100 comprises ID101 which has the identification number for identifying controllers #1 to #3, point number 103 which shows the interpolation stage by defining points P0 to P1 as a number 1 and points P1 to P2 as a number 2, an instruction type 105 that shows a controlled state and consists of an in-position instruction 105a and a control completion instruction 105b, wherein the in-position instruction 105a serves as a reaching instruction that is generated by reaching a predetermined range in the previous positioning instruction position, for example, and a synthetic locus calculation value 107.FIG.", "3 (b) shows a format of the respective axes calculation frame 200 as the second frame transmitted between controllers #1 to #3.For example, the instructions for executing the respective-axes calculation stored in control instruction unit 51a of controller #1 are transmitted to other controllers #2 and #3.The respective-axes calculation frame 200 comprises controllers #1- to #3, point number 203 which is the same as the point number 103 mentioned above, a control type 205 which shows linear interpolation, circular interpolation, etc., an interpolation target axis 207 which specifies servo motors M1, M6, for example, as a target used for an interpolation operation, and a total shift amount 209 used as the distance between the positioning points of the respective axes and total shift amount 211 of each of the X and Y axes.", "The total shift amount 209 of controller #1 generates the distance between the first point P0 and the points P1 and P2 with respect to positioning points of the respective axes to specify servo-motors M1, M6 as the interpolation target axis 207.On the other hand, the total shift amount 209 of controller #2 generates the distance between the first point P0 and the points P11 and P12 with respect to positioning points of the respective axes to specify servo motors M4, M5 as the interpolation target axis 207.Referring to FIGS.", "1 to 6, the following description will discuss an operation for driving a tire molding machine using the servo-control system mentioned above.", "FIG.", "5 is a flowchart that shows the operation of the servo control system shown in FIG.", "1, and FIG.", "6 is a time chart thereof.", "In the tire molding apparatus 20, the drum 1 and belt conveyors B4, B5, B6 are synchronously operated each other, and in addition to this synchronous operation, the belt conveyors B10 and B11, and the belt conveyors B8 and B9 are also synchronously operated respectively.", "Here, an explanation will be given of a simplified case in which the servo-motors M1, M6 serving as the first and second motors and the servo-motors M4, M5 serving as the third and fourth motors are synchronously controlled between the respective controllers #1 and #2.In the case when the servo-motors M1, M6 for driving the drum 21 and the belt conveyor B6, as well as the servo-motors M4, M5 for driving the belt conveyors B4, B5, are synchronously controlled between the respective controllers #1 and #2, the first respective axes (corresponding to the X axis) of the servo-motors M1, M5 are respectively rotated with predetermined amounts, while the second respective axes (corresponding to the Y axis) of the servo-motors M6, M4 are respectively rotated with predetermined amounts; therefore, as shown in FIG.", "4, the synchronous control of this type may be regarded as interpolation control.", "Therefore, an explanation will be given on the assumption that, as shown in FIG.", "4, the servo-motors M1, M4, M5, M6 are controlled to shift positioning points from P0 to P2 through P1, and from P0 to P12 through P11 on the biaxial plane of the X and Y axes.", "Referring to FIGS.", "1 to 6, the operation of the servo-control system mentioned above will be explained below.", "Here, calculation clocks are inputted to the bus-control-use I/Fs 31 to 33 by the clock synchronous circuits 45 to 47.When, at time t1, a starting instruction for interpolation is inputted to each of controllers #1, #2 from an external device (not shown) (step 201), the CPUs 41, 42 of controllers #1, #2 generate respective-axes calculation-use frames 200 for calculating the respective-axes instructions for points P1, P11 based upon stored information from the control instruction units 51a, 52a (respective-axes instruction generating means, step S203).", "At time t2, the CPU 41(42) transmits the respective-axes calculation-use frame 200 to the bus-control-use I/F 31(32)→>the bus 30→the bus-control-use I/F 32(31) (the second transmission means, step S205), and the CPU 42(41) of controllers #2, #1 receives the respective-axes calculation-use frame 200 through the bus-control-use I/F 32 (31) as a slave process (the second receiving means), and stores this in the respective-axes calculation unit 52c(51c) of the memory 52(51) (step S221).", "At time t3, the CPU 41(42) of controllers #1, #2 activates an interpolation control signal serving as an interpolation instruction so that an interrupt signal is generated from the interrupt control circuit 35(36) to transmit the interrupt signal to the bus-control-use I/F31(32)→bus30→bus-control-use I/F 32(31) (step S207).", "The CPU 42(41) of controller #2 (#1) receives the interrupt signal through the bus-control-use I/F 32(31) as a slave process (step 223), and determines whether or not the corresponding axis is managed by its own controller #1, #2 through the interpolation target axis 207 of therespective-axes calculation-use frame 200 (step S225).", "When the axis is managed by its own controller #1, #2, a flag for making the respective-axes calculation-use frame 200 of the respective-axes calculation unit 51c(51c) of the memory 51(52) effective is set to 1 (step S209, S227), it is determined whether or not the respective-axes calculation-use frame 200 is effective (step S211, S229).", "Here, since this is effective, at time t4, the CPU41(42) of controller #1(#2) functions as the first calculation means in synchronism with the calculation clock (clock signal) of the clock synchronous circuit 45, that is, it obtains a shift distance Lt1 (Lt11) of a synthetic locus as a length on the interpolation line between points P0-PL (P0-P11) at the current time, and in accordance with the controlling state of the interpolation, for example, by the fact that the target has reached a predetermined range in the previous positioning instruction position, the in-position instruction 105a, etc.", "are set, and the synthetic locus-use frame 100 containing a synthetic locus calculated value is generated (first step, step S213).", "At time t5, the CPU 41(42) of controller #1(#2) transmits this to bus-control-use I/F 31(32)→>bus 30→>bus-control-use I/F 32(31) (first transmission means, step S215), and the CPU 42(41) of controller #2 (#1) receives the synthetic locus-use frame 100 through the bus control use I/F32 (31) and executes the second step, and reads out the synthetic locus use frame 100 from the bus control use I/F 32, and stores this in the synthetic locus calculation unit 52b (51b) in the memory 52 (51) (step S231).", "At time t6, the CPU41, (42) of controllers #1, #2 functions as a second calculation means, that is, calculates to determine the coordinates of X, Y axes managed by controllers #1, #2, by using the shift distance Lt1 (Lt11) of the synthetic locus between point P0-P1(P0-P11) and the respective-axes calculation-use instruction stored in the respective-axes calculation unit 52b (52b) of the memory 52(51) (the third step, step S217, S233).", "Controllers #1, #2 repeatedly execute the above-mentioned steps S201 to S237 until the completion of the interpolation control, thereby completing the interpolation process (step S219, S235), and a flag which nullifies the respective-axes calculation-use frame 200 of the respective axes calculation unit 51c (51c) of the memory 51 (52) is set to 0 (step S221, S237), thereby completing the operation.", "The respective-axes calculation-use frame 200 is generated by controllers #1, #2 for each point, and the respective-axes calculation-use frame 200 of points P2, P12 are back-ground processed, calculated before the start of control of points P2, P12, and transmitted to controllers #1, #2.Moreover, at the time of deceleration and stop, the in-position signal 105a serving as the reaching instruction for executing the in-position process is set in the instruction type 105 so that, during the time from the start of deceleration and stop to the positioning to each point, the composite locus-use frame 100 is transmitted to controller #2, and the respective controllers #1, #2 are allowed to execute the process at the time of deceleration and stop in addition to the respective-axes calculations.", "In a servo-control system 25 having servo-motors M1, M6 and servo-motors M4, M5 that are interpolation targets between these different controllers #1, #2, the synthetic locus calculation Lt1 for interpolation controlling so as to set point P0→P1 in controller #1 and the synthetic locus calculation Lt1 for interpolation controlling so as to set point P0→P1 in controller #2 are simultaneously executed, and the synthetic locus calculation value Lt1 of controller #1 is transmitted to controller #2 while the synthetic locus calculation value Lt11 is transmitted to controller #1.Thus, controllers #1, #2 are able to carry out respective axes calculations based upon synthetic locus calculation values Lt1, Lt11, so that it becomes possible to shorten the processing time of interpolation control as the servo-control system 25.In other words, in the case when the same interpolation control calculation is carried out by the respective controllers #1, #2 as in the conventional case, the interpolation controlling time is set to 15 μsec×2+2.5 μsec 32.5 μsec, supposing that the execution time of the composite locus calculation is 15 μsec and the execution time of the respective-axes calculation is 2.5 μsec, while the interpolation controlling time of the servo-control system 25 of the present invention is 15 μsec+2.5 μsec=17.5 μsec.", "The ratio k of the interpolation controlling times is indicated as follows: K=17.5/32.5=0.54 Therefore, in the servo-control system 25 of the present embodiment, the interpolation controlling time is reduced to approximately half the conventional interpolation controlling time.", "Preferred Embodiment 2 Referring to FIG.", "7, the following description will discuss another preferred embodiment of the present invention.", "As shown in FIG.", "7, an upper controller 101 is installed in the servo-control system 25, and the upper controller 101 may have functions corresponding to control instruction units 51a to 53a in memories 51 to 53 installed in the respective controllers #1 to #3 of FIG.", "2, functions for transmitting interpolation instructions to controllers #1 to #3, and functions of the respective-axes instruction generating means for generating the respective axes instructions by using interpolation instructions and for transmitting these instructions to controllers #1, #2.In accordance with such a servo-control system 25, the processes carried out in step S203 and S205 in FIG.", "5 can be executed by the upper controller 101 in a substituting manner.", "INDUSTRIAL APPLICABILITY As described above, the servo-control system and its controlling method of the present invention are suitably applied to, for example, a tire molding apparatus." ] ]

[ [ "Contact spring assembly", "The present invention relates to a method of securing a reliable electrical contact between two adjacent surfaces by means of a contact spring (30) of a contact spring assembly (1).", "The secure and reliable contact is achieved by providing excellent conditions for maintaining the contact spring fully operable at all times and is accomplished by providing a shelter (12) for the contact spring (30) on a carrier (10) therefore, thereby protecting the contact spring by eliminating the possibility of the contact spring being overloaded and plastically deformed during installation." ], [ "1.A method of providing an effective shielding or grounding of electronic equipment while securing a reliable contact between two adjacent surfaces of the electronic equipment, comprising the steps of: providing a contact spring assembly having a carrier and a contact spring; and providing on the carrier a protection for sheltering the contact spring against overloading or permanent deformation.", "2.The method of claim 1, wherein the contact spring is formed having several individual contact spring elements provided next to each other, and having spring element receiving pockets in an upper side of the carrier, facing the spring elements in the assembled condition, wherein the pockets receive individual contact spring elements therein when the said contact spring elements are subjected to an excessive load.", "3.The method of claim 2, wherein the pockets are formed having a depth exceeding the thickness of the contact spring material.", "4.The method of claim 2, wherein lands are formed on the upper side of the carrier having interspaces between the lands forming the spring element receiving pockets, such that the lands absorb any excessive load applied to the contact spring or to individual spring elements.", "5.The method of claim 2, wherein the the contact spring is secured to the carrier by forming the contact spring so as to at least partly surround the carrier, by extending the spring element receiving pockets and the lands around the longitudinal side edges of the carrier.", "6.The method of claim 1, wherein the contact spring carrier is secured to one of the surfaces by providing attachment tabs forming a snap-in connection.", "7.The method of claim 2, wherein the spring element receiving pockets are formed having a width exceeding a width of the respectively associated spring elements, providing a clearance between respective side edges of the spring elements and of the pockets and providing free displacement of the spring elements into the respectively associated pockets in an overload situation.", "8.The method of claim 2, wherein positioning guides are formed on inner side surfaces of at least one of the pockets providing correct positioning of the contact spring in relation to the carrier by centering each spring element sideways with respect to its associated pocket.", "9.A contact spring assembly, adapted to shield or ground electronic equipment, comprising: a contact spring having several individual contact spring elements provided side by side; and a contact spring carrier, adapted to engage a surface of the electronic equipment, supporting and at least partially surrounded by the contact spring, having spring element receiving pockets formed in its upper side that face the spring elements when assembled and extend transversely to the longitudinal direction of the carrier to receive the individual contact spring elements therein when the contact spring elements are subjected to an excessive load.", "10.The assembly of claim 9, wherein the depth of the spring element receiving pockets exceeds the thickness of the contact spring material.", "11.The assembly of claim 9, further comprising transversal lands formed on an upper side of the carrier, having interspaces therebetween forming the spring element receiving pockets for taking up any excessive load applied to the contact spring.", "12.The assembly of claim 9, wherein the spring element receiving pockets and lands extend around the longitudinal side edges of the carrier.", "13.The assembly of claim 9, wherein the spring elements are each formed having an upper contact area for engagement with one of the surfaces of the electronic equipment, and having longitudinal edge portions of the spring bent downwardly and inwardly towards each other forming lower contact areas for engagement with another of the surfaces, wherein the upper and lower contact areas are joined by rounded side portions.", "14.The assembly of claim 13, wherein the width of the carrier, as measured at the longitudinal side edges of the lands, exceeds the width of the spring as measured at the rounded side edge portions.", "15.The assembly of claim 13, wherein the lower contact areas of the spring are each provided with contact bosses for enhancing the contact with the surface.", "16.The assembly of claim 9, wherein the spring element receiving pockets have a width significantly exceeding the width of the associated spring elements.", "17.The assembly of claim 9, further comprising guides, on the inner side surfaces of at least one of the pockets, positioning the contact spring correctly in relation to the carrier and centering each spring element sideways with respect to its associated pocket.", "18.The assembly of claim 9, further comprising at least one pair of attachment tabs provided a small distance apart on the underside of the carrier, having free ends resiliently displaceable towards each other under pressure so as to spring back when relieved.", "19.The assembly of claim 18, wherein the attachment tabs are each provided with a locking lug on their outer surface facing away from the other tab of each pair.", "20.A contact spring carrier of a contact spring assembly, comprising: an upper portion; and spring receiving pockets formed in the upper portion facing the contact spring when assembled.", "21.The carrier of claim 20, further comprising transversal lands on the upper side portion having interspaces between forming the spring element receiving pockets.", "22.The carrier of claim 20, wherein the spring receiving pockets and lands extend around the longitudinal side edges of the carrier.", "23.The carrier of claim 20, wherein at least one pair of attachment tabs are provided a small distance apart on the underside of the carrier, having free ends thereof resiliently displaceable towards each other under pressure so as to spring back when relieved.", "24.The carrier of claim 23, wherein the attachment tabs are each provided with a locking lug on their outer surface facing away from the other tab of each pair.", "25.The carrier of claim 20, wherein the carrier is formed from a sheet material blank.", "26.A method of forming a carrier for a contact spring assembly, comprising the steps of: providing a blank sheet material; punching an optional number of evenly spaced apertures in the blank; and folding side edge portions of the blank downwardly and inwardly towards each other along a first folding line, wherein the side edge portions are positioned underneath the apertures and the blank material remaining between the apertures form raised lands on an upper side of the carrier.", "27.The method of claim 26, further comprising the step of folding at least the major lengthwise portion of the outer side edge portions back upwardly and outwardly along a second folding line.", "28.The method of claim 26, further comprising the step of forming a number of tabs at regular intervals along each longitudinal side edge portion of the carrier blank.", "29.The method of claim 27, further comprising the step of bending the tabs down, along the second folding line, to extend approximately normal to an underside of the carrier.", "30.The method of claim 28, further comprising the steps of: forming a slit in each tab; and pressing an essentially central portion of each tab outwardly forming a locking lug of a generally arcuate shape.", "31.A method of providing attachment of a contact spring assembly, comprising a carrier and a contact spring, to a wall or panel of electronic equipment, comprising the steps of: providing at least one attachment member at the underside of the carrier facing a first, outer surface of the wall; forming at least one recess in the wall from an opposite inner surface thereof, forming a slit through the bottom of the recess; and inserting the attachment member into the slit for locking engagement with the recess.", "32.The method of claim 31, wherein the attachment member has a total length smaller than the thickness of the wall, the locked attachment member thereby being completely hidden in the wall.", "33.The method of claim 31, further comprising the step of providing at least one pair of attachment tabs on the underside of the carrier, such that the attachment tabs form a snap-in lock with the recess." ], [ "<SOH> BACKGROUND <EOH>For most electronic equipment, and not least for electronic equipment employed in telecommunication systems, it is essential to provide an effective shield or screen with regard to electromagnetic radiation that may disturb and interfere with the operation of other electronic equipment.", "In order to prevent electromagnetic coupling (EMC) between electronic components it is therefore equally important to block out ambient electromagnetic radiation on the one hand and to shield against the emission of radiation on the other hand.", "Such a shielding may be provided for individual components as well as for PCB's carrying such components, board fronts and entire casings, such as magazines, enclosing such electronic equipment.", "The most common way of providing such a shield for all of the above mentioned applications is by way of a contact spring that with a base portion is fixed to a first surface of a component, a PCB, a board front or a casing and that contacts an opposite surface with contact spring tongues extending from the base.", "The same general type of contact spring is also frequently used for grounding purposes, whether for preventing harmful electrostatic discharge between electronic equipment or for earth-connecting an electronic component, that is creating a zero potential for said equipment or component.", "In all of said applications it is vital for the contact spring to make good contact between the two surfaces and to securely maintain such contact even when a component or casing wall etc.", "is dismounted and reassembled one or several times.", "The attachment of the contact spring to the surface in question must also be secure so as to avoid that the contact spring itself falls off said surface and interferes with other components of the equipment.", "However, none of the contact springs presented so far have been able to solve all of the above-discussed problems to a satisfactory degree.", "A common design for such contact springs is to provide a clamp portion through which the contact spring is attached to the relevant surface either by being pushed over one edge thereof or by being inserted into apertures formed in said surface.", "None of these suggestions provides a definite solution to the problems of maintaining a secure contact and of fixing the contact spring to the surface, since only one longitudinal side thereof is attached to the surface.", "The other side providing the spring action may be easily deformed so that contact is no longer guaranteed for all contact tongues or may even get caught in some object so that the entire spring is torn off from the surface.", "This may even happen before the component is finally mounted if a spring tongue gets caught for instance in the clothes of an installer.", "The forming of apertures for the clamp portions is a bad solution since even a small aperture in many cases deteriorates the shielding.", "Specifically for the purpose of grounding adjacent board fronts in a magazine or other casing there is also the requirement that the attachment shall not interfere unacceptably with the standardized inner space of the normally U-shaped front.", "The reason for this latter requirement is the ever-increasing thickness of the employed PCB's, more and more commonly requiring the full available space of the board front.", "One approach to provide an attachment that does not interfere with said inner space is to attach one longitudinal side of the contact spring to the outer side surface of the board front by means of an adhesive.", "Although this solution requires no inner space it involves great efforts of cleaning the surfaces of both the board front and the contact spring before adhesion, and yet does not at all times prevent the contact spring from falling off during insertion of the respective board front.", "A further approach to provide an appropriate contact spring for the above purposes is to employ an assembly consisting of a substantially flat elongated carrier plate that is attached to the respective surface by riveting and on which a contact spring is supported.", "Examples of such solutions are disclosed in U.S. Pat.", "Nos.", "4,623,752 and 5,001,297.The two longitudinal edges of the contact spring are bent inwardly towards each other and extend below the carrier whereas the spring tongues span the carrier transversally.", "In this assembly the spring portion may be deformed completely until it engages the upper side of the carrier.", "Not only does this involve risks of permanent deformation of the spring by overloading, the full compression of the spring may also cause the spring to be disengaged from the carrier.", "In situations where an object such as a wall section is inserted incorrectly, such a design involves a great danger of the contact spring, or at least spring elements thereof, being completely cut-off by shearing action.", "The conventional attachment, such as by riveting, also takes up valuable space on the inner side of said surface and requires the use of tools." ], [ "<SOH> SUMMARY <EOH>The invention overcomes the above problems in an efficient and satisfactory manner.", "A general object of the invention is to provide a solution to the problem of providing an effective shielding and/or grounding of electronic equipment.", "In particular, it is an object of the invention to provide an improved method of securing a reliable electrical contact between two adjacent surfaces by means of a contact spring of a contact spring assembly.", "Briefly, the secure and reliable contact is achieved by providing excellent conditions for maintaining the contact spring fully operable at all times.", "Specifically, this is accomplished by providing a shelter for the contact spring on a carrier therefore, thereby protecting the contact spring by eliminating the possibility of the contact spring being overloaded and plastically deformed or even cut-off by shearing forces applied by an adjacent surface during installation.", "In accordance with an embodiment of the invention an effective protection from plastic contact spring deformation or overloading is achieved in particular by providing a contact spring assembly having a carrier for attachment to one of two opposite surfaces of electronic equipment and a contact spring supported by the carrier for making electrical contact with both said surfaces.", "Briefly the invention suggests providing sheltering pockets in the upper side of the carrier for the individual spring elements of the contact spring.", "In others words, when the contact spring experiences a load that would normally tend to produce plastic deformation thereof, the sheltering pockets will receive their associated spring elements and will protect them from further load.", "The carrier will itself take up such excessive load, in the areas thereof surrounding the pockets.", "In a further embodiment of the invention the sheltering pockets are extended around the longitudinal edges of the carrier, thereby specifically offering protection to the contact spring against the shearing action from adjacent surfaces during installation.", "In a further embodiment the longitudinal side edges of the contact spring are received and thus sheltered in the extended pockets in the installed, unloaded condition.", "Thereby, the contact spring is securely retained in the correct position on the carrier, with the spring members aligned with their associated pocket.", "The correct positioning of the contact spring on the carrier is enhanced even further by providing guides on the side edges of at least one extended sheltering pocket of the carrier, said guides fitting snugly to the sides of the associated spring element at the longitudinal side edges.", "In yet another embodiment of the invention the pockets are formed by providing mutually spaced transversal lands on the upper side of the carrier.", "Such a configuration lends itself well to forming the carrier from a sheet material in which transversal openings are cut out forming the pockets when the longitudinal edges of the sheet material are folded downwardly and inwardly towards each other.", "In such a configuration the remaining portions of the sheet material between the openings form the upper lands of the carrier, protecting the spring elements by taking up the excessive forces.", "Another object of the invention is to provide an improved and very effective contact spring assembly for shielding and grounding purposes, presenting a solution to the problem of providing a secure and reliable contact between two surfaces.", "A further object of the invention is to provide an improved carrier for a contact spring assembly.", "Yet another object of the invention is to provide an improved method of forming a carrier for a contact spring assembly.", "These and further objects of the invention are met by the invention as defined in the appended patent claims.", "A second aspect of the invention relates to a method of attaching a contact spring assembly and specifically the carrier therof, to a surface of electronic equipment.", "Briefly, the invention suggests providing attachment means on the underside of the carrier, that is the side facing such a surface in the attached condition.", "In a preferred embodiment the attachment means each consist of a pair of spaced resiliently yielding attachment tabs cooperating with a slit formed in the underlying electronic equipment surface to form a sort of snap-in attachment with the equipment surface.", "In this manner the contact spring assembly may be attached as a complete unit without the need for any specific tools or fastening means or adhesives.", "In a further embodiment of this second aspect specifically applicable in situations where the available space on the inner side of a wall or panel of the electronic equipment is restricted, a recess is formed on the inner side of said wall, the slit being formed through a bottom surface of the recess.", "With an appropriate dimensioning of the recess and the attachment tabs this approach permits the secure attachment of the contact spring assembly without interfering with the space on the inner side of said wall or panel.", "This second aspect of the invention is specifically applicable to contact spring assemblies employed for shielding PCB board fronts where the inner space of the board front is becoming more and more restricted.", "In summary, the present invention provides the following advantages over the state of the art: The contact spring maintains an effective electrical contact at all times; The risk of the contact spring collapsing or being plastically deformed is eliminated; The contact spring will not come loose from its attachment; Shearing forces will not be applied to the contact spring during installation; The contact spring is secured in an appropriate position relative to the carrier; The attachment requires minimum space; No need for tools or specific means to attach the contact spring assembly.", "Other advantages offered by the present invention will be readily appreciated upon reading the below detailed description of embodiments of the invention." ], [ "TECHNICAL FIELD The present invention relates generally to the grounding and/or shielding of electronic equipment and specifically relates to a contact spring assembly for grounding and/or shielding components, such as modules; members carrying electronic components, such as printed circuit boards; or casings enclosing such members and/or components.", "BACKGROUND For most electronic equipment, and not least for electronic equipment employed in telecommunication systems, it is essential to provide an effective shield or screen with regard to electromagnetic radiation that may disturb and interfere with the operation of other electronic equipment.", "In order to prevent electromagnetic coupling (EMC) between electronic components it is therefore equally important to block out ambient electromagnetic radiation on the one hand and to shield against the emission of radiation on the other hand.", "Such a shielding may be provided for individual components as well as for PCB's carrying such components, board fronts and entire casings, such as magazines, enclosing such electronic equipment.", "The most common way of providing such a shield for all of the above mentioned applications is by way of a contact spring that with a base portion is fixed to a first surface of a component, a PCB, a board front or a casing and that contacts an opposite surface with contact spring tongues extending from the base.", "The same general type of contact spring is also frequently used for grounding purposes, whether for preventing harmful electrostatic discharge between electronic equipment or for earth-connecting an electronic component, that is creating a zero potential for said equipment or component.", "In all of said applications it is vital for the contact spring to make good contact between the two surfaces and to securely maintain such contact even when a component or casing wall etc.", "is dismounted and reassembled one or several times.", "The attachment of the contact spring to the surface in question must also be secure so as to avoid that the contact spring itself falls off said surface and interferes with other components of the equipment.", "However, none of the contact springs presented so far have been able to solve all of the above-discussed problems to a satisfactory degree.", "A common design for such contact springs is to provide a clamp portion through which the contact spring is attached to the relevant surface either by being pushed over one edge thereof or by being inserted into apertures formed in said surface.", "None of these suggestions provides a definite solution to the problems of maintaining a secure contact and of fixing the contact spring to the surface, since only one longitudinal side thereof is attached to the surface.", "The other side providing the spring action may be easily deformed so that contact is no longer guaranteed for all contact tongues or may even get caught in some object so that the entire spring is torn off from the surface.", "This may even happen before the component is finally mounted if a spring tongue gets caught for instance in the clothes of an installer.", "The forming of apertures for the clamp portions is a bad solution since even a small aperture in many cases deteriorates the shielding.", "Specifically for the purpose of grounding adjacent board fronts in a magazine or other casing there is also the requirement that the attachment shall not interfere unacceptably with the standardized inner space of the normally U-shaped front.", "The reason for this latter requirement is the ever-increasing thickness of the employed PCB's, more and more commonly requiring the full available space of the board front.", "One approach to provide an attachment that does not interfere with said inner space is to attach one longitudinal side of the contact spring to the outer side surface of the board front by means of an adhesive.", "Although this solution requires no inner space it involves great efforts of cleaning the surfaces of both the board front and the contact spring before adhesion, and yet does not at all times prevent the contact spring from falling off during insertion of the respective board front.", "A further approach to provide an appropriate contact spring for the above purposes is to employ an assembly consisting of a substantially flat elongated carrier plate that is attached to the respective surface by riveting and on which a contact spring is supported.", "Examples of such solutions are disclosed in U.S. Pat.", "Nos.", "4,623,752 and 5,001,297.The two longitudinal edges of the contact spring are bent inwardly towards each other and extend below the carrier whereas the spring tongues span the carrier transversally.", "In this assembly the spring portion may be deformed completely until it engages the upper side of the carrier.", "Not only does this involve risks of permanent deformation of the spring by overloading, the full compression of the spring may also cause the spring to be disengaged from the carrier.", "In situations where an object such as a wall section is inserted incorrectly, such a design involves a great danger of the contact spring, or at least spring elements thereof, being completely cut-off by shearing action.", "The conventional attachment, such as by riveting, also takes up valuable space on the inner side of said surface and requires the use of tools.", "SUMMARY The invention overcomes the above problems in an efficient and satisfactory manner.", "A general object of the invention is to provide a solution to the problem of providing an effective shielding and/or grounding of electronic equipment.", "In particular, it is an object of the invention to provide an improved method of securing a reliable electrical contact between two adjacent surfaces by means of a contact spring of a contact spring assembly.", "Briefly, the secure and reliable contact is achieved by providing excellent conditions for maintaining the contact spring fully operable at all times.", "Specifically, this is accomplished by providing a shelter for the contact spring on a carrier therefore, thereby protecting the contact spring by eliminating the possibility of the contact spring being overloaded and plastically deformed or even cut-off by shearing forces applied by an adjacent surface during installation.", "In accordance with an embodiment of the invention an effective protection from plastic contact spring deformation or overloading is achieved in particular by providing a contact spring assembly having a carrier for attachment to one of two opposite surfaces of electronic equipment and a contact spring supported by the carrier for making electrical contact with both said surfaces.", "Briefly the invention suggests providing sheltering pockets in the upper side of the carrier for the individual spring elements of the contact spring.", "In others words, when the contact spring experiences a load that would normally tend to produce plastic deformation thereof, the sheltering pockets will receive their associated spring elements and will protect them from further load.", "The carrier will itself take up such excessive load, in the areas thereof surrounding the pockets.", "In a further embodiment of the invention the sheltering pockets are extended around the longitudinal edges of the carrier, thereby specifically offering protection to the contact spring against the shearing action from adjacent surfaces during installation.", "In a further embodiment the longitudinal side edges of the contact spring are received and thus sheltered in the extended pockets in the installed, unloaded condition.", "Thereby, the contact spring is securely retained in the correct position on the carrier, with the spring members aligned with their associated pocket.", "The correct positioning of the contact spring on the carrier is enhanced even further by providing guides on the side edges of at least one extended sheltering pocket of the carrier, said guides fitting snugly to the sides of the associated spring element at the longitudinal side edges.", "In yet another embodiment of the invention the pockets are formed by providing mutually spaced transversal lands on the upper side of the carrier.", "Such a configuration lends itself well to forming the carrier from a sheet material in which transversal openings are cut out forming the pockets when the longitudinal edges of the sheet material are folded downwardly and inwardly towards each other.", "In such a configuration the remaining portions of the sheet material between the openings form the upper lands of the carrier, protecting the spring elements by taking up the excessive forces.", "Another object of the invention is to provide an improved and very effective contact spring assembly for shielding and grounding purposes, presenting a solution to the problem of providing a secure and reliable contact between two surfaces.", "A further object of the invention is to provide an improved carrier for a contact spring assembly.", "Yet another object of the invention is to provide an improved method of forming a carrier for a contact spring assembly.", "These and further objects of the invention are met by the invention as defined in the appended patent claims.", "A second aspect of the invention relates to a method of attaching a contact spring assembly and specifically the carrier therof, to a surface of electronic equipment.", "Briefly, the invention suggests providing attachment means on the underside of the carrier, that is the side facing such a surface in the attached condition.", "In a preferred embodiment the attachment means each consist of a pair of spaced resiliently yielding attachment tabs cooperating with a slit formed in the underlying electronic equipment surface to form a sort of snap-in attachment with the equipment surface.", "In this manner the contact spring assembly may be attached as a complete unit without the need for any specific tools or fastening means or adhesives.", "In a further embodiment of this second aspect specifically applicable in situations where the available space on the inner side of a wall or panel of the electronic equipment is restricted, a recess is formed on the inner side of said wall, the slit being formed through a bottom surface of the recess.", "With an appropriate dimensioning of the recess and the attachment tabs this approach permits the secure attachment of the contact spring assembly without interfering with the space on the inner side of said wall or panel.", "This second aspect of the invention is specifically applicable to contact spring assemblies employed for shielding PCB board fronts where the inner space of the board front is becoming more and more restricted.", "In summary, the present invention provides the following advantages over the state of the art: The contact spring maintains an effective electrical contact at all times; The risk of the contact spring collapsing or being plastically deformed is eliminated; The contact spring will not come loose from its attachment; Shearing forces will not be applied to the contact spring during installation; The contact spring is secured in an appropriate position relative to the carrier; The attachment requires minimum space; No need for tools or specific means to attach the contact spring assembly.", "Other advantages offered by the present invention will be readily appreciated upon reading the below detailed description of embodiments of the invention.", "BRIEF DESCRIPTION OF THE DRAWINGS The invention, together with further objects and advantages thereof may best be understood by making reference to the following description taken together with the accompanying drawings, in which: FIGS.", "1A-B are top plan and end views respectively of a complete contact spring assembly according to the invention, consisting of a contact spring and a carrier; FIG.", "2A is a top plan view of a contact spring of the assembly illustrated in FIGS.", "1A-B; FIG.", "2B is a cross section through the contact spring illustrated in FIG.", "2A, along the line A-A; FIG.", "3A is a top plan view of a contact spring carrier of the assembly illustrated in FIGS.", "1A-B; FIG.", "3B is a cross section through the contact spring carrier illustrated in FIG.", "3A, along the line B-B; FIGS.", "4A-B are partially sectioned end views of a complete contact spring assembly according to the invention attached to a surface, illustrating two different load conditions that may be encountered during use; FIGS.", "5A-B are top plan and end views respectively of an embodiment of a contact spring blank for forming a contact spring of the assembly according to the invention; FIG.", "6A is a top plan view of an embodiment of a carrier blank for forming a carrier of the contact spring assembly according to the invention; FIGS.", "6B-C illustrate details of the carrier blank of FIG.", "6A; FIG.", "7A is a top plan view of a complete supplementary contact spring assembly according to the invention, likewise consisting of a contact spring and a carrier; FIG.", "7B is a top plan view of a supplementary section of a contact spring carrier for use in the supplementary assembly of FIG.", "7A; and FIG.", "7C is a top plan view of a supplementary section of a contact spring for use in the supplementary contact spring assembly of FIG.", "7A.", "DETAILED DESCRIPTION The invention will be described below with reference to drawing FIGS.", "1-7 that illustrate embodiments thereof adapted for use in combination with PCB board fronts to provide shielding and/or grounding thereof.", "However, it shall be understood that the invention is not restricted to these exemplifying embodiments or to such an application.", "The basic principles of the invention may likewise be applied for shielding and/or grounding purposes in association with other electronic equipment, such as mentioned above.", "Therefore, modifications and variations of the invention that may be required in such applications fall within the scope of the invention.", "Referring now to the drawings, and more particularly to FIGS.", "1A-B, there is shown a contact spring assembly of the present invention, in this embodiment comprising a main assembly generally designated by 1 and intended for use with a supplementary assembly illustrated in FIG.", "7A and generally designated by 101.Such a divided contact spring assembly will many times be advantageous or even required in association with a board front in order to provide access to or space for fasteners etc.", "employed on the board front for various purposes.", "The illustrated end portions 4A, 4B of the assembly 1 are configured for such purposes.", "Notwithstanding the above, the invention likewise covers producing the assembly in variable lengths or even in long length to be cut up in the appropriate lengths necessary for a specific application.", "Likewise the end portions may be designed otherwise for different applications.", "The contact spring assembly 1 of FIGS.", "1A-B is comprised generally of a contact spring carrier 10 supporting a contact spring 30 in position thereon.", "The contact spring 30 of the assembly 1 is illustrated in detail in FIGS.", "2A-B and has individual spring elements 31 provided side by side and evenly distributed along the length of the assembly 1.The spring elements 31 are separated by interspaces 32.The spring elements 31 are extended transversally to the longitudinal direction of the assembly 1.At each end the spring elements 31 are fixed to the respective longitudinal side edge portion 33A, 33B of the spring 30.With specific reference to FIG.", "2B it is illustrated that the spring elements 31 are each formed with a central portion 31A having an inverted V-shape.", "This central portion 31A forms an upper contact area for engagement with one of two surfaces 2A, 3A (see FIGS.", "4A and 4B) of the electronic equipment to be shielded.", "The longitudinal edge portions 33A, 33B of the spring 30 are bent downwardly and inwardly towards each other forming lower contact areas 31B for engagement with the other of said two surfaces 2A, 3A.", "In order to enhance the contact in this area the lower contact areas 31B are each provided with a number of contact bosses 35.Between the upper central portion 31A and the lower edge portions 31A, 31B of the spring 30 are formed rounded side portions 31C, 31D, the purpose of which will be clarified below.", "In this connection it should be noted that the interspaces 32 and thereby the spring elements 31 extend around the side portions 31C, 31D and a short distance into the lower contact areas 31B.", "The contact spring 30 is preferably manufactured from a sheet material, such as thin steel plate, having good electric conductivity and offering the appropriate spring characteristics.", "Such materials are known in the art and will therefore not be specified herein.", "Likewise the material is normally provided with a conventional surface coating, such as of tin, copper or even silver or gold, protecting it from environmental influence.", "A presently preferred method of manufacturing the contact spring of FIGS.", "2A-B from a contact spring blank 30BL will be described fewer below, with reference to FIGS.", "5A-B.", "In the assembly 1 the described contact spring 30 is supported on a contact spring carrier 10 illustrated in detail in FIGS.", "3A-B.", "The carrier 10 has an elongate, generally rectangular shape and is formed on its upper side—referring both to the illustration in FIG.", "3B and to its position relative to the associated electronic equipment surface 2A, as illustrated in FIGS.", "4A-B—with raised portions 11 in the form of lands extending transversally to the longitudinal direction of the carrier 10.Between the lands 11 are formed recessed portions forming sheltering pockets 12.In the assembled condition the spring elements 31 are aligned with an associated sheltering pocket 12, whereas the interspaces 32 between the spring elements 31 are aligned with an associated land 11.The depth of the sheltering pockets 12 exceeds the thickness of the spring elements 31, and the width W3 of the sheltering pockets 12 clearly exceeds the width W1 of the individual spring elements 31.Similarly the width W2 of the interspaces 32 between the spring members exceeds the width W4 of the lands 11.By virtue of the above described configuration it is clear that the spring members 31 can be received and sheltered in the pockets 12.Accordingly, in situations where the contact spring 30 is subjected to excessive load the individual spring elements 31 are deflected down into the pockets 12 so that the lands 11 extending up through the interspaces 32 take up the excessive load.", "Such an overload situation is represented with solid lines in FIG.", "4A that illustrates the case where a spring element 31—its unloaded condition illustrated with dash dot lines—is pressed down into a pocket 12 by an opposite surface 3A.", "Said drawing FIG.", "4A also serves to illustrate that the spring assembly 1 may be assembled by compressing the contact spring 30 so that the carrier 10 may be inserted therein from one side.", "The lands 11 and thus the sheltering pockets 12 are extended around the longitudinal side edges 18, 19 of the carrier 10 (FIG.", "3B) so that, in their unloaded condition the rounded side portions 31C, 31D of the spring elements 31 are accommodated in the extended portions of the pockets 12.Specifically the spring 30 is formed having a width L1 between the rounded side portions 31C, 31D being clearly smaller than the width L2 of the carrier 10 as measured between the side edges 18, 19.The purpose of this configuration is primarily to correctly position the spring 30 relative to the carrier 10.As was mentioned above the pockets 12 are made clearly wider than the spring elements 31 to secure free movement of the spring elements 31 into the pockets and thereby to secure protection of the spring 30 in overload situations.", "However it is essential that the spring 30 and the carrier 10 be retained in correct position relative to each other.", "This is achieved by providing guides 20 (illustrated in detail in FIG.", "6B) at the side surfaces of one or more pockets 12′, in the area of the extended portions of the pockets 12′.", "Said guides 20 will engage and position the respective spring element 31, thereby also stabilizing the complete contact spring 30 relative to the carrier 10.On the other hand the extended pockets 12 also serve to protect the spring 30 against shearing forces.", "Such shearing action may otherwise easily be the result when a board-front wall 3 (schematically represented in FIG.", "4B), or alternatively a component or casing wall, a panel etc, is forcibly inserted in a displaced or inclined position against an already installed board-front wall 2.In the worst case this may cause the contact spring 30 or individual elements 31 thereof being completely cut off.", "This risk will be eliminated by the invention, by virtue of the fact that the contact spring elements 31 will be sheltered in the pockets 12 also at the longitudinal side edges 18, 19 of the carrier 10.In this manner, the carrier lands 11 surrounding the pockets 12 in the extended portions thereof will take up the shearing forces and will rather apply a moment to the wall 3 of the board-front being installed.", "This situation is illustrated in FIG.", "4B that does not claim to be a true illustration of a pair of adjacent board fronts but schematically illustrates wall portions 2, 3 thereof, with the intention of clarifying the principles of the described function.", "The applied moment in turn tends to straighten up the board front wall 3 from the original position I, through position II and to position III, from which the appropriate installation will proceed.", "The described automatic straightening up of the board-front wall 3 being installed, has the further advantageous effect that the edges thereof will not scratch off any surface coating from the spring material.", "This is of great importance since such surface coating material must not be allowed to fall down on the sensible electronic components.", "The carrier 10 and thereby the complete spring assembly 1 are intended to be attached to a surface, such as the outer surface 2A of the board-front wall 2 illustrated in FIGS.", "4A-B, through attachment means 15, 16.In the illustrated embodiment the attachment means comprise an optional number of pairs of attachment tabs 15, 16 provided on the underside of the carrier 10.The tabs 15, 16 of each pair are provided at a small distance from each other.", "The number of pairs of attachment tabs and their positions may on the other hand be chosen as required by the specific application.", "The free ends of these tabs 15, 16 may be resiliently brought together during insertion into a slit 5 formed in the board-front wall 2.To facilitate insertion into the slit 5 the tabs 15, 16 are bent slightly inwardly towards each other at their free outer ends, thereby forming a sort of insertion taper.", "Each attachment tab is preferably formed having a locking lug 17 on its outer surface facing away from the other tab of each pair, thereby providing a positive fixing of the assembly in position as the locking lugs 17 pass through the slit 5 and the tabs 15, 16 are relieved and spring back.", "An extremely space saving or even completely hidden attachment is accomplished in accordance with the invention as illustrated in FIGS.", "4A-B.", "A recess 6 is formed in the board-front wall 2, from the inner surface 2B thereof facing away from the attached assembly 1.The slit 5 is then formed through the bottom of the recess 6.With the illustrated dimensioning of the depth of the recess 6 and the length of the tabs 15, 16, the latter will, in their locked position, end inside the recess.", "In this manner the attachment does not interfere with the inner space, which is extremely advantageous in applications for board fronts, for the reasons discussed in the introduction.", "Referring now specifically to the embodiment of FIGS.", "5A-B the contact spring 30 according to the invention is formed from a sheet material spring blank 30BL, preferably of steel.", "The blank 30BL is manufactured by punching a series of apertures 32 in the sheet material.", "Said apertures and the sheet material bands separating the apertures form the interspaces 32 and the spring elements 31 respectively, of the completed spring 30.The sheet material portions left outside each end of the apertures form the side edge portions 33A, 33B of the spring 30.The end portions 34A and 34B as well as each contact boss 35 are preferably formed in the same punching operation.", "Subsequently the spring 30 is given its final shape, as illustrated in FIG.", "2A, in a bending operation, in which is formed the inverted V-shape with the first contact area 31A of the spring elements 31 as well as the downwardly, inwardly bent edge portions 33A-B.", "The latter form the lower contact areas 31B of the spring and are connected to the spring elements 31 through the curved side portions 31C, 31D.", "A presently preferred method of producing the contact spring carrier 10 of the invention will now be described with specific reference to FIGS.", "6A-C that illustrate a sheet material blank 10BL for forming the carrier 10.The thickness of the sheet material is chosen mainly to provide the appropriate strength properties for the carrier to safely perform its supporting function, but also to be clearly greater than the thickness of the spring material blank 30BL, for reasons that will become apparent.", "An optional number of evenly spaced apertures 12, 12′ are punched in the generally rectangular blank 10BL.", "These apertures will form the sheltering pockets 12, 12′ of the finished carrier 10 and the blank material remaining between the apertures 12 will form the lands 11 of the carrier 10.As was mentioned above, one or more of the apertures 12′ are formed having inwardly protruding guides 20 formed at end portions of the respective side surfaces of the aperture.", "This is illustrated in detail in FIG.", "6B whereas FIG.", "6C illustrates a standard aperture 12.End configurations 14A, 14B are formed in the same punching operation, as well as a number of tabs 15, 16 formed at regular intervals along each longitudinal side edge portion 13A, 13B respectively.", "A slit 17A is formed in each tab 15, 16 so that an essentially central portion 17 of each tab 15, 16 may be pressed outwardly forming a locking lug 17 of a generally arcuate shape.", "The side edge portions 13A, 13B of the blank 10BL are then bent or folded downwardly and inwardly towards each other along a first folding line F1 indicated in FIG.", "6A.", "Through this first bend or fold the side edge portions 13A, 13B will be positioned generally underneath the apertures.", "The outer side edge portion is then folded back upwardly and outwardly, except for the tabs 15, 16 that are only bent down to be extended approximately normal to the underside of the carrier 10.This final bending or folding is carried out along a second folding line F2.The “double folding or bending” of the punched carrier blank 10BL provides a comparatively simple and economical method of forming a carrier 10 in one piece, having the appropriate properties.", "The first “fold” F1 forms the pockets 12 and the lands 11, whereas the second “fold” forms the attachment tabs 15, 16 and provides a further “backing” of the carrier 10, providing the necessary strength for supporting the spring 30 and for a secure attachment of the assembly 1.FIGS.", "7A-C illustrate the supplementary contact spring assembly 101, a supplementary carrier 110 and a supplementary contact spring 130 respectively, that in the illustrated embodiment are used as a separate unit joining two adjacent main assemblies 1.This supplementary assembly 101 and its parts have the same general structure as the main assembly 1 and its parts.", "However, the supplementary assembly 101 and its carrier and spring are illustrated having straight edges 104A, 104B; 114A, 114B and 134A, 134B respectively, cooperating with the profiled ends 4A, 4B of the main assemblies 1.As mentioned above said end configurations may be modified depending upon the requirements of specific applications.", "Moreover, the supplementary assembly 101 is provided with a cutout 104C along one of its side edges, said cutout providing space for a fastener etc.", "on the board front that has to be accessible with the spring assembly attached.", "In this case the adjacent carrier lands 111 are shortened so that they only extend approximately halfway across the carrier 110 and thus provide space for a specific spring element 131 configuration having a general E-shape.", "This provides for an uninterrupted spring action along the full length of the supplementary assembly 101, in spite of the provision of the cutout 104C.", "Although the carrier 10 has been described herein with reference to a specific embodiment thereof formed from a sheet material blank, it shall be emphasized that the invention is not restricted to such a design.", "Since the carrier does not participate in the actual shielding or grounding it is not required having specific electrical properties but strength properties to provide an adequately secure and stable support for the spring and a secure attachment for the assembly.", "Therefore, the invention likewise covers forming the carrier by alternative methods and from different materials, such as by injection molding of a suitable plastic material or casting of metals etc.", "Likewise, the invention shall not be restricted to the use of the illustrated attachment lugs forming the snap-in attachment, but shall also cover the use of other kinds of attachment means that are in themselves known within similar and other areas.", "This includes forming single tabs on the carrier which are inserted into slits in the wall and are then twisted so as to become locked in the recess or on the opposite side of the wall.", "It will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departure from the scope thereof, which is defined by the appended claims." ] ]

[ [ "Toothbrush", "A toothbrush head with having elongated projections made of an elastomer material, having an integral resiliently flexible elastomeric material wiper blade at the end remote from the head.", "Suitable constructions of projections and wiper blades are disclosed, and combinations with bristles.", "The wiper blades enhance oral hygiene." ], [ "1-20.", "(canceled) 21.A toothbrush head, having a face from which projects at least one projection elongated in a length direction generally perpendicular to the face and made of an elastomer material, the projection characterized by; having an integral resiliently flexible wiper blade of elastomer material at its end remote from the head, the wiper blade extending in a length direction generally parallel to the length direction of the projection, the wiper blade having a width direction generally perpendicular to the length direction of the projection and extending in its width direction across at least 50% of the cross section of the projection and having a thickness dimension in a thickness direction perpendicular to its width direction which is 50% or less of its width dimension.", "22.A toothbrush head according to claim 21 characterized in that the projection comprises an elongated cylindrical or frustro-conical body extending in a length direction from the face of the toothbrush head in a direction generally perpendicular to the face and terminating in an end surface remote from the face, the end surface being substantially planar or shallow convex, and the wiper blade is integrally formed with the body and extends in the length direction of the body from the said end surface.", "23.A toothbrush head according to claim 21 characterized by an end surface of shallow cone shaped, shallow pyramidal shaped, shallow gable shaped, shallow polyhedral shaped or shallow domed.", "24.A toothbrush head according to claim 21, characterized by a body of length ca.", "0.8-1.2 cm, and a cross section of 0.5-4 mm.", "25.A toothbrush head according to claim 21 characterized by a wiper blade of a generally planar blade, with an edge remote from the face, a length dimension being the greatest dimension of the wiper blade and extending across the end surface of the body perpendicular to the length direction of the body, and having a width dimension parallel to the length direction of the projection which is less than the length dimension, and a least dimension being the thickness perpendicular to its length and width.", "26.A toothbrush head according to claim 25 having a width: length ratio in the range 1:3 to 1:20 and a thickness:width ratio in the range 0.5 to 0.1.27.A toothbrush head according to claim 21 characterized by a shape as viewed in side view perpendicular to the length direction of the body and along the thickness direction of the wiper blade which is generally rectangular.", "28.A toothbrush head according to claim 21 made of a thermoplastic elastomer material having a hardness of 10 to about 90 Shore A.", "29.A toothbrush head according to claim 21, characterized by one or more projection arranged along one or more of the sides of the toothbrush head.", "30.A toothbrush head according to claim 21, characterized by one or more projection arranged at the end of the head remote from the handle.", "31.A toothbrush head according to claim 21, characterized by projections arranged in rows oriented across the width of the head.", "32.A toothbrush head according to claim 21, characterized by projections arranged in lines oriented along the length of the head.", "33.A toothbrush head according to claim 21, characterized by projections arranged in polygonal clusters.", "34.A toothbrush head according to claim 21, characterized by projections arranged with width direction of their wiper blade oriented generally parallel to the length direction of the head.", "35.A toothbrush head according to claim 21, characterized by projections arranged with the width direction of their wiper blade oriented generally perpendicular to the length direction of the head.", "36.A toothbrush head according to claim 21, characterized by projections arranged with the width direction of their wiper blade oriented at an angle between 0° and 90° to the length direction of the head.", "37.A toothbrush head according to claim 21, characterized by two or more of the projections with their wiper blades oriented differently to each other.", "38.A toothbrush head according to claim 21 characterized by projections combined with bristles.", "39.A toothbrush head according to claim 38 characterized by projections arranged around the edges of the head, with bristles extending from the central area of the face between the projections.", "40.A toothbrush head according to claim 38 characterized by rows, lines and/or clusters of projections combined with rows, lines or clusters of tufts of bristles." ], [ "This invention relates to toothbrush heads and to toothbrushes incorporating such a head.", "Toothbrushes generally comprise a head and a handle, the head being either an integral part with the handle e.g.", "integrally moulded of plastic material together with the handle, or replaceably mountable at an end of the handle.", "Normally the toothbrush head has a face (the “bristle face”) on which are mounted bristle filaments, generally made of a relatively stiff fibre material e.g.", "the commonly used material Nylon, and generally projecting perpendicularly, or at a near-perpendicular angle from the bristle face.", "An example of such fibres are those sold under the trademark Tynex™ by Dupont.", "Toothbrushes are known in which the ends of the bristles are profiled, for example into a point as disclosed in EP-A-0 596 633 into the shape of a flattened blade, e.g.", "as disclosed in U.S. Pat.", "No.", "4,167,794.Toothbrushes are known which in addition to or as an alternative to such bristle filaments have massage parts mounted on their head.", "Such massage parts are generally provided for massaging and stimulating the gums, and can also serve other functions such as cleaning and/or polishing the teeth.", "Often such massage parts are made of an elastomer, i.e.", "rubbery material and are elongate structures and generally project perpendicularly, or at a near-perpendicular angle from the bristle face, e.g.", "generally in the direction of the bristles when present.", "For example U.S. Pat.", "No.", "4,277,862 and DE 29512817 U1 disclose massage parts in the form of strips flanking the sides of the toothbrush head.", "WO-A-97/16995 discloses massage parts in the form of fine elastomeric filaments arranged on the face of the toothbrush head.", "EP-A-0 360 766A discloses massage parts in the form of cylinders having rounded knobs at their ends.", "GB-A-2 035 076 discloses massage parts in the form of cones having fine “horns” at their ends.", "U.S. Pat.", "No.", "5,040,210 discloses massage parts in the form of cones or obliquely cut cylinders.", "WO-A-00/49911 discloses massage parts in the form of pyramids.", "WO-A-98/03097 discloses massage parts in the form of elastomeric strips with their width oriented across the head.", "In all of these disclosures the massage parts are made of soft rubbery material and their function is to gently clean the tooth surfaces and/or to apply a gentle massage to the user's gums.", "It is an object of this invention to provide a toothbrush having an improved gum massage and stimulation and tooth cleaning effect.", "According to this invention a toothbrush head is provided, having a face from which projects at least one projection being elongated in a length direction generally perpendicular to the face and being made of an elastomer material, the projection characterised by; having at its end remote from the head an integral resiliently flexible wiper blade of elastomer material, the wiper blade extending in a length direction generally parallel to the length direction of the projection, the wiper blade having a width direction generally perpendicular to the length direction of the projection and extending in its width direction across at least 50% of the cross section of the projection and having a thickness dimension in a thickness direction perpendicular to its width direction which is 50% or less of its width dimension.", "In a preferred embodiment the projection comprises an elongated cylindrical or frustro-conical body extending in a length direction from the face of the toothbrush head in a direction generally perpendicular to the face and terminating in an end surface remote from the face, the end surface being substantially planar or shallow convex, and the wiper blade is integrally formed with the body and extends in the length direction of the body from the said end surface.", "The terms “cylindrical” and “conical” used herein include true cylinders and cones, i.e.", "having circular cross sections and straight sides (tapering in the case of a cone).", "These terms also include distorted cylindrical and distorted cone shapes, for example having distorted circular, e.g.", "oval cross sections, or polygonal cross sections, e.g.", "prism or pyramid shapes, for example with rounded corners.", "The terms also include shapes with other than longitudinally straight sides, e.g.", "concave or convex curved sides are also included.", "The long axis of the cylinder, or the base-apex direction of the cone is the longitudinal direction of the projection as referred to above.", "If the body is frustro-conical its narrow end is remote from the face.", "Typical shallow convex shapes for the end surface include for example shallow cone shaped, shallow pyramidal shaped, shallow gable shaped, shallow polyhedral shaped or shallow domed.", "A typical length for such a body is ca.", "0.8-1.2 cm, i.e.", "approximating to the typical length of toothbrush bristles.", "A typical cross section is ca.", "0.5-4 mm, such as 2-4 mm, preferably 3-4 mm.", "A frustro-conical body shape is preferred because such a shape facilitates removal of the projection from an injection mould if the projection is made by injection moulding e.g.", "of a thermoplastic elastomer (“TPE”).", "When the body is frustro conical in shape the conical slope of the taper is suitably up to 20°, preferably 10° or less.", "Suitably the end surface of the body remote from the face is planar, e.g.", "perpendicular to the length direction of the body.", "If the remote surface is shallow convex then preferably its convex projection is at most 50% of the cross section of the end of the body remote from the face, preferably 25% or less, more preferably 10% or less.", "The wiper blade is suitably a generally planar blade, with an edge remote from the face, although its thickness direction may deviate from planarity, e.g.", "by ±10%, e.g.", "the thickness dimension may bulge or the blade may be curved in section as cut across its length direction.", "Suitably the greatest dimension (“width”) of the wiper blade extends across, preferably entirely across, the end surface of the body, i.e.", "perpendicular to the length direction of the body, preferably intersecting the central longitudinal axis of the body.", "Suitably the wiper blade has a width dimension parallel to the length direction of the projection which is less than the length direction of the wiper blade parallel to the length direction of the body, and its least dimension being its thickness perpendicular to its length and width.", "A suitable width: length ratio is in the range 1:3 to 1:20, preferably 1:3 to 1:10.A suitable thickness:width ratio is in the range 0.5 to 0.1, preferably 0.25-0.1.Typically in shape as viewed in side view perpendicular to the length direction of the body and along the thickness direction of the wiper blade, the wiper blade may be generally rectangular.", "The cross section of the wiper cut across its thickness direction may be generally parallel sided, so that the wiper may be of a generally tetragonal shape.", "Alternative shapes, e.g.", "an arc such as a chord of a circle as viewed in side view perpendicular to the length direction of the body and along the thickness direction of the blade, may also be used.", "Also, the cross section of the wiper cut across its thickness direction may taper, e.g.", "being of a wedge shape narrowing away from the face of the toothbrush.", "The projections may be made of known TPE materials which are known for use for the massage projections of known toothbrushes e.g.", "as discussed above.", "Suitable materials are soft elastomeric materials, e.g.", "having a hardness of less than 90 Shore A. Preferably the elastomeric material has a hardness from about 10 to about 90 Shore A, more preferably 50 Shore A or less, e.g.", "from about 14 to about 35 Shore A.", "Suitable materials are for example natural rubber, crosslinked polybutadiene, etc., an example of such a material being Santoprene™.", "Suitable elastomeric materials include those available under the trade names Megol and Santoprene, and silicone elastomeric materials may also be used.", "Other suitable elastomeric materials are disclosed in the state of the art referred to above.", "The projections may be made by injection moulding.", "The projections may be made individually or may be made in groups, e.g.", "linked by an integrally moulded linker of the elastomeric material.", "It is known to make two-component toothbrushes in which a plastic material “skeleton” is made first and then this skeleton is enclosed in a second injection mould cavity in which elastomer parts are then made in a second injection moulding step so that the elastomer and plastic materials bond.", "The elastomer projections of this invention may be made via such a two stage process.", "For example a plastic material skeleton comprising at least the part to become the head of the completed toothbrush may first be made, having locations where one or more projection as described above is to be located in the completed toothbrush.", "This skeleton may have then be enclosed in a second injection mould cavity having cavity parts defining the shape of the one or more projection to be formed, and then fluid elastomeric material may be injected into the second injection mould, so that the one or more projection is formed as the elastomer sets solid.", "The conditions of pressure and temperature within the second mould may be such that the elastomer material bonds to the plastic material.", "The one or more projection(s) of this invention may be positioned at any location on the toothbrush head.", "For example one or more projection may for example be arranged along one or more of the sides of the toothbrush head, at the end of the head remote from the handle, or a plurality of the projections may be arranged randomly, in rows oriented across the width of the head (i.e.", "perpendicular to the head-handle “length” axis) in lines oriented along the length of the head (i.e.", "parallel to the head—handle axis) in clusters, or otherwise.", "Such clusters may for example comprise arrays, e.g.", "rectangular arrays, or polygonal clusters, and may be located at any place on the head, for example a cluster at the tip end of the head remote from the handle.", "The wiper blade may be oriented in any orientation relative to the length and width direction of the toothbrush head.", "For example the projection(s) may be arranged with their width direction oriented generally parallel to the length direction of the head, or generally perpendicular to the length direction of the head, or at an angle between 0° and 90°, e.g.", "45° to the length direction of the head.", "Two or more of the projections may have their wiper blades oriented differently, for example at 90° to each other.", "The projections may be combined with bristles of a generally conventional nature.", "For example the toothbrush may have the above-described projections arranged around the edges of the head, with bristles extending from the face of the head, i.e.", "from the central area of the face between the projections.", "Alternatively the toothbrush may have the above-mentioned rows, lines and/or clusters of projections combined with rows, lines or clusters of tufts of bristles.", "For example rows or lines or groups of rows or lines of the said projections may alternate respectively with rows or lines, or groups of rows or lines of tufts of bristles.", "In such a combination the one or more projection may be substantially the same length as the conventional bristles, or alternatively the one or more projection may be longer or shorter than the conventional bristles.", "The projections of the invention may also be combined with elastomer massage parts of other shapes, e.g.", "as conventionally known in the art.", "The invention will now be described by way of example only with reference to the accompanying drawings.", "FIG.", "1 shows part of a projection of the invention having a generally cylindrical body.", "FIG.", "2 shows part of a projection of the invention having a generally frustro-conical body.", "FIG.", "3 shows toothbrushes having the projections of this invention.", "Referring to FIG.", "1 a projection is shown generally 10.FIGS.", "1A and 1B are perspective views at right angles to each other, and FIG.", "1C is a downwards view in the opposite direction to arrow designating the length direction “L” in FIG.", "1A.", "The projection 10 is elongated in the length direction “L” generally perpendicular to the face (not shown in FIG.", "1) of a toothbrush (not shown in FIG.", "1).", "FIGS.", "1D, 1E and 1F show side views perpendicular to the length direction “L” of alternate constructions of the part of the projection 10 remote from the face of the toothbrush.", "The projection 10 is made of an elastomer material such as Santoprene™.", "The projection 10 comprises an elongated cylindrical body 11 extending in the length direction L and terminates in an end surface 12 remote from the face.", "The end surface 12 as shown in FIG.", "1A is substantially planar.", "The length of the body i.e.", "the dimension from the face of the toothbrush head (not shown) to the surface 12 is ca.", "0.8-1.2 cm, and its cross section, i.e.", "the diameter of its circular section, is ca 2-4 mm, preferably 3-4 mm.", "A wiper blade 13 is integrally formed with the body 11 and extends in the length direction “L” of the body from the said remote surface 12.The wiper blade as shown in FIGS.", "1A and 1B is a generally planar blade having its greatest dimension (“width”) extending entirely across the remote surface 12 of the body 11 perpendicular to the length direction of the body 11 and intersecting the central longitudinal axis in the length direction “L” of the body 11.The view in FIG.", "1A is generally looking in the thickness direction of the wiper 13, the view in FIG.", "1B is in the width direction of the wiper 13, and the view in FIG.", "1C is looking in the length direction of the wiper 13.Therefore as viewed in side view perpendicular to the length direction of the body 11 and along the thickness direction of the blade 13, the wiper blade 13 is generally rectangular.", "Referring to FIGS.", "1D and 1E an alternative construction is shown in which the remote surface 12 of the body 11 is a shallow convex shape, i.e.", "shallow domed, bulging in the length direction “L” of the body 11.The wiper blade 13 is of a generally rectangular shape similar to that as shown in FIGS.", "1A, 1B and 1C.", "The view in FIG.", "1D is a side view perpendicular to the length direction “L” of the body 11 and perpendicular to the width direction of the blade 13, and along the thickness direction of the blade 13.The view in FIG.", "1E is a side view perpendicular to the length direction “L” of the body 11 and perpendicular to the thickness direction of the blade 13, and along the width direction of the blade 13.Referring to FIG.", "1F an alternative construction is shown in which the remote surface of the body 11 is planar, and the wiper 13 is in the shape of an arc being a chord of a circle.", "The view in FIG.", "1F is a side view perpendicular to the length direction “L” of the body 11 and along the thickness direction of the blade 13.Referring to FIG.", "2 a projection is shown generally 20.FIGS.", "2A and 2B are perspective views at right angles to each other, and FIG.", "2C is a downwards view in the opposite direction to arrow designating the length direction “L” in FIG.", "2A.", "The projection 10 has a body 21 which is frustro-conical in shape elongated in the length direction “L”, with its narrow end remote from the face (not shown in FIG.", "2) of a toothbrush (not shown in FIG.", "2), the length direction “L” again being generally perpendicular to the face (not shown) of the toothbrush.", "The projection 20 is made of an elastomer material such as Santoprene™.", "The projection 20 terminates in a surface 22 remote from the face, being the narrow end of the frustro-conical shape.", "The remote surface 22 as shown in FIG.", "2A is substantially planar.", "The length of the body 21 i.e.", "the dimension from the face of the toothbrush head (not shown) to the surface 22 is ca.", "0.8-1.2 cm, and its cross section, i.e.", "the diameter of its circular section at its base end longitudinally opposite the surface 22, is ca 2-4 mm, preferably 3-4 mm.", "A wiper blade 23 is integrally formed with the body 21 and extends in the length direction “L” of the body from the said remote surface 22.The wiper blade as shown in FIGS.", "2A and 2B is again a generally planar blade having its greatest dimension (“width”) extending entirely across the remote surface 22 of the body 21 perpendicular to the length direction “L” of the body 21 and intersecting the central longitudinal axis in the length direction “L” of the body 21.The view in FIG.", "2A is generally looking in the thickness direction of the wiper 23, the view in FIG.", "2B is in the width direction of the wiper 23, and the view in FIG.", "2C is looking in the length direction of the wiper 23, i.e.", "in the apex-base direction of the cone shape toward the base of the cone shape.", "Therefore in shape as viewed in side view perpendicular to the length direction “L” of the body 23 and along the thickness direction of the blade 23, the wiper blade 23 is generally rectangular.", "In FIGS.", "1B, 1E and 2B it is seen that the cross section of the wiper 13, 23 cut across its thickness direction is generally parallel sided, so that the wiper 13 of FIGS.", "1A to 1C is generally tetragonal in shape.", "In FIG.", "1G the cross section of the wiper 13 cut across its thickness direction tapers, so that the wiper 13 is generally wedge shaped.", "Referring to FIG.", "3, FIGS.", "3A to 3I show toothbrushes 30 generally, having projections 10, 20 of the invention.", "Each toothbrush 30 comprises a handle 31 and a head 32, moulded integrally with the handle 31, with a neck region 33 in between.", "FIGS.", "3A to 3G are plan views looking along the length direction “L” of the projections 10, 20.FIGS.", "3H and 3I are side views looking generally perpendicular to the length direction “L” and to the longitudinal head-handle direction of the toothbrush.", "Each head has a face 34 from which projects at least one projection 10, 20 as described above.", "In each of the heads 32 shown, tufts 35 of conventional toothbrush bristles, e.g.", "known Tynex™ filaments project in a direction generally perpendicular to the face 34.As shown in FIG.", "3A, projections 10, 20 are arranged in rows perpendicular to the longitudinal head-handle axis of the toothbrush 30.A row of tufts of bristles 35 is located between a pair of rows of the projections 10, 20, so that rows of projections 10, 20 and tufts 35 alternate longitudinally.", "As shown in FIG.", "3B, projections 10, 20 are arranged along the side edges of the head 32, with tufts 35 between the projections 10, 20 in the middle part of the face 34.As shown in FIG.", "3C, tufts 35 are arranged along the side edges of the head 32, with projections 10, 20 between the tufts 35 in the middle part of the face 34.As shown in FIG.", "3D, projections 10, 20 are arranged in a polygonal cluster 36, i.e.", "a hexagon of six projections surrounding a central projection, at the end of the head 32 remote from the handle 21.Between the cluster 36 and the handle 31 are rows of tufts of bristles 35.In the toothbrushes 30 shown in FIGS.", "3A to 3D the wiper blade 13, 23 of the projections 10, 20 are arranged with their width direction parallel to the longitudinal head-handle direction of the toothbrush 30.In FIG.", "3E an arrangement of projections 10, 20 and tufts 35 similar to that of FIG.", "3D is shown, with the difference that the wiper blade 13, 23 of each projection 10, 20 is oriented with its width direction perpendicular to the longitudinal head-handle direction of the toothbrush 30.In the toothbrushes 30 shown in FIGS.", "3F and 3G the arrangement of projections 10, 20 and tufts 35 is similar in plan to that shown in FIG.", "3A.", "In FIG.", "3F the wiper blade 13, 23 of the projections 10A, 20A are arranged with their width direction parallel to the longitudinal head-handle direction of the toothbrush 30, but the projections 10B, 20B are arranged with their width direction perpendicular to the longitudinal head-handle direction of the toothbrush 30.In FIG.", "3G the wiper blade 13, 23 of the projections 10A, 20A are arranged with their width direction parallel to the longitudinal head-handle direction of the toothbrush 30, but the projections 10C, 20C are arranged with their width direction at an angle of 45° to the longitudinal head-handle direction of the toothbrush 30.FIGS.", "3H and 3I show side views of toothbrushes according to this invention, the arrangement of projections 10, 20 and tufts 35 being similar to that shown in plan in FIG.", "3A.", "In FIG.", "3H the wiper blades 13, 23 of the projections 10A, 20A are arranged with their width direction parallel to the longitudinal head-handle direction of the toothbrush 30.In FIG.", "31 the wiper blades 13, 23 of the projections 10A, 20A are arranged with their width direction perpendicular to the longitudinal head-handle direction of the toothbrush 30.In FIGS.", "3H and 3I the alignment of the projections 10, 20 generally perpendicular to the face 34 is seen, and the length of the projections 10, 20 being substantially the same as the bristles 35 is also seen.", "In use the toothbrush 30 of the invention is used with a conventional toothbrushing technique, e.g.", "the known Bass technique.", "The wiper blades 13, 23 are flexible relative to the body 11, 21 and can assist in the removal of dirt etc from tooth surfaces." ] ]

[ [ "Deflector devices", "A deflector for offsetting a streamer being towed by its lead-in by a seismic survey vessel comprises a wing-shaped body which is coupled to the lead-in and shaped to produce a sideways force which urges the lead-in laterally with respect to the direction of movement of the vessel.", "A boom extends rearwardly from the wing-shaped body, and forms part of an arrangement by which the angle of the wing-shaped body in the water, and therefore the sideways force produced by it, can be varied.", "The upper end of the wing-shaped body is directly connected to the underside of an elongate float." ], [ "1.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body adapted to be coupled to the tow line and shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the wing-shaped body, the end of the boom remote from the wing-shaped body being adapted to be connected to the tow, and the angle between the boom and the wing-shaped body being remotely adjustable to vary the sideways force produced by the wing-shaped body, and an elongate float member whose underside is directly connected to the upper end of the wing-shaped body, 2.A deflector device as claimed in claim 1, wherein the centre of buoyancy of the float member is near the trailing edge of the wing-shaped body.", "3.A deflector device as claimed in claim 1 or claim 2, wherein the angle between the longitudinal axis of the float member and the chord of the wing-shaped body is selected such that, in use, the longitudinal axis of the float member is aligned with the towing direction when the chord of the wing-shaped body is at its mean or normal angle to the towing direction.", "4.A deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a principal wing-shaped body adapted to be coupled to the tow line and shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the principal wing-shaped body, an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and an elongate float member whose underside is directly connected to the upper end of the principal wing-shaped body, 5.A deflector device as claimed in claim 4, wherein the angle between the boom and the principal wing-shaped body is remotely adjustable, to vary the sideways force produced by the principal wing-shaped body.", "6.A deflector device as claimed in claim 4, wherein the angle between the boom and the principal wing-shaped body is substantially fixed, and further comprising remotely operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.", "7.A deflector device as claimed in any one of claims 4 to 6, wherein the centre of buoyancy of the float member is near the trailing edge of the principal wing-shaped body.", "8.A deflector device as claimed in any one of claims 4 to 7, wherein the angle between the longitudinal axis of the float member and the chord of the principal wing-shaped body is selected such that, in use, the longitudinal axis of the float member is aligned with the towing direction when the chord of the principal wing-shaped body is at its mean or normal angle to the towing direction.", "9.A deflector device as claimed in any one of claims 4 to 8, wherein the end of the boom remote from the wing-shaped body is adapted to be connected to the tow.", "10.A deflector device as claimed in any preceding claim, wherein the or each wing-shaped body is made from titanium.", "11.A deflector device as claimed in claim 10, wherein the float member is made from titanium.", "12.A deflector device as claimed in any one of claims 1 to 10, wherein the float member is made from a fibre-reinforced composite material.", "13.A method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic streamers over an area to be surveyed, wherein the lateral position of at least one of the streamers is controlled by a deflector device in accordance with any one of the preceding statements of invention." ], [ "This invention relates to deflector devices of the kind used between a towing vessel and a tow located in water, for example a seismic streamer or streamer array, or a seismic source array, in order to pull the tow out to one side of the vessel, so as to position it at a desired lateral offset from the course followed by the vessel.", "A deflector device of this kind is described in detail in our U.S. Pat.", "No.", "5,357,892, and comprises a wing-shaped deflector body having a remotely-operable pivotal lever or “boom” which extends rearwardly from a point near the middle of the trailing edge of the wing-shaped body.", "In use, the wing-shaped deflector body is suspended beneath a float so as to be completely submerged and positioned generally vertically in the water, and is connected to the towing vessel by means of a tow line, while the tow is connected to the end of the boom remote from the wing-shaped body.", "As the device is pulled through the water, the wing-shaped body produces a sideways force, or “lift”, which moves the tow laterally.", "This lift can be varied by adjusting the angle of the boom from the vessel, thus permitting the lateral offset of the tow from the course of the vessel to be varied in use.", "The deflector device of U.S. Pat.", "No.", "5,357,892 has been successfully commercialised by the Applicant as its MONOWING deflector device.", "In use, rolling stability of the device is provided by the connection to the float, while stability of the device about a vertical axis is provided by the drag produced by the tow.", "The deflectors in current use are very large, typically 7.5 m high by 2.5 m wide, and weigh several tonnes.", "They are usually suspended around 2 m to 8 m below the float by means of a fibre rope, and are also provided with a safety chain intended to prevent separation of the float and deflector wing in the event that the rope breaks.", "In bad weather, the upper part of the deflector wing may rise up out of the water, allowing the rope connecting the deflector wing and the float to go slack.", "If the deflector wing then drops abruptly, the rope, and possibly the safety chain, may break, and/or the attachment points may be damaged.", "One solution to this problem is disclosed in PCT Patent Application No.", "PCT/IB98/01946 (WO 99/33700).", "It is an object of the present invention to provide an alternative and simpler solution.", "According to one aspect of the present invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a wing-shaped body adapted to be coupled to the tow line and shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the wing-shaped body, the end of the boom remote from the wing-shaped body being adapted to be connected to the tow, and the angle between the boom and the wing-shaped body being remotely adjustable to vary the sideways force produced by the wing-shaped body, and an elongate float member whose underside is directly connected to the upper end of the wing-shaped body, According to another aspect of the invention, there is provided a deflector device for use with a tow line between a towing vessel and a tow in water behind the vessel, the device comprising a principal wing-shaped body adapted to be coupled to the tow line and shaped to produce in use a sideways force which urges the tow line laterally with respect to the direction of movement of the towing vessel, a boom extending rearwardly from the principal wing-shaped body, an auxiliary wing-shaped body, smaller than the principal wing-shaped body, secured to the end of the boom remote from the principal wing-shaped body and shaped so as to produce in use a sideways force in generally the opposite direction to that produced by the principal wing-shaped body, and an elongate float member whose underside is directly connected to the upper end of the principal wing-shaped body, In this second aspect of the invention, the angle between the boom and the principal wing-shaped body may be remotely adjustable, to vary the sideways force produced by the principal wing-shaped body.", "Alternatively and preferably, the angle between the boom and the principal wing-shaped body may be substantially fixed, and the deflector device may further comprise remotely operable means for varying the angle of the auxiliary wing-shaped body to vary the sideways force produced by the auxiliary wing-shaped body, and thereby vary the sideways force produced by the principal wing-shaped body.", "In either case, the end of the boom remote from the principal wing-shaped body may be adapted to be connected to the tow.", "In preferred implementations of both aspects of the invention, the centre of buoyancy of the float member is near the trailing edge of the firstmentioned (or principal) wing-shaped body, and the angle between the longitudinal axis of the float member and the chord of the principal wing-shaped body is selected such that, in use, the longitudinal axis of the float member is aligned with the towing direction when the chord of the principal wing-shaped member is at its mean or normal angle to the towing direction.", "The or each wing-shaped body is preferably made from titanium, while the float member may be made either from titanium or from a fibre-reinforced composite material.", "The invention also includes a method of performing a marine seismic survey, the method including towing a plurality of laterally spaced seismic streamers over an area to be surveyed, wherein the lateral position of at least one of the streamers is controlled by a deflector device in accordance with any one of the preceding statements of invention.", "The invention will now be described by way of example only, with reference to the accompanying drawings, of which: FIG.", "1 is a partial schematic view of a seismic survey vessel carrying out a marine seismic survey; FIG.", "2 is a perspective view of a first embodiment of a deflector device in accordance with the invention, for use in carrying out the survey of FIG.", "1; FIG.", "3 is a somewhat schematic part sectional view of the deflector device FIG.", "2; FIGS.", "4 and 5 are views similar to that of FIG.", "3 and showing different operating positions of part of the deflector device of FIGS.", "2 and 3; FIG.", "6 is a somewhat schematic part-sectional view of a second embodiment of a deflector device in accordance with the present invention, for use in carrying out the survey of FIG.", "1; and FIG.", "7 is a front view of part of the deflector device of FIG.", "6.The seismic survey vessel shown in FIG.", "1 is indicated generally at 10, and is preferably as described in our PCT Patent Application No.", "PCT/GB98/01832 (WO 99/00295).", "The vessel 10 is shown towing a seismic source 15, typically a TRISOR multiple air gun source of the kind described in our U.S. Pat.", "No.", "4,757,482, and an array 16 of four substantially identical streamers 18.However, it will be appreciated that, in practice, many more than four streamers can be towed, for example by using the techniques described in our PCT Patent Application No.", "PCT/IB98/01435 (WO 99/15913).", "The streamers 18 are towed by means of their respective lead-ins 20 (ie the high strength steel- or fibre-reinforced electrical or electro-optical cables which convey electrical power, control and data signals between the vessel 10 and the streamers), and their spread is controlled by two deflector devices, indicated at 22, connected to the respective forward ends 24 of the two outermost streamers.", "The deflector devices 22 act in co-operation with respective spreader lines 26 connected between the forward end 24 of each outermost streamer 18 and the forward end 24 of its adjacent streamer to maintain a substantially uniform spacing between the streamers.", "One of the deflector devices 22 is shown in more detail in FIGS.", "2 to 5.The deflector device 22 is similar in general principle to the deflector device of our U.S. Pat.", "No.", "5,357,892, but is a much improved version of it.", "In particular, the deflector device 22 has a main wing-shaped body 28 which is coupled in use to a respective outer lead-in 20 via a towing bridle 27, and which corresponds to the deflector body 2 of U.S. Pat.", "No.", "5,357,892.However, the main wing-shaped body 28 is of improved hydrodynamic cross-sectional shape and includes a fixed-angle trailing edge flap 30, both of which features enhance lift.", "Also, the main wing-shaped body 28 is provided at its lower end with a vortex-controlling end plate 31 of the kind described in more detail in our PCT Patent Application No.", "PCT/FR99/02272, to reduce drag and improve stability, and is largely made of titanium to reduce weight.", "Additionally, the angle lever 10 of U.S. Pat.", "No.", "5,357,892 is replaced by a rearwardly extending boom 32, which is pivotally connected at one end 34 to the low pressure side 36 of the body 28 near the midpoint of that side of the body, at a mounting bracket 38.The other end 40 of the boom 32 has a towing eye (not shown) which is coupled to the forward end 24 of a respective one of the two outermost streamers 18.Pivotal movement of the boom 32 is controlled by a mechanism comprising first and second struts 41, 42, which are pivotally connected to each other at 44 and to each end of the boom at 34 and 46, forming with the boom a triangle, and an extending hydraulic actuator strut 48 pivotally connected between the apex of the triangle, ie the pivotal connection point 44 of the struts 41, 42, and a pivotal connection point 50 positioned on the low pressure side 36 of the body 28 between its midpoint and its trailing edge.", "It will be appreciated that extension of the hydraulic actuator strut 48, from its unextended position of FIG.", "3, will move the boom 32 outwardly from the low pressure side 36 of the body 28, from its closest position shown in FIG.", "3.The extent of the outward movement is preferably about 20°, as shown in FIGS.", "4 and 5.An auxiliary wing-shaped body 52, which is much smaller than the body 28 in length, thickness and chord, is secured to the end 40 of the boom 32 with its longitudinal axis (which lies in a plane perpendicular to the plane of FIG.", "3) extending parallel to the longitudinal axis of the body 28.The body 52 is fixedly secured to the boom 32 at or near the midpoint of its trailing edge 54, and its leading edge 56 is inclined away from the body 28 such that the chord of the body 52 (ie the line connecting its leading edge 56 and its trailing edge 54) is at an angle of about 10° to the boom 32.This angle is chosen because it is about half the angular extent of the movement of the boom 32.The shape of the body 52 is designed to produce, in use, a sideways force in a direction approximately opposite to that produced by the body 28 (approximately opposite, because it will be appreciated that the direction of the force varies in use as the boom 32 moves).", "This sideways force is increased by providing the body 52 with a fixed trailing edge flap 58, angled away from the boom 32 at an angle of about 35°.", "As the boom 32 is pivoted away from the body 28, the sideways force produced by the body 52 acts as a restoring force, and thus varies the angle of the body 28 with respect to the direction of tow, so changing the lift produced by the body 28.This restoring force augments the restoring force produced by the drag of the towed streamers 18 (and in particular, reduces the effect of any stability-reducing variations or reductions in that drag).", "Indeed, the deflector device 22 will remain stable with no streamer attached, eg if its streamer 18 breaks or is severed at its forward end 24.In accordance with the present invention, and as shown in FIG.", "2, the body 28 is provided at its upper end with an elongate streamlined float 80, which is directly and rigidly secured to the body 28, so that the latter depends downwardly from the float like the keel of a boat.", "When the body 28 is made from titanium, the float 80 is preferably also made from titanium; otherwise, the float may be made from a fibre-reinforced composite material.", "The float 80 typically has a volume of about 5000 litres, and is designed to give the whole deflector 22 a slightly positive buoyancy.", "The centre of lift of the float 80 is substantially aligned with a line parallel to and near the trailing edge of the body 28.The angle between the longitudinal axis of the float 80 and the chord of the body 28 is selected such that, in use, the longitudinal axis of the float is aligned with the towing direction when the chord of the member 28 is at its most commonly used angle to the towing direction (or alternatively, the mean of its range of possible angles to the towing direction).", "This has the effect of tending to minimize the range of possible misalignments in use between the longitudinal axis of the float 80 and the towing direction.", "FIGS.", "6 and 7 show at 60 an alternative implementation of part of the deflector device 22 of FIGS.", "2 to 5, with corresponding parts having the same reference numbers as were used in FIGS.", "2 to 5.The principal difference between this alternative embodiment and the embodiment of FIGS.", "2 to 5 is that in the deflector device 60, the boom 32 is secured at a fixed angle, typically about 10°, to the low pressure side 36 of the main wing-shaped body 28, while the angle of the chord of the auxiliary wing-shaped body 52 with respect to the boom 32 is variable by means of a remotely operable electric stepper motor 62.As best seen in FIG.", "7, the electric stepper motor 62 is secured to the boom 32 with its axis extending laterally of the boom, and is disposed in a cut-out or aperture 64 in the auxiliary wing-shaped body 52.Coaxial drive shafts 66 protrude from each axial end of the motor 62 and are secured to the body 52 to rotate it about the common axis of the drive shafts.", "A slot 65 is provided in the body 52 between the aperture 64 and the trailing edge of the body, to accommodate the boom 32 as the body 52 is rotated by the motor 62.As an alternative to the apertured and slotted implementation of the body 52, the body 52 can be implemented in two separate but symmetrical halves disposed on respective sides of the boom 32 and each attached to a respective one of the drive shafts 66 of the motor 62.The boom 32 is of sandwich construction: it is made of two similarly shaped plates 68 which are bolted together at intervals along their length and which sandwich between them both a mounting flange 70 of the motor 62 and the boom mounting bracket 38 secured to the low pressure side of the main wing-shaped body 28.Typically, the boom 32 is detached from the bracket 38 whenever the deflector device 60 is on the vessel 10, for ease of stowage.", "As in the embodiment of FIGS.", "2 to 5, the end 40 of the boom is provided with a towing eye, indicated at 74 in FIGS.", "6 and 7, for connection to a streamer 18.However, as mentioned earlier, since stability is no longer dependent upon a streamer 18 being connected to the end 40 of the boom 32, the towing eye 74 can be omitted, and the streamer 18 can be towed from the lead-in 20 at a point near the attachment point of the deflector device 60.The same is true for the deflector device 22.It will be appreciated that varying the angle of the auxiliary wing-shaped body 52 of the deflector device 60 has the same effect as varying the angle of the boom 32 of the deflector device 22, ie it changes the angle of the main wing-shaped body 28 with respect to the direction of tow and so changes the lift produced by the main wing-shaped body.", "However, for the deflector device 60, less power is required to produce a given change in angle of the main wing-shaped body 28, because of the increased leverage provided by the position of the auxiliary wing-shaped body 52 towards the end 40 of the boom 32 (as opposed to the position of the hydraulically-operated actuator strut 48 of the deflector device 22).", "It is this which permits the use of the relatively low-powered electric stepper motor 62 in the deflector device 60, in place of the relatively more powerful hydraulic system which operates the mechanism based on the strut 48.However, if desired, the electric stepper motor 62 can be replaced by a simple hydraulic actuator secured to the boom 32, since this also would not need to be as powerful as the hydraulic system which operates the mechanism based on the strut 48.An additional advantage of replacing the hydraulic system and the mechanism based on the strut 48 with the electric stepper motor 62 or a simple hydraulic actuator is the considerable weight saving which can be achieved.", "It will be appreciated that many modifications can be made to the described embodiment of the invention.", "For example, the auxiliary wing-shaped body 52 of the deflector device 60 can be fixed, as in the deflector device 22.Additionally, the deflector devices 22 and 60 can be used with tows other than streamers, for example seismic sources.", "And the invention can be applied to a deflector device similar to the MONOWING deflector device of U.S. Pat.", "No.", "5,357,892." ] ]

[ [ "A gambling apparatus and method of monitoring a ganbling event", "A gambling apparatus comprising at least one sensor (2, 3) for monitoring the progress of a live gambling event (1) having a set up period and an end-result period and generating at least a still representation of the live gambling event.", "The apparatus also comprises a display operable to be in communication with the at least one camera or sensor (2, 3) and capable of showing the at least one still representation of the live gambling event (1).", "A communication link between the at least one camera or sensor (2, 3) and the display is provided.", "The apparatus further comprises a processor operable to be in communication with the display and to provide an animation and/or still or moving live representation of the live gambling event (1) on the display." ], [ "1-28.", "(canceled) 29.A gambling apparatus comprising: at least one sensor for monitoring the progress of a live gambling event having a set-up period and an end-result period and generating at least a still representation of the live gambling event; a display operable to be in communication with the at least one sensor and capable of showing the at least one still representation of the live gambling event; a communication link between the at least one sensor and the display; a processor operable to be in communication with the display and to provide an animation corresponding to the status of the live gambling event on the display; and, a bandwidth monitor operable to determine information concerning the bandwidth of the communication link between the at least one sensor and the display and to vary the quantity of the data from the at least one sensor displayed by the display in response to the bandwidth information from the bandwidth monitor.", "30.A gambling apparatus according to claim 29, wherein the at least one sensor is capable of generating at least a still representation of the live gambling event at the end-result period.", "31.A gambling apparatus according to claim 29, wherein the display provides a combination of the at least one still representation and the animation.", "32.A gambling apparatus according to claim 29, wherein the processor is provided on the communication link between the at least one sensor and the display.", "33.A gambling apparatus according to claim 32, wherein the bandwidth monitor comprises; a high frequency signal generator provided on the communication link between the at least one sensor and the processor; and signal detection means operably connected to the processor, the high frequency signal having a series of harmonics such that, in response to the high frequency signal, the value of the frequency of the highest harmonic received is determined and communication of data to the processor at that frequency is enabled.", "34.A gambling apparatus according to claim 33, wherein the value of the frequency of the highest harmonic received is determined periodically during communication of data between the at least one sensor and the display.", "35.A gambling apparatus according to claim 34, wherein the value of the frequency of the highest harmonic received is determined every 100th of a second so as to allow variation of the frequency of data transmission between the at least one sensor and the display every 100th of a second.", "36.A gambling apparatus according to claim 29, wherein the communication link between the at least one sensor and the display comprises the Internet.", "37.A gambling apparatus according to claim 29, wherein the communication link between the at least one sensor and the display comprises a telephone line.", "38.A gambling apparatus according to claim 29, wherein the display and the processor are comprised in a personal computer.", "39.A gambling apparatus according to claim 29, wherein the at least one sensor comprises a camera for capturing an image of the live gambling event, the representation of the live gambling event being the image.", "40.A gambling apparatus according to claim 39, wherein the image of the event shown by the display is a live video image of the event.", "41.A gambling apparatus according to claim 39, wherein the image is a video image and means are provided to determine changing parts of the video image, the data transmitted to the display, via the processor, being supplemented only with changing parts of the video image.", "42.A gambling apparatus according to claim 39, wherein the at least one sensor comprises first and second cameras, each directable at a different view of the live gambling event.", "43.A gambling apparatus according to claim 42, wherein the display shows an image from the first camera during the set up period and an image from the second camera during the end-result period.", "44.A gambling apparatus according to claim 29, further comprising data entry means locatable adjacent the live gambling event and operable to be in communication with the processor for entry of data relating to the status and/or end-result of the event and transmission to the processor.", "45.A method of remotely monitoring a live gambling event having a set up period and an end-result period comprising the steps of: monitoring the progress of the live gambling event and generating at least a still representation of the event; producing an animation of the gambling event, the animation corresponding to a period within which the live gambling event is at that time; presenting the gambling event at a location remote from the gambling event as a combination of the animation and the generated representation of the live gambling event; determining the bandwidth of the communication between the gambling event and the presentation location and varying the quantity of data transmitted dependent on the bandwidth available.", "46.A method according to claim 45, wherein the at least one still representation of the live gambling event comprises a still representation at the end-result period.", "47.A method according to claim 45, wherein the animation and the generated image are presented as a superimposition.", "48.A method according to claim 45, wherein the step of monitoring the progress of the live gambling event and generating at least a still representation of the event comprises capturing at least a still image of the live gambling event.", "49.A method according to claim 48, wherein the step of capturing at least a still image comprises capturing at least a first still image of a first view of the event in the setting up period and capturing at least a second image of a second view of the event in the end-result period, the first view being different from the second view.", "50.A method according to claim 48, wherein the captured image comprises a captured video image.", "51.A method according to claim 45, conducted using the apparatus of claim 29.52.A gambling apparatus comprising: at least one camera for capturing an image of a live gambling event having a set up period and an end-result period; a display operable to be in communication with the at least one camera and capable of showing at least a still image of the live gambling event; a communication link between the at least one camera and the display; a processor operable to be in communication with the display and to provide an animation corresponding to the status of the live gambling event on the display; and, a bandwidth monitor operable to determine information concerning the bandwidth of the communication link between the at least one sensor and the display and to vary the quantity of the data from the at least one sensor displayed by the display in response to the bandwidth information from the bandwidth monitor." ], [ "THE PRESENT INVENTION relates to an apparatus and method for remotely monitoring a live gambling event.", "For many years it has been customary for legalised gambling to take place in casinos.", "Typically, a casino will operate a number of games, such as roulette or various card games, in which one or more players may take part and place bets on the outcome of the game.", "For example, in the game of roulette, a spinnable wheel is provided, having a series of numbered slots.", "The wheel is set spinning and a ball placed within the wheel such that as the wheel comes to a halt the ball falls into one of the slots.", "Prior to the spinning of the wheel, each individual playing the game places a bet on a numbered board, against the number corresponding to the numbered slot in which he predicts that the ball will fall.", "If an individual's prediction is correct, and he wins the bet, he is credited with an amount based on the amount of his original bet.", "The problem with this type of gambling casino is that it is essential for each individual to be present within the casino while placing the bet.", "This can be inconvenient, especially as some jurisdictions do not permit the presence of casinos and therefore it is necessary for individuals to travel to a casino in another jurisdiction, if they wish to place a bet.", "It is known to provide so-called “online gambling” arrangements in which individuals may access a central computer remotely, such as via the Internet.", "The central computer runs a “virtual game”, simulating the games that regularly exist in a real casino, and on which individuals may place bets electronically in a manner corresponding to that at a real casino.", "In such an online gambling arrangement, the central computer generates one or more “random” variables on which the result of the game is based.", "The problem with this type of online gambling arrangement is twofold.", "Firstly, it is not possible for a computer to generate a truly random variable and therefore there is a risk that individuals may eventually be able to calculate accurately or model the variable that the computer will generate, before placing their bet.", "Such individuals would then be able to cheat when placing bets.", "Secondly, and more importantly, because the individual is presented with a computer generated result which may have been produced in any way, individuals may be suspicious that the gambling arrangement is not being conducted fairly and that they do not have a reasonable chance of their bet winning.", "Therefore, individuals may be reluctant to use such online gambling arrangements.", "It has been proposed that such online gambling arrangements be conducted around a live gambling event, which is transmitted to the individual, remotely, using a video camera and display.", "For example, U.S. Pat.", "No.", "5,800,268 discloses a gambling arrangement in which an individual is able to place bets on a live event whilst watching the event from a remote position via a telephone line connection.", "Thus, the individual is able to view the proceedings surrounding the live gambling event and is therefore re-assured of the fairness of the gambling arrangement.", "The problem with this proposal is that sending video pictures remotely requires a large bandwidth of communication.", "The bandwidth required is not presently available to most users of the Internet.", "For example, to send a normal picture requires a baud rate of 256 kb.", "However, a typical domestic Internet connection has a maximum baud rate of 56 kb and, in practice, usually achieves a baud rate of only 28 kb.", "The present invention seeks to alleviate one or more of the above problems.", "According to a first aspect of the invention, there is provided a gambling apparatus comprising: at least one sensor for monitoring the progress of a live gambling event having a set up period and an end-result period and generating at least a still representation of the live gambling event; a display operable to be in communication with the at least one sensor and capable of showing the at least one still representation of the live gambling event; a communication link between the at least one sensor and the display; and a processor operable to be in communication with the display and to provide an animation corresponding to the status of the live gambling event on the display.", "Conveniently, the at least one sensor is capable of generating at least a still representation of the live gambling event at the end-result period.", "Preferably, the display provides a combination of the at least one still representation and the animation.", "Advantageously, the apparatus further comprises a bandwidth monitor operable to determine information concerning the bandwidth of the communication link between the at least one sensor and the display and to vary the quantity of the data from the at least one sensor, displayed by the display in response to the bandwidth information from the bandwidth monitor.", "Conveniently, the processor is provided on the communication link between the at least one sensor and the display.", "Preferably, the bandwidth monitor comprises; a high frequency signal generator provided on the communication link between the at least one sensor and the processor; and signal detection means operably connected to the processor, the high frequency signal having a series of harmonics such that, in response to the high frequency signal, the value of the frequency of the highest harmonic received is determined and communication of data to the processor at that frequency is enabled.", "Advantageously, the value of the frequency of the highest harmonic received is determined periodically during communication of data between the at least one sensor and the display.", "Conveniently, the value of the frequency of the highest harmonic received is determined every 100th of a second so as to allow variation of the frequency of data transmission between the at least one sensor and the display every 100th of a second.", "Preferably, the communication link between the at least one sensor and the display comprises the Internet.", "Advantageously, the communication link between the at least one sensor and the display comprises a telephone line.", "Conveniently, the display and the processor are comprised in a personal computer.", "Preferably, the at least one sensor comprises a camera for capturing an image of the live gambling event, the representation of the live gambling event being the image.", "Advantageously, the image of the event shown by the display is a live video image of the event.", "Conveniently, the image is a video image and means are provided to determine changing parts of the video image, the data transmitted to the display, via the processor, being supplemented only with changing parts of the video image.", "Preferably, the at least one sensor comprises first and second cameras, each directable at a different view of the live gambling event.", "Advantageously, the display shows an image from the first camera during the set up period and an image from the second camera during the end-result period.", "Conveniently, the gambling apparatus further comprises data entry means locatable adjacent the live gambling event and operable to be in communication with the processor for entry of data relating to the status and/or end-result of the event and transmission to the processor.", "According to a second aspect of the invention, there is provided a method of remotely monitoring a live gambling event having a set up period and an end-result period comprising the steps of: monitoring the progress of the live gambling event and generating at least a still representation of the event; producing an animation of the gambling event; presenting the gambling event at a location remote from the gambling event as a combination of the animation and the generated representation of the live gambling event.", "Conveniently, the at least one still representation of the live gambling event comprises a still representation at the end-result period.", "Preferably, the animation and the generated image are presented as a superimposition.", "Advantageously, the step of monitoring the progress of the live gambling event and generating at least a still representation of the event comprises capturing at least a still image of the live gambling event.", "Conveniently, the step of capturing at least a still image comprises capturing at least a first still image of a first view of the event in the setting up period and capturing at least a second image of a second view of the event in the end-result period, the first view being different from the second view.", "Preferably, the captured image comprises a captured video image.", "Advantageously, the step of determining the bandwidth of the communication between the gambling event and the presentation location and varying the quantity of data transmitted dependent on the bandwidth available.", "Conveniently, the method is conducted using the apparatus described above.", "According to a third aspect of the invention, there is provided a gambling apparatus comprising: at least one camera for capturing an image of a live gambling event having a set up period and an end-result period; a display operable to be in communication with the at least one camera and capable of showing at least a still image of the live gambling event; a communication link between the at least one camera and the display; and a processor operable to be in communication with the display and to provide an animation corresponding to the status of the live gambling event on the display.", "In order that the invention may be more readily understood and so that further features thereof may be appreciated, embodiments thereof will now be described, by way of example, with reference to the accompanying drawings in which: FIG.", "1 is a schematic view of one embodiment of the invention; and FIG.", "2 is a pictorial view of a display according to one embodiment of the invention.", "Referring to FIG.", "1 in which a schematic view of one embodiment of the invention is shown, a live gambling event 1 is conducted in a casino.", "The live gambling event described in relation to this embodiment of the invention is roulette but it is to be appreciated that any game that could be played in a casino could be used instead, such as card games, for example blackjack.", "As explained earlier, it is to be appreciated that each game of roulette has a setting-up period in which bets may be placed; a spinning period in which the roulette wheel is spun and an end-result period which begins when the wheel stops spinning and the ball has fallen into one of the slots in the wheel.", "Accordingly, -the status of the game may be defined as being within one of these three periods.", "The roulette game 1 is played in the usual way, and bets are placed by individuals who are physically present at the roulette table.", "A first camera 2 is directed at the roulette table and also the surrounding area such that the casino pit staff and any players at the table are within its field of vision.", "A second camera 3 is also provided adjacent the roulette table and is directed upon the roulette wheel itself such that the roulette wheel fills its field of vision.", "It is preferred that that the second camera 3 is directly above the roulette wheel and therefore provides a downward view of the roulette wheel.", "Also provided in the casino is a pit terminal 4 which may be a personal computer, and into which one of the casino pit staff may enter the status of each game and the end-result of each game.", "Each of the first camera 2, the second camera 3, and the pit terminal 4 are connected by respective first, second and third communication lines 5, 6 & 7 to a server 8 resident in the casino.", "In this embodiment of the invention, the server 8 runs the Linux operating system but it is to be appreciated that, in other embodiments of the invention, different operating systems may be used.", "The server 8 receives the images from the first and second cameras 2 and 3 and also the data from the pit terminal 4.The server 8 also receives instructions from an operational terminal 9 via a fourth communication line 10.The casino staff enter a variety of data into the operational terminal 9.Firstly, there is data relating to betting information on the basis of which the server 8 determines whether or not to accept large bets.", "Secondly, there is accounting information on the basis of which the server 8 determines the viability of the live gambling event 1.Thirdly, there is statistical information on the basis of which the server 8 detects any cheating that is being undertaken.", "In response to these inputs, the server 8 generates the data required to display a web page relating to the roulette game 1.The server 8 also tests the bandwidth available, in a manner described below, and calculates the quantity of data which can be reliably and efficiently sent on the basis of this calculation, again in a manner described below.", "The data is sent from the server 8 via a fifth communication line 11 to an Internet service provider 12, which in turn transmits the data via the Internet 13 to a personal computer 14 running web browser software capable of displaying the data as a web page 15 to a remote individual operating the personal computer 14.Referring now to FIG.", "2, in which an image of the web page 15 is shown, this embodiment of the invention will be further described.", "The web page 15 displays an animated roulette wheel 16 which has the appearance of spinning when the roulette game is in the spinning period and continues to appear to rotate until the end-result period is reached.", "It is preferred that the animated roulette wheel appears to be spinning in a clockwise direction in all even numbered roulette games and in an anti-clockwise direction for all odd numbered games.", "Also displayed in the web page 15 is a computer generated image of the roulette table layout 17.The image of the roulette table layout 17 shows the numbers on the real roulette table and allows the user of the personal computer 14 to place bets using a “point and click” interface.", "The image of the roulette table layout 17 also shows each of the bets that have been placed in relation to the roulette game, pictorially.", "An area of the web page 15 depicts a live image 18 of the roulette game 1, as transmitted by the first camera 2 or the second camera 3.As is described in greater detail below, the live image 18 may be a stationary, still image of the roulette game 1 or may be a continuously moving video image of the roulette game, depending upon the bandwidth available via the connection between the server 8 and the personal computer 14.Also displayed on the web page 15 are a number of other indications and buttons to assist the user of the personal computer 14 to place bets in relation to the roulette game 1.In particular, a timer 19, displays the time remaining in the set-up period in which bets may be placed.", "The game status indication 20 indicates whether the game is open for bets (i.e.", "the roulette game is in the set-up period) or is closed for bets (i.e.", "the game is in the spinning period or in the end-result period).", "In a results indication 21 a list of the last ten game results is displayed, with the most recent game result at the top.", "A lock-in button 22 can be “clicked” by the user of the personal computer 14 in order to lock in his bet.", "Various other indications and buttons are provided on the web page 15, as will be apparent to those of skill in the art.", "In use, a game of roulette is played according to this embodiment of the present invention, as follows.", "At the roulette game 1, the first camera 2 is directed at the roulette table and surrounding area and this image is sent via the first communication line 5, the server 8, the fifth communication line 11, the Internet service provider 12, and the Internet 13 to the personal computer 14 and is displayed in the live image 18.The roulette game 1 is in its setting up period in which bets may be placed.", "The pit staff enter the status of the game in the pit terminal 4, and any bets that are placed by the individuals at the roulette game I and this information is transmitted via the communication line 7 to the server 8 and then, in turn, to the personal computer 14 as for the image from the first camera 2.The web page 15 thus displays the status of the game in the game status indication 20 and the animation of the roulette wheel 16 is stationary.", "Any bets that are placed are shown on the roulette board 17.The user of the personal computer 14 may place any bets using the “point and click” interface on the roulette board 17 and when satisfied with the bets may click the lock-in button 22 in order to lock in the bets.", "Information regarding the bets placed by the remote user is transmitted to the server 8 via the Internet 13, the Internet service provider 12 and the fifth communication line 11.The server 8 then records the information regarding the bet until the end-result period.", "When the duration of the set up period has expired (usually 4 minutes) no more bets are taken from individuals at the roulette game 1, the roulette wheel of the roulette game 1 is set spinning and the information regarding the change in status of the game is entered by the pit staff in the pit terminal 4.This information is transmitted to the server 8 via the third communication line 7, in response to which the server 8 switches the camera image sent to the personal computer 14 to the image from the second camera 3.The server 8 also, simultaneously, sends to the personal computer 14, the data regarding the change of status of the game.", "Accordingly, substantially simultaneously with the spinning of the roulette wheel on the roulette table 1, the web page 15 is updated with the change of status information such that the game status indication 20 changes to “closed” and no new bets are allowed to be placed on the roulette board 17.Furthermore, the roulette wheel animation 16 begins its spinning animation and the roulette table live image 18 displays the image from the second camera 3.When the roulette wheel of the roulette game 1 stops spinning and the ball falls in the slots, the game enters its end-result period.", "The pit staff enter the end-result (i.e.", "the number of the slot into which the ball fell) in the pit terminal 4 and this information is transmitted to the server 8 which, in turn, sends the appropriate data to the personal computer 14.The web page 15 is thus updated, such that the new result is shown in the results indication 21, the animation of the roulette wheel 16 stops and the live image 18 of the roulette wheel of the roulette game 1 in the end-result position is shown.", "If the individual using the personal computer 14 placed a winning bet then the winnings are transmitted accordingly.", "After a predetermined interval, the pit staff commence a new roulette game, by clearing the table of the roulette game 1 and entering in the pit terminal 4 that a new setting up period has begun.", "This information is sent to the server 8, in response to which the server 8 transmits the image from the first camera 2 to the personal computer 14, together with the data regarding the change of status of the game to the personal computer 14.The web page 15 then updates the game status indication 20 to “open” to show that bets may be placed on the roulette board 17 and the image 18 of the roulette game 1 is switched to that of the view from the first camera 2.The process, as described above, may then be repeated.", "In relation to the above process, it is to be appreciated that, in order to ensure that the user of the personal computer 14 is convinced of the fairness of the gambling arrangement, the live image 18 of the roulette table 1 shows the end-result of the roulette table substantially simultaneously with the end-result occurring at the live gambling event.", "Similarly, in order to avoid possible cheating by a user of the personal computer 14, by placing bets after the spinning period has ended, the data and images regarding the roulette game on the roulette table 1 are transmitted substantially instantaneously to the personal computer 14 and displayed on the web page 15.In order to achieve this, a system is provided to monitor the bandwidth of the connection between the server 8 and the personal computer 14 and the quantity of video information from the first camera 2 or the second camera 3 that is transmitted from the server 8 is varied according to the bandwidth available.", "In particular, a signal generator (not shown) attached to the server 8 generates a high frequency signal which is transmitted via the fifth communication line 11, to the Internet service provider 12 and then to the Internet 13 and subsequently to the personal computer 14, in addition to the data needed to update the web page 15.The high frequency signal has a series of harmonics that correspond to the exact frequencies that the server 8 has the capability to send.", "The personal computer 14 receives the signal and records the highest harmonic that has been received.", "The personal computer 14 then transmits to the server 8 the value of the highest frequency received.", "The server 8 then transmits data to the personal computer 14 using this frequency.", "The high frequency signal is transmitted to the personal computer 14 every 100th of a second and, similarly, the personal computer 14 returns to the server 8 the value of the highest harmonic received every 100th of a second.", "Accordingly, the frequency of which the server 8 transmits data to the personal computer 14 is continually changeable, depending upon the available bandwidth of the connection between the server 8 and the personal computer 14.In response to the bandwidth calculated by the server 8 to be available for transmitting data to the personal computer 14, the amount of information relating to the image from either the first camera 2 or second camera 3 that is transmitted from the server 8 is varied.", "In particular, if there is insufficient bandwidth for the entire video image to be sent to the personal computer 14 then only those parts of the image which are changing significantly are sent to the personal computer 14 which then updates the corresponding part of the live image 18 of the roulette game 1.If the bandwidth available for transmitting information between the server 8 and the personal computer 14 is insufficient for any form of live video image then a series of still images are displayed in the live image 18.However, it is to be appreciated that the image 18 of the roulette table 1 must, at least, show a still image of the roulette table at the beginning of the end-result period.", "While this embodiment of the invention has been described in relation to single personal computer 14, it is to be understood that in further embodiments, a plurality of personal computers could be provided, each separately in communication with the server 8.In this way, a plurality of users may place bets remotely.", "As has been explained earlier, in some other embodiments of the invention, the live gambling event 1 is a game other than roulette.", "For example, the game may be the card game blackjack (also known as pontoon) which, in general terms, is played as follows.", "A plurality of card hands are dealt, each comprising two cards.", "In accordance with the rules of the card game, each card has a numerical value.", "Once each hand of two cards has been dealt, the setting up period begins in which a bet may be placed in relation to any of the card hands.", "Further cards are then dealt to each hand in turn in the subsequent dealing period.", "Finally, there is the end-result period, when the dealing for each hand is finished and the winning hand is determined as that which has the highest total value of cards, without exceeding a value of twenty one.", "Accordingly, in this embodiment of the invention, the first and second cameras 2,3 are directed at a card game table on which the cards are dealt.", "The first camera 2 is directed at the card game table and also the surrounding area such that the casino pit staff and any players at the table are within its field of vision.", "The second camera 3 is directed upon the card game table itself such the any dealt card hands fill its field of vision.", "On the web page 15, an animation of cards being dealt is displayed instead of the animated roulette table 16 of the first embodiment of the invention.", "Similarly, the computer generated image of the roulette table image 17 is replaced by a computer generated image of the dealt cards allowing the user of the personal computer 14 to place bets using a “point and click” interface.", "Furthermore, the live image 18 depicts the image of the cards on the table captured by the first or second camera 2,3.In other respects, the gambling apparatus is arranged substantially the same as for the first embodiment of the invention.", "In use of this embodiment of the invention, the image captured by the first camera 2 is transmitted to the personal computer 14 and is displayed in the live image 18 as described in relation to the first embodiment of the invention.", "In accordance with the rules of the blackjack game, a plurality of card hands are dealt on the card game table and these are visible both to individuals physically present at the card game table and to the user of the personal computer 14 who may observe the image 18 of the dealt card hands.", "Once the card hands have been dealt, the game is in its setting up period in which bets may be placed.", "The pit staff enter the status of the game in the pit terminal 4, and any bets that are placed by the individuals at the card game table and this information is transmitted to the personal computer 14 as described in relation to the first embodiment of the invention.", "The animation of the dealt cards that is displayed corresponds to this status of the game.", "The user of the personal computer 14 may place bets on one or more of the dealt card hands in a manner equivalent to that described in the first embodiment.", "When the duration of the set up period has expired, no more bets are accepted from individuals at the blackjack game.", "The pit staff prepare to deal further cards which commences the dealing period of the game that corresponds to the spinning period of the roulette game.", "The information regarding the change in status of the game is entered by the pit staff in the pit terminal 4.This information is transmitted to the personal computer 14 which then updates the web page 15 in a corresponding manner as for the first embodiment of the invention.", "The pit staff then proceed to deal any further cards in relation to each previously dealt card hand in accordance with the rules of blackjack until each hand is finished.", "When the dealing in relation to each card hand is finished, the game enters its end-result period.", "The pit staff enter the end-result (i.e.", "the winning card hand or hands) as for the first embodiment of the invention and if the individual using the personal computer 14 placed a winning bet then the winnings are transmitted accordingly.", "As for the first embodiment of the invention, after a predetermined interval, the pit staff commence a new blackjack game, by clearing the card game table, dealing new hands and entering in the pit terminal 4 that a new setting up period has begun.", "The process previously described may then be repeated.", "In a further embodiment of the present invention, the live gambling event 1 comprises a video slot machine.", "The slot machine is one generally known in the art comprising three independently spinnable wheels, a series of symbols being provided at regular intervals along the outer rim of each wheel.", "The three wheels are arranged, with their respective axes in line, in a housing.", "The housing has an elongate window, parallel to the axes of the three wheels, through which at least one symbol on each wheel is visible.", "The symbol on each wheel that is visible changes as the wheel spins.", "The normal function of such a video slot machine is generally as follows.", "A bet is placed in relation to the video slot machine in a setting up period.", "The wheels of the slot machine are then set spinning which commences the spinning period of the game.", "Each of the three wheels of the video slot machine is stopped at a random moment such that a particular symbol on each wheel is visible through the window in the housing of the video slot machine.", "Once each wheel is stopped, the end-result period of the game begins and winnings are determined on the basis of the particular combination of symbols on the three wheels visible through the window in the housing of the video slot machine.", "In this embodiment of the invention, instead of the first and second cameras 2, 3, a sensor detects the movement of each of the three wheels of the video slot machine and, in particular, which of the symbols is visible on each wheel through the window in the housing of the video slot machine.", "Furthermore, instead of the pit terminal 4, a further sensor automatically detects the status of the game.", "In use of this embodiment of the invention, the sensor detects the position of each of the three wheels of the video slot machine and generates a corresponding digitised representation of the image that would be visible of the three wheels of the video slot machine through the window in the housing by an individual physically present at the video slot machine.", "The generated representation is transmitted to the personal computer 14 and is displayed in the live image 18 as described in relation to the first embodiment of the invention.", "The user of the personal computer 14 may then place a bet in relation to the video slot machine during this setting up period.", "When the duration of the set up period (usually 10 seconds) has expired the wheels of the video slot machine are set spinning.", "The spinning of the wheels is detected by the further sensor, which, in turn, transmits the information regarding the change of status of the game to the personal computer 14.The personal computer 14 then updates the web page 15 in a corresponding manner as for the first embodiment of the invention.", "When each of the three wheels of the video slot machine has stopped, the end-result period begins.", "The generated representation of the symbols that are visible on the video slot machine is transmitted to the personal computer 14 and is displayed in the live image 18.If the individual using the personal computer 14 placed a winning bet then the winnings are transmitted accordingly.", "A new game on the video slot machine is then begun with the further sensor transmitting to the personal computer 14 an indication of the start of the setting up period in which new bets may be taken.", "The process previously described may then be repeated.", "It is to be appreciated that in other embodiments of the invention, a different number of wheels may be provided in the video slot machine instead of the three wheels described above.", "In particular, it is envisaged that video slot machines having one, five or even nine wheels may be provided.", "In the present specification “comprise” means “includes or consists of” and “comprising” means “including or consisting of”.", "The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof." ] ]

[ [ "Integrated optical transmitter, receiver for free space optical communication and network system and application apparatus thereof", "The present invention relates to the optical transmitter, receiver and application apparatus thereof for OWLL (Optical WireLess Link) which transmits and receives the optical signals through the free space and FSON (Free Space Optical Network) system using OWLL.", "Photonic devices such as laser diode and photo detector and integrated circuits for driving the photonic devices are formed directly into a single chip and the chip is assembled with optical instrument which is manufactured as a standardized optical module.", "Then, the optical transmitter, receiver and application apparatus thereof becomes small, light, cost-effective, multi-functional and reliable." ], [ "1.A transmitter for Free Space Optical Communication comprising: a semiconductor substrate; a light source formed on said substrate; a photo detector formed on said substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said substrate for driving said light source using the input signals from the outside and controlling the output power of said light source using the signals from said photo detector; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with said frame for receiving the light from said light source and transmitting the received light to the external free space.", "2.The transmitter of claim 1, wherein said light source is a laser diode or a light emitting diode.", "3.The transmitter of claim 1, wherein said optics module comprises: a lens; and a lens holder being, able to adjust the focal length of said lens.", "4.The transmitter of claim 1, wherein said lens is an aspheric lens or a Fresnel lens.", "5.The transmitter of claim 1, further comprising: a first screw unit formed to be integrated or assembled with said frame; and a second screw unit formed to be integrated or assembled with said optics module; wherein said frame and said optics module are assembled using said first and second screw units.", "6.The transmitter of claim 5, wherein said first and second screw units are standardized whereby various optics modules having lenses of different sizes can be assembled with said frame.", "7.The transmitter of claim 1, wherein the light from said transmitter is eye-safe.", "8.A receiver for Free Space Optical Communication comprising: a semiconductor substrate having a first and a second faces being opposite to each other; a photo detector formed on said first face of said substrate; an optical receiver circuit integrally formed on said first face of said substrate for transforming and outputting the signals received from said photo detector; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with said frame for receiving the light from the external free space and transmitting the received light to said photo detector.", "9.The receiver of claim 8, wherein said optical receiver circuit comprises a terminal for monitoring the magnitude of input signal at the outside of said optical receiver circuit.", "10.The receiver of claim 9, further comprising: a display unit connected to said terminal via at least one of said plurality of pins of said frame for displaying said magnitude of input signal to the outside of said receiver.", "11.The receiver of claim 9, wherein said magnitude of input signal can be transferred to the base station at the outside of said receiver.", "12.The receiver of claim 8, wherein said optics module comprises: a lens; and a lens holder being able to adjust the focal length of said lens.", "13.The receiver of claim 12, wherein said lens is an aspheric tens or a Fresnel lens.", "14.The receiver of claim 8, further comprising: a first screw unit formed to be integrated or assembled with said frame; and a second screw unit formed to be integrated or assembled with said optics module; wherein said frame and said optics module are assembled using said first and second screw, units.", "15.The receiver of claim 14, wherein said first and second screw unit are standardized whereby various optics modules having lenses of different sizes can be assembled with said frame.", "16.The receiver of claim 8, wherein said optics module is arranged in a row with said optical receiver circuit and said photo detector.", "17.The receiver of claim 8, wherein said optics module is arranged parallel to said second face on or above said second face side; and said frame has an aperture exposing a part of said second face opposite to the part of said first face where said light source is formed.", "18.The receiver of claim 17, wherein said optics module is a tens formed on said second face of said substrate; and said aperture exposes a part where said lens is formed.", "19.The receiver of claim 18, wherein said lens is formed by etching said semiconductor substrate.", "20.The receiver of claim 18, wherein said lens is formed by coating.", "21.A transceiver for Free Space Optical Communication comprising: a semiconductor substrate; a light source formed on said substrate; a first photo detector formed on said substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said substrate for driving said light source using the input signals from the outside and controlling the output power of said light source using the signals from said first photo detector; a second photo detector formed on said substrate; an optical receiver circuit integrally formed on said substrate for transforming and outputting the signals received from said second photo detector; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with said frame for receiving the light from said light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with said frame for receiving the light from the external free space and transmitting the received light to said second photo detector.", "22.The transceiver of claim 21, further comprising: a first screw unit formed to be integrated or assembled with said frame and adjacent with the part of said substrate where said light source is formed; a second screw unit formed to be integrated or assembled with said frame and adjacent with the part of said substrate where said second photo detector is formed; a third screw unit formed to be integrated or assembled with said transmitting optics module; and a fourth screw unit formed to be integrated or assembled with said receiving optics module; wherein said frame and said transmitting optics module are assembled using said first and third screw units; and wherein said frame and said receiving optics module are assembled using said second and fourth screw units.", "23.The transceiver of claim 21, wherein said transmitting optics module and said receiving optics module face to the same side.", "24.The transceiver of claim 21, wherein said transmitting optics module and said receiving optics module have the same configuration.", "25.The transceiver of claim 21, wherein said transmitting optics module and said receiving optics module have different configurations from each other.", "26.A transceiver for Free Space Optical Communication comprising: a first semiconductor substrate; a light source formed on said first substrate; a first photo detector formed on said first substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said first substrate for driving said light source using the input signals from the outside and controlling the output power of said light source using the signals from said first photo detector; a first frame, where said first substrate is fixed, having a plurality of pins for electrical connection to the outside; a second semiconductor substrate; a second photo detector formed on said second substrate; an optical receiver circuit integrally formed on said second substrate for transforming and outputting the signals received from said second photo detector; a second frame, where said second substrate is fixed, having a plurality of pins for electrical connection to the outside; a printed circuit board where said first and second frames are fixed at a predetermined interval; a transmitting optics module formed to be assembled with said printed circuit board for receiving the light from said light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with said printed circuit board for receiving the light from the external free space and transmitting the received light lo said second photo detector.", "27.The transceiver of claim 26, further comprising: a first screw unit formed to be integrated or assembled with said printed circuit board and adjacent with the part of said first substrate where said light source is formed; a second screw unit formed to be integrated or assembled with said printed circuit board and adjacent with the part of said second substrate where said second photo detector is formed; a third screw unit formed to be integrated or assembled with said transmitting optics module; and a fourth screw unit formed to be integrated or assembled with said receiving optics module; wherein said printed circuit board and said transmitting optics module are assembled using said first and third screw units; and wherein said printed circuit board and said receiving optics module are assembled using said second and fourth screw units.", "28.A transceiver for Free Space Optical Communication comprising: a semiconductor substrate; a first light source formed on said substrate; a first photo detector formed on said substrate for detecting the light from said first light source; a first current driver and automatic output controller circuit integrally formed on said substrate for driving said first light source using the input signals from the outside and controlling the output power of said first light source using the signals from said first photo detector; a first optical receiver circuit integrally formed on said substrate and connected to said first current driver and automatic output controller circuit for providing said first current driver and automatic output controller circuit with input signals; a second photo detector connected to said first optical receiver circuit for providing said first optical receiver circuit with input signal; a first optical fiber adaptor connected to said second photo detector for connecting said second photo detector to an optical fiber; a third photo detector formed on said substrate; a second optical receiver circuit integrally formed on said substrate for transforming and outputting the signals received from said third photo detector; a second current driver and automatic output controller circuit integrally formed on said substrate for receiving signals from said second optical receiver circuit; a second light source connected to said second current driver and automatic output controller circuit and driven bar said second current driver and automatic output controller circuit; a second optical fiber adaptor connected to said second light source for connecting said second tight source to an optical fiber; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with said frame for receiving the Light from said first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with said frame for receiving the light from the external free space and transmitting the received light to said third photo detector.", "29.The transceiver of claim 28, wherein said second photo detector and said second light source are packaged in TO-cans respectively.", "30.The transceiver of claim 28, wherein said second photo detector and said second light source are formed on said substrate.", "31.A transceiver for Free Space Optical Communication comprising: a semiconductor substrate; a light source formed on said substrate; a first photo detector formed on said substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said substrate for driving said light source using the input signals from the outside and controlling the output power of said light source using the signals from said first photo detector; a second photo detector formed on said substrate; an optical receiver circuit integrally formed on said substrate for transforming and outputting the signals received from said second photo detector; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with said frame for receiving the light from said first light source and transmitting the received light to the external free space; a receiving optics module formed to be assembled with said frame for receiving the light from the external free space and transmitting the received light to said second photo detector; and a media converter circuit integrally formed on said substrate and connected to said current driver and automatic output controller circuit and said optical receiver circuit, for transforming the signals transmitted from said optical receiver circuit to Ethernet signals and for transforming Ethernet signals received from the outside to said current driver and automatic output controller circuit and transmitting it, and having UTP (unshielded twisted-pair) port for transmitting and receiving Ethernet signals to and from the outside.", "32.A transponder for Free Space Optical Communication comprising: a semiconductor substrate; a light source formed on said substrate; a first photo detector formed on said substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said substrate and connected to said light source for driving said light source using the input signals from the outside and controlling the output power of said light source using the signal from said first photo detector; a multiplexer circuit integrally formed on said substrate and connected to said current driver and automatic output controller circuit for multiplexing the input signals from the outside and outputting the multiplexed signals to said current driver and automatic output controller circuit; a second photo detector formed on said substrate; an optical receiver circuit integrally formed on said substrate for transforming and outputting the signals received from said second photo detector; a demultiplexer circuit integrally formed on said substrate and connected to said optical receiver circuit for receiving signals from said optical receiver circuit and outputting demultiplexed signals; a frame, where said substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with said frame for receiving the light from said first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with said frame for receiving the light from the external free space and transmitting the received light to said second Photo detector.", "33.A transponder for Free Space Optical Communication comprising: a first semiconductor substrate; a first photo detector formed on said first substrate; an optical receiver circuit integrally formed on said first substrate for transforming and outputting the signals received from said first photo detector; a demultiplexer circuit, integrally formed on said first substrate, having an input port connected to said optical receiver circuit for receiving signals from said optical receiver circuit, a drop port for distributing a part of demultiplexed signals, and an output port for outputting the rest of said demultiplexed signals; a first frame, where said first substrate is fixed, having a plurality of pills for electrical connection to the outside; a second semiconductor substrate; a light source formed on said second substrate; a second photo detector formed on said substrate for detecting the light from said light source; a current driver and automatic output controller circuit integrally formed on said second substrate and connected to said light source for driving said light source using the input signals from the outside and controlling the output power of said light source using the signals received from said second photo detector; a multiplexer circuit, integrally formed on said second substrate, having an input port for receiving signals from said output port of said demultiplexer, an add port for receiving additional signals from the outside, and an output port for outputting multiplexed signal to said current driver and automatic output controller circuit; a second frame, where said second substrate is fixed, having a plurality of pins for electrical connection for the outside; a printed circuit board where said first and second frames are fixed at a predetermined interval; a transmitting optics module formed to be assembled with said printed circuit board for receiving the light from said first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with said printed circuit board for receiving the light from the external free space and transmitting the received light to said second photo detector." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The 21th century information communication society requires a social environment in which the subscribers can exchange the large amount of information at high speed, and such high speed communication becomes possible due to the improvements of the wireless communication technique of high frequency band and high speed optical communication technique using optical fibers.", "The study of optical communication which started in 1970s has progressed recent ten and some years to minimize the transmission loss to extend the transmission distance and to transmit a large amount of information at high speed, and now the optical communication system is in the stage of practical use, that is, the band width of the core optical communication network is over 100 Gbps, and it may reach some Tbps by 2000s.", "However, the technique providing the information at over tens of Mbps speed for the final user or subscriber is not developed so much.", "Roles of optical communication technique, which secure the high speed, parallelism, and large capacity, are very important to establish very high speed broadband integrated services communication network.", "The conventional wireless communication system, which transmits data at tens of kbps speed in PCS system of 2 GHz, is not enough to provide wireless multimedia service.", "In this regard, studies about IMT-2000 having maximum data transmission rate of 2 Mbps, which is called as the third generation wireless communication, are in progress, and now it is in the stage of practical user.", "However, the next generation multimedia system for very high rate data transmission such as HDTV requires tens to hundreds Mbps rate data transmission for the subscribers, therefore, the IMT-2000 cannot be a final solution.", "The next generation multimedia is a system and service which make various information such as text, data, audio, graphic, photo, animation, image, etc.", "to produce, collect, transmit, and process integrally, and the multimedia industry means the industrial field related to those activities.", "Recently, the multimedia information industry goes in the direction of digitalization, bi-directionization, asynchronization, and integrallization of image, sound, etc.", "in the content, form, and exchange method due to the development of the technologies in computer and communication fields.", "The effect of the technology development to the industrial structure is evolutional.", "For the most important obstacle to the present multimedia service, the performance of the communication network having insufficient capacity is pointed out, and the role of locomotive to progressive reproduction of the next generation multimedia is given to providing the communication network of very high speed and large capacity for individual subscribers economically.", "It is considered that the only network technology which able to provide the very high speed and large capacity information for individual subscribers is the fiber-to-the-home (“FTTH”), however, in case of the FTTH, the installation is difficult, and the cost of installation is large because additional cost is required to lay the optical fiber underground as well as the communication device.", "Moreover, it requires additional steps of aligning between the optical fiber and laser diode (“LD”) or photo detector (“PD”) for the optical transmitting/receiving module.", "The present invention pursues very economical and easily installable optical transmitting/receiving module which enabling FSON which can solve the problems of the FTTH instead of the wireless communication network using coaxial cables and microwave (“MW”) transmitting/receiving device such as high frequency oscillator, modulator, etc.", "to connect the base station (“BS”) and the central base station (“CBS”) such as mobile service switching center.", "Until now, the FSON is used as the back-up system for the existing wire network utilizing the advantages that the service can be provided instantly because the installation is easy and fast and that the communication protection is guaranteed physically, or most efforts are concentrated on development of high power transceiver focusing point-to-point connection considering quick installation, therefore, it is not used so practically.", "Therefore, the present invention suggests economical transmitting/receiving modules for FSON suitable to provide the very high speed and large capacity information for a plurality of users or subscribers stably using OWLL and FSON system using OWLL different from the existing simple point-to-point type." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The new OWLL and FSON system leaded to resolve the problems and limits of the above described convention technology has differences to the conventional wire/wireless communication network in that they can provide the complex multimedia communication service such as high-speed internet, point-to-point and point-to-multiple point data, audio, and image transmission with very high speed, large capacity, stability, and efficiency preparing the next generation multimedia era.", "The OWLL and FSON system in which basic blocks are set according to the transmission distance and transmission rate and such blocks are combined in various way to provide very high speed and large capacity information without being affected by the position and distance of the subscriber is the communication system of completely new concept for very high speed and large capacity communication system.", "The OWLL and FSON system should be robust to the turbulence of the air, temperature gradient, snow, rain, fog, etc.", "and able to change the intensity and direction of the optical output, bit-rate, etc.", "adaptively according to the surrounding environments.", "In addition, it should be constituted as a system able to monitor, control, and operate the transmitting/receiving status integrally.", "The necessities for OWLL and FSON system are the economical transmitter, receiver, and various application apparatuses thereof enabling the OWLL and FSON system.", "Therefore, the object of the present invention is to provide the transmitter, receiver, and various application apparatuses thereof for OWLL and FSON.", "Another object of the present invention is to provide the transmitter, receiver, and various application apparatuses thereof for OWLL, which are small, light, cheap, stable, and reliable.", "To achieve the above objects, the present invention provides transmitting/receiving apparatuses for providing OWLL and FSON information communication service in which light source(s) such as laser diode, photo-electric device(s) for optical transmission and reception such as photo detector, and related circuit(s) are formed on one printed circuit board, and the printed circuit board and the optics modules are manufactured as standardized modules to be easily assembled with each other.", "To achieve the above objects, the present invention provides transmitting/receiving apparatuses for providing OWLL and FSON information communication service in which light source(s) such as laser diode, photo-electric device(s) for optical transmission and reception such as photo detector, and related circuit(s) are formed on one printed circuit board, and the printed circuit board and the optics modules are manufactured as standardized modules to be easily assembled with each other.", "That is, a transmitter for free space optical communication according to the present invention comprises: a semiconductor substrate; a light source formed on the substrate; a photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with the frame for receiving the light from the light source and transmitting the received light to the external free space.", "Here, the light source is preferably a laser diode or a light emitting diode.", "The optics module comprises: a lens; and a lens holder being able to adjust the focal length of the lens, and an aspheric lens or a Fresnel lens can be used for the lens.", "In addition, the transmitter of the present invention further includes a first screw unit formed to be integrated or assembled with the frame; and a second screw unit formed to be integrated or assembled with the optics module to make the frame and the optics module be assembled using the first and second screw units.", "The light from the transmitter is eye-safe.", "A receiver for free space optical communication according to the present invention comprises: a semiconductor substrate having a first and a second faces being opposite to each other; a photo detector formed on the first face of the substrate; an optical receiver circuit integrally formed on the first face of the substrate for transforming and outputting the signals received from the photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the photo detector.", "Here, the optical receiver circuit comprises a terminal for monitoring the magnitude of input signal at the outside of the optical receiver circuit, and it is preferable that the receiver further includes a display unit connected to the terminal via at least one of the plurality of pins of the frame for displaying the magnitude of input signal to the outside of the receiver or the magnitude of input signal can be transferred to the base station at the outside of the receiver.", "Also, the receiver of the present invention has a first screw unit formed to be integrated or assembled with the frame; and a second screw unit formed to be integrated or assembled with the optics module to make it possible for the frame and the optics module to be assembled using the first and second screw units.", "On the other hand, the optics module is arranged in a row with the optical receiver circuit and the photo detector or parallel to the second face on or above the second face side.", "In case of the latter, the frame has an aperture exposing a part of the second face opposite to the part of the first face where the light source is formed, the optics module is a lens formed on the second face of the substrate, and the aperture exposes a part where the lens is formed.", "The lens can be formed by etching or coating.", "A transceiver for free space optical communication according to the present invention comprises: a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the first photo detector; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector.", "Here, the transceiver further includes a first screw unit formed to be integrated or assembled with the frame and adjacent with the part of the substrate where the light source is formed; a second screw unit formed to be integrated or assembled with the frame and adjacent with the part of the substrate where the second photo detector is formed; a third screw unit formed to be integrated or assembled with the transmitting optics module; and a fourth screw unit formed to be integrated or assembled with the receiving optics module, and it is preferable that the frame and the transmitting optics module are assembled using the first and third screw units and the frame and the receiving optics module are assembled using the second and fourth screw units.", "The transmitting optics module and the receiving optics module can face to the same side, and the transmitting optics module and the receiving optics module have the same configuration or different configurations from each other.", "Here, it is possible to fix a first and a second frames on one printed circuit board after fixing a first and a second substrates on the first and second frames after forming the light source, first photo detector, current driver and automatic output controller circuit on the first substrate and forming the second photo detector and optical receiver circuit for optical communication on the second substrate.", "The transceiver of the present invention may provide a connection with an optical fiber link.", "That is, a transceiver according to another embodiment of the present invention comprises: a semiconductor substrate; a first light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the first light source; a first current driver and automatic output controller circuit integrally formed on the substrate for driving the first light source using the input signals from the outside and controlling the output power of the first light source using the signals from the first photo detector; a first optical receiver circuit integrally formed on the substrate and connected to the first current driver and automatic output controller circuit for providing the first current driver and automatic output controller circuit with input signals; a second photo detector connected to the first optical receiver circuit for providing the first optical receiver circuit with input signal; a first optical fiber adaptor connected to the second photo detector for connecting the second photo detector to an optical fiber; a third photo detector formed on the substrate; a second optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the third photo detector; a second current driver and automatic output controller circuit integrally formed on the substrate for receiving signals from the second optical receiver circuit; a second light source connected to the second current driver and automatic output controller circuit and driven by the second current driver and automatic output controller circuit; a second optical fiber adaptor connected to the second light source for connecting the second light source to an optical fiber; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the third photo detector.", "Here, the second photo detector and the second light source may be packaged in TO-cans, respectively, or formed directly on the substrate.", "Moreover, the transceiver of the present invention provides a connection to the Ethernet using a media converter, and a transceiver of another embodiment for this purpose comprises: a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally, formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the first photo detector; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector; and a media converter circuit, integrally formed on the substrate and connected to the current driver and automatic output controller circuit and the optical receiver circuit, for transforming the signals transmitted from the optical receiver circuit to Ethernet signals and for transforming Ethernet signals received from the outside to the current driver and automatic output controller circuit and transmitting it, and having UTP (unshielded twisted-pair) port for transmitting and receiving Ethernet signals to and from the outside.", "A transponder for free space optical communication according to the present invention comprises a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the tight source; a current driver and automatic output controller circuit integrally formed on the substrate and connected to the light source for driving the light source using the input signals from the outside and controlling the output power of the light source using the signal from the first photo detector; a multiplexer circuit integrally formed on the substrate and connected to the current driver and automatic output controller circuit for multiplexing the input signals from the outside and outputting the multiplexed signals to the current driver and automatic output controller circuit; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a demultiplexer circuit integrally formed on the substrate and connected to the optical receiver circuit for receiving signals from the optical receiver circuit and outputting demultiplexed signals; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector.", "A transponder for free space optical communication according to another embodiment of the present invention comprises: a first semiconductor substrate; a first photo detector formed on the first substrate; an optical receiver circuit integrally formed on the first substrate for transforming and outputting the signals received from the first photo detector; a demultiplexer circuit, integrally formed on the first substrate, having an input port connected to the optical receiver circuit for receiving signals from the optical receiver circuit, a drop port for distributing a part of demultiplexed signals, and an output port for outputting the rest of the demultiplexed signals; a first frame, where the first substrate is fixed, having a plurality of pins for electrical connection to the outside; a second semiconductor substrate; a light source formed on the second substrate; a second photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the second substrate and connected to the light source for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals received from the second photo detector; a multiplexer circuit, integrally formed on the second substrate, having an input port for receiving signals from the output port of the demultiplexer, an add port for receiving additional signals from the outside, and an output port for outputting multiplexed signal to the current driver and automatic output controller circuit; a second frame, where the second substrate is fixed, having a plurality of pins for electrical connection for the outside; a printed circuit board where the first and second frames are fixed at a predetermined interval; a transmitting optics module formed to be assembled with the printed circuit board for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the printed circuit board for receiving the light from the external free space and transmitting the received light to the second photo detector." ], [ "FIELD OF THE ART The present invention relates to a transmitter, receiver and application apparatuses thereof enabling an optical wireless link (“OWLL”) using communication method in which optical signals are transmitted/received through the free space, i.e., the air, and a free space optical network (“FSON”) system using the OWLL.", "BACKGROUND OF THE INVENTION The 21th century information communication society requires a social environment in which the subscribers can exchange the large amount of information at high speed, and such high speed communication becomes possible due to the improvements of the wireless communication technique of high frequency band and high speed optical communication technique using optical fibers.", "The study of optical communication which started in 1970s has progressed recent ten and some years to minimize the transmission loss to extend the transmission distance and to transmit a large amount of information at high speed, and now the optical communication system is in the stage of practical use, that is, the band width of the core optical communication network is over 100 Gbps, and it may reach some Tbps by 2000s.", "However, the technique providing the information at over tens of Mbps speed for the final user or subscriber is not developed so much.", "Roles of optical communication technique, which secure the high speed, parallelism, and large capacity, are very important to establish very high speed broadband integrated services communication network.", "The conventional wireless communication system, which transmits data at tens of kbps speed in PCS system of 2 GHz, is not enough to provide wireless multimedia service.", "In this regard, studies about IMT-2000 having maximum data transmission rate of 2 Mbps, which is called as the third generation wireless communication, are in progress, and now it is in the stage of practical user.", "However, the next generation multimedia system for very high rate data transmission such as HDTV requires tens to hundreds Mbps rate data transmission for the subscribers, therefore, the IMT-2000 cannot be a final solution.", "The next generation multimedia is a system and service which make various information such as text, data, audio, graphic, photo, animation, image, etc.", "to produce, collect, transmit, and process integrally, and the multimedia industry means the industrial field related to those activities.", "Recently, the multimedia information industry goes in the direction of digitalization, bi-directionization, asynchronization, and integrallization of image, sound, etc.", "in the content, form, and exchange method due to the development of the technologies in computer and communication fields.", "The effect of the technology development to the industrial structure is evolutional.", "For the most important obstacle to the present multimedia service, the performance of the communication network having insufficient capacity is pointed out, and the role of locomotive to progressive reproduction of the next generation multimedia is given to providing the communication network of very high speed and large capacity for individual subscribers economically.", "It is considered that the only network technology which able to provide the very high speed and large capacity information for individual subscribers is the fiber-to-the-home (“FTTH”), however, in case of the FTTH, the installation is difficult, and the cost of installation is large because additional cost is required to lay the optical fiber underground as well as the communication device.", "Moreover, it requires additional steps of aligning between the optical fiber and laser diode (“LD”) or photo detector (“PD”) for the optical transmitting/receiving module.", "The present invention pursues very economical and easily installable optical transmitting/receiving module which enabling FSON which can solve the problems of the FTTH instead of the wireless communication network using coaxial cables and microwave (“MW”) transmitting/receiving device such as high frequency oscillator, modulator, etc.", "to connect the base station (“BS”) and the central base station (“CBS”) such as mobile service switching center.", "Until now, the FSON is used as the back-up system for the existing wire network utilizing the advantages that the service can be provided instantly because the installation is easy and fast and that the communication protection is guaranteed physically, or most efforts are concentrated on development of high power transceiver focusing point-to-point connection considering quick installation, therefore, it is not used so practically.", "Therefore, the present invention suggests economical transmitting/receiving modules for FSON suitable to provide the very high speed and large capacity information for a plurality of users or subscribers stably using OWLL and FSON system using OWLL different from the existing simple point-to-point type.", "SUMMARY OF THE INVENTION The new OWLL and FSON system leaded to resolve the problems and limits of the above described convention technology has differences to the conventional wire/wireless communication network in that they can provide the complex multimedia communication service such as high-speed internet, point-to-point and point-to-multiple point data, audio, and image transmission with very high speed, large capacity, stability, and efficiency preparing the next generation multimedia era.", "The OWLL and FSON system in which basic blocks are set according to the transmission distance and transmission rate and such blocks are combined in various way to provide very high speed and large capacity information without being affected by the position and distance of the subscriber is the communication system of completely new concept for very high speed and large capacity communication system.", "The OWLL and FSON system should be robust to the turbulence of the air, temperature gradient, snow, rain, fog, etc.", "and able to change the intensity and direction of the optical output, bit-rate, etc.", "adaptively according to the surrounding environments.", "In addition, it should be constituted as a system able to monitor, control, and operate the transmitting/receiving status integrally.", "The necessities for OWLL and FSON system are the economical transmitter, receiver, and various application apparatuses thereof enabling the OWLL and FSON system.", "Therefore, the object of the present invention is to provide the transmitter, receiver, and various application apparatuses thereof for OWLL and FSON.", "Another object of the present invention is to provide the transmitter, receiver, and various application apparatuses thereof for OWLL, which are small, light, cheap, stable, and reliable.", "To achieve the above objects, the present invention provides transmitting/receiving apparatuses for providing OWLL and FSON information communication service in which light source(s) such as laser diode, photo-electric device(s) for optical transmission and reception such as photo detector, and related circuit(s) are formed on one printed circuit board, and the printed circuit board and the optics modules are manufactured as standardized modules to be easily assembled with each other.", "To achieve the above objects, the present invention provides transmitting/receiving apparatuses for providing OWLL and FSON information communication service in which light source(s) such as laser diode, photo-electric device(s) for optical transmission and reception such as photo detector, and related circuit(s) are formed on one printed circuit board, and the printed circuit board and the optics modules are manufactured as standardized modules to be easily assembled with each other.", "That is, a transmitter for free space optical communication according to the present invention comprises: a semiconductor substrate; a light source formed on the substrate; a photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with the frame for receiving the light from the light source and transmitting the received light to the external free space.", "Here, the light source is preferably a laser diode or a light emitting diode.", "The optics module comprises: a lens; and a lens holder being able to adjust the focal length of the lens, and an aspheric lens or a Fresnel lens can be used for the lens.", "In addition, the transmitter of the present invention further includes a first screw unit formed to be integrated or assembled with the frame; and a second screw unit formed to be integrated or assembled with the optics module to make the frame and the optics module be assembled using the first and second screw units.", "The light from the transmitter is eye-safe.", "A receiver for free space optical communication according to the present invention comprises: a semiconductor substrate having a first and a second faces being opposite to each other; a photo detector formed on the first face of the substrate; an optical receiver circuit integrally formed on the first face of the substrate for transforming and outputting the signals received from the photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; and an optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the photo detector.", "Here, the optical receiver circuit comprises a terminal for monitoring the magnitude of input signal at the outside of the optical receiver circuit, and it is preferable that the receiver further includes a display unit connected to the terminal via at least one of the plurality of pins of the frame for displaying the magnitude of input signal to the outside of the receiver or the magnitude of input signal can be transferred to the base station at the outside of the receiver.", "Also, the receiver of the present invention has a first screw unit formed to be integrated or assembled with the frame; and a second screw unit formed to be integrated or assembled with the optics module to make it possible for the frame and the optics module to be assembled using the first and second screw units.", "On the other hand, the optics module is arranged in a row with the optical receiver circuit and the photo detector or parallel to the second face on or above the second face side.", "In case of the latter, the frame has an aperture exposing a part of the second face opposite to the part of the first face where the light source is formed, the optics module is a lens formed on the second face of the substrate, and the aperture exposes a part where the lens is formed.", "The lens can be formed by etching or coating.", "A transceiver for free space optical communication according to the present invention comprises: a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the first photo detector; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector.", "Here, the transceiver further includes a first screw unit formed to be integrated or assembled with the frame and adjacent with the part of the substrate where the light source is formed; a second screw unit formed to be integrated or assembled with the frame and adjacent with the part of the substrate where the second photo detector is formed; a third screw unit formed to be integrated or assembled with the transmitting optics module; and a fourth screw unit formed to be integrated or assembled with the receiving optics module, and it is preferable that the frame and the transmitting optics module are assembled using the first and third screw units and the frame and the receiving optics module are assembled using the second and fourth screw units.", "The transmitting optics module and the receiving optics module can face to the same side, and the transmitting optics module and the receiving optics module have the same configuration or different configurations from each other.", "Here, it is possible to fix a first and a second frames on one printed circuit board after fixing a first and a second substrates on the first and second frames after forming the light source, first photo detector, current driver and automatic output controller circuit on the first substrate and forming the second photo detector and optical receiver circuit for optical communication on the second substrate.", "The transceiver of the present invention may provide a connection with an optical fiber link.", "That is, a transceiver according to another embodiment of the present invention comprises: a semiconductor substrate; a first light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the first light source; a first current driver and automatic output controller circuit integrally formed on the substrate for driving the first light source using the input signals from the outside and controlling the output power of the first light source using the signals from the first photo detector; a first optical receiver circuit integrally formed on the substrate and connected to the first current driver and automatic output controller circuit for providing the first current driver and automatic output controller circuit with input signals; a second photo detector connected to the first optical receiver circuit for providing the first optical receiver circuit with input signal; a first optical fiber adaptor connected to the second photo detector for connecting the second photo detector to an optical fiber; a third photo detector formed on the substrate; a second optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the third photo detector; a second current driver and automatic output controller circuit integrally formed on the substrate for receiving signals from the second optical receiver circuit; a second light source connected to the second current driver and automatic output controller circuit and driven by the second current driver and automatic output controller circuit; a second optical fiber adaptor connected to the second light source for connecting the second light source to an optical fiber; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the third photo detector.", "Here, the second photo detector and the second light source may be packaged in TO-cans, respectively, or formed directly on the substrate.", "Moreover, the transceiver of the present invention provides a connection to the Ethernet using a media converter, and a transceiver of another embodiment for this purpose comprises: a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally, formed on the substrate for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals from the first photo detector; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector; and a media converter circuit, integrally formed on the substrate and connected to the current driver and automatic output controller circuit and the optical receiver circuit, for transforming the signals transmitted from the optical receiver circuit to Ethernet signals and for transforming Ethernet signals received from the outside to the current driver and automatic output controller circuit and transmitting it, and having UTP (unshielded twisted-pair) port for transmitting and receiving Ethernet signals to and from the outside.", "A transponder for free space optical communication according to the present invention comprises a semiconductor substrate; a light source formed on the substrate; a first photo detector formed on the substrate for detecting the light from the tight source; a current driver and automatic output controller circuit integrally formed on the substrate and connected to the light source for driving the light source using the input signals from the outside and controlling the output power of the light source using the signal from the first photo detector; a multiplexer circuit integrally formed on the substrate and connected to the current driver and automatic output controller circuit for multiplexing the input signals from the outside and outputting the multiplexed signals to the current driver and automatic output controller circuit; a second photo detector formed on the substrate; an optical receiver circuit integrally formed on the substrate for transforming and outputting the signals received from the second photo detector; a demultiplexer circuit integrally formed on the substrate and connected to the optical receiver circuit for receiving signals from the optical receiver circuit and outputting demultiplexed signals; a frame, where the substrate is fixed, having a plurality of pins for electrical connection to the outside; a transmitting optics module formed to be assembled with the frame for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the frame for receiving the light from the external free space and transmitting the received light to the second photo detector.", "A transponder for free space optical communication according to another embodiment of the present invention comprises: a first semiconductor substrate; a first photo detector formed on the first substrate; an optical receiver circuit integrally formed on the first substrate for transforming and outputting the signals received from the first photo detector; a demultiplexer circuit, integrally formed on the first substrate, having an input port connected to the optical receiver circuit for receiving signals from the optical receiver circuit, a drop port for distributing a part of demultiplexed signals, and an output port for outputting the rest of the demultiplexed signals; a first frame, where the first substrate is fixed, having a plurality of pins for electrical connection to the outside; a second semiconductor substrate; a light source formed on the second substrate; a second photo detector formed on the substrate for detecting the light from the light source; a current driver and automatic output controller circuit integrally formed on the second substrate and connected to the light source for driving the light source using the input signals from the outside and controlling the output power of the light source using the signals received from the second photo detector; a multiplexer circuit, integrally formed on the second substrate, having an input port for receiving signals from the output port of the demultiplexer, an add port for receiving additional signals from the outside, and an output port for outputting multiplexed signal to the current driver and automatic output controller circuit; a second frame, where the second substrate is fixed, having a plurality of pins for electrical connection for the outside; a printed circuit board where the first and second frames are fixed at a predetermined interval; a transmitting optics module formed to be assembled with the printed circuit board for receiving the light from the first light source and transmitting the received light to the external free space; and a receiving optics module formed to be assembled with the printed circuit board for receiving the light from the external free space and transmitting the received light to the second photo detector.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 is a schematic diagram showing a transmitter for free space optical communication according to an embodiment of the present invention.", "FIG.", "2 is a block diagram showing an example of a current driver and automatic output controller circuit used in the transmitter shown in FIG.", "1.FIGS.", "3 and 4 are schematic diagrams showing transmitters for free space optical communication according to another embodiments of the present invention.", "FIG.", "5 is a schematic diagram showing a receiver for free space optical communication according to an embodiment of the present invention.", "FIG.", "6 is a block diagram showing an example of an optical receiver circuit used in the receiver shown in FIG.", "5.FIGS.", "7 and 8 are schematic diagrams showing transmitters for free space optical communication according to another embodiments of the present invention.", "FIG.", "9 shows a transceiver for free space optical communication according to an embodiment of the present invention.", "FIG.", "10 shows a transceiver for free space optical communication according to another embodiment of the present invention.", "FIG.", "11 shows a transceiver for free space optical communication accessible via optical fiber link according to an embodiment of the present invention: FIG.", "12 shows a transceiver for free space optical communication accessible via optical fiber link according to another embodiment of the present invention.", "FIG.", "13 is a schematic diagram showing a transceiver for free space optical communication able to connect to the Ethernet according to another embodiment of the present invention.", "FIG.", "14 shows an example of a transponder for free space optical communication according to the present invention.", "FIGS.", "15 and 16 are schematic diagrams showing the transmitting and receiving parts of a transponder for free space optical communication whose transmitting and receiving parts are separated according to an embodiment of the present invention, respectively.", "FIG.", "17 is a layout diagram showing a receiver for free space optical communication according to another embodiment of the present invention.", "FIGS.", "18 through 20 are sectional diagrams showing receivers for free space optical communication according to another embodiments of the present invention.", "BEST MODE FOR CARRYING OUT THE INVENTION Now, preferred embodiments of the present invention will be described in detail with reference to accompanying drawings.", "First, a structure of a transmitter for free space optical communication will be described.", "FIG.", "1 is a schematic diagram showing a transmitter 100 for free space optical communication according to an embodiment of the present invention, and FIG.", "2 is a block diagram showing an example of a current driver and automatic output controller circuit used in the transmitter shown in FIG.", "1.As shown in FIG.", "1, a current driver and automatic output controller integrated circuit (“IC”) 130 is formed on a semiconductor substrate 101 made of silicon (“Si”), etc.", "in a transmitter 100.The current driver and automatic output controller IC 130 can be formed in various ways, and an example thereof is shown in FIG.", "2.That is, it includes an input amplifier 1302 receiving an input signal from the outside and amplifying the signal and a LD driver circuit 1304 driving the LID 110, the light source, using the signal amplified through the input amplifier 1302, and the signal detected through the PD 120 is amplified by the light detecting amplifier 1306, transmitted to the automatic output control circuit 1308, and used to control the LD driver circuit 1304.In addition, the current driver and automatic output controller IC 130 is manufactured according to known IC manufacturing process.", "On the substrate 101 on which the current driver and automatic output controller IC 130 is formed, a laser diode (“LD”) 110, which is a light source to transmit a light carrying an free space optical communication signal to the free space outside of the transmitter 100 is formed.", "A light emitting diode (“LED”) can be used as the light source as well as LD.", "For LDs, various kinds of LDs such as Febry-Perot LD, distributed feedback LD (“DFB-LD”), vertical cavity surface emitting laser (“VCSEL”), etc.", "can be used.", "The light from the LD 110 is collimated through an optics module 140 and transmitted to the free space.", "It is related to the transmission distance of the transmitter which kind of light sources is used.", "Transmitters can be classified for very short distance (less than 100 m), short distance (50-300 m), middle distance (150-500 m), and long distance (500-2000 m), and, for example, a VCSEL having a nominal wavelength of 0.85*10-6 m is preferably used for the very short distance transmitter as the light source.", "In addition, the nominal wavelength of the light from the LD can be 1.3*10-6 m or 1.55*10-6 m if the transmitter according to the present invention is used for the middle distance of less than 500 m or short distance of less than 300 m free space optical communication.", "It is preferable that the light from the light source satisfies the safety standard for human body including the eyes.", "Moreover, a photo detector (“PD”) 120 is formed on the PCB 101 adjacent to the LD 110 having a little bit of space between them to detect the light from the LD 110.For PD 120, various kinds of devices such as MSM (metal-semiconductor-metal) PD, PIN (inversely biased P—N junction) PD, APD (avalanche photodiode), etc.", "can be used.", "The PD 120 detects the light from the LD 110 and uses it as a signal to control the output of the LD 110.The current driver and automatic output controller IC 130 formed on the substrate 101 has a plurality of bonding pads 103 to provide the connection with the circuit, and the LD 110 and PD 120 are connected to the parts providing connections to corresponding connecting parts in the current driver and automatic output controller IC 130 among bonding pads 103.That is, the LD 110 is connected to the LD driver circuit 1304 of FIG.", "2, and the PD 120 is connected to the light detecting amplifier 1306 in FIG.", "2.Since the LD 110 is placed on the part connected to an optics module, a separate connecting part 109 can be formed to connect to the current driver and automatic output controller IC 130.The process of forming the current driver and automatic output controller IC 130 on the substrate 101 follows a general semiconductor manufacturing process, and the PD 120 can be formed together in the circuit manufacturing process if needed.", "In case of LD 110, an LD device formed separately is attached on the substrate 101.Manufactured substrate 101 is fixed on an IC frame 107, and bonding pads 103 provided for the IC 130 are wire bonded with bonding pads 104 of the ID frame 107 to be connected to the outside via pins 108 of the IC frame 107.On the other hand, the optics module 140 is constituted of a lens 141 and a lens holder 142, and it is fixed on the IC frame 107 where the substrate 101 on which the light source 110, PD 120, and IC 130 are formed is fixed.", "The lens 141 may be an aspheric lens or a Fresnel lens.", "Since a Fresnel lens can be manufactured easily by using an injection method, etc., it has an advantage to reduce the manufacturing cost of the transmitter.", "At this tinge, it is preferable that the lenses are standardized for transmission distances to manufacture the transmitter.", "In addition, the lens holder 142 is formed to adjust the position of the lens 141 before and behind in the optics module 140 to adjust the focal distance according to the use of the transmitter.", "The light from the light source 110 is collimated by the lens 141 to a proper extent to be received by a receiver, and the nominal beam divergence of the light from the transmitter is 1*10-3 radian.", "On the other hand, the optics module 140 and IC freme 107 are formed as standardized blocks to be assembled with each other easily, and they are fixed together after assembling.", "FIGS.", "3 and 4 show examples of the transmitter which have screw units to assemble the optics module and IC frame.", "As shown in FIG.", "3 or 4, screw units 350 in FIG.", "3 and 450 in FIG.", "4 are formed on both sides of the optics modules 340 in FIG.", "3 and 440 in FIG.", "4 and the IC frames 307 in FIG.", "3 and 407 in FIG.", "4 to assemble two parts by turning the screws.", "The screw units can be formed integrally with the IC frame or optics module, or they can be formed to be assembled with the IC frame or optics module.", "In FIGS.", "3 and 4, the assembled forms by turning the screws are shown.", "In FIGS.", "3 and 4, other components have similar structures as described with reference to FIG.", "1, the similar components are indicated as similar symbols.", "To form screw units for the optics module and IC frame, it is possible to form frame surrounding the optics module or IC frame and form screw units therein.", "When the screw units are formed, it is preferable that the screw units of the standardized gauge are formed in optics module having lenses of various sizes and IC frames including ICs which are also standardized for each of the transmission distances are formed, two parts of which can be assembled according to the needs.", "Then, it is possible to optionally mount lenses of small or large diameter according to the needs such as the transmission distance, reliability, etc.", "for the same IC frame.", "That is, according to the present invention, it is very easy to manufacture a transmitter of proper standard because the IC frame and optics module can be easily assembled by a method of forming screw units, etc.", "In addition, it is preferable that an output window transparent to the wavelength of the light source is provided outside of the optics module to install the transmitter outdoors.", "A protective cover or heater to confront the change of humidity or temperature can also be provided.", "Now, a structure of a receiver free space optical communication will be described.", "FIG.", "5 is a schematic diagram showing a receiver for free space optical communication according to an embodiment of the present invention, and FIG.", "6 is a block diagram showing an example of an optical receiver circuit used in the receiver shown in FIG.", "5.In the receiver 500, a optical receiver IC 530 having an example structure shown in FIG.", "6 is formed on a substrate 510 made of Si, etc.", "The optical receiver IC 530 can be constituted of a pre-amplifier (“TIA” which is a trans-impedance amplifier) 5302 to amplify the signal from a PD 510, a signal amplifier 5304 to amplify the signal transmitted from the pre-amplifier 5302, an automatic gain controller 5306 to control the gain of the received signal, a data recovery circuit 5308 to recover the data from the received signal, a clock generation circuit 5310 to extract the clock from the received signal and transmit it to the data recovery circuit 5308, etc.", "The optical receiver IC 530 is also manufactured according to known IC manufacturing process.", "On the substrate 501, the PD 510 to detect a light received from the free space outside of the receiver is formed.", "For PD 510, various kinds of devices such as MSM PD, PIN PD, APD, etc.", "can be used as used in the transmitter 100.A connecting part 509 to connect the PD 510 to the optical receiver IC 530 is also formed.", "The process of forming the PD 510 and the optical receiver IC 530 on the substrate 501 follows a general semiconductor manufacturing process, and the PD 510 and the optical receiver IC 530 can be formed together in the same manufacturing process.", "Completed substrate 501 is fixed on an IC frame 507, and bonding pads 503 provided for the IC 530 are wire bonded with bonding pads 504 of the ID frame 507 for the optical receiver IC 530 to be connected to the outside via pins 508 of the IC frame 507.The light received from the outside is collected via an optics module 540 and transmitted to the PD 510.The optics module 540 is constituted of a lens 541 and a lens holder 542 similar to the transmitter 100.For lens 521, an aspheric lens or Fresnel lens can be used as in the transmitter 100.The efficiency of the beam collection can be maximized if a Fresnel lens 5411 is used.", "In addition, since the Fresnel lens can be easily manufactured by using a very economical way such as an injection method, etc., it is more advantageous to secure economical efficiency of transmitter and/or receiver for FSON than any other lenses.", "Moreover, since the Fresnel lens has a large numerical aperture, which makes the acceptance angle large, it is possible to receive the light signal easily and effectively.", "It is preferable to make the optics module and IC frame of the receiver as standardized blocks to be assembled with each other easily as in the transmitter.", "FIGS.", "7 and 8 show examples of the receiver which have screw units to assemble the optics module and IC frame.", "As shown in FIG.", "7 or 8, screw units 750 in FIG.", "7 and 850 in FIG.", "8 are formed on both sides of the optics modules 740 in FIG.", "7 and 840 in FIG.", "8 and the IC frames 701 in FIG.", "7 and 801 in FIG.", "8 to assemble two parts by turning the screws.", "As in the transmitter, the screw units can be formed integrally with the IC frame or optics module, or they can be formed to be assembled with the IC frame or optics module.", "Screw units may be formed to have a standard gauge able to assemble the lens of a proper size according to needs.", "In FIGS.", "7 and 8, the assembled form by turning the screws is shown.", "In FIGS.", "7 and 8, other components have similar structures as described with reference to FIG.", "5, the similar components are indicated as similar symbols.", "To form screw units for the optics module and IC frame, it is possible to form frames surrounding the optics module or IC frame and form screw units therein.", "The fact that the transmitter and receiver should constantly have reliability is a very important function of the free space optical communication system.", "In case of OWLL, there is a possibility for the intensity of a signal to be degraded if the alignment between the transmitter and the receiver becomes wrong different from the optical fiber communication link.", "Therefore, the alignment between the transmitter and the receiver should be monitored constantly if it maintains good condition or not.", "For this purpose, a monitoring terminal 539 to monitor the intensity of the received signal constantly can be provided according to the embodiment of the present invention as shown in FIG.", "5.In addition, it is possible to display the intensity of the signal received to the receiver by connecting the monitoring terminal 330 to a display device (not shown).", "As the display device, an LED of a visible ray can be used.", "Addition to the displaying the intensity externally, it is possible to report the extent of degradation of the signal obtained on the optical receiver circuit to the central base station which manages and administrates the whole FSON system.", "The conventional transceiver for fiber optical communication using optical fiber needs a precise packaging which spends a long time to align and pig-tail between the LD and the fiber or between the PD and the fiber to an extent of minuteness of some μm.", "Therefore, the cost of manufacturing the conventional transceiver is very high.", "On the other hand, the transceiver for OWLL and FSON as suggested in the present invention has a advantage to be manufactured very economically.", "That is, since the transceiver for OWLL and FSON as suggested in the present invention is very economical, the FSON system can be more economical than FTTH (fiber-to-the-home) system.", "In case of the receiver, it is preferable that it accepts only the light in which the transmitter outputs selectively.", "The output light of the transmitter is the light having nominal wavelength of 0.85*10-6 m, 1.3*10-6 m, 1.55*10-6 m, etc.", "as described above.", "For this purpose, it is preferable to provide an input window transparent only to the light in which the transmitter outputs and able to shield the normal light in front of the optics module of the receiver.", "To install the receiver outdoors, it may also need to provide a protective cover or heater.", "FIG.", "9 shows an all-in-one transceiver (“TRX”) for OWLL and FSON system in which a transmitter and a receiver are formed as one module.", "Since the OWLL and FSON system is basically a bi-directional communication system, the transmitter and the receiver tend to be used together other than used separately.", "The transmitter in FIG.", "9 is that the transmitter and the receiver shown in FIGS.", "1 and 5, respectively, are formed integrally for this purpose.", "As shown in FIG.", "9, a transmitting/receiving IC 930 is formed integrally on a semiconductor substrate 901, and an LD 910 and a PD 920 for light transmitting module and a PD 960 for light receiving module are formed together on the substrate 901.An IC frame on which the semiconductor substrate 901 is fixed is assembled with a transmitting optics module 940 and a receiving optics module 990.The other structures are similar to those of the transmitter 100 and the receiver 500.If the transceiver is formed like this, the light transceiver becomes very small.", "Therefore, this structure is useful when the size of the optics module can be very small.", "However, transceivers often face to each other when an OWLL is constituted.", "In this case, the light signal may input to the light source of the transmitting optics module of the transceiver as well as the receiving optics module, and sometimes, large optics modules of a few to several tens cm scale are needed.", "Therefore, a prescribed space should be maintained between the transmitting part and the receiving part of the transceiver, and it is advantageous to constitute the circuits of transmitting and receiving part separately.", "In FIG.", "10, an example of the transceiver in which the circuits of the transmitting and receiving parts are separated is shown.", "As shown in FIG.", "10, a current driver and automatic output controller IC 1030 for transmitting part and an optical receiver IC 1080 for receiving part are formed on different semiconductor substrates 1001 and 1051.An LD 1010 and a PD 1020 are formed together on the substrate 1001 for transmitting part and connected to the current driver and automatic output controller IC 1030, and a PD 1060 is formed on the substrate 1051 for receiving part and connected to the optical receiver IC 1080.Each substrate 1001 or 1051 is fixed on an IC frame 1007 or 1057, and those IC frames are placed on another substrate 1050 having an appropriate interval between them.", "The interval between transmitting and receiving parts can be determined properly by the size of the optics module forming the transceiver, etc.", "A transmitting optics module 1040 and a receiving optics module 1090 are assembled to the light source1010 of transmitting part and the PD 1060 of receiving part, respectively.", "In the transceivers 900 and 1000 shown in FIGS.", "9 and 10, the transmitting optics modules 940 and 1040 and receiving optics modules 990 and 1090 can be manufactured as standardized modules and assembled with IC frames or substrates as in the transmitter 100 and the receiver 500 described above, and assembling method can also be same as used in the transmitter 100 or receiver 500.In addition, The transceivers 900 and 1000 shown in FIGS.", "9 and 10 can have all characteristics of the transmitter 100 and receiver 500 described above For the transmitting and receiving optics modules 940 and 1040, it is possible to use the same standard or different standards.", "Moreover, in the transceivers shown in FIGS.", "9 and 10, the transmitting optics module 940 and 1040 and receiving optics modules 990 and 1090 are installed in the same direction, however, they can be installed in different directions.", "For this purpose, the positions of the circuits and optical devices formed on the substrate can be properly adjusted.", "On the other hand, OWLL and FSON system of the present invention can be effectively used by combining with the existing optical communication system using optical fibers.", "For this purpose, the transceiver of the present invention may include the constitution of the transceiver for optical fiber communication to provide the optical fiber link.", "FIG.", "11 shows a structure of a transceiver for free space optical communication accessible via an optical fiber link according to an embodiment of the present invention.", "As shown in FIG.", "11, in addition to a first current driver and automatic output controller circuit and a first optical receiver circuit for free space optical communication, a second current driver and automatic output controller circuit and a second optical receiver circuit for optical fiber communication are formed integrally on one semiconductor substrate 1101.An LD 1110 and a PD 1120 connected to the first current driver and automatic output controller circuit and a PD 1160 connected to the first optical receiver circuit are also formed on the substrate 1101.The substrate 1101 is fixed on an IC frame 1107 and wire bonded 1105.A transmitting optics module 1140 and a receiving optics module 1190 are assembled to the free space optical communication side of the IC frame 1107, and a PD 1172 for the second optical receiver circuit and an LD 1176 for the second current driver and automatic output controller circuit are connected to optical fiber communication side to receive the signal transmitted from the optical fiber link and transfer to the first current driver and automatic output controller circuit and to transfer the signal transmitted from the first optical receiver circuit to the optical fiber link, respectively.", "For this purpose, the PD 1172 and the LD 1176 are connected to the optical fiber links via optical fiber adapters 1174 and 1178, respectively.", "At this time, the PD 1172 and the LD 1176 for optical fiber communication can be packages mounted on TO-cans.", "It is possible to form the photo devices such as PD and LD for optical fiber communication together on the semiconductor substrate instead of connecting from the outside of the substrate.", "FIG.", "12 shows a structure of a transceiver in which the photo devices for optical fiber communication are formed on the semiconductor substrate as described above.", "As shown in FIG.", "12, a light source 1276 and a photo detector 1277 of transmitting part and a photo detector 1272 of receiving part for optical fiber communication are formed on a semiconductor substrate 1201 on which a circuit part 1230 is formed.", "Next, the light source 1276 and the photo detector 1272 are connected to optical fiber links via optical fiber adapters 1278 and 1274, respectively.", "It is possible to form the transmitting and receiving parts of the optical transceivers 1100 and 1200 shown in FIGS.", "11 and 12 on separate semiconductor substrates and fix them on PCB substrates to have prescribed intervals as in the transceiver in FIG.", "10.That is, after forming the first current driver and automatic output controller circuit and the second optical receiver circuit on one semiconductor substrate and he first optical receiver circuit and the second current driver and automatic output controller circuit on the other semiconductor substrate, each part is fixed on another substrate.", "As described above, this structure is advantageous when an appropriate interval should be maintained between the transmitting and receiving parts.", "In addition, OWLL and FSON system of the present invention can be effectively used by combining with the existing Ethernet or LAN.", "For this purpose, Ethernet signals and signals of the optical transceiver of the present invention are transformed to each other using a media converter.", "The device for this purpose is shown in FIG.", "13.That is, a media converter circuit for data transformation is formed integrally on a semiconductor substrate 1301 together with the current driver and automatic output controller circuit and the optical receiver circuit.", "The media converter circuit is connected to an unshielded twisted-pair (“UTP”) port 1370 for connection to the Ethernet via a pin 1308 connected to the media converter circuit part of the IC 1330.However, sometimes the transceiver for OWLL and the media converter should be connected using an optical fiber link because the UTP cable for Ethernet is not able to use for long distance.", "For example, it is the case that the position of the transceiver for OWLL is far from the position of the subscriber such as a roof of the building.", "Then, the data signal of the transceiver should be conveyed to the media converter near the subscriber via light.", "In this case, the optical transceiver having communication function with the optical fiber link described with reference to FIGS.", "11 and 12 can be used to connect to the external media converter.", "The subscriber network using FSON can be tried in various forms.", "Both ring type network and star type network using ATM (asynchronous transfer mode) are possible, and tree, bus, and mesh type networks are also possible.", "When the network is formed, sometimes there is a case that a node uses some data by itself and relays the other data to another node after transmitting/receiving data of large bandwidth from/to the central base station.", "In this case, a transmitting/receiving module needs a function of multiplexing/demultiplexing.", "FIG.", "14 shows an example of a transponder for OWLL according to the present invention having multiplexing/demultiplexing function.", "As shown in FIG.", "14, an IC 1430 including a MUX/DEMUX circuit as well as a current driver and automatic output controller circuit of the transmitting side and an optical receiver circuit of the receiving side is formed on a semiconductor substrate 1401 The MUX/DEMUX circuit multiplexes the data transmitted from the input pin, transmits them to the current driver and automatic output controller circuit of the transmitting part, demultiplexes the signals received from the optical receiver circuit, and transmits them to the output pin.", "An LD 1410 and PDs 1420 and 1460 are formed on the semiconductor substrate 1401, and the other structures are similar to those in the transceiver 1000 shown in FIG.", "10.In case that the subscriber network is constituted as a ring network using ATM method, it is necessary to have add/drop function in which signals of some bandwidths among transmitted signals are distributed to the subscriber and signals received from the subscriber are added and transmitted with transmitted signals.", "FIGS.", "15 and 16 show examples of the transponder for FSON having the above-described function.", "However, in case of FSON system of ring network, directions of transmission and reception are generally different.", "Therefore, if the transceiver is manufactured as all-in-one type, it may be difficult to use for FSON system.", "In this regard, the transponder having separate transmitting part and receiving part is formed according to an embodiment of the present invention, and FIGS.", "15 and 16 show the structures of the transmitting and receiving parts of the transponder described above.", "As shown in FIG.", "15, the transmitting part includes an LD 1510, a PD 1520, and an IC 1530 including a current driver and automatic output controller circuit for transmission and a MUX circuit formed on a semiconductor substrate 1501.The IC frame 1507 on which the IC 1530 is fixed is provided with a Data In pin provided with data from the receiving part and a Data ADD pin provided with data to be added on the place where the transceiver is installed.", "The IC frame 1507 is assembled with the transmitting optics module 1540.The receiving part is shown in FIG.", "16.A PD 1610 and an IC 1630 including an optical receiver circuit for reception and a DEMUX circuit are formed on a semiconductor substrate 1601.An IC frame 1606 on which the IC 1630 is fixed is provided with a Data Out pin providing data to the transmitting part and a Data DROP pin providing data to the place where the transceiver is installed.", "The IC frame 1607 is assembled with a receiving optics module 1640.As described above, if the transmitting part and the receiving part are formed as separate modules, it can be easily installed though the directions of transmission and reception are different.", "On the other hand, receivers according to embodiments described above have a constitution in which a receiving optics module, photo detector, and optical receiver circuit are arranged serially, but it is possible to place the receiving optics module to be perpendicular with the substrate on which the photo detector and the optical receiver circuit are formed.", "If so, the substrate may have an additional function of filtering the visible rays, and it is possible to form the lens directly on the semiconductor substrate by etching or coating.", "Now, those embodiments are described in detail.", "FIG.", "17 is a layout diagram showing a structure of a receiver according to another embodiment of the present invention in the direction of the substrate on which a photo detector and an optical receiver circuit are formed, and FIG.", "18 is a sectional view of the receiver taken along the line XVIII-XVIII in FIG.", "17.As shown in FIG.", "17, an optical receiver IC 1730 and a photo detector 1710 are formed on a semiconductor substrate 1701, and the substrate 1701 is fixed on an IC frame 1707 and wire bonded 1705.The structure of the substrate is similar to the receiver of FIG.", "5 described above.", "On the other hand, in case of the embodiment shown in FIGS.", "17 and 18, a lens 1840 of an optics module 1840 is formed in perpendicular direction with the substrate 1701, which can be known from the sectional structure thereof.", "In addition, the IC frame 1707 have an aperture 1850 to expose a semiconductor area on which the PD 1710 is formed.", "Then, the light concentrated via the lens 1840 is transferred to the PD 1710 through the substrate 1701 made of Si, etc.", "Therefore, it is possible to pass a desired light selectively by selecting the substrate material.", "Moreover, a lens can be formed directly using a semiconductor substrate without installing a separate lens outside of the substrate.", "In this case, the manufacturing process of the optical receiver becomes simple, and the size of the receiver becomes smaller.", "FIG.", "19 shown a sectional view of a receiver in which a lens is formed by etching on the opposite side of the semiconductor substrate on which an optical receiver circuit and a PD are formed according to the present invention.", "The layout structure of the receiver shown in FIG.", "19 is similar to that shown in FIG.", "17.A lens 1940 is formed by etching on the opposite surface (lower surface in the drawing) of the substrate 1901 to the surface (upper surface in the drawing) on which an optical receiver circuit 1930 and a PD 1910 are formed.", "The lens 1940 can be manufactured using etching process of the conventional semiconductor manufacturing process.", "An aperture to expose the lens 1940 is formed in an IC frame 1907 on which the substrate 1901 is fixed.", "Since the lens 1940 is formed using the substrate 1901 on which the IC is formed without using separate material, the size of the receiver becomes smaller and the manufacturing process becomes simple.", "A lens can be formed using coating method.", "According to an embodiment shown in FIG.", "20, a lens 2040 is formed by coating on the opposite surface (lower surface in the dragging) of the substrate 2001 to the surface (upper surface in the drawing) on which a PD 2010 and an IC 2030 are formed.", "The lens 2040 can be manufactured using coating process of the conventional semiconductor manufacturing process.", "The layout structure of the receiver is similar to that shown in FIG.", "17, and an aperture to expose the lens 2040 is formed in an IC frame 2007 on which the substrate 2001 is fixed.", "It is apparent that the characteristics of the receivers described with reference to FIGS.", "17 through 20 can be applicable to the receiver and the application apparatuses thereof.", "While the present invention has been described in detail with reference to the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.", "[Industrial Applicability] The OWLL and FSON system having various advantages comparing to the conventional optical fiber communication system can be established using the transmitter, receiver, and application devices thereof according to the present invention.", "In addition, the transmitter, receiver, and application devices thereof according to the present invention are small, light, cheap, and standardized.", "At the same time, the transmitter, receiver, and application devices thereof according to the present invention can provide various functions required in the FSON system, and the provide those functions stably and reliably." ] ]

[ [ "Method for evaluating formation properties", "Methods for evaluating the properties of formations surrounding a borehole, include the steps of measuring electrical properties of the formation from within the borehole; deriving a model of parameters of the formation surrounding the borehole; and estimating the properties of the formation using the model to interpret the measured electrical properties.", "In one aspect of the invention in which the formation includes a series of distributed beds, each bed having specific properties.", "Another aspect includes grouping similar beds, assigning one or more properties to all of the beds in a group, and using the assigned properties to estimate the properties of the formation for measurements relating to the beds of that group.", "Another aspect includes making several different measurements of formation electrical properties and simultaneously estimating the formation properties from the different measurements to provide a joint inversion.", "A still further aspect includes the use of a complex model incorporating electrical properties with other petrophysical information to estimate the properties of the formation from the electrical measurements." ], [ "1.A method for evaluating the properties of an underground formation surrounding a borehole, comprising: (i) measuring electrical properties of the formation from within the borehole; (ii) deriving a model of parameters of the formation surrounding the borehole; and (iii) estimating the properties of the formation using the model to interpret the measured electrical properties.", "2.A method as claimed in claim 1, characterised in that the formation includes a series of distributed beds, each bed having specific properties, the method comprising estimating the positions of the beds, simulating measurements of the electrical properties using the estimated bed positions, comparing the simulated measurements to the measured electrical properties, and using the comparison to optimise the bed positions and estimate the formation properties from the measured electrical properties.", "3.A method as claimed in claim 2, further comprising: (a) determining the variation of the measured electrical properties across the series of beds; (b) analysing the measured properties to obtain a first estimate of the position of each bed in the series; (c) simulating the variation of the measured electrical properties across the series using the model, the specific bed properties and the first estimated positions of the beds; (d) comparing the simulated variation of electrical properties with the determined variation of measured electrical properties; (e) using the comparison to adapt the estimate of the position of the beds such that the estimated variation and the determined variation are substantially the same; and (f) using the adapted estimate of the bed positions in the estimation of the formation properties.", "4.A method as claimed in claim 2, wherein the bed position estimated is the bed boundary position.", "5.A method as claimed in claim 1, further comprising grouping similar beds, assigning one or more properties to all of the beds in a group, and using the assigned properties to estimate the properties of the formation for measurements relating to the beds of that group.", "6.A method as claimed in claim 5, further comprising: (a) identifying sequences of beds that are below the resolution of the electrical measurement; (b) assigning one or more properties to the beds in those sequences; and (c) using the assigned properties to estimate the properties of the formation including those sequences.", "7.A method as claimed in claim 1, further comprising making several different measurements of formation electrical properties and simultaneously estimating the formation properties from the different measurements to provide a joint inversion.", "8.A method as claimed in claim 7, further comprising: (a) obtaining several measurements of electrical properties of a formation of interest, each measurement being of a different nature; (b) simultaneously estimating the formation properties using the model and the different measurements of electrical properties.", "9.A method as claimed in claim 3, wherein the bed position estimated is the bed boundary position.", "10.A method as claimed in claim 1, further comprising the use of a complex model incorporating electrical properties with other petrophysical information to estimate the properties of the formation from the electrical measurements." ], [ "The present invention relates to methods for evaluating underground formation properties, in particular for evaluating the properties of an underground formation surrounding a borehole using measurements of electrical properties of the formation from within the borehole.", "In formation evaluation, deep resistivity tool measurements are commonly used to provide an estimation of the virgin zone resistivity Rt, which is strongly dependent on the hydrocarbon content of the rock.", "Oil saturation is linked to virgin zone resistivity, porosity and other formation parameters through different saturation equations: Archie, Waxman-Smits.", "However, current deep resistivity measurements have limited vertical resolution and are sensitive to important parasitic effects.", "Significant corrections have to be applied prior to estimating a usable value of Rt, e.g.", "borehole effects, adjacent-bed (shoulder) effects and step-profile invasion corrections.", "These corrections are generally assumed independently and applied in sequence, a valid assumption when the bed thickness is relatively large.", "The assumption that these effects are independent may not be valid in certain conditions, preventing an accurate estimation of reserves.", "For example, in thin beds, the depth of investigation of laterolog deep and shallow measurements becomes similar.", "Without differentiation between the measurements, standard invasion correction is no longer possible.", "In case of high resistivity contrast between neighbouring beds, the Born approximation used in processing measurements from induction tools such as the Array Induction Tool (AIT) of Schlumberger is no longer valid, possibly causing spurious curve separation and inappropriate invasion correction.", "Recently developed focusing (Barber, T. et al.", ": “Optimal Evaluation of Formation Resistivities Using Array Induction and Array Laterolog Tools” 2000 SPWLA Annual Logging Symposium, Dallas, Jun.", "4-7, 2000) will reduce these artifacts on field logs, while maximum-entropy inversion of array induction logs (Barber, T. D. et al.", ": “Interpretation of Multiarray Induction Logs in Invaded Formations at High Relative Dip Angles”, paper A presented at the 1998 SPWLA Annual Logging Symposium, Keystone, May 26-29) allows removal of these artifacts on legacy array induction data.", "These drawbacks can be overcome by simultaneously taking into account borehole, shoulder and invasion effects through a forward-model based inversion technique.", "This processing scheme consists of two steps: building a geometrical model of the formation, as a sequence of beds, invaded or not.", "simulating the tool response for this pre-defined formation model by using a forward modeling code, comparing the predicted measurements and the actual tool measurements, and updating the formation parameters until a good match is obtained between the two.", "The application of inversion techniques to resistivity logging has been already proposed by Mezzatesta et al (Mezzatesta, A. G., Eckard, M. H. and Strack, K.-M.: “Integrated Interpretation of Galvanic and Induction Measurements by Inversion Methods”, paper E presented at the 1995 SPWLA Annual Logging Symposium, Paris, Jun.", "26-29).", "This proposes that a simultaneous 2D inversion of galvanic and induction data could significantly improve the estimation of the virgin zone resistivity.", "A later paper (Frenkel, M. A. and Mezzatesta, A. G.: “Minimum and maximum pay estimation using resistivity log data inversion”, paper Z presented at the 1998 Annual Logging Symposium, Keystone, May 26-29) discusses quality-control aspects (such as measurement sensitivities to formation properties estimation of uncertainties and minimum and maximum bounds).", "With the development of robust minimization algorithms and the availability of fast forward-modeling codes, inversion techniques have become a usable tool for the interpreter.", "Thin-bed analysis has been among the first applications (Warrilow, I. M. et al.", ": “Application of Resistivity Inversion Software To Thin-Bed Evaluation”, presented at the SPE Asia Pacific Oil & Gas Conference, Kuala Lumpur, 1995).", "In such environments, the sensitivity to Rt of deep-resistivity tools degrades drastically and hydrocarbon-bearing beds are often by-passed.", "Several authors have shown that the layering of the formation can be introduced as a priori geometrical information in the inversion scheme to stabilize the ill-posed “resolution enhancement” problem.", "More recently, a fast and automatic inversion scheme has been made available to analyze measurements provided by a new array laterolog (Griffiths, R. et al.", ": “Better Saturation from New Array Laterolog”, 1999 SPWLA Annual Logging Symposium, Oslo, June 1-4).", "This application requires little expertise and can be used for routine analysis.", "The service now couples more recent hardware technology such as the High Resolution Laterolog Array tool (HRLA) of Schlumberger and modern processing techniques which bring significant improvement in reserves delineation.", "The present invention seeks to provide improved methods for interpreting electrical log data that allow some or all of the problems identified above to be handled and to provide a more reliable determination of Rt to aid in evaluation of formation properties.", "Methods according to the invention comprise: (i) measuring electrical properties of the formation from within the borehole; (ii) deriving a model of parameters of the formation surrounding the borehole; and (iii) estimating the properties of the formation using the model to interpret the measured electrical properties.", "One aspect of the invention comprises such a method in which the formation includes a series of distributed beds, each bed having specific properties, the method comprising estimating the positions of the beds, preferably the position of the bed boundaries, simulating measurements of the electrical properties using the estimated bed positions and comparing the simulated measurements to the measured electrical properties, and using the comparison to optimise the bed positions and estimate the formation properties from the measured electrical properties.", "This aspect further comprises: (a) determining the variation of the measured electrical properties across the series of beds; (b) analysing the measured properties to obtain a first estimate of the position of each bed in the series; (c) simulating the variation of the measured electrical properties across the series using the model, the specific bed properties and the first estimated positions of the beds; (d) comparing the simulated variation of electrical properties with the determined variation of measured electrical properties; (e) using the comparison to adapt the estimate of the position of the beds such that the estimated variation and the determined variation are substantially the same; and (f) using the adapted estimate of the bed positions in the estimation of the formation properties.", "Another aspect of the invention comprises grouping similar beds, assigning one or more properties to all of the beds in a group, and using the assigned properties to estimate the properties of the formation for measurements relating to the beds of that group.", "This aspect further comprises: (a) identifying sequences of beds that are below the resolution of the electrical measurement; (b) assigning one or more properties to the beds in those sequences; and (c) using the assigned properties to estimate the properties of the formation including those sequences.", "Another aspect of the invention comprises making several different measurements of formation electrical properties and simultaneously estimating the formation properties from the different measurements to provide a joint inversion.", "This further aspect comprises: (a) obtaining several measurements of electrical properties of a formation of interest, each measurement being of a different nature; (b) simultaneously estimating the formation properties using the model and the different measurements of electrical properties.", "A still further aspect of the invention comprises the use of a complex model incorporating electrical properties with other petrophysical information to estimate the properties of the formation from the electrical measurements.", "The present invention will now be described by way of examples, with reference to the accompanying drawings, in which: FIG.", "1 shows a schematic bed description of a type useful in methods according to the invention; FIG.", "2 shows a processing flow-chart applicable to methods according to the invention; FIG.", "3 shows log plots demonstrating optimisation of bed boundaries; FIG.", "4 shows log plots demonstrating parameter grouping; FIG.", "5 shows log plots of joint inversion; and FIG.", "6 shows log plots of complex model interpretations.", "Methods according to the invention are based on the log-simulation capability for a wide range of wireline and logging-while-drilling (LWD) tools.", "The 2D log simulators for the wireline array and dual induction tools and 2-MHz LWD resistivity tools all use a semianalytic mode-matching method developed by Liu and Chew in the mid 1980s (Chew, W.: “Waves and Fields in Inhomogeneous Media”, Van Nostrand Reinhold, N.Y., 1990).", "For wireline induction tools, the log-simulation outputs are the raw and skin-effect corrected apparent resistivities for each log station.", "Different post-processing options, which provide vertical-resolution enhancement and/or resolution-matching, are available to the user to simulate exactly the data acquired and post-processed by the acquisition platform (Barber, T. D.: “Phasor Processing of Induction Logs Including Shoulder and Skin Effect Correction”, U.S. Pat.", "No.", "4,513,376, 1984; Barber, T. D., and Rosthal, R.:“Using a Multiarray Induction Tool to Achieve Logs with Minimum Environmental Effects”, paper SPE 22725, presented at the 1991 SPE Annual Technical Conference and Exhibition, Dallas).", "Compensated LWD resistivity tools are available in different sizes, some with different transmitter-receiver spacings.", "The log simulators compute the raw, complex-valued voltages for each transmitter-receiver combination and condense them into attenuation and phase shifts between the receivers.", "These raw data are converted to apparent resistivities by post-processing.", "The numerical code used for laterologs is specifically designed for simulate logs of tools made of long electrodes carrying galvanic currents in axisymmetric formations.", "The numerical scheme used is a finite-element method.", "Methods according to the invention follow the inversion-processing methodology below: 1.Depthl-interval selection: A zone of interest is first selected, where the traditional sequence of 1D processing does not give a sufficiently accurate answer (borehole, shoulder and invasion effects cannot be separated).", "2.Formation model creation: The formation is represented as a sequence of parallel beds, according to the geometrical model.", "An invasion model (such as step, ramp, annulus.", ".", ". )", "is assigned to each bed, together with a specific set of physical/petrophysical properties necessary to describe it.", "3.Formation properties estimation: The optimization (inversion) routine is run to estimate user-selected parameters (any sub-set of the formation properties can be fixed).", "The formation model is defined as a sequence of parallel layers.", "The first step consists of estimating bed-boundary positions.", "This detection is performed by a segmentation algorithm, which identifies and positions boundaries on a selected set of logs.", "A simple logic can be used to distinguish between invaded and noninvaded beds, based on a user-defined invasion flag, triggered from any log.", "The output of this task is a formation model described by a limited set of parameters.", "The second step consists of refining the bed description, if needed.", "For specific beds, the user can select from a range of complex invasion profiles (radial resistivity ramp, annulus, invasion vertical ramp) or default to a simple step profile.", "It is also possible to link resistivities and petrophysical properties together, through a saturation equation with preset parameters.", "One complete set of properties (geometrical and physical) needed to describe a bed model in the most general case is given in FIG.", "1, in which: Dh=Hole Diameter, L ra=Annulus Zone Radius, L ri=Invasion Radius, L Ra=Annulus Zone Resistivity, ohm.m Rm=Mud Resistivity, ohm.m Rmf=Mud Filtrate Resistivity, ohm.m Rt=Virgin Zone Resistivity, ohm.m Rw=Formation Water Resistivity, ohm.m Rwb=Bound Water Resistivity, ohm.m Rwe=Equivalent Formation Water Resistivity, ohm.m Rxa=Invaded Zone Resistivity, ohm.m Sxo(t)=Invaded Zone (Total) Water Saturation (%) Sw(t)=Virgin Zone (Total) Water Saturation (%) Vcl=Shale Volume (%) Φ(t)=(Total) Porosity (%) The description includes both geometrical (radii, bed boundaries) and physical/petrophysical parameters (such as resistivities, porosity, and shale volume), u stands for upper, l stands for lower.", "For each bed, the set of parameters to be estimated can be selected from among those used in the description.", "The estimation of the selected parameters is performed by minimizing a cost function (or penalty function) C(p), defined as the weighted squared difference between selected measurements and corresponding modeled logs: C ⁡ ( p ) = ∑ i ⁢ a i ⁢  M i - f i ⁡ ( p )  2 σ i 2 ( 1 ) where p is the parameter vector (unknowns), Mi is a measurement channel and fi the corresponding theoretical tool response computed by a forward model; σi is the estimation of the confidence on measurement Mi, and αi a user-selected weight.", "In the first version of the product, σi is assumed to be a fraction of the signal level, identical for each of the measurements.", "The inversion is an iterative process, which starts with an initial guess of the formation model.", "A forward model then computes the theoretical response of each tool to this formation model and compares it with the actual measurements.", "If a significant mismatch occurs between the two (high cost function), the formation property values are refined to reduce the difference, until the match becomes acceptable with respect to specific convergence criteria (see FIG.", "2).", "The minimization is performed using a version of the Levenberg-Marquardt algorithm (other minimization algorithms may be used).", "It is also possible to use a priori information in the parameter estimation.", "The principle processing quality control indicator is the value of the cost function, together with the individual log reconstruction errors: Re ⁢ ⁢ c i = 100 * ( M i - f i ⁡ ( p ) ) f i ⁡ ( p ) ( 2 ) Methods according to the invention allow four different functionalities for accurate formation description and parameter estimation: The optimization of bed-boundary positions.", "The possibility to link together parameters to be determined.", "The joint analysis of different resistivity tools measurements.", "A link between resistivity variations and petrophysical parameters using a saturation equation (complex models).", "For clarity, these specific features will be described below separately, but can be used jointly.", "The user has the possibility to optimize the bed-boundary positions, which are considered as bed-model parameters.", "A positivity constraint is set on the bed thickness, to ensure that the boundary positions do not crossover during the optimization process.", "FIG.", "3 shows a synthetic example where the estimation of Rt from a Dual Laterolog Tool benefits from a reoptimization of the bed boundary locations.", "The synthetic resistivity profiles are plotted in the left logarithmic track, together with a simulated dual laterolog tool response (Rm=0.2 ohm.m, Dh=8.5″).", "Inaccurate bed boundary positions translate into inaccurate Rt, Rxo and ri estimations, together with poor reconstruction errors (middle logarithmic track).", "When bed-boundary positions and formation properties are jointly optimized, the inversion converges back to the original model (right logarithmic track).", "The true resistivity profiles (Rt, Rxo) are plotted in the left logarithmic track.", "In the first run, the values of Rxo, Rt and ri are estimated using a poor initial guess of the bed-boundary positions.", "The bed boundary positions are estimated in the normal way, for example by determining the inflection points of the LLS or LLD logs.", "Since the inflection points can be affected by a number of parameters, not just the position of the bed boundary, estimating the position of the boundary in this way can lead to large errors.", "The very large errors in the positioning of bed-boundaries results in large reconstruction errors in the corresponding areas, which propagates into poor Rt, Rxo, ri estimations in invaded beds (middle logarithmic track RUN 1).", "In the second run, bed boundary positions are optimized by comparison of the model profile and the reconstruction and the bed boundary position and model parameters are adjusted to minimise the difference.", "The further processing leads to accurate Rt, Rxo, ri estimations, in both squeeze and anti-squeeze conditions (right logarithmic track RUN 2).", "Another way to overcome the problem of poor bed boundary detection would be to over-segment the log up to the vertical resolution of the tool.", "This strategy has been preferred for the High-Resolution Laterolog Array (HRLA) automatic inversion scheme (Griffiths, R. et al.", ": “Better Saturation from New Array Laterolog”, 1999 SPWLA Annual Logging Symposium, Oslo, June 1-4).", "Methods according to the invention also provide the possibility to link together unknown formation parameters in an action called “parameter grouping”.", "One particular application is in thinly laminated sand-shale formations, where the tool resolution does not permit independent analysis of each bed: the user can assume that Rt is the same in each sand bed.", "The inversion will check the compatibility of resistivity data with this assumption.", "The second synthetic case illustrates this capability (FIG.", "4).", "In this example, noisy synthetic data have been generated, corresponding to a Dual Laterolog response in thin-bed sequences (Rm=0.7 ohm.m, Dh=8.5″).", "The bed boundaries have been defined by analysis of a high-resolution Rxo measurement.", "Stability of the results is greatly improved by assuming that Rt is the same within each sequence.", "The laterolog response in a thin-bed formation is simulated and a Gaussian noise of 2% is added to both shallow and deep measurements.", "The sequence of invaded/noninvaded beds has been determined a priori, with an accurate positioning of bed-boundaries and a known value of Rxo (from a microresistivity tool response for instance).", "The upper section is made of several beds of various thicknesses, poorly resolved by the tool when each of their properties is determined independently.", "As a consequence of the limited sensitivity of the measurements to Rt (laterolog measurements become shallow in thin-bed conditions, because of high shoulder-bed effect), the noise is amplified and leads to large errors in Rt and ri (left logarithmic track No Grouping).", "Using the assumption that the different bed properties, i.e.", "Rt and ri are equal across the invaded beds, the processing converges back to the original model (right logarithmic track Grouping).", "Once again, in the analysis of a real dataset, good log reconstruction would not validate such a “grouping” assumption but would simply indicate its compatibility with the input data.", "The formation model chosen for the analysis has to be determined and validated prior to the inversion (such as, by a petrophysical analysis using a set of high-resolution sensor measurements).", "The lower section is made of beds much thinner than the vertical resolution of the tool.", "As a result the measurements indicate a uniform formation.", "In this case, the prior information on bed boundary positions is crucial for the analysis, and the inversion can be seen as a resolution enhancement process.", "The invention allows simultaneous processing of the measurements from different tools recorded in the same borehole.", "This feature has two complementary advantages: The possibility to automatically exploit the measurement complementarity to improve formation property estimation.", "To determine a model of the formation consistent with all the measurements analyzed.", "The benefits of simultaneously analyzing galvanic (laterolog) and induction tools has already been proposed.", "Using the concept of resolution and statistical analysis, the size of the ellipsoid of uncertainties can be significantly reduced in a joint analysis of different tool measurements, for some specific cases.", "It is known that laterolog measurements are generally more sensitive to the properties of an oil-bearing bed (where Rxo<Rt), while induction measurements are the best to identify the water zone properties (if Rmf>Rw).", "Although less critical with the extension of the operating range of new logging tools, this complementarity helps to improve formation resistivity estimation.", "The second advantage of a joint analysis of different tool types resides in the improved description of the formation that can be obtained.", "Resistivity anisotropy for instance, cannot be determined from laterolog or induction tools alone, but requires a comparison of the two responses.", "Complementarity will be automatically exploited in a multitool inversion, providing that depth-matching is performed before inversion.", "FIG.", "5 shows the results of a 2D joint inversion of noisy synthetic laterolog and induction data, for a formation model representing an oil/water contact and two single beds (oil-bearing bed above and water-bearing below, Rm=0.5 ohm.m, Dh=8.5″, reconstructed measurements are plotted together with the estimation of Rt, Rxo and ri.).", "The measurements were perturbed by noise with two components: a zero-mean gaussian noise proportional to the signal (2%) on each measurement and an additive bias from 2% of the signal (deepest channels) to 5% (shallowest channels).", "Two single-tool inversions are made, with laterolog and induction data, followed by a joint inversion of the tools.", "In each case, all bed properties are estimates, including the bed boundary positions.", "The error on Sw estimation in the oil zone is at a minimum in the case of a joint analysis of laterolog and induction data.", "Results are summarized in the table below: True HRLA AIT HRLA + AIT Sw (%) Sw (%) Sw (%) Sw (%) Bed 1: Oil 0.18 0.16 0.20 0.195 Bed 2: Oil 0.18 0.165 0.19 0.175 Bed 2: 1.0 0.98 1.0 1.0 Water Bed 3: 1.0 0.82 1.0 1.0 Water The properties of the bed with an oil/water contact are generally better resolved than those of the homogeneous beds (Beds 1 and 3), whatever tool is analyzed.", "In the case of the laterolog, the resistive section of Bed 2 deflects the current flow towards the water zone.", "Without noise, the inversion performed with any combination of tool(s) converges to the exact solution.", "The methods of the invention allow use petrophysical information to better describe vertical resistivity variations and thus reduce the number of unknown parameters.", "The formation is generally represented as a sequence of parallel beds, resulting in a squared estimation of the virgin zone resistivity.", "This representation is clearly not adequate in transition zones such as sand/silt/shale sequences in turbidite environments or with grain-size changes.", "The invention allows modeling of vertical variations of properties such as shale volume Vcl, or total porosity Φt, to account for smooth resistivity changes within a bed.", "In this case the vertical resistivity profile is predicted using Archie's saturation equation.", "R t ⁡ ( z ) = 1 ( ϕ t 2 * S wt 2 ) ⁢ R we ( 3 ) where Swt and Rwe are functions of Vcl.", "One can assume a linear vertical variation for Φt or Vcl , which will result in a depth-dependence of Rt.", "Depending on the assumptions, the inversion algorithm will directly determine water saturation's [Sw(t) and Sxo(t)], always assumed constant within a bed.", "FIG.", "6 presents the results of an analysis using complex models with transition zones.", "The upper section is made of three beds with changing porosity (from 0.1 to 0.3 P.U.", "), the middle and lower sections shows beds with variations of shale fractional volume (from 0.1 to 0.5).", "In the middle (resp.", "lower) section, the free (resp.", "total) water saturation of each bed is assumed constant.", "For this example, AIT-B* tool data have been modeled with Rm=1 ohm.m and Dh=8.5″.", "In an analysis without additional petrophysical input, the the different logs are squared to form a squared formation model.", "A ramp will be approximated by a staircase profile, causing a couple of important difficulties: (1) A significant number of additional parameters have to be determined, with the risk of having several equivalent solutions or even an unstable inversion, (2) Too coarse segmentation (with respect to the tool resolution and the vertical resistivity gradient) will not allow accurate reconstruction of the measurements, creating artifacts (oscillations) in the reconstruction errors.", "This can be seen on FIG.", "6 (left logarithmic track RUN 1).", "Correct analysis of the origin of the resistivity variation, resulting in the correct petrophysical model in the inversion scheme will allow better representation of the data, with fewer parameters and increased confidence (right logarithmic track RUN 2)." ] ]

[ [ "Oligonucleotides, agents containing these oligonucleotides, and the use thereof", "The invention relates to particular oligonucleotides, pharmaceutical agents that contain these oligonucleotides, and to the therapeutic use thereof.", "The oligonucleotides are, in particular, capable of inhibiting the proliferation of pancreatic tumors.", "These oligonucleotides thus have a therapeutic potential for the treatment of pancreatic tumors.", "This can involve, in the broadest sense, an antisense therapy." ], [ "1-34.", "(cancelled) 35.Oligonucleotide, characterized in that more than 40% of the nucleobases are cytosine groups or mimetics thereof, the oligonucleotide has an arrangement of at least 8 consecutive nucleobases of sequence SEQ ID NO:1 and that the oligonucleotide is modified at the 3′ terminal with polyethyleneglycol (MW 1500) or a tocopheryl group and at the 5′ terminal with a tocopheryl group.", "36.The oligonucleotide according to claim 35, characterized in that at least a portion of the nucleobases is arranged according to the sequence SEQ ID NO:1.37.The oligonucleotide according to claim 35, characterized in that the ratio of cytosine, or mimetics thereof, to guanine or mimetics thereof is at least 2:1.38.The oligonucleotide according to claim 37, characterized in that less than 15% of the nucleobases are adenine or mimetics thereof.", "39.The oligonucleotide according to claim 35, characterized in that the oligonucleotide has 8 to 30 nucleobases.", "40.The oligonucleotide according claim 35, characterized in that both terminals have a tocopheryl group.", "41.The oligonucleotide according to claim 35, namely 5′-tocopheryl-TGC TCC CCC CTG GCT-3′-PEG1500; 5′-tocopheryl-TGC TCC CCC CTG GCT-3′-tocopheryl.", "42.Oligonucleotide, characterized in that more than 40% of the nucleobases are cytosine groups, or mimetics thereof, and the oligonucleotide has an arrangement of at least 8 consecutive nucleobases of sequence SEQ ID NO:2.43.The oligonucleotide according to claim 42, characterized in that at least a portion of the nucleobases are arranged according to the sequence SEQ ID NO:2.44.The oligonucleotide according to claim 42, characterized in that the ratio of cytosine, or mimetics thereof, to guanine or mimetics thereof is at least 2:1.45.The oligonucleotide according to claim 44, characterized in that less than 15% of the nucleobases are adenine or mimetics thereof.", "46.The oligonucleotide according to claim 42, characterized in that the oligonucleotide has 8 to 30 nucleobases.", "47.The oligonucleotide having the sequence SEQ ID NO:2.48.The oligonucleotide according to claim 42, characterized in that the oligonucleotide has terminal modifications.", "49.The oligonucleotide according to claim 48, characterized in that the oligonucleotide is modified at the 3′ and/or 5′ terminal.", "50.The oligonucleotide according to claim 49, characterized in that at least one polyalkyleneglycol, a lipophilic group or a peptide is bound terminally.", "51.The oligonucleotide according to claim 50, characterized in that the polyalkyleneglycol is a polyethyleneglycol.", "52.The oligonucleotide according to claim 51, characterized in that the polyethyleneglycol has a mean molecular weight of 150 to 3000 Daltons.", "53.The oligonucleotide according to claim 52, characterized in that the polyethyleneglycol is hexaethyleneglycol.", "54.The oligonucleotide according to claim 50, characterized in that the lipophilic group is a fatty acid group, cholesterol, a steroid group, a tocopheryl group, or a derivative of those groups.", "55.The oligonucleotide according to claim 54, characterized in that the lipophilic group is α-tocopheryl, preferably D-tocopheryl.", "56.The oligonucleotide according to claim 48, characterized in that one terminal has a polyethyleneglycol group and the other terminal has a tocopheryl group.", "57.The oligonucleotide according to claim 56, characterized in that the polyethyleneglycol group is bound to the 3′ terminal and the tocopheryl group to the 5′ terminal.", "58.The oligonucleotide according to claim 48, characterized in that both terminals have a tocopheryl group.", "59.The oligonucleotide according to claim 42, namely 5′-tocopheryl-CCT CGC TTC GCC CGT-3′-PEG; 5′-tocopheryl-CCT CGC TTC GCC CGT-3′-tocopheryl.", "60.The oligonucleotide according to claim 35, characterized in that the terminal group is bound through an amide or ester linkage.", "61.The oligonucleotide according to claim 35, characterized in that the oligonucleotide has at least one phosphorothioate linkage.", "62.The oligonucleotide according to claim 61, characterized in that all nucleosides are linked via phosphorothioates.", "63.Pharmaceutical agent containing at least one oligonucleotide according to claim 35, and optionally pharmaceutically acceptable auxiliary agents.", "64.Method for the treatment of tumors, in particular human tumors, which comprises administering an oligonucleotide according to claim 35 to a subject in need thereof.", "65.The method according to claim 64, in which the treatment is directed towards pancreatic tumors.", "66.The method according to claim 64, in which the treatment is adjuvant.", "67.The method according to claim 64, wherein the treatment is in combination with at least one cytostatic agent and/or radiation therapy.", "68.Method for inhibition of the proliferation of tumor cells, in which at least one oligonucleotide according to claim 35 is allowed to act on tumor cells in vitro or ex vivo.", "69.The oligonucleotide according to claim 42, characterized in that the terminal group is bound through an amide or ester linkage.", "70.The oligonucleotide according to claim 42, characterized in that the oligonucleotide has at least one phosphorothioate linkage.", "71.The oligonucleotide according to claim 70, characterized in that all nucleosides are linked via phosphorothioates.", "72.Pharmaceutical agent containing at least one oligonucleotide according to claim 42, and optionally pharmaceutically acceptable auxiliary agents.", "73.Method for the treatment of tumors, in particular human tumors, which comprises administering an oligonucleotide according to claim 42 to a subject in need thereof.", "74.The method according to claim 73, in which the treatment is directed towards pancreatic tumors.", "75.The method according to claim 73, in which the treatment is adjuvant.", "76.The method according to claim 73, wherein the treatment is in combination with at least one cytostatic agent and/or radiation therapy.", "77.Method for inhibition of the proliferation of tumor cells, in which at least one oligonucleotide according to claim 42 is allowed to act on tumor cells in vitro or ex vivo.", "78.Pharmaceutical agent containing at least one oligonucleotide according to claim 47, and optionally pharmaceutically acceptable auxiliary agents." ], [ "The present invention relates to certain oligonucleotides, pharmaceutical agents containing these oligonucleotides, and the use thereof.", "The oligonucleotides are in particular able to inhibit the proliferation of pancreatic tumors.", "These oligonucleotides therefore have a therapeutic potential for the treatment of pancreatic tumors.", "This can involve, in the broadest sense, an antisense therapy.", "Approximately 25,000 US citizens die from pancreatic cancer every year (A. Warshaw and C. Fernandez-del Castillo, New England J. Med.", "326, 455 (1992)).", "Little is known about risk factors and the failure of radiation therapy and chemotherapy leads to a low 5-year survival rate after diagnosis.", "Modified oligodeoxyribonucleotides (ODNs) have been found to be a group of very promising medicinal products in recent years and various mechanisms have been proposed for their mode of action.", "High expectations exist for the so-called antisense oligodeoxyribonucleotides (ASODNs) that suppress the expression of a specific protein, for example an oncogene.", "Despite these high expectations and the in vitro demonstration that a specific gene is expressed to a lesser degree, only a single ASODN—Vitravene (ISIS, Carlsbad)—has been approved for use as a medicinal product.", "On the other hand, oligodeoxyribonucleotides that do not have the sequence of an ASODN have been reported to have non-specific biological effects.", "The mechanism of action is not initially important for the development of a medicinal product, as long as the action can be clearly demonstrated in vivo and no side effects, or only justifiable ones, are seen.", "Although a large number of modifications have been proposed and implemented in vitro for cell cultures (J. P. Shaw, K. Kent, J. Bird, J. Fischback, B. Froehler, Nucl.", "Acid Res.", "19, 747 (1991), clinical studies and in vivo studies have been conducted almost exclusively with phosphorothioates in which an oxygen molecule has been replaced by a sulfur molecule in the phosphodiester linkages, on the assumption that such thioates will have a greater stability in the presence of nucleases, whilst being readily transported into cells.", "The half-life of normal phosphorodiester ODNs is on average 20-40 minutes, whereas the half-life of thioates is approximately 2-5 hours.", "This low increase in the stability of thioates may be the reason for the otherwise so promising model of antisense strategies in vivo not yet having led to a medicinal product.", "With a half-life of 2-5 hours, the bioavailability is too low and it remains to be seen what effects the shorter fragments, resultant upon degradation, will have.", "It is known that ODNs with terminal modifications at the 3′ and 5′ position have a greater resistance to attack by exonucleases (M. Maier, K. Bleicher, H. Kalthoff, E. Bayer, Biomed.", "Peptd., Proteins & Nucl.", "Acids 1, 235 (1995)).", "Such modified ODNs have been used to date in cell cultures, but not in pre-clinical investigation models or clinical studies.", "In addition, most in vitro studies did not use phosphorothioates modified in the 3′ and 5′ positions, but instead the diester, so that endonucleases can still attack them.", "Lipophilic, cationic tensides, such as LipofektinR are often added in vitro to improve uptake by cells.", "Such methods have not been adopted in vivo, or are markedly limited in scope, for various reasons, including the toxicity of such adjuvants.", "It has now been found that the proliferation of human pancreatic tumors can be suppressed in vitro in cell cultures and in vivo in orthotopic xenotransplantation models using certain oligodeoxyribonucleotides, and in particular those oligonucleotides that have covalently-bound groups in the terminal 3′ and/or 5′ positions.", "In particular it was found that those sequences rich in C and G, or at least one CG motive, are especially effective in inhibiting the proliferation of pancreatic tumors in vivo.", "The the present invention therefore relates to the oligonucleotides described in the claims.", "The term “oligonucleotide” refers to an oligomer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof.", "These include oligonucleotides that comprise naturally-occurring nucleobases, sugars and covalent internucleoside linkages (backbone), as well as oligonucleotides with moieties that are not found in nature, but which have a similar function.", "Such modified oligonucleotides are preferred over native forms in accordance with the invention since they have desirable properties, for instance an elevated uptake by cells, a higher affinity for target nucleic acids and a greater stability in the presence of nucleases.", "In particular, the nucleotides may have modifications or substitutions to the nucleobases (herein also referred to simply as “base”).", "The unmodified, or natural, nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).", "The modified nucleobases (mimetics of natural nucleobases) include synthetic nucleobases such as 5-methylcytosine (5Me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and further alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propinyluracil and 5-propinylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo-, 8-amino, 8-thiol-, 8-thioalkyl-, 8-hydroxyl- and further 8-substituted adenines and guanines, 5-halo-, in particular 5-bromo-, 5-trifluoromethyl- and further 5-substituted uracils and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.", "Certain of these nucleobases are of particular use for increasing the binding affinity.", "These include 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted, N-6 substituted and O-6 substituted purines, for example 2-aminopropyladenine, 5-propinyluracil and 5-propinylcytosine.", "It was shown, for example, that 5-methylcytosine increases the stability of a nucleic acid duplex by 0.6-1.2° C. C-rich oligonucleotides in which more than 40%, and preferably more than 50%, of the nucleobases are cytosine groups, or mimetics thereof, are suitable in accordance with the invention.", "Mimetics of cytosine groups in the above sense are modified nucleobases that have similar binding characteristics, i.e., above all the same specificity, but quite possibly a different affinity for complementary nucleobases.", "The same holds for mimetics of other nucleobases.", "A suitable percentage of guanine, or mimetics thereof, is expedient in accordance with the invention.", "A ratio of C:G of 2:1 or above, and preferably of 3:1 or more, is an advantage.", "In accordance with a special embodiment, oligonucleotides in accordance with this invention have at least one GC/CG motive, i.e., at least one cytosine group or a mimetic thereof is arranged in consecutive sequence to a guanine or a mimetic thereof.", "Furthermore, a relatively low proportion, preferably below 15%, and in particular below 10%, of adenine or a mimetic thereof is expedient in accordance with this invention.", "In a special embodiment, oligonucleotides in accordance with this invention do not possess any adenine.", "In general it is expedient if the oligonucleotides in accordance with this invention have 8 to 30, preferably 12 to 25 and in particular approximately 15 nucleobases.", "The arrangement of nucleobases in oligonucleotides in accordance with this invention is generally guaranteed by the linkage of the nucleobases to one another in a suitable manner.", "In general this yields an oligomer with a consecutive sequence of nucleobases that are linked to one another through a backbone forming a main chain.", "Although linear oligomers are preferred, in certain cases branched, cyclic or even cross-linked structures are also suitable.", "The oligonucleotides are generally nucleosides linked to one another, i.e., base-sugar combinations.", "The base component of the nucleotides is normally a heterocyclic base, in most cases a purine or pyrimidine.", "The nucleosides are generally linked to one another through a group covalently bound to the sugar portion of the nucleoside.", "In those nucleosides that have a pentofuranosyl sugar, this group may be bound to the 2′, 3′ or 5′ hydroxyl group of the sugar.", "In general these groups covalently link neighboring nucleosides to one another to form a linear, oligomeric compound.", "The corresponding ends of this linear, oligomeric structure can in turn be linked together to form circular structures.", "Open, linear structures are preferred, however.", "The linking groups within the oligonucleotide structure are generally the internucleoside backbone of the oligonucleotide.", "The normal linkage or normal backbone of RNA and DNA are 3′ to 5′ phosphodiester linkages, i.e., the linking groups are phosphate groups.", "Oligonucleotides that have a modified backbone or non-natural internucleoside linkages are preferred, though, in accordance with this invention.", "Preferred, modified oligonucleotide backbones in particular include phosphorothioates, partially or completely sulfurated, for instance chiral phosphorothioates, phosphoromonothioates and phosphorodithioates.", "Further modified backbones are phosphorotriesters, aminoalkylphosphorotriesters, methylphosphonates and further alkylphosphonates, e.g., 3′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidates, e.g., 3-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and borophosphates with normal 3′-5′ linkages, 2′-5′ linked analogs thereof and those having an inverted polarity, in which neighboring pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′.", "Various salts, mixed salts and the free acid forms are also included.", "Special modified oligonucleotide backbones without a phosphorus atom are generally formed by short-chain alkyl or cycloalkyl internucleoside linkages that may also embrace heteroatoms or heterocycles.", "These include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones: formacetyl and thioformacetyl acetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.", "In further special oligonucleotides both the sugar and internucleoside linkage, i.e., the backbone, of natural nucleotide units, are modified.", "In antisense applications the bases are generally retained for hybridization with a suitable target nucleic acid.", "One such oligomeric compound, i.e., an oligonucleotide with exceptional hybridization properties, is referred to as a “Peptide Nucleic Acid” (PNA).", "In PNA compounds the sugar backbone of an oligonucleotide is replaced with an amide-like backbone, in particular an aminoethylglycine backbone.", "The nucleobases are retained and are bound directly or indirectly to the nitrogen atoms of the amide portion of the backbone.", "Particularly-preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and, further, oligonucleosides with heteroatom backbones, in particular based on structural units such as —CH2—NH—O—CH2—, —CH2N(CH3)—O—CH2— [known as a methylene (methylimino) backbone or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— or —O—N(CH3)—CH2—CH2—.", "Modified oligonucleotides may also contain one or more substituted sugar moieties.", "For example, the 2′ position may be substituted with OH, F, O-, S- or N-alkyl, O-, S- or N-alkenyl, O-, S- or N-alkinyl, or O-alkyl-O-alkyl, wherein the alkyl, alkanyl and alkinyl are preferably substituted or unsubstituted C1-C10-alkyl or C2-C10-alkenyl and alkinyl.", "Particularly preferred are substituents such as O[(CH2)nO]mCH3, O(CH2)nOCH2, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nNH2 and O(CH2)nON[(CH2)nCH3)]2, where n and m are whole numbers from 1 to 10.Preferred modifications comprise an alkoxy-alkoxy group, for example 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxymethyl) or 2′-MOE).", "A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH2)2 group, also known as 2′-DMAOE.", "Further preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F).", "Similar modifications may also be made at other positions on the oligonucleotide, especially the 3′ position of the sugar on the 3′ terminal nucleotide, or in 2′-5′ linked oligonucleotides, and the 5′ position of the 5′ terminal nucleotide.", "Oligonucleotides in accordance with this invention may also contain sugar mimetics such as cyclobutyl groups in place of the pentofuranosyl sugar.", "Further modifications to oligonucleotides of the invention include the linkage of one or more groups or conjugates to the oligonucleotide to increase the activity, cellular distribution or cellular uptake of the oligonucleotides.", "Such groups include, above all, polyglycols, in particular polyalkyleneglycols, preferably polyethyleneglycols, peptides and, above all, lipophilic groups such as fatty acid groups with preferably 8 to 32 carbon atoms that may be saturated, monounsaturated or polyunsaturated, cholesterol, tocopherols, especially α-tocopherol and, above all, the naturally-occurring D-enantiomer thereof, or steroids, as well as derivatives thereof, in particular cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, aliphatic chains, e.g., dodecandiol or undecyl groups, phospholipids, e.g., dihexyldecyl-rac-glycerol or triethylammonium-1,2-die-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine chains, adamantane acetic add, palmityl groups or octadecylamine or hexylamino-carbonyl-oxicholesterol groups.", "The conjugates include for example, nanoparticles that may expediently have a positive charge.", "Diameters in the range 100 to 500 nm may be used.", "Polymers are used especially in this connection.", "Particles with a polystyrene or polyacrylate base are mentioned in particular.", "Modification with lipophilic groups, above all in the 3′ and 5′ positions, increases bioavailability, because the half-life is extended by orders of magnitude against the attack of nucleases.", "Similar effects are achieved through the use of certain hydrophilic groups such as polyethyleneglycols, above all in the 3′-position.", "If the oligonucleotides in accordance with this invention are conjugated to nanoparticles that have a positive charge then the stability towards the attack of nucleases is also increased, both for unmodified phosphorothioate linkages and for oligonucleotides with terminal modifications and an excellent uptake in tumor cells observed.", "Oligonucleotides in accordance with this invention that are particularly preferred are those with terminal modifications.", "Moreover, further modifications to the oligonucleotides of this invention may affect individual nucleosides, with a plurality of nucleosides modified differently or uniformly.", "Mixtures of oligonucleotides with different modifications are especially useful in terms of use in accordance with the invention.", "The compounds in accordance with this invention can be made in a manner that is known.", "Known solid-phase synthesis systems may in particular be used.", "Suitable systems for synthesis are available commercially, for instance from Applied Biosystems (Foster City, Calif.) Accordingly, the compounds are synthesized in vitro.", "Pharmaceutically acceptable salts, esters, a salts of such esters, or any other compounds which, upon administration in an animal, in particular humans, are capable of providing, directly or indirectly, the biologically active metabolites or a part thereof are also useful.", "For instance, “Prodrugs” may be used.", "These are inactive forms of the active ingredient that are converted in vivo to an active form.", "Prodrug versions of oligonucleotides may be made available, for instance, as S-(acetyl-2-thioethyl)phosphate derivatives.", "Pharmaceutically acceptable base addition salts include salts with metals or amines, such as alkali and alkaline earth metals or organic amines, especially cations such as sodium, potassium, ammonium, magnesium and calcium, or polyamines such as spermine and spermidine.", "The pharmaceutically acceptable acid addition salts include salts with inorganic bases, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like, as well as with organic adds, e.g., acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalinesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalinedisulfonic acid, polygalacturonic acid, and the like.", "Oligonucleotides with the sequences given below are examples that exhibit an excellent inhibition of the proliferation of human pancreas tumor cells: 5′-TGC TCC CCC CTG GCT-3′ (SEQ ID NO: 1) 5′-CCT CGC TTC GCC CGT-3′ (SEQ ID NO: 2) Of particular advantage are the corresponding phosphorothioates.", "Modification at the 3′ position with polyethyleneglycol (MW 1500) and at the 5′ position with α-tocopherol brings additional advantages.", "SEQ ID NO: 1 is an example of an antisense oligonucleotide for the suppression of gene expression of the mutated p53 protein, whilst SEQ ID NO: 2 is an example of a sequence chosen from a randomized oligonucleotide library.", "Further sequences may be chosen from antisense sequences or randomized oligonucleotide libraries within the framework of the invention and modified if necessary, in particular through application of one or more of the following selection criteria: Inhibition of the proliferation of growth of pancreas tumor cells through determination of 3H thymidine incorporation; Stability of the construct against attack by endonucleases and/or exonucleases by measurement of the half-life of the oligonucleotides using capillary electrophoresis; Determination of the uptake of the oligonucleotides in cells using confocal Laser Scanning microscopy; Induction of apoptosis.", "A further object of the present invention is the use of a compound in accordance of the invention to inhibit the proliferation of tumor cells.", "This use also embraces a method for inhibiting the proliferation of tumor cells, in which at least one oligonucleotide in accordance with the invention is allowed to act on tumor cells.", "This method can be performed in principle in vitro, ex vivo or in vivo.", "Corresponding systems, e.g., in the form of cells or tissue, can be made available as a culture in vitro or ex vivo.", "Usage in vivo generally involves the administration of the oligonucleotide to the target individual.", "Such tumor cell systems can be made available by an expert in the usual way.", "In particular, techniques are known to bring about corresponding degeneration of organisms and cells, for instance via recombinant techniques.", "Culture of organisms, cells or tissues in a suitable manner and assessment of proliferation in particular qualitatively, and if desired quantitatively, on the basis of suitable assays is also generally known to those skilled in the art.", "In particular it is a question of optimization of the method and the choice of suitable conditions under which the action of oligonucleotides in accordance with this invention bring about a modulation of proliferation.", "The administration of oligonucleotides in accordance with this invention to an individual can be accomplished by those skilled in the art.", "As a rule, the individual is given a certain quantity of at least one of the oligonuclotides in accordance with this invention, generally formulated for corresponding pharmaceutical or veterinary use.", "Consequently, the present invention also relates to agents, and in particular pharmaceutical agents, that comprise at least one oligonucleotide in accordance with this invention, as well as suitable auxiliary agents where desired, that in particular form a pharmaceutically acceptable formulation basis.", "The manufacture of such agents is known to someone skilled in the art.", "The oligonucleotides in accordance with this invention can be used within the framework of these uses and processes, for instance for scientific purposes.", "Above all, the compounds in accordance with this invention can be used for therapeutic purposes.", "Therapeutic uses of particular importance relate to the treatment of tumors.", "Treatment here means preventive/prophylactic avoidance or at least delay in terms of time, or alleviating or treating acute or chronic disease, i.e., in particular reducing, and optionally suppressing, the proliferation of the tumor and thus reducing the dissemination of the tumor and thus the risk of, or incidence of, metastases that derive from the primary tumor.", "The subject of the present invention is therefore also the use of at least one compound in accordance with this invention for the treatment of tumors.", "This usage also embraces a method by which an effective quantity of at least one compound in accordance with this invention is administered to an individual to be treated, especially a human or an animal of commercial importance or a pet.", "Administration is generally once or several times a day, optionally at the same time as other active ingredients, or preparations containing active ingredients, or alternating with them.", "In this sense, the therapy may embrace the use of further treatment measures, for instance chemotherapy through the administration of cytostatic agents or radiotherapy.", "Special advantages are seen in the treatment of pancreatic tumors, particularly in humans.", "The present invention is now discussed in more detail by reference to the examples below.", "EXAMPLE 1 Method Section Syntheses The modified ODNs were produced using an Applied Biosystem Model 394 DNA/RNA Synthesizer with TentaGel (Rapp Polymere Tübingen) as the carrier.", "Modified protocols for synthesis using Tentagel have been described in the literature (E. Bayer, M. Maier.", "K. Bleicher.", "H.-J Gaus, Z. Naturforsch.", "50b, 671-676 (1995).", "PEG, fatty acid groups, cholesterol and tocopherol groups were introduced on the 5′ terminal as phosphoramidates in CH2Cl2/acetonitrile (0.1 M).", "TETD (ABI, Welterstadt) was used primarily as the sulfurating agent.", "The DMT-protected ODNs were purified using HPLC: 15 min.", "linear gradient 20-80% acetonitrile in 0.1 M trimethylammoniumacetate, flow rate 1 ml/min, column Nucleosil 100 C18, 250×4 mm, a flow rate of 15 m/min, column GROM-SIL 100 ODS2 FE, 250×20 mm (Grom, Herrenberg).", "Cell Cultures and Cell Uptake The cell lines Panc-Tu-I, Panc-Tu-II, A818-4 and HPAF were used above all as human pancreatic tumor cells.", "The uptake of modified ODN by the cells was evaluated using confocal laser microscopy.", "Measurement of Proliferation In Vitro Cell suspensions (7×104 cells ml−1) were introduced into each well of a 96-well microtiter plate (Nunc, Wiesbaden).", "The medium was changed after 24 hours and the cell cultures (with corresponding ODN treatment and the controls without treatment) were labeled in 100 μl runs with 10 μl medium containing 7.4 kBq methyl-3H-thymidine (Amersham, Braunschweig) during the last 3 hours of the specified incubation times.", "The cells were then harvested and the radioactivity measured using a liquid scintillation counter.", "The p53 protein was also determined in vitro using ELISA or Western Blot.", "In Vitro Investigations in SCID Mice Female SCID mice aged up to 8 weeks were used.", "A laparotomy was performed and 1×106 PancTu-I cells were injected into the pancreas under anesthesia.", "One group was given the ODN subcutaneously (using an implanted continuous ALZET® pump) and the other group was given ODN intraperitoneally once a day at a concentration of 6/18 mg/g body weight.", "The mice were then euthanized after 14 or 21 days.", "The pancreatic tumors were weighed, and measured to calculate the volume using the formula π/6×height×length×width.", "The liver, lungs, omentum and spleen were removed to allow checking of the typical routes of metastasis.", "Inhibition of Proliferation In Vivo The increase in volume and the increase in weight of the tumor for the 2 groups treated with the ODN 5′tocopheryl-TGC TCC CCC CTG GCT-3′-PEG1500 (as phosphorothioate) are shown in FIGS.", "1a and 1b respectively.", "The increase in weight and volume of the 2 groups is markedly less than that of the control groups.", "The volume of the tumors fell by 64% in the group that received intraperitoneal injection.", "Treatment using the subcutaneous continuous pump appears to be more effective still.", "The volume of the tumors had fallen by 70% after 21 days.", "Histological and histochemical investigation of the organs did not reveal any conspicuous findings.", "FIG.", "2 shows the dose-dependence of the effect.", "The volume of the tumor and weight of the tumor of the groups treated with ODN 5′-tocopheryl-CCT CGC TTC GCC CGT-3-PEG1500 are shown in FIGS.", "3a and 3b respectively.", "Treatment with ODN was commenced immediately after implantation of the human pancreas tumor.", "The volume and weight of the tumor similarly declined by approximately two-thirds.", "This shows that the growth of human pancreatic tumors is not just stopped in vivo, but a marked reduction in the size of the tumor is achieved within 2-3 weeks.", "This is of exceptional importance since the pancreas adenocarcinoma cell line Panc-Tu-I used in vivo is a tumor cell line that is very resistant to apoptosis and is entirely resistant to numerous other treatment strategies, such as the triggering of programmed cell death by Fas/CD95 activation.", "TRAIL or chemotherapeutics." ] ]

[ [ "Moisture management double face woven fabric", "A process for manufacturing a differential function, double-face fabric, comprises the steps of a) providing two different yarns, the first yarn exhibiting hydrophilic behavior and the second yarn exhibiting hydrophobic behavior; b) providing a knot yarn; and c) producing a fabric by simultaneously weaving said two yarns, and linking them with said knot yarn." ], [ "1.A process for manufacturing a differential function, double-face fabric, comprising the steps of: providing two different yarns, the first yarn exhibiting hydrophilic behavior and the second yarn exhibiting hydrophobic behavior; providing a knot yarn; producing a fabric by simultaneously weaving said two yarns, and linking them with said knot yarn.", "2.A process according to claim 1, wherein the weaving is performed in a warp and weft structure.", "3.A process according to claim 1, wherein the first of the two different yarns is made from wool alone, or from wool wrapped with a nylon filament or from cellulose alone.", "4.A process according to claim 1, wherein the first of the two different yarns is made from wool wrapped with covered elastane or from cellulose with elastane.", "5.A process according to claims 3 and 4, wherein the yarn is treated with fluorocarbons selected from fluoroalkanes, fluoroalcohols, fluoroglycols, fluoroanhydrides, fluoroacrylates, fluoroesters, and brominated fluoroalkanes.", "6.A process according to claim 1, wherein the second of the two different yarns is made from a polyester selected from polyethyleneterephthalate, polymethyleneterephthalate, polybutyleneterephthalate, modified polyesters, or their mixtures.", "7.A process according to claim 5, wherein the yarn is treated with polysiloxanes selected from functional linear or network polysiloxanes.", "8.A process according to claims 5 or 7, wherein the treatment is carried out at about 40° C. 9.A process according to claim 5 or 7, wherein the yarns are dyed prior to the chemical treatment.", "10.A process according to claim 1, wherein the knot yarn is made from polyamides selected from nylon 66, or polyesters selected from polyethyleneterephthalates.", "11.A process according to any one of claims 1 to 10, wherein the fabric comprises a face side of wool or cellulose and a back side of polyester.", "12.A process according to claim 11, wherein the back side is hydrophilic and water-absorbent.", "13.A process according to claim 11, wherein the face side is hydrophobic and water-repellent.", "14.A double-faced fabric, comprising two layers made by simultaneously weaving two different yarns and linking them with a knot yarn.", "15.A fabric according to claim 14, wherein one of the two different yarns is a wool or wool-based yarn, or cellulose or cellulose-based yarn.", "16.A fabric according to claim 15, wherein the second of the two different yarns is a polyester yarn.", "17.A garment made of a fabric according to any one of claims 14 to 16." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Clothing fabrics may be produced for various objectives and needs.", "Traditionally, it is common in the art to provide formal and casual garments for outwear as monofunctional fabrics which have one dominant property, for example; heat retention or wind protection provided by natural or synthetic yarns of weaves and knits.", "Another traditional approach is to provide fabrics with additive function; to improve the functionality of fabrics by the application of various chemicals/polymers and by adding certain features to the garment, for example: water repellence, antibacterial or UV protection, flame retardancy, shrink-proofing, etc.", "Another common approach is to modify the fabric properties by coating or lamination by using various polymers.", "However, fabric that is single-layered, may offer only one function, or additive functions, but not contrasting chemical function such as hydrophobicity and hydrophilicity.", "A known method used to achieve such result is to provide a multilayer fabric by introducing a membrane between two layers, leading to different functionality of the two sides of the multilayer fabric.", "U.S. Pat.", "No.", "5,787,503 teaches the joining together of two different layers, one hydrophobic and the other hydrophilic, by knitting, to form a double-layer sweater, wherein the linking between the two layers is a polygonal-shaped quilting stitch pattern forming insulating pockets between the layers.", "The purpose of this configuration is to manufacture a sweater that keeps the wearer dry as well as warm.", "A different approach for a double-layered garment is described in U.S. Pat.", "No.", "6,128,783, which teaches the production of a reversible sweater having one layer composed of knit fabric, and the other of micro-fiber fabric.", "According to this patent, the wearer may select which side of the sweater to wear according to weather conditions.", "The use of a dual-function garment has been considered also for medical applications.", "U.S. Pat.", "No.", "6,148,444 discloses the use of a sweater for hemodialysis patients.", "According to this patent, the sweater is warm and keeps the patient dry at the same time, and contains a series of openings that will allow access to grafts or fistulas, as well as catheters, during medical procedures.", "It is a purpose of the present invention to provide a combination of desired properties in a garment generating a double-layered fabric, without introducing any membrane between the two layers, choosing two compatible layers, wherein one face of the fabric has a desired property, and the other has a different desirable property, and then superimpose the two layers together.", "It is another purpose of the present invention to provide an outwear that combines desired properties in a comfortable, economic and simple to manufacture manner.", "It is still another purpose of the invention to provide a two-layered fabric, comprising an external face, designated as face-side, made from wool alone or wool wrapped with nylon or cellulosic yarn, and an internal layer, designated as back-side, made from polyester.", "The face-side is water-repellent, and prevents permeability of water droplets inside the fabric while the back-side is hydrophilic, water-absorbent allowing the perspiration to diffuse through the fabric outside, and then to evaporate.", "It is a further object of the invention to provide a breathable fabric, moving moisture away from the skin.", "It is a further object of the invention to provide a process for the manufacture of a light clothing or garment possessing the above mentioned properties.", "Other purposes and advantages of this invention will become apparent as the description proceeds." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention relates to a process of manufacturing a differential function fabric, which can be further used to prepare a breathable garment, which can aid moisture evaporation from the skin.", "In one aspect, the invention is directed to the preparation of a double face fabric comprising the steps of dyeing, chemically treating two different yarns, simultaneously weaving the yarns in a warp and weft, and linking them by a knot yarn.", "The knot yarn is preferably—but non limitatively—made from nylon 66.According to a preferred embodiment of the invention, the first of the two yarns is made from wool alone, or from wool wrapped with nylon 66 filament, or cellulosic yarn, or wool wrapped with covered elastane or cellulosic yarn with elastane.", "In this specification, the term “elastane” (also known as “Spandex”) is used to designate man-made elastic fiber, consisting of polyester diol, or polyether diol with diisocyanate.", "According to another preferred embodiment, the second yarn is made from spun or filament polyester selected from polyethyleneterephthalate (PET), polymethyleneterephthalate (PTT), or polybutyleneterephthalate (PBT).", "According to a preferred embodiment of the invention, a double face fabric which has differential functionality is provided, comprising two layers, a face side which is hydrophobic and water-repellent, and a back side which is hydrophilic, and water-absorbent.", "Preferably, the face side is wool or wool-based or cellulosic-based, and the back face is polyester.", "According to another aspect of the invention, the wool yarn or cellulosic yarn is treated with fluorocarbons, and the polyester yarn is treated with polysiloxanes.", "The invention will be further understood through the following illustrative and non-limititative examples." ], [ "FIELD OF THE INVENTION The present invention relates to the manufacture of a novel differential-function woven clothing article.", "More particularly, the invention relates to a breathable fabric comprising a water-repellent hydrophobic external layer, and internal hydrophilic layer, wherein water or sweat moisture is diffused out through the internal layer.", "The invention further relates to garments made of such fabric.", "BACKGROUND OF THE INVENTION Clothing fabrics may be produced for various objectives and needs.", "Traditionally, it is common in the art to provide formal and casual garments for outwear as monofunctional fabrics which have one dominant property, for example; heat retention or wind protection provided by natural or synthetic yarns of weaves and knits.", "Another traditional approach is to provide fabrics with additive function; to improve the functionality of fabrics by the application of various chemicals/polymers and by adding certain features to the garment, for example: water repellence, antibacterial or UV protection, flame retardancy, shrink-proofing, etc.", "Another common approach is to modify the fabric properties by coating or lamination by using various polymers.", "However, fabric that is single-layered, may offer only one function, or additive functions, but not contrasting chemical function such as hydrophobicity and hydrophilicity.", "A known method used to achieve such result is to provide a multilayer fabric by introducing a membrane between two layers, leading to different functionality of the two sides of the multilayer fabric.", "U.S. Pat.", "No.", "5,787,503 teaches the joining together of two different layers, one hydrophobic and the other hydrophilic, by knitting, to form a double-layer sweater, wherein the linking between the two layers is a polygonal-shaped quilting stitch pattern forming insulating pockets between the layers.", "The purpose of this configuration is to manufacture a sweater that keeps the wearer dry as well as warm.", "A different approach for a double-layered garment is described in U.S. Pat.", "No.", "6,128,783, which teaches the production of a reversible sweater having one layer composed of knit fabric, and the other of micro-fiber fabric.", "According to this patent, the wearer may select which side of the sweater to wear according to weather conditions.", "The use of a dual-function garment has been considered also for medical applications.", "U.S. Pat.", "No.", "6,148,444 discloses the use of a sweater for hemodialysis patients.", "According to this patent, the sweater is warm and keeps the patient dry at the same time, and contains a series of openings that will allow access to grafts or fistulas, as well as catheters, during medical procedures.", "It is a purpose of the present invention to provide a combination of desired properties in a garment generating a double-layered fabric, without introducing any membrane between the two layers, choosing two compatible layers, wherein one face of the fabric has a desired property, and the other has a different desirable property, and then superimpose the two layers together.", "It is another purpose of the present invention to provide an outwear that combines desired properties in a comfortable, economic and simple to manufacture manner.", "It is still another purpose of the invention to provide a two-layered fabric, comprising an external face, designated as face-side, made from wool alone or wool wrapped with nylon or cellulosic yarn, and an internal layer, designated as back-side, made from polyester.", "The face-side is water-repellent, and prevents permeability of water droplets inside the fabric while the back-side is hydrophilic, water-absorbent allowing the perspiration to diffuse through the fabric outside, and then to evaporate.", "It is a further object of the invention to provide a breathable fabric, moving moisture away from the skin.", "It is a further object of the invention to provide a process for the manufacture of a light clothing or garment possessing the above mentioned properties.", "Other purposes and advantages of this invention will become apparent as the description proceeds.", "SUMMARY OF THE INVENTION The invention relates to a process of manufacturing a differential function fabric, which can be further used to prepare a breathable garment, which can aid moisture evaporation from the skin.", "In one aspect, the invention is directed to the preparation of a double face fabric comprising the steps of dyeing, chemically treating two different yarns, simultaneously weaving the yarns in a warp and weft, and linking them by a knot yarn.", "The knot yarn is preferably—but non limitatively—made from nylon 66.According to a preferred embodiment of the invention, the first of the two yarns is made from wool alone, or from wool wrapped with nylon 66 filament, or cellulosic yarn, or wool wrapped with covered elastane or cellulosic yarn with elastane.", "In this specification, the term “elastane” (also known as “Spandex”) is used to designate man-made elastic fiber, consisting of polyester diol, or polyether diol with diisocyanate.", "According to another preferred embodiment, the second yarn is made from spun or filament polyester selected from polyethyleneterephthalate (PET), polymethyleneterephthalate (PTT), or polybutyleneterephthalate (PBT).", "According to a preferred embodiment of the invention, a double face fabric which has differential functionality is provided, comprising two layers, a face side which is hydrophobic and water-repellent, and a back side which is hydrophilic, and water-absorbent.", "Preferably, the face side is wool or wool-based or cellulosic-based, and the back face is polyester.", "According to another aspect of the invention, the wool yarn or cellulosic yarn is treated with fluorocarbons, and the polyester yarn is treated with polysiloxanes.", "The invention will be further understood through the following illustrative and non-limititative examples.", "BRIEF DESCRIPTION OF THE DRAWINGS In the drawings: FIG.", "1 is a flow-chart of the manufacturing steps of a differential-function fabric; FIG.", "2 is a schematic illustration of a cross-section of a double-layer fabric.", "DETAILED DESCRIPTION OF THE INVENTION As stated, the invention relates to a process for the manufacturing of a fabric comprising two layers, to a fabric made thereby and to breathable garments made of the fabric.", "In order to achieve the desired effect, a fabric is produced from two different yarns through spinning, weaving, dyeing and chemical treatments, and through the weaving process these two layers are knotted by a third yarn to produce the desired product.", "The production steps of an illustrative and non-limitative process are schematically illustrated in FIG.", "1, and are as follows: a) A fabric consists of two basic yarns 1 and 2 each made from a different material (in the example of FIG.", "1, wool and polyester); b) The knot yarn is made from a third material 3, which can be the same as, or different from, the material of yarn 2; c) All three yarns are dyed (4) separately by known methods for wool, polyester and nylon; d) The two yarns are woven together (5) in a double-face weave, in a warp and weft fashion, and are joined simultaneously by knot yarn 3, which combines the warp yarns and introduce it into the weft direction in a certain sequence to form woven fabric 5; e) The woven fabric is then processed through a finishing step (6), consisting, e.g., of wet treatment of the fabric through washing, drying and continuous decatizing steps; f) A light double-layered garment (7) is made from the fabric by methods known in the art.", "The wool yarn used in the present invention can be a yarn made from wool alone, or wrapped with nylon, particularly nylon 66 or can be made from cellulosic yarn.", "The polyester yarn can be made of any suitable polyester, and is preferably, but non limitatively, made of polyethyleneterephthalate (PET).", "The wool yarn or cellulosic yarn serves as the face-side of the fabric, and the polyester yarn is provided for the back-side of the fabric.", "According to a preferred embodiment of the invention the wool yarn is dyed in an acid dye bath at about 100° C. (or any other suitable temperature) with an acid dye, or the known metal complex 1:2 dyestuf, or by a reactive dyestuff, such as alpha-bromoacrylamide, or vinylsulfone derivatives, followed by cleaning in order to improve the wet fastness properties of the yarn, and then rinsed with water.", "According to a preferred embodiment of the invention the cellulosic yarn is dyed in an alkaline bath mainly with cold or warm reactive dyes (mono or bifunctional dyes) at a temperature range of 50° C. -80° C., followed by multiple rinsing and soaping cycle to improve the wet fastness properties of the yarn.", "In order to achieve the desired water-repellency of the final product, it is important to chemically treat the wool or cellulosic yarn.", "This is obtained by immersing the wool or cellulosic yarn in a water bath, and then by adding 4-5 wt % of a fluorocarbon derivative.", "In an illustrative and non-limitative process this step is carried out at about 40° C. for about 20 minutes, and then the solution is drained.", "Thus, by this process, the yarn becomes hydrophobic.", "The yarn is checked for hydrophobicity by methods known in the art, which are not discussed herein, for the sake of brevity.", "The polyester yarn is scoured prior to the dyeing process by immersion in a solution of a mixture of non-ionic surface-active material and soda ash.", "In an illustrative and non-limitative process the solution is then heated to about 60° C. at a rate of 2° C. per minute, and kept under such conditions for about 20 minutes.", "The scouring removes the majority of the spin finishes and waxes formed in the spun polyester yarn.", "The yarn is then dyed in a dyeing bath, at 115-130° C. by employing cationic/disperse dyestuffs.", "In order to achieve a better water-permeability of the polyester yarn, it is important to increase its hydrophilicity.", "This can be done by immersing the polyester yarn in a solution of 4-5 wt % of a functional polysiloxane derivative, prepared at ambient temperature.", "In an illustrative and non-limitative process the treatment is carried out at about 40° C. for about 20 minutes, and the solution is then drained.", "Finally, the yarn is checked for its hydrophilicity and absorbency by methods known in the art.", "The knot yarn is made from polyamides, preferably (but non limitatively) from Nylon 66, and it is scoured prior to dyeing as described previously.", "The dyeing is performed typically by a mixture of metal complex 1:2, or an acid dyestuff.", "The dyeing is typically carried out at about 100° C. In order to achieve dark shades, the nylon yarn is fixed, by immersing it in a solution of 2-3% on weight of fiber (owf) of a fixative agent, in an acetic acid medium at pH 5.In an illustrative and non-limitative process the fixation step is carried out at about 50° C., for about 20 minutes.", "Finally, the solution is drained.", "Since the fixation can sometimes interfere with the wicking force of the yarn, it is desirable to monitor this property, following the completion of the fixation.", "The wool or cellulose and the polyester yarns are then woven on the Rapier looms to form a double-layered fabric.", "The two layers are linked together by the nylon knot yarn that combines the warp yarns of the two layers, introducing it into the weft.", "In order to achieve a fabric (constructed from wool and polyester) that is machine-washable, the wool fiber can be treated by subtractive antifelt finish, for applications like oxidizing agents, and/or by additive antifelt finish (of polymers such as, polyurethanes, polysiloxanes).", "The scouring process of the fabric consists of the application of 1-2 wt % non-ionic surfactant and is typically carried out at about 60° C., in an open-width washing machine having several compartments at an illustrative fabric speed of 15 m/min.", "It is important to dry the fabric at a temperature of less than 110° C., since higher temperatures may induce change in the fabric color and affect the functionality of the fabric.", "The process is been finalized by continuous decatizing.", "This step is carried out in a decatizing machine, which does not contain metal surfaces.", "FIG.", "2 schematically illustrates a cross-section of the formed double-layer fabric, in which the face-side 21 is hydrophobic, thus causing it to repel water in the direction indicated by arrow 22, and the back-side 23 is hydrophilic, water-absorbent, causing body moisture to diffuse out through the two layers, in the direction indicated by arrow 24, and then to evaporate.", "The two layers of the fabric are linked together by knot yarn 25.According to a preferred embodiment of the invention, a wool or cellulosic yarn is woven together with a polyester yarn, and a knot yarn made of nylon is used to link the two yarns, to produce a double-layered fabric, in which the fabric external face is a wool or cellulosic layer, while its internal face is polyester, and these layers are knotted together by the nylon yarn.", "The following examples serve to illustrate the invention, and are not intended to limit it in any way.", "EXAMPLE 1 A double-layer fabric with differential functionality was manufactured by the procedures described above, using the following conditions: The weave was: double face plain weave, color woven, rigid fabric.", "Fabric weight was 200 gr/m2.The fabric composition was: 50% wool, 41% polyester, 9% nylon 66.The yarns were as follows: first yarn: worsted wool yarn, count 40/1 Nm.", "This yarn was wrapped with nylon 66 filament, 11 dtex at 880 turns per meter (tpm).", "The wool micron was 20.5 The second yarn was made from polyester (Dacron 702, Coolmax Ex.", "Dupont) with yarn count of 50/1 Nm.", "The two yarns were woven together with a knit yarn, polyamide 66.The knot was introduced in the weft and combined with warp yarns.", "EXAMPLE 2 A double-layer fabric, weft stretch was manufactured by the procedures described above, using the following conditions: The weave of the fabric; double face plain weave with weft stretch.", "The fabric weight was 220 gr/m2.The fabric composition was: 58% wool, 37% polyester, 2% elastane, and 3% polyamide.", "The yarns were: in the warp direction: worsted warp wool yarn count 40/1 Nm.", "This yarn was wrapped with nylon 66 filament at 880 turns per meter (tpm).", "The second yarn was polyester, Dacron 702 in a yarn count of 50/1 Nm at 1270 tpm.", "The knot yarn, polyamide 66, 78 dtex was used to combine the two yarns in the weaving step.", "Weft direction: the weft consisted of wool yarn, 40/1 Nm and polyester yarn, 50/1 Nm.", "Both yarns were wrapped with elastane.", "EXAMPLE 3 A bistretch double layer fabric was manufactured by the procedures described above, using the following conditions: The weave of the fabric was double face plain weave with bistretch elasticity.", "The fabric weight was 265 gr/m2 and the fabric composition: 60% wool, 35% polyester (Dacron 702), 11% nylon 66, and 4% elastane.", "Yarns in a warp and weft as follows: worsted wool 40/1 Nm wrapped with elastane and polyester 50/1 Nm wrapped with elastane.", "While embodiments of the invention have been described by way of illustration, it will be understood that the invention can be carried out by persons skilled in the art with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims." ] ]

[ [ "Method and device for the electrical zero balancing for a micromechanical component", "A method for the electrical zero balancing of a micro mechanical component including a first capacitor electrode rigidly suspended over a substrate, a second capacitor electrode rigidly suspended over the substrate, and a third capacitor electrode disposed there between, resiliently and deflectably suspended over the substrate, as well as a differential-capacitance detector for measuring a differential capacitance of the capacitances of the variable capacitors configured in this manner.", "In this context, a first electric potential is applied to the first capacitor electrode; a second electric potential is applied to the second capacitor electrode; a third electric potential is applied to the third capacitor electrode; and a fourth electric potential is applied to the substrate.", "The fourth electrical potential applied to the substrate for the electrical zero-point balancing is changed for the operation of the differential-capacitance detector." ], [ "1-6.Cancelled 7.A method for electrical zero balancing a micro-mechanical component which includes a first capacitor electrode rigidly suspended over a substrate, a second capacitor electrode rigidly suspended over the substrate, a third capacitor electrode arranged between the first capacitor electrode and the second capacitor electrode and resiliently and deflectably suspended over the substrate, and a differential-capacitance detector for measuring a differential capacitance of the capacitances of a plurality of variable capacitors: applying a first electric potential to the first capacitor electrode; applying a second electric potential to the second capacitor electrode; and applying a third electric potential to the third capacitor electrode; and applying a fourth electric potential to the substrate, wherein the fourth electrical potential applied to the substrate for the electrical zero-point balancing is changed for operation of the differential-capacitance detector.", "8.The method of claim 7, wherein the first electric potential, the second electric potential, the third electric potential, and the fourth electric potential required for measuring the differential capacitance are applied in a clocked cycle.", "9.The method of claim 7, wherein the micro-mechanical component includes an interdigital capacitor device with movable capacitor electrodes and fixed capacitor electrodes.", "10.A device for electrical zero balancing of a micro-mechanical component, which includes a first capacitor electrode rigidly suspended over a substrate, a second capacitor electrode rigidly suspended over the substrate, a third capacitor electrode arranged between the first capacitor electrode and the second capacitor electrode and resiliently and deflectably suspended over the substrate, and a differential-capacitance detector for measuring a differential capacitance of a plurality of capacitances of a plurality of variable capacitors, the device comprising: a potential-supplying device to apply a first electric potential to the first capacitor electrode, to apply a second electric potential to the second capacitor electrode, to apply a third electric potential to the third capacitor electrode, and to apply a fourth electric potential to the substrate, wherein the potential-supplying device is able to vary the fourth electrical potential applied to the substrate for the electrical zero-point balancing for operation of the differential-capacitance detector.", "11.The device of claim 10, wherein the first electric potential, the second electric potential, the third electric potential, and the fourth electric potential required for measuring the differential capacitance are applied in a clocked cycle.", "12.The device of claim 10, wherein the micro-mechanical component includes an" ], [ "<SOH> BACKGROUND INFORMATION <EOH>Acceleration sensors in general and micro mechanical acceleration sensors in particular, based on the technology of surface or volume micromechanics, are gaining ever greater market shares in the automotive equipment sector and are increasingly replacing the piezoelectric acceleration sensors that have been standard till now.", "Micro mechanical acceleration sensors of other systems may function in such a manner that the resiliently supported seismic mass device, which is deflectable in response to an external acceleration in at least one direction, when deflected, effects a change in capacitance at a differential-capacitor device connected thereto, this change is a measure of the acceleration.", "It is customary for these elements to be structurally formed in polysilicon, e.g., epitaxial polysilicon, over a sacrificial layer of oxide.", "However, micro mechanical sensor elements are not only generally used to detect linear and rotative accelerations, but also to detect gradients and rotational speeds.", "In this context, the differential-capacitive measuring principle may apply, according to which the measured quantity, for example the acceleration, causes a positional change in a movable capacitor electrode of a micro mechanical sensor structure, which induces two corresponding fixed capacitor electrodes, positioned on both sides of the movable capacitor electrode, to change their electrical measurement capacitance values in the opposite sense.", "In other words, the capacitance of the one capacitor increases by a specific amount, and the capacitance of the other capacitor formed in such a manner, decreases by a corresponding value, and, in fact, due to corresponding changes in the capacitor electrode distances.", "The smallest asymmetries in the zero position of such measuring structures or in the parasitic capacitance components of the micro mechanical sensor element in question lead, in the process, to an electrical offset or an electrical zero-point displacement at the output of the sensor element.", "Such an offset may be compensated when balancing an individual sensor by adding an appropriate voltage or an appropriate current in the relevant signal path of the differential-capacitance detector.", "By intervening in this manner in the relevant signal path when balancing the sensor offset, it may happen that other functional parameters are negatively influenced, for example, temperature sensitivities may arise in the offset or the signal amplifications and the sensor sensitivity or the like may change simultaneously.", "This leads then to further compensation and balancing requirements and substantially increases the outlay for sensor balancing.", "In addition, the gradation of such an offset balancing is dependent upon the total amplification of the signal path in question, for example upon the nominal sensitivity to be balanced, provided that at least some of the amplification balancing is not performed until after the offset-compensation point." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The exemplary method according to the present invention for the electrical zero balancing of a micro mechanical component may provide that the offset balancing or the zero-point balancing of a micromechanical, capacitively evaluated sensor element may be performed outside of the sensitive signal path, i.e., independently of amplification factors, and without introducing parasitic signal distortions, caused, for example, by responses to temperature changes.", "The idea underlying the present invention is that the fourth electrical potential applied to the substrate for the electrical zero-point balancing, is changed for the operation of the differential-capacitance detector.", "In accordance with an exemplary embodiment, the potentials required for measuring differential capacitance are able to be applied in a clocked cycle.", "In accordance with another exemplary embodiment, the micro mechanical component includes an interdigital capacitor device including a multiplicity of movable and fixed capacitor electrodes." ], [ "FIELD OF THE INVENTION The present invention relates to a method and a device for the electrical zero balancing of a micro mechanical component including a first capacitor electrode rigidly suspended over a substrate, a second capacitor electrode rigidly suspended over the substrate, and a third capacitor electrode disposed there between, resiliently and deflectably suspended over the substrate, as well as a differential-capacitance detector for measuring the differential capacitance of the capacitances of the variable capacitors configured in this manner.", "Although applicable to any number of micro mechanical components and structures, particularly sensors and actuators, the present invention, as well as its basic underlying problem definition are explained with reference to a micro mechanical Coriolis acceleration sensor of a rotation-rate sensor that is manufacturable using the technology of silicon surface micromechanics.", "BACKGROUND INFORMATION Acceleration sensors in general and micro mechanical acceleration sensors in particular, based on the technology of surface or volume micromechanics, are gaining ever greater market shares in the automotive equipment sector and are increasingly replacing the piezoelectric acceleration sensors that have been standard till now.", "Micro mechanical acceleration sensors of other systems may function in such a manner that the resiliently supported seismic mass device, which is deflectable in response to an external acceleration in at least one direction, when deflected, effects a change in capacitance at a differential-capacitor device connected thereto, this change is a measure of the acceleration.", "It is customary for these elements to be structurally formed in polysilicon, e.g., epitaxial polysilicon, over a sacrificial layer of oxide.", "However, micro mechanical sensor elements are not only generally used to detect linear and rotative accelerations, but also to detect gradients and rotational speeds.", "In this context, the differential-capacitive measuring principle may apply, according to which the measured quantity, for example the acceleration, causes a positional change in a movable capacitor electrode of a micro mechanical sensor structure, which induces two corresponding fixed capacitor electrodes, positioned on both sides of the movable capacitor electrode, to change their electrical measurement capacitance values in the opposite sense.", "In other words, the capacitance of the one capacitor increases by a specific amount, and the capacitance of the other capacitor formed in such a manner, decreases by a corresponding value, and, in fact, due to corresponding changes in the capacitor electrode distances.", "The smallest asymmetries in the zero position of such measuring structures or in the parasitic capacitance components of the micro mechanical sensor element in question lead, in the process, to an electrical offset or an electrical zero-point displacement at the output of the sensor element.", "Such an offset may be compensated when balancing an individual sensor by adding an appropriate voltage or an appropriate current in the relevant signal path of the differential-capacitance detector.", "By intervening in this manner in the relevant signal path when balancing the sensor offset, it may happen that other functional parameters are negatively influenced, for example, temperature sensitivities may arise in the offset or the signal amplifications and the sensor sensitivity or the like may change simultaneously.", "This leads then to further compensation and balancing requirements and substantially increases the outlay for sensor balancing.", "In addition, the gradation of such an offset balancing is dependent upon the total amplification of the signal path in question, for example upon the nominal sensitivity to be balanced, provided that at least some of the amplification balancing is not performed until after the offset-compensation point.", "SUMMARY OF THE INVENTION The exemplary method according to the present invention for the electrical zero balancing of a micro mechanical component may provide that the offset balancing or the zero-point balancing of a micromechanical, capacitively evaluated sensor element may be performed outside of the sensitive signal path, i.e., independently of amplification factors, and without introducing parasitic signal distortions, caused, for example, by responses to temperature changes.", "The idea underlying the present invention is that the fourth electrical potential applied to the substrate for the electrical zero-point balancing, is changed for the operation of the differential-capacitance detector.", "In accordance with an exemplary embodiment, the potentials required for measuring differential capacitance are able to be applied in a clocked cycle.", "In accordance with another exemplary embodiment, the micro mechanical component includes an interdigital capacitor device including a multiplicity of movable and fixed capacitor electrodes.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1 shows a part-sectional view of an acceleration sensor according to an exemplary embodiment of the present invention, in the context of a first substrate potential.", "FIG.", "2 shows a part-sectional view of the acceleration sensor according to an exemplary embodiment of the present invention, in the context of a second substrate potential.", "DETAILED DESCRIPTION In the figures, components which are the same or functionally equivalent are denoted by the same reference numerals.", "FIG.", "1 shows a part-sectional view of an acceleration sensor according to an exemplary embodiment of the present invention, in the context of a first substrate potential.", "The schematized sectional view shown in FIG.", "1 illustrates three capacitor electrodes for a differential-capacitive signal evaluation.", "In this context, in FIG.", "1, F1 denotes a first capacitor electrode rigidly suspended over a substrate SU; F2 a second capacitor electrode rigidly suspended over substrate SU; and B a third capacitor electrode disposed there between, deflectably suspended over substrate SU.", "Third capacitor electrode B is able to be returned to its neutral position via a spring device.", "The three electrodes F1, B, F2 are connected to a differential-capacitance detector (not shown) to measure a differential capacitance of the capacitances C1, C2 of variable capacitors F1, B; B, F2 configured in this manner.", "Electric potential VF1 is applied to first fixed capacitor electrode F1; electric potential VF2 is applied to second capacitor electrode F2; and electric potential VB is applied to the third capacitor electrode.", "For example, VF1 is=5 V, VF2 =0 V, and VB=2.5 V. In addition, electric potential VS=V1 of, e.g., 2.5 V is applied to substrate SU.", "Furthermore, the electric field line pattern derived therefrom is schematically indicated in FIG.", "1.The double arrow in the figure indicates the detection directions for deflections of movable third capacitor electrode B.", "In this context, the potentials required for capacitance measurement, in practice, are not statically applied to the capacitor electrodes, but rather in a clocked cycle.", "To facilitate the description, one assumes a full symmetry of the distances of capacitor electrodes F1, F2, B to one another, but an asymmetry for the parasitic capacitances, so that a zero-point balancing is required.", "The resulting force acting in detecting direction S on movable third capacitor electrode B is assumed to be zero for electric potentials V1, VB, VF2, VS applied in accordance with FIG.", "1.FIG.", "2 shows a part-sectional view of an acceleration sensor according to an exemplary embodiment of the present invention, in the context of a first substrate potential.", "If electric potential VS of substrate SU is now changed from V1=2.5 V to V2=3 V, then, depending on the direction of change, as the result of a developing asymmetrical lateral field-line distribution, a small electric force K may be exerted in a detecting direction S on movable capacitor electrode B.", "In other words, due to the change in electric potential VS of substrate SU from V1=2.5 V to V2 =3 V, the electric field lines are distorted, which leads to resulting force K. This force K leads to a lateral deflection of movable capacitor electrode B and thus to an adjustment of capacitance values C1, C2 to new capacitance values C1′, C2′ from both capacitors F1, B; B, F2 and, thus, to a change in the zero point at the output of the differential-capacitance detector.", "For an electric potential VS of substrate SU that would be lower than that of movable capacitor electrode B, a force would result in a direction opposite to that of FIG.", "2.What is important in this context is that electric potential VS of substrate SU be variable, completely independently of the signal-amplification path.", "Although the present invention is described above on the basis of an exemplary embodiment, it is not limited thereto, and may be modified in numerous manners.", "In the above examples, the acceleration sensor according to the present invention is explained in order to elucidate its basic principles.", "One may, of course, conceive of combinations of the examples and substantially more complicated refinements, while employing the same elements or method steps.", "Any micro mechanical base materials may also be used, and not only the silicon substrate cited here exemplarily." ] ]

[ [ "Electrical connector module with housings for receiving forwardly-inserted female contacts", "The object of the invention is an electrical connector module comprising housings (16) for receiving forwardly-inserted female contacts (18), each contact comprising a forward cage (20) and a rear blade (24) provided with pins (26) to be crimped on a flexible circuit (12).", "The object of the arrangement according to the present invention is to permit a forward mounting of the contact with an immobilization in its housing." ], [ "1.Electrical connector module comprising housings (16) each for receiving a forwardly-inserted female contact (18), characterized in that each housing (16) has the shape of a truncated pyramid with a clearance angle and in that each female contact (18) comprises on its cage bosses (36) for immobilizing and preventing gap, by exerting forces perpendicular to the wall of the said housing.", "2.Electrical connector module according to claim 1, characterized in that the truncated pyramid shape of each housing (16) is at a slight clearance angle on only two opposite surfaces which receive the immobilization bosses (36).", "3.Electrical connector module according to claim 1, characterized in that each female contact (18) comprises a cage (20) which carries at least one retention catch (38) so as to assure an immobilization in the forward/reverse direction in the housing.", "4.Electrical connector module according to claim 3, characterized in that each retention catch (38) is disposed facing parallel opposing surfaces of each housing.", "5.Electrical connector module according to claim 4, characterized in that each retention catch (38) is disposed downstream of the said contact.", "6.Electrical connector module according to claim 3, characterized in that each female contact (18) comprises in addition to the forward cage (20), a rear blade (24), provided with pins (26) to be crimped on a flexible circuit (12) and means (32) for retaining the flexible circuit (12) when the pins (26) are crimped on the flexible circuit.", "7.Electrical connector module according to claim 6, characterized in that the retaining means (32) comprise a flap (28) which is pivotable about an axis (30) with a tongue/groove assembly (32) adapted to generate an offset portion in the flexible circuit (12) and to immobilize this latter upon lowering the flap (28).", "8.Electrical connector module according to claim 1, characterized in that it comprises means (44) for snap-locking in a module-carrier." ], [ "The present invention concerns an electrical connector module with housings for receiving forwardly-inserted contacts.", "These contacts are female contacts.", "This electrical connector module is also adapted to be introduced into a module-carrier.", "The female contacts comprise a body with a cage at the front and a blade at the rear.", "This blade carries pins to be seated on the flexible circuit.", "This module is equipped with a moveable flap, known generally but described according to a preferred embodiment in a related application in the name of the same applicant.", "Such a flap assures a mechanical blockage of the flexible circuit so as to prevent its tearing away perpendicular to the seatings during inadvertent pulling forces exerted not only on the connector but also on the cables themselves.", "This flap also prevents peeling, that is to say delaminating of the sheets constituting the flexible circuit, as well as transmission of undulations via the flexible circuit, which would also tend to damage the seatings.", "During operation of the connector assembly, this flap is advantageously arranged to assume three fully-defined angular positions, one being as manufactured for insertion of the contacts, another being a delivery position for insertion of the flexible circuit into the modules, and the last, completely lowered, being closure of the flap effected simultaneously with the operation of seating the flexible circuit on the blades of the contacts.", "The flexible circuits described above, more generally designated “flex” in the connector field, are sheets formed of a complex of layers of insulating material between which are built up wiring lines of conductive material constituting both conductors and contacts for these circuits.", "These conductive wiring lines are protected and electrically insulated.", "Such flexible circuits are used in the case of multiple contacts to be disposed in a small clearance, and to be rejoined at the point of exit as they were upon arrival.", "The width and thickness of the wiring lines determines the conductive capacity, which in turn leads to dispose, on a same flexible circuit and in a same connector, contacts of varying dimensions.", "Nevertheless, for purposes of simplifying the present description, the wiring lines and contacts have the same dimensions.", "The numerous advantages of these flexible circuits have led designers to use them more and more, especially in the automotive industry.", "In practice, the connection of the set of contacts is effected in a single operation, by seating after insertion of the flexible circuit into the connector.", "Generally, locking means assure in a complementary manner the mechanical retention of the sheet in the said connector.", "There remains the problem of inserting the contacts into the module and the present invention proposes an appropriate response by resorting to an arrangement which permits insertion of the contacts forwardly with a locking that prevents the retraction of each of these contacts out of their housing.", "To that end, the electrical connector module comprises housings for receiving forwardly introduced contacts, and is characterized in that each housing has the shape of a truncated pyramid with a clearance angle, and in that each contact comprises bosses on the cage for immobilization and suppression of gap.", "There is also provided at least one retention catch so as to assure an immobilization in the forward/reverse direction.", "This module according to the present invention is also adapted to be introduced into a module-carrier, and it comprises to that end snap-locking means.", "The present invention will now be described with reference to the accompanying drawings, which correspond to a preferred but non-limiting embodiment, with reference to the accompanying drawings, which show: FIG.", "1, a perspective view of an electrical connector module according to the present invention, FIG.", "2, a perspective view partially broken-away of this same module, FIG.", "3A, a perspective view of a contact provided with retaining means, FIG.", "3B, a perspective view of a contact of greater width, FIGS.", "4A and 4B, a sectional view of a housing receiving a contact provided with retaining means and a sectional view but in a perpendicular plane, and, FIG.", "5, a perspective view of a module according to the present invention inserted into a module-carrier.", "In FIG.", "1, there is shown the contact-carrying electrical connector module 10, as well as a flexible circuit 12 about to be mounted.", "The module 10 comprises a body 14 with housings 16 adapted to receive contacts 18, in this case female contacts.", "Each contact 18 comprises a forward portion forming a cage 20, a transitional zone 22, and a rear blade 24, provided with pins 26 to be seated, as is best seen in FIGS.", "2, 3A and 3B.", "The preferred embodiment of this module body is described in a detailed manner in a related application in the name of the same applicant.", "Apart from the body 12, an important component is the means for restraining the flexible circuit, which comprise a flap 28, which pivots about an axis 30, with a tongue/groove assembly 32 adapted to generate an offset portion in the flexible circuit and to mechanically immobilize this latter upon lowering the flap.", "Such a position is shown in detail in FIG.", "5.It will be noted that this flap comprises a window 34 which permits assuring the seating of the pins on the flexible circuit, simultaneous to the lowering motion of the flap, through the said window.", "This seating is obtained by means of a tool adapted to the type of pins, and whose manufacture is within the capability of the skilled artisan.", "The problem of mounting the contacts 18 in the housing 16 is solved by characteristics of the contact and characteristics of the housing.", "Specifically, the housing 16 has the shape of a truncated pyramid, with a clearance angle of slight slope, on two opposed faces among the four faces.", "The large base is the inlet for connection of the housing and this housing narrows from the front to the rear.", "Moreover, each cage 20 of each female contact 18 is provided with bosses 36 which come to bear on the walls in facing relation of this housing with a clearance angle as is shown in the section of FIG.", "4A.", "Consequently, the bosses 36 are present facing and perpendicularly bearing on non-parallel walls.", "Moreover, this same cage carries at least one retention catch 38, disposed in the perpendicular plane and visible in FIG.", "4B.", "Each catch is dimensioned and disposed so as to cooperate with the wall 40 of the housing when the contact is completely inserted.", "In this case, the catches come into contact on the two opposing surfaces, other than those facing the bosses 36.The two surfaces receiving the catches 38 are parallel.", "The number of catches is variable as a function of the type of contact and for a contact of large dimensions, as shown in FIG.", "3B, it is suitable to provide two pairs.", "In the preferred embodiment, the catches are disposed at the rear of the cage 20, whereas the bosses are positioned forwardly.", "The contact is thus immobilized, without gap, in the housing via support on the walls of this housing in the transverse direction and by coupling in the direction of forward/reverse retraction.", "The bosses 36 also participate in retaining the contact in its housing.", "It is specifically necessary that the contact be maintained in its housing up to the seating operation.", "For the security of the retention, it is provided to dispose an abutment 41 rearwardly of each contact.", "The contact is lodged in the housing upstream of this abutment by the relative dimensions of the contact and of the housing.", "Thus, when a pulling force might be exerted on the contact, the contact is forced rearwardly into the housing up to the abutment, the resisting force being calculated to safeguard the integrity of the electrical connection until abutment occurs.", "Once the module 10 is itself inserted into a module-carrier 42, it is verified that the contacts 18 also remain accessible from the front, so as to permit the connection.", "It is thus necessary to retain these contacts in their respective housings, at the level of the module, during disconnection or during an accidental pulling force on the module carrier.", "The module and the module carrier are equipped with means 44 for snap-locking of the module into the module-carrier.", "In FIG.", "1, only one of the two elastic locks is visible, each provided to cooperate with an opening arranged in the module-carrier.", "It will also be noted that the shape with clearance angle of the housing permits generating a progressive insertion force of the contact into the housing during its positioning.", "Moreover, after complete insertion, each contact is force-fitted such that no gap remains, contrary to the mountings of the prior art.", "There nevertheless remains a safeguard in the case of significant pulling force prior to coming in contact with the abutment 41.In the case of a substantial pulling force, the module is forcibly extracted from the module-carrier and it is this connection which serves as the weak link to safeguard the electrical connection, even if the locking by the tongue/groove on the flexible circuit would be insufficient.", "Such an arrangement permits facilitating the mounting of contacts on the one hand, while safeguarding the electrical connection even in the case of a substantial pulling force, on the other hand." ] ]

[ [ "Self-encoding sensor with microspheres", "A micro-sphere-based analytic chemistry system is disclosed in which self-encoding microspheres having distinct characteristic optical response signatures to specific target analytes may be mixed together while the ability is retained to identify the sensor type and location of each sensor in a random dispersion of large numbers of such sensors in a sensor array using an optically interrogatable encoding scheme.", "An optical fiber bundle sensor is also disclosed in which individual microsphere sensors are disposed in microwells at a distal end of the fiber bundle and are optically coupled to discrete fibers or groups of fibers within the bundle.", "The identities of the individual sensors in the array are self-encoded by exposing the array to a reference analyte while illuminating the array with excitation light energy.", "A single sensor array may carry thousands of discrete sensing elements whose combined signal provides for substantial improvements in sensor detection limits, response times and signal-to-noise ratios." ], [ "1.A method of detecting a target analyte the method comprising: a. providing a first classifier for the response of a first population of sensors from a first pool of sensors to a first target analyte; b. distributing a second population of sensors from said first pool of sensors on an array; and c. determining the response of said second population of sensors to a sample, wherein the response of said second population resembles said first classifier for the response of said first population to said first target analyte, thereby indicating the presence of said first target analyte in said sample.", "2.The method according to claim 1, wherein said sensors are microspheres.", "3.The method according to claim 2, wherein said microspheres comprise dyes.", "4.The method according to claim 1, 2 or 3, wherein said second population of sensors is distributed on a substrate.", "5.The method according to claim 1, 2, 3 or 4, wherein said second population of sensors is distributed on a patterned substrate with wells.", "6.The method according to claims 4 or 5, wherein said substrate is a fiber optic bundle.", "7.The method according to claim 4 or 5, wherein said substrate is selected from the group consisting of glass and plastic.", "8.The method according to claims 1, 2, 3, 4, 5, 6, 7 or 8, further comprising providing a second classifier for the response of a third population of sensors from said first pool of sensors to a second target analyte, wherein at least a second and a third response to said first and second target analytes, respectively, of said third population of sensors from said first pool of sensors is determined, and wherein said response of said third population of sensors from said first pool of sensors resembles at least one of said first and second classifiers for the response to said first and second target analytes, thereby indicating the presence of said at least one of said first and second target analytes.", "9.The method according to claims 1, 2, 3, 4, 5, 6, 7 or 8, wherein said response of said first population of sensors to said first target analyte is indicative of the response of at least a second and third population of sensors from said first pool to said first target analyte.", "10.A method of detecting a target analyte the method comprising: a. providing a first and second classifier for the response of a first and a second population of sensors from first and second pools of sensors, respectively, to a first target analyte; b. distributing third and fourth populations of sensors from said first and second pools of sensors, respectively, on an array; and c. determining the response of said third and fourth population of sensors to a sample, wherein the response of said third and fourth populations resembles said first and second classifiers for the response of said first and second populations, respectively, to said first target analyte, thereby indicating the presence of said first target analyte in said sample.", "11.A method of making an array the method comprising: a. providing a population of microspheres, wherein said microspheres comprise an optical signature; b. contacting said microspheres with a sample comprising a target analyte; c. recording the response of said microspheres to said target analyte, d. generating a classifier for said response of said microspheres to said target analyte; and d. distributing said microspheres on a substrate with a surface comprising discrete sites." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The use of optical fibers and optical fiber strands in combination with light absorbing dyes for chemical analytical determinations has undergone rapid development, particularly within the last decade.", "The use of optical fibers for such purposes and techniques is described by Milanovich et al., “Novel Optical Fiber Techniques For Medical Application”, Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators and Fiber Optics” in C.R.C.", "Critical Reviews In Analytical Chemistry, Vol.", "19, 1988, pp.", "135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors In Analytical Chemistry” in Molecular Luminescence Spectroscopy, Methods and Applications (S. G. Schulman, editor), Wiley & Sons, New York (1988); Angel, S. M., Spectroscopy 2(4):38 (1987); Walt, et al., “Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.", "403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed.", "CRC Press, Boca Raton, Fla., 1991, 2nd Volume.", "When using an optical fiber in an in vitrolin vivo sensor, one or more light absorbing dyes are located near its distal end.", "Typically, light from an appropriate source is used to illuminate the dyes through the fiber's proximal end.", "The light propagates along the length of the optical fiber; and a portion of this propagated light exits the distal end and is absorbed by the dyes.", "The light absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest; and may or may not be retainable for subsequent use in a second optical determination.", "Once the light has been absorbed by the dye, some light of varying wavelength and intensity returns, conveyed through either the same fiber or collection fiber(s) to a detection system where it is observed and measured.", "The interactions between the light conveyed by the optical fiber and the properties of the light absorbing dye provide an optical basis for both qualitative and quantitative determinations.", "Of the many different classes of light absorbing dyes which conventionally are employed with bundles of fiber strands and optical fibers for different analytical purposes are those more common compositions that emit light after absorption termed “fluorophores” and those which absorb light and internally convert the absorbed light to heat, rather than emit it as light, termed “chromophores.” Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light (photons) at specified wavelengths and then emit light of a longer wavelength and at a lower energy.", "Substances able to fluoresce share a number of common characteristics: the ability to absorb light energy at one wavelength; reach an excited energy state; and subsequently emit light at another light wavelength.", "The absorption and fluorescence emission spectra are individual for each fluorophore and are often graphically represented as two separate curves that are slightly overlapping.", "The same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits; but the light emitted by the fluorophore will always provide the same emission spectrum.", "Finally, the strength of the fluorescence signal may be measured as the quantum yield of light emitted.", "The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore.", "For more detailed information regarding each of these characteristics, the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; “Molecular Luminescence Spectroscopy Methods and Applications: Part I” (S. G. Schulman, editor) in Chemical Analysis, vol.", "77, Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London, 1968.Many of the recent improvements employing optical fiber sensors in both qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the distal end of the optical fiber.", "In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations and applications such as pH measurement, oxygen detection, and carbon dioxide analyses.", "These developments are exemplified by the following publications: Freeman, et al., Anal Chem.", "53:98 (1983); Lippitsch et al., Anal.", "Chem.", "Acta.", "205:1, (1988); Wolfbeis et al., Anal.", "Chem.", "60:2028 (1988); Jordan, et al., Anal.", "Chem.", "59:437 (1987); Lubbers et al., Sens.", "Actuators 1983; Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal.", "Chem.", "58:1427 (1986); Seitz, W. R., Anal.", "Chem.", "56:16A-34A (1984); Peterson, et al., Anal.", "Chem.", "52:864 (1980): Saari, et al., Anal.", "Chem.", "54:821 (1982); Saari, et al., Anal.", "Chem.", "55:667 (1983); Zhujun et al., Anal.", "Chem.", "Acta.", "160:47 (1984); Schwab, et al., Anal.", "Chem.", "56:2199 (1984); Wolfbeis, O. S., “Fiber Optic Chemical Sensors”, Ed.", "CRC Press , Boca Raton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal.", "Chem., 481A-487A, Vol.", "67, (1995).", "More recently, fiber optic sensors have been constructed that permit the use of multiple dyes with a single, discrete fiber optic bundle.", "U.S. Pat.", "Nos.", "5,244,636 and 5,250,264 to Walt, et al.", "disclose systems for affixing multiple, different dyes on the distal end of the bundle, the teachings of each of these patents being incorporated herein by this reference.", "The disclosed configurations enable separate optical fibers of the bundle to optically access individual dyes.", "This avoids the problem of deconvolving the separate signals in the returning light from each dye, which arises when the signals from two or more dyes are combined, each dye being sensitive to a different analyte, and there is significant overlap in the dyes' emission spectra.", "Most recently, fiber optic sensors have been employed in arrays of semi-selective chemical sensors and pattern recognition schemes to discriminate and quantify odors.", "Such approaches have been useful in implementing the principles of biological olfaction in the design of sensing devices or systems.", "In this field of biomimetry, various technologies have been applied to the sensor transduction mechanism.", "For example, surface acoustic wave, conducting polymer, metal oxide sensor field-effect transistor (MOSFET), piezo-electric, and quartz crystal microbalance sensor arrays have been pursued.", "While such technologies provide inventive approaches utilizing a variety of physical and chemical phenomena to odor sensing, there are a number of limitations to these methods which restrict the efficacy of such devices.", "Firstly, element-to-element reproducibility both within a single array and between sensor arrays is typically unsatisfactory and thus requires recalibration and network retraining from sensor to sensor.", "Secondly, most of these methods have a relatively slow response time, frequently requiring several minutes to respond to the presence of an odor.", "Thirdly, such methods have relatively high detection limits and low sensitivity, typically not functioning at odor levels below 10 ppm.", "Fourthly, devices which embody such technologies typically require a relatively large inherent size, thereby restricting miniaturization of the sensor array for use in remote sensing applications.", "Finally, construction of multi-sensor arrays by these methods is complex and involves expensive and tedious preparation and placement of individual sensors within a well-defined array.", "Most recently, many of these shortcomings have been overcome through the application of fiber optic sensor arrays in an artificial nose sensor device and system.", "U.S. Pat.", "Nos.", "5,320,814 and 5,512,490 to Walt, et al., the teachings of each of these patents being incorporated herein by reference, disclose a fiber optic array formed of heterogeneous, semi-selective thin films which function as sensing receptor units and are able to detect a variety of different analytes and ligands using spectral recognition patterns.", "This technology has been applied to a vapor-sensing system which utilizes arrays of polymer-dye combinations which coat the ends of select optical fibers in a fiber optic bundle.", "These developments are further described in Dickinson, et al, Nature 382:697 (1996) and White, et al, Anal.", "Chem.", "68:2191 (1996).", "An innovative feature of the four previously referenced patents to Walt, et al., was the placement of multiple chemical functionalities at the end of a single optical fiber bundle sensor.", "This configuration yielded an analytic chemistry sensor that could be remotely monitored via the typically small bundle.", "The drawback, however, was the difficulty in applying the various chemistries associated with the chemical functionalities at the sensor's end; the functionalities were built on the sensor's end in a step-wise serial fashion.", "This was a slow process, and in practice, only tens of functionalities could be applied.", "U.S. patent application Ser.", "No.", "08/818,199 to Walt, et al, the teachings of which are incorporated herein by this reference, discloses the use of dye infiltrated polymer microspheres as a substitute for polymer-dye coating layers in optical fiber array sensors.", "With this approach, a fiber optic bundle serves as a substrate for dye-polymer microsphere array which contains a variety of microsphere bead sensors having different chemical and optical responses to the presence of target analytes.", "One innovative feature of this invention is in providing for a bead-based analytic chemistry system in which beads or microspheres carrying different chemical functionalities may be mixed together while retaining the ability to identify the functionality of each bead using an optically interrogatable encoding scheme.", "Additionally, this invention provides for an optical fiber bundle sensor in which the separate beads or microspheres may be optically coupled to discrete fibers or groups of fibers within the bundle.", "While the innovative features of this invention have separate applications, when implemented together, the invention provides for an optical fiber sensor that can support large numbers, thousands or more, of separate chemical sensor elements, which can be incorporated into a chemical sensor array and chemical analysis system.", "This approach provides for rapid fabrication and assembly of individual sensors and complex sensor arrays containing a multitude of discrete sensor types.", "The method also provides for a high degree of reproducibility and conformity within a batch of sensors and sensor arrays.", "Additional advantages are realized due to the ultrafine sizing available in microspheres.", "The overall size of the sensor array can be substantially reduced to submillimeter scale.", "This reduction in scale is particularly advantageous for remote sensing arrays.", "While the method of applying microsphere sensor elements in chemical sensor arrays as taught in U.S. patent application Ser.", "No.", "08/818,199 to Walt, et al, has many innovative features, this method has certain limitations.", "The method requires a complex multi-step bead encoding process to identify the type and location of bead subpopulations used in the sensor array.", "Beads are encoded by employing combinations of fluorescent dyes in varying ratio.", "The choice of encoding dyes is limited to those dyes which emit light at different wavelengths upon exposure to excitation light energy.", "While combinations of dyes in different ratios provide for encoding subpopulations of beads, the number of dye ratios available for encoding beads with a given dye pair or combination is significantly limited due to crowding the emission spectrum from peak overlap.", "In addition, a separate reporting dye is necessary for obtaining a unique characteristic optical response signature for a target analyte.", "Thus, the encoding dye choice is further limited by selecting dyes whose emission wavelengths do not overlap or interfere with the reporting dye which is uniquely responsive to the presence of an analyte.", "Another limiting feature of this invention is that the process of encoding beads requires a series of measurements which calibrate and train the sensors and the sensor array.", "Encoding is initially accomplished by first illuminating the beads with excitation light energy and monitoring and recording the type and location of the specific bead subpopulation within the sensor array having a given encoding dye ratio.", "Next, the array is exposed to an analyte while illuminating the array with excitation light energy in the presence of a reporter dye.", "Those beads which are responsive to the analyte in the presence of the reporter dye are monitored and mapped on the sensor array.", "In addition, the characteristic optical response signature is stored in a library.", "This step is repeated for each analyte of interest in combination with a reporter dye.", "Once all bead subpopulations are encoded and their response characteristics monitored and recorded, the entire sensor array must be decoded for each analyte by indexing each sensor element with the stored optical response signature for each analyte.", "This process of decoding individual subpopulations of beads may be require additional steps when a large number of subpopulations are deployed in the array, thereby increasing the training time required for each array.", "Other alternative approaches to bead encoding, utilizing molecular tagging, capillary gas chromatography and electron capture detection have been disclosed by Still, et al, Acc.", "Chem.", "Res.", "29:155 (1996).", "However, such methods are limited in scope and have been applied only to a narrow class of bead materials having specific chemical functionality and molecular tags which are readily analyzable." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In general, the invention provides self-encoding analytic chemical sensor arrays comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises at least one reporter dye.", "The reporting dye has a first characteristic optical response signature when subjected to excitation light energy in the presence of a reference analyte, and the microspheres are distributed on the surface.", "The beads may further comprise a bioactive agent.", "In an additional aspect, the invention provides methods of detecting a target analyte in a sample comprising contacting the sample with an sensor array.", "The sensor array comprises a substrate with a surface comprising discrete sites and a population of microspheres.", "The microspheres comprise at least a first and a second subpopulation, each subpopulation comprising a bioactive agent and at least one reporter dye.", "The reporting dye has a first characteristic optical response signature when subjected to excitation light energy in the presence of a reference analyte and the microspheres are distributed on the surface.", "The presence or absence (or quantity) of the analyte is then detected.", "The methods may further comprise identifying the location of each bioactive agent on said substrate by adding the reference analyte.", "In a further aspect, the invention provides methods for reducing the signal-to-noise ratio in the characteristic optical response signature of a sensor array having a subpopulations of array elements.", "The methods comprise decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array and measuring the characteristic optical response signature of each sensor element in the array.", "The baseline of the optical response signature is then adjusted for each sensor element in said array, and the baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations is summed.", "The characteristic optical response signature of each sensor subpopulation as a summation of said baseline-adjusted characteristic optical response signatures of all sensor elements within each of said subpopulations is then reported.", "In an additional aspect, the invention provides methods for amplifying the characteristic optical response signature of a sensor array having subpopulations of array elements.", "The methods comprise decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array and measuring a characteristic optical response signature of each sensor element in the array.", "The baseline of the optical response signature for each sensor element in said array is then adjusted.", "The baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations is then summed and the characteristic optical response signature of each sensor subpopulation as a summation of the baseline-adjusted characteristic optical response signatures of all sensor elements within each of the subpopulations is reported.", "The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims.", "It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention.", "The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.", "In an additional aspect the invention provides a method of detecting a target analyte comprising providing a first classifier for the response of a first population of sensors from a first pool of sensors to a first target analyte and distributing a second population of sensors from the first pool of sensors on an array.", "The method further includes and determining the response of the second population of sensors to a sample, wherein the response of the second population resembles the first classifier for the response of the first population to the first target analyte, thereby indicating the presence of the first target analyte in the sample.", "In addition the invention provides a method of detecting a target analyte comprising providing a first and second classifier for the response of a first and a second population of sensors from first and second pools of sensors, respectively, to a first target analyte and distributing third and fourth populations of sensors from the first and second pools of sensors, respectively, on an array.", "The method further includes determining the response of the third and fourth population of sensors to a sample, wherein the response of the third and fourth populations resembles the first and second classifiers for the response of the first and second populations, respectively, to the first target analyte, thereby indicating the presence of the first target analyte in the sample.", "In addition the invention provides a method of making an array comprising providing a population of microspheres, wherein the microspheres comprise an optical signature, contacting the microspheres with a sample comprising a target analytem recording the response of the microspheres to the target analyte, and generating a classifier for the response of the microspheres to the target analyte.", "The method further includes distributing the microspheres on a substrate with a surface comprising discrete sites." ], [ "The present application claims the benefit of U.S.", "Provisional application 60/238,866, filed Oct. 6, 2000, which is expressly incorporated herein by reference.", "FIELD OF THE INVENTION The present invention is generally concerned with chemical sensors, sensor arrays and sensing apparatus for the detection of gaseous and liquid analytes.", "More particularly, the invention is directed to optical chemical sensors and the detection and evaluation of optical data generated by sensing receptor units.", "BACKGROUND OF THE INVENTION The use of optical fibers and optical fiber strands in combination with light absorbing dyes for chemical analytical determinations has undergone rapid development, particularly within the last decade.", "The use of optical fibers for such purposes and techniques is described by Milanovich et al., “Novel Optical Fiber Techniques For Medical Application”, Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators and Fiber Optics” in C.R.C.", "Critical Reviews In Analytical Chemistry, Vol.", "19, 1988, pp.", "135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors In Analytical Chemistry” in Molecular Luminescence Spectroscopy, Methods and Applications (S. G. Schulman, editor), Wiley & Sons, New York (1988); Angel, S. M., Spectroscopy 2(4):38 (1987); Walt, et al., “Chemical Sensors and Microinstrumentation”, ACS Symposium Series, Vol.", "403, 1989, p. 252, and Wolfbeis, O. S., Fiber Optic Chemical Sensors, Ed.", "CRC Press, Boca Raton, Fla., 1991, 2nd Volume.", "When using an optical fiber in an in vitrolin vivo sensor, one or more light absorbing dyes are located near its distal end.", "Typically, light from an appropriate source is used to illuminate the dyes through the fiber's proximal end.", "The light propagates along the length of the optical fiber; and a portion of this propagated light exits the distal end and is absorbed by the dyes.", "The light absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest; and may or may not be retainable for subsequent use in a second optical determination.", "Once the light has been absorbed by the dye, some light of varying wavelength and intensity returns, conveyed through either the same fiber or collection fiber(s) to a detection system where it is observed and measured.", "The interactions between the light conveyed by the optical fiber and the properties of the light absorbing dye provide an optical basis for both qualitative and quantitative determinations.", "Of the many different classes of light absorbing dyes which conventionally are employed with bundles of fiber strands and optical fibers for different analytical purposes are those more common compositions that emit light after absorption termed “fluorophores” and those which absorb light and internally convert the absorbed light to heat, rather than emit it as light, termed “chromophores.” Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light (photons) at specified wavelengths and then emit light of a longer wavelength and at a lower energy.", "Substances able to fluoresce share a number of common characteristics: the ability to absorb light energy at one wavelength; reach an excited energy state; and subsequently emit light at another light wavelength.", "The absorption and fluorescence emission spectra are individual for each fluorophore and are often graphically represented as two separate curves that are slightly overlapping.", "The same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits; but the light emitted by the fluorophore will always provide the same emission spectrum.", "Finally, the strength of the fluorescence signal may be measured as the quantum yield of light emitted.", "The fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore.", "For more detailed information regarding each of these characteristics, the following references are recommended: Lakowicz, J. R., Principles of Fluorescence Spectroscopy, Plenum Press, New York, 1983; Freifelder, D., Physical Biochemistry, second edition, W. H. Freeman and Company, New York, 1982; “Molecular Luminescence Spectroscopy Methods and Applications: Part I” (S. G. Schulman, editor) in Chemical Analysis, vol.", "77, Wiley & Sons, Inc., 1985; The Theory of Luminescence, Stepanov and Gribkovskii, Iliffe Books, Ltd., London, 1968.Many of the recent improvements employing optical fiber sensors in both qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the distal end of the optical fiber.", "In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations and applications such as pH measurement, oxygen detection, and carbon dioxide analyses.", "These developments are exemplified by the following publications: Freeman, et al., Anal Chem.", "53:98 (1983); Lippitsch et al., Anal.", "Chem.", "Acta.", "205:1, (1988); Wolfbeis et al., Anal.", "Chem.", "60:2028 (1988); Jordan, et al., Anal.", "Chem.", "59:437 (1987); Lubbers et al., Sens.", "Actuators 1983; Munkholm et al., Talanta 35:109 (1988); Munkholm et al., Anal.", "Chem.", "58:1427 (1986); Seitz, W. R., Anal.", "Chem.", "56:16A-34A (1984); Peterson, et al., Anal.", "Chem.", "52:864 (1980): Saari, et al., Anal.", "Chem.", "54:821 (1982); Saari, et al., Anal.", "Chem.", "55:667 (1983); Zhujun et al., Anal.", "Chem.", "Acta.", "160:47 (1984); Schwab, et al., Anal.", "Chem.", "56:2199 (1984); Wolfbeis, O. S., “Fiber Optic Chemical Sensors”, Ed.", "CRC Press, Boca Raton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R., Anal.", "Chem., 481A-487A, Vol.", "67, (1995).", "More recently, fiber optic sensors have been constructed that permit the use of multiple dyes with a single, discrete fiber optic bundle.", "U.S. Pat.", "Nos.", "5,244,636 and 5,250,264 to Walt, et al.", "disclose systems for affixing multiple, different dyes on the distal end of the bundle, the teachings of each of these patents being incorporated herein by this reference.", "The disclosed configurations enable separate optical fibers of the bundle to optically access individual dyes.", "This avoids the problem of deconvolving the separate signals in the returning light from each dye, which arises when the signals from two or more dyes are combined, each dye being sensitive to a different analyte, and there is significant overlap in the dyes' emission spectra.", "Most recently, fiber optic sensors have been employed in arrays of semi-selective chemical sensors and pattern recognition schemes to discriminate and quantify odors.", "Such approaches have been useful in implementing the principles of biological olfaction in the design of sensing devices or systems.", "In this field of biomimetry, various technologies have been applied to the sensor transduction mechanism.", "For example, surface acoustic wave, conducting polymer, metal oxide sensor field-effect transistor (MOSFET), piezo-electric, and quartz crystal microbalance sensor arrays have been pursued.", "While such technologies provide inventive approaches utilizing a variety of physical and chemical phenomena to odor sensing, there are a number of limitations to these methods which restrict the efficacy of such devices.", "Firstly, element-to-element reproducibility both within a single array and between sensor arrays is typically unsatisfactory and thus requires recalibration and network retraining from sensor to sensor.", "Secondly, most of these methods have a relatively slow response time, frequently requiring several minutes to respond to the presence of an odor.", "Thirdly, such methods have relatively high detection limits and low sensitivity, typically not functioning at odor levels below 10 ppm.", "Fourthly, devices which embody such technologies typically require a relatively large inherent size, thereby restricting miniaturization of the sensor array for use in remote sensing applications.", "Finally, construction of multi-sensor arrays by these methods is complex and involves expensive and tedious preparation and placement of individual sensors within a well-defined array.", "Most recently, many of these shortcomings have been overcome through the application of fiber optic sensor arrays in an artificial nose sensor device and system.", "U.S. Pat.", "Nos.", "5,320,814 and 5,512,490 to Walt, et al., the teachings of each of these patents being incorporated herein by reference, disclose a fiber optic array formed of heterogeneous, semi-selective thin films which function as sensing receptor units and are able to detect a variety of different analytes and ligands using spectral recognition patterns.", "This technology has been applied to a vapor-sensing system which utilizes arrays of polymer-dye combinations which coat the ends of select optical fibers in a fiber optic bundle.", "These developments are further described in Dickinson, et al, Nature 382:697 (1996) and White, et al, Anal.", "Chem.", "68:2191 (1996).", "An innovative feature of the four previously referenced patents to Walt, et al., was the placement of multiple chemical functionalities at the end of a single optical fiber bundle sensor.", "This configuration yielded an analytic chemistry sensor that could be remotely monitored via the typically small bundle.", "The drawback, however, was the difficulty in applying the various chemistries associated with the chemical functionalities at the sensor's end; the functionalities were built on the sensor's end in a step-wise serial fashion.", "This was a slow process, and in practice, only tens of functionalities could be applied.", "U.S. patent application Ser.", "No.", "08/818,199 to Walt, et al, the teachings of which are incorporated herein by this reference, discloses the use of dye infiltrated polymer microspheres as a substitute for polymer-dye coating layers in optical fiber array sensors.", "With this approach, a fiber optic bundle serves as a substrate for dye-polymer microsphere array which contains a variety of microsphere bead sensors having different chemical and optical responses to the presence of target analytes.", "One innovative feature of this invention is in providing for a bead-based analytic chemistry system in which beads or microspheres carrying different chemical functionalities may be mixed together while retaining the ability to identify the functionality of each bead using an optically interrogatable encoding scheme.", "Additionally, this invention provides for an optical fiber bundle sensor in which the separate beads or microspheres may be optically coupled to discrete fibers or groups of fibers within the bundle.", "While the innovative features of this invention have separate applications, when implemented together, the invention provides for an optical fiber sensor that can support large numbers, thousands or more, of separate chemical sensor elements, which can be incorporated into a chemical sensor array and chemical analysis system.", "This approach provides for rapid fabrication and assembly of individual sensors and complex sensor arrays containing a multitude of discrete sensor types.", "The method also provides for a high degree of reproducibility and conformity within a batch of sensors and sensor arrays.", "Additional advantages are realized due to the ultrafine sizing available in microspheres.", "The overall size of the sensor array can be substantially reduced to submillimeter scale.", "This reduction in scale is particularly advantageous for remote sensing arrays.", "While the method of applying microsphere sensor elements in chemical sensor arrays as taught in U.S. patent application Ser.", "No.", "08/818,199 to Walt, et al, has many innovative features, this method has certain limitations.", "The method requires a complex multi-step bead encoding process to identify the type and location of bead subpopulations used in the sensor array.", "Beads are encoded by employing combinations of fluorescent dyes in varying ratio.", "The choice of encoding dyes is limited to those dyes which emit light at different wavelengths upon exposure to excitation light energy.", "While combinations of dyes in different ratios provide for encoding subpopulations of beads, the number of dye ratios available for encoding beads with a given dye pair or combination is significantly limited due to crowding the emission spectrum from peak overlap.", "In addition, a separate reporting dye is necessary for obtaining a unique characteristic optical response signature for a target analyte.", "Thus, the encoding dye choice is further limited by selecting dyes whose emission wavelengths do not overlap or interfere with the reporting dye which is uniquely responsive to the presence of an analyte.", "Another limiting feature of this invention is that the process of encoding beads requires a series of measurements which calibrate and train the sensors and the sensor array.", "Encoding is initially accomplished by first illuminating the beads with excitation light energy and monitoring and recording the type and location of the specific bead subpopulation within the sensor array having a given encoding dye ratio.", "Next, the array is exposed to an analyte while illuminating the array with excitation light energy in the presence of a reporter dye.", "Those beads which are responsive to the analyte in the presence of the reporter dye are monitored and mapped on the sensor array.", "In addition, the characteristic optical response signature is stored in a library.", "This step is repeated for each analyte of interest in combination with a reporter dye.", "Once all bead subpopulations are encoded and their response characteristics monitored and recorded, the entire sensor array must be decoded for each analyte by indexing each sensor element with the stored optical response signature for each analyte.", "This process of decoding individual subpopulations of beads may be require additional steps when a large number of subpopulations are deployed in the array, thereby increasing the training time required for each array.", "Other alternative approaches to bead encoding, utilizing molecular tagging, capillary gas chromatography and electron capture detection have been disclosed by Still, et al, Acc.", "Chem.", "Res.", "29:155 (1996).", "However, such methods are limited in scope and have been applied only to a narrow class of bead materials having specific chemical functionality and molecular tags which are readily analyzable.", "SUMMARY OF THE INVENTION In general, the invention provides self-encoding analytic chemical sensor arrays comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises at least one reporter dye.", "The reporting dye has a first characteristic optical response signature when subjected to excitation light energy in the presence of a reference analyte, and the microspheres are distributed on the surface.", "The beads may further comprise a bioactive agent.", "In an additional aspect, the invention provides methods of detecting a target analyte in a sample comprising contacting the sample with an sensor array.", "The sensor array comprises a substrate with a surface comprising discrete sites and a population of microspheres.", "The microspheres comprise at least a first and a second subpopulation, each subpopulation comprising a bioactive agent and at least one reporter dye.", "The reporting dye has a first characteristic optical response signature when subjected to excitation light energy in the presence of a reference analyte and the microspheres are distributed on the surface.", "The presence or absence (or quantity) of the analyte is then detected.", "The methods may further comprise identifying the location of each bioactive agent on said substrate by adding the reference analyte.", "In a further aspect, the invention provides methods for reducing the signal-to-noise ratio in the characteristic optical response signature of a sensor array having a subpopulations of array elements.", "The methods comprise decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array and measuring the characteristic optical response signature of each sensor element in the array.", "The baseline of the optical response signature is then adjusted for each sensor element in said array, and the baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations is summed.", "The characteristic optical response signature of each sensor subpopulation as a summation of said baseline-adjusted characteristic optical response signatures of all sensor elements within each of said subpopulations is then reported.", "In an additional aspect, the invention provides methods for amplifying the characteristic optical response signature of a sensor array having subpopulations of array elements.", "The methods comprise decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array and measuring a characteristic optical response signature of each sensor element in the array.", "The baseline of the optical response signature for each sensor element in said array is then adjusted.", "The baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations is then summed and the characteristic optical response signature of each sensor subpopulation as a summation of the baseline-adjusted characteristic optical response signatures of all sensor elements within each of the subpopulations is reported.", "The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims.", "It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention.", "The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.", "In an additional aspect the invention provides a method of detecting a target analyte comprising providing a first classifier for the response of a first population of sensors from a first pool of sensors to a first target analyte and distributing a second population of sensors from the first pool of sensors on an array.", "The method further includes and determining the response of the second population of sensors to a sample, wherein the response of the second population resembles the first classifier for the response of the first population to the first target analyte, thereby indicating the presence of the first target analyte in the sample.", "In addition the invention provides a method of detecting a target analyte comprising providing a first and second classifier for the response of a first and a second population of sensors from first and second pools of sensors, respectively, to a first target analyte and distributing third and fourth populations of sensors from the first and second pools of sensors, respectively, on an array.", "The method further includes determining the response of the third and fourth population of sensors to a sample, wherein the response of the third and fourth populations resembles the first and second classifiers for the response of the first and second populations, respectively, to the first target analyte, thereby indicating the presence of the first target analyte in the sample.", "In addition the invention provides a method of making an array comprising providing a population of microspheres, wherein the microspheres comprise an optical signature, contacting the microspheres with a sample comprising a target analytem recording the response of the microspheres to the target analyte, and generating a classifier for the response of the microspheres to the target analyte.", "The method further includes distributing the microspheres on a substrate with a surface comprising discrete sites.", "BRIEF DESCRIPTION OF THE DRAWINGS In the accompanying drawings, reference characters refer to the same parts throughout the different views.", "The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention.", "Of the drawings: FIG.", "1 is a schematic diagram illustrating the self-encoding microsphere sensor according to the present invention; FIG.", "2 is a process flow diagram of the preparation, encoding and incorporation of microspheres into a sensor array of the present invention; FIGS.", "3A and 3B is a schematic process diagram illustrating the preparation and placement of self-encoded microsphere subpopulations in fiber optic sensor array of the present invention; FIG.", "4 is a process flow diagram illustrating microwell formation in the fiber optic bundle and placement of the microspheres in the microwells according to the method of the present invention; FIGS.", "5A and 5B are micrographs illustrating the microwells formed on the distal end of a fiber optic bundle and microspheres inserted in the microwell cavities; FIGS.", "6A and 6B are micrographs showing the array of microspheres in their corresponding microwells prior to and subsequent to agitation by tapping and an air pulse, demonstrating the electrostatic binding of the microspheres in the microwell cavities; FIG.", "7 is a schematic diagram of the inventive fiber optic sensor and associated instrumentation and control system; FIG.", "8 is a schematic diagram illustrating the experimental apparatus used in the optical measurements of Examples 7 through 17; FIG.", "9 illustrates the characteristic optical response signature of porous silica beads infiltrated with Nile Red dye upon exposure to toluene vapor; FIG.", "10 illustrates the characteristic optical response signature of PMS beads infiltrated with Nile Red dye upon exposure to methanol vapor; FIGS.", "11A and 11B illustrate the characteristic optical response signature of a PS802 coated porous silica bead infiltrated with Nile Red dye upon exposure to toluene and methanol vapor; FIGS.", "12A and 12B illustrate the characteristic optical response signature of a PDPO coated porous silica beads infiltrated with Nile Red dye upon exposure to toluene and methanol vapor; FIG.", "13 illustrates the characteristic optical response signature of porous silica beads infiltrated with Nile Red dye upon exposure to ethyl acetate vapor; FIG.", "14 illustrates the innovation of optical response signal summing for reducing signal-to-noise ratios in Nile Red infiltrated PMS bead subpopulation measurements of methanol vapor; FIG.", "15 illustrates the innovation of optical response signal summing for signal enhancement in PMS bead subpopulation measurements of methanol vapor; FIG.", "16 compares the characteristic optical response signatures of two PS802 coated porous silica beads infiltrated with Nile Red dye upon exposure to toluene and methanol vapor; FIG.", "17 compares the characteristic optical response signatures to methanol vapor which are used for decoding Nile Red infiltrated porous silica and PMS bead subpopulations in a self-encoded fiber optic sensor array of the present invention; FIG.", "18 compares the characteristic optical response signatures of Nile Red infiltrated porous silica and PMS bead subpopulations to n-proponal vapor in a self-encoded fiber optic sensor array of the present invention; FIG.", "19 compares the characteristic optical response signatures of Nile Red infiltrated porous silica and PMS bead subpopulations to toluene vapor in a self-encoded fiber optic sensor array of the present invention; FIG.", "20 compares the differences in bead swelling response of PS802 coated porous silica, poly methyl styrene, and poly methyl styrene/divinyl benzene bead subpopulations upon exposure to toluene vapor; FIG.", "21 depicts sequences used in the array (Table 1).", "Each probe has a 5′-(NH2—(CH2)6—) functionality for cyanuric chloride activation and attachment to the microspheres.", "Each complementary target has a 5′-fluorescein label; FIG.", "22 depicts Table 2, microsphere code and target identification.", "The left most column lists the names of the seven probes.", "The middle columns list the dye concentrations (mM) used to encode the microspheres.", "Each microsphere type incorporated two encoding dyes for identification of the probe on the bead.", "The right column lists the percentage of beads that correctly identified the target solution.", "FIG.", "23 depicts Table 3, microsphere code and target identification.", "The left most column lists the numbers from Table 1 which identify the probes.", "The middle columns list the dye concentrations (mM) used to encode the microspheres.", "Each microsphere type incorporated at least one encoding dye for identification of the probe on the bead.", "The right column lists the percentage of beads that correctly identified the target solution; FIG.", "24 depicts Table 4, microsphere array sensitivity.", "The sensitivity of the system using an intensified CCD camera; and FIG.", "25 depicts a number of individual experiments and the coefficient of variances.", "FIG.", "26.The concatenated 60 time point (frame) responses of the five sensor types to 4-NT (open symbols) and ethanol (filled symbols) vapors are shown for the three arrays.", "These temporal response profiles demonstrate how a nitroaromatic compound can be distinguished from a non-nitroaromatic compound based on different response features.", "Analytes are denoted by the following symbols: Ethanol: training array (▪), 1st testing array (●) and 2nd testing array (▴).", "4-NT: training array (), 1st testing array (◯) and 2nd testing array (Δ).", "FIG.", "27.Graph of the GWMW (1,1) scores vs. the GWMW (2,2) scores for the first test data set collected one month after the training data.", "Analytes are denoted with the following symbols: 4-NT (n), 1,3-DNB (▪), all VOCs (x) and false negative observations (◯).", "A 98.2% correct classification rate was achieved.", "FIG.", "28.Graph of the GWMW (1,1) scores vs. the GWMW (2,2) scores for the second test data set collected six months after the training data.", "Analytes are denoted with the following symbols: 4-NT (n), 1,3-DNB (▪), all VOCs (x), false negative observations (◯) and false positive observations (⋄).", "A 93.8% correct classification rate was achieved.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides an analytic chemistry system that comprises a self-encoding sensor array comprising a population of beads or microspheres on discrete locations on the surface of a substrate.", "Within the bead population are separate bead subpopulations, each of which provides a characteristic optical response signature when illuminated by excitation light energy in the presence of a reference analyte, which may in some cases be the target analyte.", "Although the subpopulations may be randomly mixed together, the identity and location of each bead is determined via a characteristic optical response signature when illuminated by excitation light energy in the presence of a reference analyte.", "This allows the decoding of the array, i.e.", "the identification of the location of each subpopulation of beads on an array, to proceed very simply.", "In a preferred embodiment, the beads are encoded with one or more reporter dyes that exhibit a characteristic, i.e.", "unique, optical response signature to a reference analyte, generally a fluid such as a vapor.", "Thus, in this embodiment, exposure of the entire array to a reference analyte will allow the identification of the location of each bead of each subpopulation.", "As a result, by comparing the response of the entire sensor array to a known analyte, the individual sensor elements of the array are conveniently decoded simultaneous in one simple measurement.", "The self-encoding feature of the present invention eliminates the need for a more complex, multi-step encoding system.", "The sensor array can then be used to detect the presence of target analytes, for example when the beads also comprise bioactive agents such as oligonucleotides, by looking for changes in the optical signature of the beads upon binding of the target analyte, for example a substantially complementary labelled oligonucleotide.", "As will be appreciated by those in the art, this may be done in a variety of ways, generally through the use of a change in an optical signal.", "This change can occur via many different mechanisms.", "A few examples include the binding of a dye-tagged analyte to the bead, the production of a dye species on or near the beads, the destruction of an existing dye species, a change in the optical signature upon analyte interaction with dye on bead, or any other optical interrogatable event.", "Thus, once the location of each species of oligonucleotide probe has been identified, the array can then be used to detect the presence of unknowns that will preferably specifically associate with the bioactive agents on the beads.", "In an alternate preferred embodiment, when the target analyte is not labeled, the optical response of each element in the array can be compared to a library of characteristic optical response signatures for its corresponding bead subpopulation type, where the characteristic optical response signature to various analytes has been previously measured and recorded, and either the identity of the unknown can be determined or the sensor array can be trained to associate the measured response with a particular analyte which is then added to the library of response signatures.", "In one embodiment, a plurality of sensors is trained to respond to target analytes.", "This training is then transferable to subsequent arrays containing equivalent sensor elements.", "That is, the method includes training of the sensors on one array and then using the training model to identify analyte responses from any other array containing equivalent sensor elements.", "In one embodiment the library of sensors are trained or exposed to various target analytes on the same array.", "That is, different sensors may be applied to the same array and the different responses evoked by different target analytes are detected.", "In a preferred embodiment, multiple equivalent sensors are trained on the same array.", "This allows for increased confidence in the recorded responses that will serve as a library of responses for analysis of subsequent arrays.", "Preferably these responses are saved, for example, in a database where they can be accessed.", "In an alternative embodiment sensors are trained in separate or homogenous arrays.", "That is, all of the sensors on an array are the same and are exposed to a single target analyte at a time.", "The signal from each of the sensors can then be detected and saved as described above, and in some embodiments, is averaged or otherwise analyzed statistically.", "In a preferred embodiment, the signal from each population of sensors on an array is analyzed statistically.", "This results in the reduction of highly complex signals obtained from an array to a “classifier” that can be used to analyze subsequent arrays.", "That is, once a sensor is trained, a classifier for a particular bead type and target analyte is used to analyze equivalent bead types.", "By “classifier” is meant a numerical or mathematical description of the response of a particular sensor to a particular analyte.", "That is, there is a discrete classifier for each combination of sensor/target analyte.", "That is, for each sensor, i.e.", "each microsphere with a particular bioactive agent, there is a distinct classifier for each target analyte.", "Alternatively, where multiple different sensors detect a single target analyte, there is a distinct classifier for each combination.", "The classifier allows for identification of the target analyte in subsequent arrays that contain beads that are equivalent to the bead used for the initial training and from which the classifier is obtained.", "In a preferred embodiment, each population of sensors on an array is obtained from a pool of equivalent sensors.", "By “pool” is meant a collection of equivalent sensors.", "By “equivalent” is meant that the beads contain the same chemical functionality.", "As such, the pool of sensors will respond similarly to the same target analyte.", "In a preferred embodiment, the pool of sensors are prepared in quantities such that populations or aliquots from a pool of sensors can be used in or applied to a plurality of arrays.", "In a preferred embodiment, the populations of sensors from a pool can be applied to greater than 100's of arrays; more preferably, populations of sensors from a pool can be applied to greater than 1000's of arrays.", "Having trained the sensors to detect particular analytes, the sensors are randomly distributed on an array as described herein.", "That is, once a first population of a particular pool of sensors is trained to respond to a particular target analyte, a second population of the same pool is distributed on a subsequent array.", "In a preferred embodiment, the subsequent array(s) are formed from populations of sensors obtained from a plurality of pools of sensors.", "Accordingly, the array contains multiple sensors randomly distributed on the array.", "In a preferred embodiment the array contains redundant sensors from a particular pool of sensors.", "That is, a plurality of sensors from each pool of sensors is distributed on the array.", "The present invention overcomes certain limitations of the current art by embodying the innovation of a self-encoding sensor array wherein a characteristic optical response signature is produced by the interaction of specific bead subpopulation compositions with a reporter dye.", "In the self-encoding sensor array of the present invention, the response signal to a target analyte serves both as a response signature for the target analyte and as the encoding signal for the entire sensor array and subpopulations within the array.", "The decoding of the array is thus accomplished in a one-step process during the array response measurement of a target analyte and utilizes the very response which is used to identify the target analyte.", "The bead encoding is thus incorporated into the array by the nature of the bead subpopulation responses to target analytes.", "In the present invention, each bead-dye combination of a subpopulation has a characteristic optical response signature when exposed to a given fluid, usually a vapor.", "The self-encoding concept is provided by the unique response characteristics of the dye in combination with a specific bead matrix material.", "Thus the bead subpopulations which are randomly dispersed in a sensor array can be rapidly identified and located after placement in the array simply by exposing the sensor array to a known test fluid and matching the resulting optical response signature to those obtained for each bead subpopulation.", "With this approach, the beads are self encoding and the response characteristics of the entire sensor array are rapidly determined and stored for measurement of a target analyte.", "The method of the present invention is particularly useful in applications of sensor arrays containing thousands of sensors having distinctive optical response signature characteristics.", "An additional benefit of the present invention is that it allows the synthesis of the bioactive agents (i.e.", "compounds such as nucleic acids and antibodies) to be separated from their placement on an array, i.e.", "the bioactive agents may be synthesized on the beads, and then the beads are randomly distributed on a patterned surface.", "Since the beads are self-encoded by having dyes present that have known responses to a reference analyte, this means that the array can later be “decoded”, i.e.", "after the array is made, a correlation of the location of an individual site on the array with the bead or bioactive agent at that particular site can be made.", "This means that the beads may be randomly distributed on the array, a fast and inexpensive process as compared to either the in situ synthesis or spotting techniques of the prior art.", "Once the array is loaded with the beads, the array can be decoded, or can be used, with full or partial decoding occuring after testing, as is more fully outlined below.", "In a preferred embodiment of the present invention, ultra-fine, porous microbeads or microspheres are utilized as individual sensors.", "The utilization of porous micron-scale sensors provides for improved sensor response and sensitivity.", "The reduction in sensor dimension substantially reduces the diffusion length and time for analyte interaction with individual sensors and significantly shortens the sensor response time, while simultaneously enhancing sensor sensitivity and lowering detection limits.", "In another preferred embodiment of the present invention, the sensor array is comprised of subpopulations of beads or microspheres which are disposed on a distal end of an optical fiber bundle wherein the separate beads or microspheres may be optically coupled to discrete fibers or groups of fibers within the bundle.", "Since typically, such fiber optic bundles comprise thousands of discrete fibers, the present invention thus provides for an optical fiber sensor which can support a large number, thousands or more, of sensor array elements of distinct and varying subpopulations each having a characteristic optical response signature when exposed to an analyte while being illuminated by excitation light energy.", "In one preferred embodiment, the distal end of a fiber optic bundle substrate is chemically etched so as to create a cavity or micro-well at the end of a discrete fiber.", "In the preferred embodiment, each one of the beads is located within separate microwells formed at terminal ends of optical fibers of the bundle.", "These microwells are formed by anisotropic etching of the cores of the optical fibers with respect to the cladding.", "The resultant etched cavity is dimensioned for accommodating an individual microbead sensor and for providing optical coupling of the individual bead sensor with the discrete optical fiber in the fiber bundle.", "Since typical fiber optic bundles contain thousands of discrete fibers, this embodiment provides for the individual optical coupling of thousands of sensors in a sensor array, thereby providing for a large number of independent sensor measurements for each bead subpopulation within the array.", "Due to both the large number of bead sensor subpopulations available and the correspondingly large number of sensor elements within each subpopulation, a significant innovation of the present invention is in providing for thousands of independent sensor response measurements in a single sensor array.", "This enables another significant innovation of the present invention by providing for the summing and amplification of the characteristic optical response signatures of multiple independent measurements taken from sensor beads within each sensor array bead subpopulation.", "This approach directly mimics the actual behavior of the human olfactory where the combined signals from thousands of receptor cells in each of grouping of nearly a thousand different receptor cell types found in the epithelium layer, none of which are particularly sensitive in themselves, lead to a highly amplified sensory response to odors [see J. S. Kauer, Trends Neurosci.", "14:79-95(1991)].", "The present invention thus embodies the evolutionary scent amplification process found in the human olfactory system in order to significantly enhance sensor array sensitivity to analytes by summing the low-level responses of a large number of sensor array elements.", "By summing the responses from several beads at low vapor concentrations, a substantial improvement in signal-to-noise ratios is achieved, exceeding a factor of ten or more.", "This innovation has led to reducing the detection limit of the sensor array by over an order of magnitude.", "The enhancement in sensitivity provided by the sensor array of the present invention is generally known to be directly proportional to the square root of the number of independent sensor bead responses available for summing.", "With such enhancements, detection limits approaching parts per billion are achievable.", "In preferred embodiments, the sensor beads are self-encoded using a reporter dye that is preferably infiltrated or entrapped within the beads.", "The reporter dye may be a chromophore or phosphor but is preferably a fluorescent dye, which due to characteristically strong optical signals provide a good signal-to-noise ratio for decoding.", "Although not necessary, the self-encoding can also be accomplished by utilizing the ratios of two or more reporting dyes having characteristic and discrete emission peaks and measuring the peak intensity ratios upon illumination with excitation light energy.", "According to another embodiment, the invention also concerns a chemical sensor array designed with a predetermined chemical specificity.", "In this embodiment, additional chemical functionality can be incorporated into each sensor subpopulation by attaching a desired moiety to the surfaces of the beads.", "In another embodiment, the sensor array has a population of beads carrying chemical functionality at, on or near, a distal end of the bundle.", "The ability to monitor optical signature changes associated with individual or multiple beads interacting with a target analyte is provided by optically coupling those signature changes into separate optical fibers or groups of fibers of a fiber optic bundle for transmission to the proximal end where analysis is performed either manually, by the user, or automatically, using image processing techniques.", "Although each sensor is different insofar that it has a different distribution of the subpopulations of beads within its microwells, only those beads that exhibit a positive optical response or signature change to a target analyte of interest need to be decoded.", "Therefore, the burden is placed on the analysis rather than on sensor manufacture.", "Moreover, since the beads and fibers in the array can be monodisperse, the fluorescent regions arising from signal generation are extremely uniform and can be analyzed automatically using commercially available microscopy analysis software.", "Such image processing software is capable of defining different spectral regions automatically and counting the number of segments within each region in several seconds.", "Accordingly, the present invention provides array compositions comprising at least a first substrate with a surface comprising individual sites.", "By “array” herein is meant a plurality of bioactive agents in an array format; the size of the array will depend on the composition and end use of the array.", "Arrays containing from about 2 different bioactive agents (i.e.", "different beads) to many millions can be made, with very large fiber optic arrays being possible.", "Generally, the array will comprise from two to as many as a billion or more, depending on the size of the beads and the substrate, as well as the end use of the array, thus very high density, high density, moderate density, low density and very low density arrays may be made.", "Preferred ranges for very high density arrays are from about 10,000,000 to about 2,000,000,000, with from about 100,000,000 to about 1,000,000,000 being preferred.", "High density arrays range about 100,000 to about 10,000,000, with from about 1,000,000 to about 5,000,000 being particularly preferred.", "Moderate density arrays range from about 10,000 to about 50,000 being particularly preferred, and from about 20,000 to about 30,000 being especially preferred.", "Low density arrays are generally less than 10,000, with from about 1,000 to about 5,000 being preferred.", "Very low density arrays are less than 1,000, with from about 10 to about 1000 being preferred, and from about 100 to about 500 being particularly preferred.", "In some embodiments, the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single bioactive agent may be made as well.", "In addition, in some arrays, multiple substrates may be used, either of different or identical compositions.", "Thus for example, large arrays may comprise a plurality of smaller substrates.", "In addition, one advantage of the present compositions is that particularly through the use of fiber optic technology, extremely high density arrays can be made.", "Thus for example, because beads of 200 nm can be used, and very small fibers are known, it is possible to have as many as 250,000 different fibers and beads in a 1 mm2 fiber optic bundle, with densities of greater than 15,000,000 individual beads and fibers per 0.5 cm2 obtainable.", "The compositions comprise a substrate.", "By “substrate” or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of beads and is amenable to at least one detection method.", "As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.", "), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers.", "In general, the substrates allow optical detection and do not appreciably fluorescese.", "Generally the substrate is flat or planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well; for example, three dimensional configurations can be used, for example by embedding the beads in a porous block of plastic that allows sample access to the beads and using a confocal microscope for detection.", "Similarly, the beads may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.", "Preferred substrates include optical fiber bundles as discussed below, and flat planar substrates such as glass, polystyrene and other plastics and acrylics.", "At least one surface of the substrate is modified to contain discrete, individual sites for later association of microspheres.", "These sites may comprise physically altered sites, i.e.", "physical configurations such as wells or small depressions in the substrate that can retain the beads, such that a microsphere can rest in the well, or the use of other forces (magnetic or compressive), or chemically altered or active sites, such as chemically functionalized sites, electrostatically altered sites, hydrophobically/hydrophilically functionalized sites, spots of adhesive, etc.", "The sites may be a pattern, i.e.", "a regular design or configuration, or randomly distributed.", "A preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-Y coordinate plane.", "“Pattern” in this sense includes a repeating unit cell, preferably one that allows a high density of beads on the substrate.", "However, it should be noted that these sites may not be discrete sites.", "That is, it is possible to use a uniform surface of adhesive or chemical functionalities, for example, that allows the attachment of beads at any position.", "That is, the surface of the substrate is modified to allow attachment of the microspheres at individual sites, whether or not those sites are contiguous or non-contiguous with other sites.", "Thus, the surface of the substrate may be modified such that discrete sites are formed that can only have a single associated bead, or alternatively, the surface of the substrate is modified and beads may go down anywhere, but they end up at discrete sites.", "In a preferred embodiment, the surface of the substrate is modified to contain wells, i.e.", "depressions in the surface of the substrate.", "This may be done as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques.", "As will be appreciated by those in the art, the technique used will depend on the composition and shape of the substrate.", "In a preferred embodiment, physical alterations are made in a surface of the substrate to produce the sites.", "In a preferred embodiment, the substrate is a fiber optic bundle and the surface of the substrate is a terminal end of the fiber bundle.", "In this embodiment, wells are made in a terminal or distal end of a fiber optic bundle comprising individual fibers.", "In this embodiment, the cores of the individual fibers are etched, with respect to the cladding, such that small wells or depressions are formed at one end of the fibers.", "The required depth of the wells will depend on the size of the beads to be added to the wells.", "Generally in this embodiment, the microspheres are non-covalently associated in the wells, although the wells may additionally be chemically functionalized as is generally described below, cross-linking agents may be used, or a physical barrier may be used, i.e.", "a film or membrane over the beads.", "In a preferred embodiment, the surface of the substrate is modified to contain chemically modified sites, that can be used to attach, either covalently or non-covalently, the microspheres of the invention to the discrete sites or locations on the substrate.", "“Chemically modified sites” in this context includes, but is not limited to, the addition of a pattern of chemical functional groups including amino groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach microspheres, which generally also contain corresponding reactive functional groups; the addition of a pattern of adhesive that can be used to bind the microspheres (either by prior chemical functionalization for the addition of the adhesive or direct addition of the adhesive); the addition of a pattern of charged groups (similar to the chemical functionalities) for the electrostatic attachment of the microspheres, i.e.", "when the microspheres comprise charged groups opposite to the sites; the addition of a pattern of chemical functional groups that renders the sites differentially hydrophobic or hydrophilic, such that the addition of similarly hydrophobic or hydrophilic microspheres under suitable experimental conditions will result in association of the microspheres to the sites on the basis of hydroaffinity.", "For example, the use of hydrophobic sites with hydrophobic beads, in an aqueous system, drives the association of the beads preferentially onto the sites.", "As outlined above, “pattern” in this sense includes the use of a uniform treatment of the surface to allow attachment of the beads at discrete sites, as well as treatment of the surface resulting in discrete sites.", "As will be appreciated by those in the art, this may be accomplished in a variety of ways.", "The compositions of the invention further comprise a population of microspheres.", "By ”population” herein is meant a plurality of beads as outlined above for arrays.", "Within the population are separate subpopulations, which can be a single microsphere or multiple identical microspheres.", "That is, in some embodiments, as is more fully outlined below, the array may contain only a single bead for each bioactive agent; preferred embodiments utilize a plurality of beads of each type.", "By “microspheres” or “beads” or “particles” or grammatical equivalents herein is meant small discrete particles.", "The composition of the beads will vary, depending on the class of bioactive agent and the method of synthesis.", "Suitable bead compositions include those used in peptide, nucleic acid and organic moiety synthesis, including, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphited, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and teflon may all be used.", "Synthetic beads may be fabricated by polymerizing or copolymerizing a variety of condensation or vinyl precursor monomers or by way of combinatorial polymer synthesis.", "Such polymers can be further modified by the addition of plasticizers, such as tritolyl phosphate (TTP), triphenyl phospate (TTP) or dibutyl phthalate (DBP).", "Particularly useful dye-encoding bead candidates for use in sensor array subpopulations are polymer and copolymer materials which exhibit either a characteristic swelling upon exposure to various vapor analytes, a characteristic polarity difference due to their chemical structure, or a characteristic chemical adsorption response with various vapor analytes.", "In prescreening candidate polymers as bead materials and evaluating candidates based on desirable swelling, polarity and adsorption characteristics, two particularly useful references are: R. A. McGill, et al., Chemtech, Sep. 24, 1996, p27-37 and J. W. Grate, et al., Anal.", "Chem.", "68:913-7 (1996).", "A variety of bead chemistries may be utilized in fabricating a wide diversity of sensor bead subpopulations.", "For example, the following compositions have been found to be particularly useful as candidate bead materials: silica, poly(ethylene glycol), polycaprolactone, poly(1,4-butylene adipate), PDPO [poly(2,6-dimethyl-1,4-phenyleneoxide)], PS078.5 [triethoxysilyl-modified polybutadiene (50% in toluene)], PS078.8 [diethoxymethylsilyl-modified polybutadiene in toluene], CPS2067 [acryloxypropylmethyl-cyclosiloxane], PS802 [(80-85%) dimethyl-(15-20%) (acryloxypropyl)methylsiloxane copolymer], PS901.5 poly(acryloxypropyl-methyl)siloxane], PS851 [(97-98%) dimethyl-(2-3%) (methacryloxypropyl)methylsiloxane copolymer], PABS [poly(acrylonitrile-butadiene-styrene)], poly(methyl methacrylate), poly(styrene-acrylonitrile 75:25), acryloxypropylmethylsiloxane-dimethylsiloxane copolymer, methylstyrene, polystyrene, acrylic polymers, and poly(methyl styrene/divinyl benzene).", "Other adsorbents, such as commercially available silica beads adapted with a variety of bonded phases for use in phenomenex columns, such as beads comprising C8, C18 and phenyl hexyl, are useful as sensor bead matrices.", "Inorganic materials such as alumina and zeolites may also be utilized.", "Other polymers and copolymers having distinguishable and suitable swelling behavior, polarity and chemical adsorption characteristics are also anticipated as likely bead candidate materials.", "Particularly useful bead candidate materials include the polymers, copolymers, and polymerized monomers listed in Table 7, Table 8 and Table 10 of U.S. Pat.", "No.", "5,512,490 to Walt, et al, which are herein incorporated by reference.", "In alternative embodiments, any synthesized or commercially available bead materials may be further modified by applying either a surface treatment or coating to modify the characteristic optical response signature.", "For example, where porous silica beads are utilized, N-octadecyltriethyoxysilane or 3-(trimethoxysilyl)propyl methacrylate may be applied as a silanization treatment.", "In general, “Microsphere Detection Guide” from Bangs Laboratories, Fishers Ind.", "is a helpful guide.", "The choice of subpopulations used to form the sensor array elements in a particular sensor array is primarily determined based on the analytical purposes of the sensor and the specific analytes which are targeted for detection.", "Typically, bead subpopulations are selected based on distinguishable differences in their characteristic optical response signatures when illuminated by excitation light energy in the presence of a target analyte.", "In fabricating self-encoding sensor arrays, bead subpopulations are selected which have characteristic optical response signatures when infiltrated with a reporting dye and illuminated by excitation light energy in the presence of both a reference analyte and target analyte.", "Thus, preferred bead materials for the sensor array are preselected based on either physical or chemical differences in bead subpopulations which produce a characteristic optical response signature in the presence of the analyte when illuminated by excitation light energy.", "Features such bead material polarity, chemical structure, chemical functionality, bead surface area, bead pore size, bead swelling characteristics, or chemical adsorption behavior, either separately or in combination, contribute to the characteristic optical response signature of a given bead subpopulation.", "In one embodiment, bead materials which are permeable or semi-permeable to fluids including vapors and liquid analytes are preferred.", "In another embodiment, bead materials that swell upon contact with fluids such as vapor or liquid analytes are preferred.", "In general, bead materials which have unique polarity, structure, pore size, surface area, functionality or adsorption characteristics are particularly useful for sensor bead matrices of the present invention.", "The microspheres comprise a reporting dye that, in combination with the characteristic bead matrix material, provides an optical response signature that can be used to identify the bead, and thus the attached bioactive agent, upon exposure to a reference analyte.", "That is, each subpopulation of microspheres (i.e.", "each sensor element) comprises a unique optical response signature or optical tag, that can be used to identify the unique bioactive agent of that subpopulation of microspheres; a bead comprising the unique optical response signature may be distinguished from beads at other locations with different optical response signatures.", "As is outlined herein, each bioactive agent will have an associated unique optical response signature such that any microspheres comprising that bioactive agent will be identifiable on the basis of the signature upon exposure to a reference analyte or fluid.", "As is more fully outlined below, it is possible to reuse or duplicate optical response signatures within an array, for example, when another level of identification is used, for example when beads of different sizes are used, or when the array is loaded sequentially with different batches of beads.", "The selection of chemical dye indicators is equally important to the design of a fiber optic sensor array system of the present invention.", "In the preferred embodiment, at least one dye 11 is incorporated into the microsphere 10.In the preferred embodiment, this dye 11 acts as both an encoding dye, for identifying the bead subpopulation location in the sensor array, and a reporting dye, for detecting a target analyte of interest.", "In an alternative embodiment, two or more dyes may be utilized as encoding-reporter dyes.", "In a preferred embodiment, at least one dye is used solely as an encoding dye and a separate dye is added during analysis as a reporting dye.", "In one embodiment, where two or more encoding dyes are used, the ratio of peak intensities for dye pairs may be used for encoding the bead subpopulation and a separate reporter dye may be added during analysis.", "In an alternative embodiment, conjugated dyes, such as acrlyoyl fluorescein and others, may be utilized where it is desirable to incorporate the dye directly into a synthesized polymer or copolymer bead material.", "While the reporter dye 11 may be either a chromophore-type or a fluorophore-type, a fluorescent dye is preferred because the strength of the fluorescent signal provides a better signal-to-noise ratio when decoding.", "In the most preferred embodiment, polarity-sensitive dyes or solvatochromic dyes are utilized.", "Solvatochromic dyes are dyes whose absorption or emission spectra are sensitive to and altered by the polarity of their surrounding environment.", "Typically, these dyes exhibit a shift in peak emission wavelength due to a change in local polarity.", "Polarity changes which cause such wavelength shifts can be introduced by the bead matrix used for a particular sensor bead subpopulation or, the presence of a target analyte.", "The change in polarity creates a characteristic optical response signature which is useful for both encoding subpopulations of bead types and for detecting specific target analytes.", "One preferred solvatochromic dye, Nile Red (Eastman Kodak, Rochester, N.Y.), exhibits large shifts in its emission wavelength peak with changes in the local environment polarity.", "In addition, Nile Red is soluble in a wide range of solvents, is photochemically stable, and has a relatively strong fluorescence peak.", "Additional dyes which are conventionally known in the art and may be used as dyes in the present invention are those found in U.S. Pat.", "No.", "5,512,490 to Walt, et al., of which Table 3, Table 4, Table 5, Table 6 and Table 11 are incorporated herein by reference.", "Different subpopulations of bead sensing elements can be fabricated for the sensor array of the present invention by immobilizing Nile Red in polymer matrices of varying composition.", "By incorporating Nile Red in bead subpopulations made from different polymer matrices of varying polarity, hydophobicity, pore size, flexibility and swelling tendency, unique subpopulations of sensor beads are produced that react differently with molecules of individual fluids, giving rise to different fluorescence responses when exposed to organic fluids.", "This results in each bead subpopulation having a characteristic optical response signature when exposed to a variety of analytes.", "In a preferred embodiment, the dyes are covalently attached to the surface of the beads.", "This may be done as is generally outlined below for the attachment of the bioactive agents, using functional groups on the surface of the beads.", "As will be appreciated by those in the art, these attachments are done to minimize the effect on the dye.", "In a preferred embodiment, the dyes are non-covalently associated with the beads, generally by entrapping the dyes in the bead matrix or pores of the beads.", "In one embodiment, the dyes are added to the bioactive agent, rather than the beads, although this is generally not preferred.", "FIG.", "2 is a process diagram illustrating the preparation of the sensor bead subpopulations and sensor bead array.", "In step 50, suspensions of the various bead subpopulations are individually prepared from either commercial bead materials or synthesized bead materials which have been made from preferred polymeric materials.", "In this step, the beads may be prewashed, surface treated with a coupling agent, such as a silanizing solution as used in Example 2 and Example 3, or treated with a plasticizer, such as TTP, TPP or DBP as used in Example 6.In preparing the bead subpopulations, each bead grouping is typically dispersed in an appropriate solvent which may comprise additions of surfactants or dispersants to enhance dispersion.", "For example, Tween 20 (J. T. Baker, Cleveland, Ohio), a polyoxyethylenesorbitan monolaurate, has been found to be particularly useful as a surfactant.", "A dye solution is prepared 51 for tagging or encoding each of the bead subpopulations for subsequent identification and indexing subpopulations in the sensor array in a later decoding step.", "In the most preferred embodiment, a single dye serves both as a sensor bead subpopulation encoding dye and as an analyte reporting dye that is used to detect the presence of a target analyte.", "In another embodiment, the dye serves solely to encode the sensor bead subpopulation and an additional dye is used as a reporter dye for detection of a target analyte.", "In one embodiment, two or more dyes may be incorporated into the bead subpopulation and the peak intensity ratios of dye pairs may be used for encoding the sensor bead subpopulation.", "Typically, a single solvatochromic dye is used as both the encoding dye and reporting dye.", "In a preferred embodiment, Nile Red dye (Aldrich, Milwaukee, Wis.) is used.", "For incorporating dye into each bead subpopulation, suspensions of the beads prepared in step 50 are mixed in step 52 with dye solutions prepared in step 51.Preferably, in step 52, the beads or microspheres are placed in a dye solution comprising dye dissolved in an organic solvent that will swell the microspheres.", "In step 54, the beads are washed, centrifuged or filtered to remove excess dye.", "The microspheres are typically washed in water, methanol, or any suitable solvent that does not swell the microspheres, but in which the dyes are still soluble.", "This allows the residual dye to be rinsed off without rinsing the dye out of the microspheres.", "In an alternative embodiment, a chemical moiety or functional group may be attached to the bead surface following removal of excess dye.", "The beads need not be spherical; irregular particles may be used.", "While both porous and non-porous beads may be utilized, porous beads are preferred for infiltrating the reporter dye and enhancing the responsivity and sensitivity of the microsphere sensor due to an increase in surface area for attachment of the reporter dye, bioactive agents, etc.", "The bead sizes range from nanometers, i.e.", "100 nm, to millimeters, i.e.", "1 mm, with beads from about 0.2 micron to about 200 microns being preferred, and from about 0.5 to about 5 micron being particularly preferred, although in some embodiments smaller beads may be used.", "FIG.", "1 illustrates the construction of a typical bead or microsphere sensor 10 comprising a reporting dye 11 entrapped within bead pores 12.It should be noted that a key component of the invention is the use of a substrate/bead pairing that allows the association or attachment of the beads at discrete sites on the surface of the substrate, such that the beads do not move during the course of the assay.", "The present invention embodies a bead-based analytical chemistry system in which beads or microspheres are fabricated from various inorganic or organic materials wherein each material can be identified by a characteristic temporal optical response signature which enables verifying both the identity and location of a particular bead within a sensor array upon exposure to a reference analyte while illuminating with excitation light energy.", "The invention provides for utilization of any source of excitation light energy and is not limited to a specific wavelength.", "The principal requirement of the excitation light is that it produces emitted light of a characteristic wavelength upon illumination of a reporter dye associated with a given bead composition.", "In a preferred embodiment, the microspheres further comprise a bioactive agent.", "By “candidate bioactive agent” or “bioactive agent” or “chemical functionality” or “binding ligand” herein is meant as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc.", "which can be attached to the microspheres of the invention.", "It should be understood that the compositions of the invention have two primary uses.", "In a preferred embodiment, as is more fully outlined below, the compositions are used to detect the presence of a particular target analyte; for example, the presence or absence of a particular nucleotide sequence or a particular protein, such as an enzyme, an antibody or an antigen.", "In an alternate preferred embodiment, the compositions are used to screen bioactive agents, i.e.", "drug candidates, for binding to a particular target analyte.", "Bioactive agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.", "Bioactive agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.", "The bioactive agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.", "Bioactive agents are also found among biomolecules including peptides, nucleic acids, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.", "Particularly preferred are nucleic acids and proteins.", "Bioactive agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds.", "For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.", "Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.", "Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.", "Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification and/or amidification to produce structural analogs.", "In a preferred embodiment, the bioactive agents are proteins.", "By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.", "The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.", "Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids.", "For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention.", "The side chains may be in either the (R) or the (S) configuration.", "In the preferred embodiment, the amino acids are in the (S) or L-configuration.", "If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.", "In one preferred embodiment, the bioactive agents are naturally occurring proteins or fragments of naturally occuring proteins.", "Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used.", "In this way libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein.", "Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.", "In a preferred embodiment, the bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.", "The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides.", "By “randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively.", "Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.", "The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized bioactive proteinaceous agents.", "In a preferred embodiment, a library of bioactve agents are used.", "The library should provide a sufficiently structurally diverse population of bioactive agents to effect a probabilistically sufficient range of binding to target analytes.", "Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that gives it affinity for the target analyte.", "Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response: a diversity of 107-108 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism.", "Published in vitro selection techniques have also shown that a library size of 107 to 108 is sufficient to find structures with affinity for the target.", "Thus, in a preferred embodiment, at least 106, preferably at least 107, more preferably at least 108 and most preferably at least 109 different bioactive agents are simultaneously analyzed in the subject methods.", "Preferred methods maximize library size and diversity.", "In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position.", "In a preferred embodiment, the library is biased.", "That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.", "For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.", "In a preferred embodiment, the bioactive agents are nucleic acids (generally called “probe nucleic acids” or “candidate probes” herein).", "By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.", "A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) and references therein; Letsinger, J. Org.", "Chem., 35:3800 (1970); Sprinzl, et al., Eur.", "J.", "Biochem., 81:579 (1977); Letsinger, et al., Nucl.", "Acids Res., 14:3487 (1986); Sawai, et al., Chem.", "Lett., 805 (1984), Letsinger, et al., J.", "Am.", "Chem.", "Soc., 110:4470 (1988); and Pauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag, et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat.", "No.", "5,644,048), phosphorodithioate (Briu, et al., J.", "Am.", "Chem.", "Soc., 111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J.", "Am.", "Chem.", "Soc., 114:1895 (1992); Meier, et al., Chem.", "Int.", "Ed.", "Engl., 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207 (1996), all of which are incorporated by reference)).", "Other analog nucleic acids include those with positive backbones (Denpcy, et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 92:6097 (1995)); non-ionic backbones (U.S. Pat.", "Nos.", "5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi, et al., Angew.", "Chem.", "Intl.", "Ed.", "English, 30:423 (1991); Letsinger, et al., J.", "Am.", "Chem.", "Soc.", "110:4470 (1988); Letsinger, et al., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed.", "Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al., Bioorganic & Medicinal Chem.", "Lett., 4:395 (1994); Jeffs, et al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat.", "Nos.", "5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed.", "Y. S. Sanghui and P. Dan Cook.", "Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, et al., Chem.", "Soc.", "Rev., (1995) pp.", "169-176).", "Several nucleic acid analogs are described in Rawls, C & E News, Jun.", "2, 1997, page 35.All of these references are hereby expressly incorporated by reference.", "These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.", "In addition, mixtures of naturally occurring nucleic acids and analogs can be made.", "Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.", "The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.", "The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, and basepair analogs such as nitropyrrole and nitroindole, etc.", "As described above generally for proteins, nucleic acid bioactive agents may be naturally occuring nucleic acids, random nucleic acids, or “biased” random nucleic acids.", "For example, digests of procaryotic or eukaryotic genomes may be used as is outlined above for proteins.", "In general, probes of the present invention are designed to be complementary to a target sequence (either the target analyte sequence of the sample or to other probe sequences, as is described herein), such that hybridization of the target and the probes of the present invention occurs.", "This complementarity need not be perfect; there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.", "However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.", "Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under the selected reaction conditions.", "High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed.", "Ausubel, et al., both of which are hereby incorporated by reference.", "Stringent conditions are sequence-dependent and will be different in different circumstances.", "Longer sequences hybridize specifically at higher temperatures.", "An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).", "Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.", "The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).", "Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g.", "10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.", "greater than 50 nucleotides).", "Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.", "In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.", "The term “target sequence” or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid.", "The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others.", "It may be any length, with the understanding that longer sequences are more specific.", "As will be appreciated by those in the art, the complementary target sequence may take many forms.", "For example, it may be contained within a larger nucleic acid sequence, i.e.", "all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others.", "As is outlined more fully below, probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample.", "Generally speaking, this term will be understood by those skilled in the art.", "In a preferred embodiment, the bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.", "In a preferred embodiment, each bead comprises a single type of bioactive agent, although a plurality of individual bioactive agents are preferably attached to each bead.", "Similarly, preferred embodiments utilize more than one microsphere containing a unique bioactive agent; that is, there is redundancy built into the system by the use of subpopulations of microspheres, each microsphere in the subpopulation containing the same bioactive agent.", "As will be appreciated by those in the art, the bioactive agents may either be synthesized directly on the beads, or they may be made and then attached after synthesis.", "In a preferred embodiment, linkers are used to attach the bioactive agents to the beads, to allow both good attachment, sufficient flexibility to allow good interaction with the target molecule, and to avoid undesirable binding reactions.", "In a preferred embodiment, the bioactive agents are synthesized directly on the beads.", "As is known in the art, many classes of chemical compounds are currently synthesized on solid supports, such as peptides, organic moieties, and nucleic acids.", "It is a relatively straightforward matter to adjust the current synthetic techniques to use beads.", "In a preferred embodiment, the bioactive agents are synthesized first, and then covalently attached to the beads.", "As will be appreciated by those in the art, this will be done depending on the composition of the bioactive agents and the beads.", "The functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc.", "is generally known in the art.", "Accordingly, “blank” microspheres may be used that have surface chemistries that facilitate the attachment of the desired functionality by the user.", "Some examples of these surface chemistries for blank microspheres are listed in Table I.", "TABLE I Surface chemistry Name: NH2 Amine COOH Carboxylic Acid CHO Aldehyde CH2—NH2 Aliphalic Amine CO NH2 Amide CH2—C1 Chloromethyl CONH—NH2 Hydrazide OH Hydroxyl SO4 Sulfate SO3 Sulfonate ArNH2 Aromatic Amine These functional groups can be used to add any number of different bioactive agents to the beads, generally using known chemistries.", "For example, bioactive agents containing carbohydrates may be attached to an amino-functionalized support; the aldehyde of the carbohydrate is made using standard techniques, and then the aldehyde is reacted with an amino group on the surface.", "In an alternative embodiment, a sulfhydryl linker may be used.", "There are a number of sulfhydryl reactive linkers known in the art such as SPDP, maleimides; α-haloacetyls, and pyridyl disulfides (see for example the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference) which can be used to attach cysteine containing proteinaceous agents to the support.", "Alternatively, an amino group on the bioactive agent may be used for attachment to an amino group on the surface.", "For example, a large number of stable bifunctional groups are well known in the art, including homobifunctional and heterobifunctional linkers (see Pierce Catalog and Handbook, pages 155-200).", "In an additional embodiment, carboxyl groups (either from the surface or from the bioactive agent) may be derivatized using well known linkers (see the Pierce catalog).", "For example, carbodiimides activate carboxyl groups for attack by good nucleophiles such as amines (see Torchilin et al., Critical Rev.", "Therapeutic Drug Carrier Systems, 7(4):275-308 (1991), expressly incorporated herein).", "Proteinaceous bioactive agents may also be attached using other techniques known in the art, for example for the attachment of antibodies to polymers; see Slinkin et al., Bioconj.", "Chem.", "2:342-348 (1991); Torchilin et al., supra; Trubetskoy et al., Bioconj.", "Chem.", "3:323-327 (1992); King et al., Cancer Res.", "54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem.", "5:220-235 (1994), all of which are hereby expressly incorporated by reference).", "It should be understood that the bioactive agents may be attached in a variety of ways, including those listed above.", "What is important is that manner of attachment does not significantly alter the functionality of the bioactive agent; that is, the bioactive agent should be attached in such a flexible manner as to allow its interaction with a target.", "Specific techniques for immobilizing enzymes on microspheres are known in the prior art.", "In one case, NH2 surface chemistry microspheres are used.", "Surface activation is achieved with a 2.5% glutaraldehyde in phosphate buffered saline (10 mM) providing a pH of 6.9.", "(138 mM NaCl, 2.7 mM, KCl).", "This is stirred on a stir bed for approximately 2 hours at room temperature.", "The microspheres are then rinsed with ultrapure water plus 0.01% tween 20 (surfactant) −0.02%, and rinsed again with a pH 7.7 PBS plus 0.01% tween 20.Finally, the enzyme is added to the solution, preferably after being prefiltered using a 0.45 μm amicon micropure filter.", "After the desired number of bead subpopulations are prepared by the method of steps 50 through 54, discussed above, the subpopulations are typically combined in step 55 to provide a random mixture of subpopulations for use as sensor array elements prior to dispersing the subpopulation mixture on the array substrate in step 56.In a preferred embodiment, FIG.", "3.shows a schematic process diagram which illustrates the preparation and placement of self-encoded sensor bead subpopulations in fiber optic bundle sensor array.", "In an alternative embodiment, step 55 may be omitted and each of the sensor bead subpopulations may be separately and sequentially positioned on the array substrate in predetermined locations.", "Thus, once the microspheres are made comprising at least one bioactive agent and a self-encoding reporter dye, the microspheres are added to discrete sites on the surface of the substrate.", "This can be done in a number of ways, but generally comprises adding the beads to the surface under conditions that will allow the association of the microspheres on or at the discrete sites.", "The association of the beads on the surface may comprise a covalent bonding of the bead to the surface, for example when chemical attachment sites are added to both the substrate and the bead; an electrostatic or hydroaffinity, when charge, hydrophobicity or hydrophilicity is used as the basis of the binding; a physical yet non-covalent attachment such as the use of an adhesive; or a spatial attachment, for example the localization of a bead within a well.", "In some embodiments it may be preferable to effect a more permanent attachment after the initial localization, for example through the use of cross-linking agents, a film or membrane over the array.", "The microsphere system may be attached to the distal end of the optical fiber bundle using a variety of compatible processes as outlined below.", "It is important that the microspheres are located close to the end of the bundle.", "This ensures that the light returning in each optical fiber predominantly comes from only a single microsphere.", "This feature is necessary to enable the interrogation of the optical response signature of individual microspheres to identify reactions involving the microsphere's functionality and also to decode the dye/bead sets contained in those microspheres.", "The adhesion or affixing technique, however, must not chemically insulate the microspheres from the analyte.", "FIG.", "7 is a schematic block diagram showing the inventive optical fiber sensor 200 and associated control system 210.The fiber optic sensor 200 comprises a fiber optic bundle 202 (Galileo Electro-Optics, Sturbridge, Mass.", "), that is typically constructed from many thousands of separately clad discrete fibers, each only a few microns in diameter, so that light does not mix between the individual fibers.", "Any suitable fiber optic bundle 202 may be employed having a range in the number of individual fibers or a range of individual fiber diameters.", "The microsphere or bead sensor array 100 is attached to the bundle's distal end 212, with the proximal end 214 being received by a conventional z-translation microscope stage 216, for vertical positioning of the array 100 for focusing, and an x-y micropositioner 218 (Burleigh Instruments, Fishers, N.Y.), for horizontal alignment of the array 100 with the optical train.", "These two components act in concert to properly position the proximal end 214 of the bundle 202 for a microscope objective lens 220.Light collected by the objective lens 220 is passed to a reflected light fluorescence attachment with a four position dichromic cube wheel 222.The attachment 222 allows insertion of light from a 75 Watt Xenon arc lamp 224 through the objective lens 220 to be coupled into the fiber bundle 202.The light from the source 224 is condensed by condensing lens 226, then filtered and/or shuttered by filter and shutter wheel 228.Light returning from the distal end 212 of the bundle 202 is passed by the attachment 222 and is then shuttered and filtered by a second wheel 234.The light is then imaged on a charge coupled device (CCD) camera 236.A conventional computer 238 executes imaging processing software to process the information from the CCD camera 236 and also possibly control the first and second shutter and filter wheels 228, 234.Either a Macintosh or, alternatively, an IBM-compatible personal computer may be utilized for controlling the instrumentation and data acquisition.", "The instrumentation and optical apparatus deployed at the proximal end 214 of the fiber optic sensor 200, exclusive of the fiber optic sensor 200, are discussed more completely by Bronk, et al., Anal.", "Chem.", "67(17):2750-2752(1995) and Bronk, et al., Anal.", "Chem.", "66:3519 (1994).", "The bead sensor array 100 may be attached to the distal end of the optical fiber bundle 202 using a variety of compatible processes.", "It is important that the microspheres 10 are located close to the end of the bundle.", "This ensures that the light returning in each discrete optical fiber predominantly comes from only a single microsphere.", "This feature is necessary to decode the self-encoded bead subpopulations for the purpose of identifying both bead type and location, to enable the interrogation of the optical signature of individual microspheres within a bead subpopulation, and to provide for the summing of individual bead responses within each subpopulation for reducing signal to noise and improving signal enhancement.", "The bead adhesion or affixing technique, however, must not chemically insulate the microspheres from the analyte or interfere with the optical measurement.", "FIGS.", "5A and 5B are micrographs illustrating the preferred method for attaching beads to a sensor array substrate.", "Microwells 250 are formed on the distal end 212 of a fiber optic bundle 202 and microspheres 10 are inserted in the microwell cavities 250.The microwells 250 are formed at the center of each optical fiber 252 of the fiber optic bundle 202.As shown in FIG.", "5B, the size of the microwells 250 are coordinated with the size of the microspheres 10 so that the microspheres 10 can be placed within the microwells 250.Thus, each optical fiber 252 of the bundle 202 conveys light from the single microsphere 10 contained in its well.", "Consequently, by imaging the end of the bundle 202 onto the CCD array 236, the optical signatures of the microspheres 10 are individually interrogatable.", "FIG.", "4 illustrates how the microwells 250 are formed and microspheres 10 placed in the microwells.", "In one embodiment, a 1 mm hexagonally-packed imaging fiber bundle 202 was employed comprising approximately 20,600 individual optical fibers having cores approximately 3.7 μm across (Part No.", "ET26 from Galileo Fibers, Sturbridge, Mass.).", "Typically, the cores of each fiber are hexagonally shaped as a result of glass hardness and drawing during fiber fabrication.", "In some cases, the shape can be circular, however.", "In step 270, both the proximal 214 and distal 212 ends of the fiber bundle 202 are successively polished on 12 μm, 9 μm, 3 μm, 1 μm, and 0.3 μm lapping films.", "Subsequently, the ends can be inspected for scratches on a conventional atomic force microscope.", "In step 272, etching is performed on the distal end 212 of the bundle 202.A solution of 0.2 grams NH4F (ammonium fluoride) with 600 μl dH2O and 100 μl of HF (hydrofluoric acid), 50% stock solution, may be used.", "The distal end 212 is etched in this solution for a specified time, preferably approximately 80 seconds.", "Upon removal from this solution, the bundle end is immediately placed in deionized water to stop any further etching in step 274.The fiber is then rinsed in running tap water.", "At this stage, sonication is preferably performed for several minutes to remove any salt products from the reaction.", "The fiber is then allowed to air dry.", "As illustrated in FIGS.", "5A and 5B, the foregoing procedure produces microwells by the anisotropic etching of the fiber cores 254 favorably with respect to the cladding 256 for each fiber of the bundle 202.The microwells have approximately the diameter of the cores 254, 3.7 μm.", "This diameter is selected to be slightly larger than the diameters of the microspheres used, 3.1 μm, in the example.", "The preferential etching occurs because the pure silica of the cores 254 etches faster in the presence of hydrofluoric acid than the germanium-doped silica claddings 256.The microspheres are then placed in the microwells 250 in step 276 according to a number of different techniques.", "The placement of the microspheres may be accomplished by dripping a solution containing the desired randomly mixed subpopulations of the microspheres over the distal end 212, sonicating the bundle to settle the microspheres in the microwells, and allowing the microsphere solvent to evaporate.", "Alternatively, the subpopulations could be added serially to the bundle end.", "Microspheres 10 may then be fixed into the microwells 250 by using a dilute solution of sulfonated Nafion that is dripped over the end.", "Upon solvent evaporation, a thin film of Nafion was formed over the microspheres which holds them in place.", "This approach is compatible for fixing microspheres for pH indication that carry FITC functionality.", "The resulting array of fixed microspheres retains its pH sensitivity due to the permeability of the sulfonated Nafion to hydrogen ions.", "This approach, however, can not be employed generically as Nafion is impermeable to most water soluble species.", "A similar approach can be employed with different polymers.", "For example, solutions of polyethylene glycol, polyacrylamide, or polyhydroxymethyl methacrylate (polyHEMA) can be used in place of Nafion, providing the requisite permeability to aqueous species.", "In an another embodiment, an alternative fixation approach employs microsphere swelling to entrap each microsphere 10 in its corresponding microwell 250.In this approach, the microspheres are first distributed into the microwells 250 by sonicating the microspheres suspended in a non-swelling solvent in the presence of the microwell array on the distal end 212.After placement into the microwells, the microspheres are subsequently exposed to an aqueous buffer in which they swell, thereby physically entrapping them in the microwells.", "By way of example of this particular embodiment, one commonly known microsphere material is tentagel, a styrene-polyethylene glycol copolymer.", "These microspheres can be unswollen in nonpolar solvents such as hexane and swell approximately 20-40% in volume upon exposure to a more polar or aqueous media.", "In certain embodiments, this fixation approach may be desirable since it does not significantly compromise the diffusional or permeability properties of the microspheres themselves.", "FIGS.", "6A and 6B show typical microspheres 10 in microwells 250 after their initial placement and then after tapping and exposure to air pulses.", "FIGS.", "7A and 7B illustrate that there is no appreciable loss of microspheres from the microwells due to mechanical agitation even without a specific fixing technique.", "This effect is probably due to electrostatic forces between the microspheres and the optical fibers.", "These forces tend to bind the microspheres within the microwells.", "Thus, in most environments, it may be unnecessary to use any chemical or mechanical fixation for the microspheres.", "It should be noted that not all sites of an array may comprise a bead; that is, there may be some sites on the substrate surface which are empty.", "In addition, there may be some sites that contain more than one bead, although this is not preferred.", "In some embodiments, for example when chemical attachment is done, it is possible to attach the beads in a non-random or ordered way.", "For example, using photoactivatible attachment linkers or photoactivatible adhesives or masks, selected sites on the array may be sequentially rendered suitable for attachment, such that defined populations of beads are laid down.", "In addition, since the size of the array will be set by the number of unique optical response signatures, it is possible to “reuse” a set of unique optical response signatures to allow for a greater number of test sites.", "This may be done in several ways; for example, by using a positional coding scheme within an array; different sub-bundles may reuse the set of optical response signatures.", "Similarly, one embodiment utilizes bead size as a coding modality, thus allowing the reuse of the set of unique optical response signatures for each bead size.", "Alternatively, sequential partial loading of arrays with beads can also allow the reuse of optical response signatures.", "In a preferred embodiment, a spatial or positional coding system is done.", "In this embodiment, there are sub-bundles or subarrays (i.e.", "portions of the total array) that are utilized.", "By analogy with the telephone system, each subarray is an “area code”, that can have the same tags (i.e.", "telephone numbers) of other subarrays, that are separated by virtue of the location of the subarray.", "Thus, for example, the same unique dye/bead combinations can be reused from bundle to bundle.", "Thus, the use of 50 unique tags in combination with 100 different subarrays can form an array of 5000 different bioactive agents.", "In this embodiment, it becomes important to be able to identify one bundle from another; in general, this is done either manually or through the use of marker beads, i.e.", "beads containing unique tags for each subarray.", "In alternative embodiments, additional encoding parameters can be added, such as microsphere size.", "For example, the use of different size beads may also allow the reuse of sets of optical response signatures; that is, it is possible to use microspheres of different sizes to expand the encoding dimensions of the microspheres.", "Optical fiber arrays can be fabricated containing pixels with different fiber diameters or cross-sections; alternatively, two or more fiber optic bundles, each with different cross-sections of the individual fibers, can be added together to form a larger bundle; or, fiber optic bundles with fiber of the same size cross-sections can be used, but just with different sized beads.", "With different diameters, the largest wells can be filled with the largest microspheres and then moving onto progressively smaller microspheres in the smaller wells until all size wells are then filled.", "In this manner, the same dye/bead combinations could be used to encode microspheres of different sizes thereby expanding the number of different oligonucleotide sequences or chemical functionalities present in the array.", "Although outlined for fiber optic substrates, this as well as the other methods outlined herein can be used with other substrates and with other attachment modalities as well.", "In a preferred embodiment, the coding and decoding is accomplished by sequential loading of the microspheres into the array.", "As outlined above for spatial coding, in this embodiment, the optical response signatures can be “reused”.", "In this embodiment, the library of microspheres each comprising a different bioactive agent (or the subpopulations each comprise a different bioactive agent), is divided into a plurality of sublibraries; for example, depending on the size of the desired array and the number of unique tags, 10 sublibraries each comprising roughly 10% of the total library may be made, with each sublibrary comprising roughly the same unique tags.", "Then, the first sublibrary is added to the fiber optic bundle comprising the wells, and the location of each bioactive agent is determined, using its optical response signature.", "The second sublibrary is then added, and the location of each optical response signature is again determined.", "The signal in this case will comprise the “first” optical response signature and the “second” optical response signature; by comparing the two matrices the location of each bead in each sublibrary can be determined.", "Similarly, adding the third, fourth, etc.", "sublibraries sequentially will allow the array to be filled.", "Thus, arrays are made of a large spectrum of chemical functionalities utilizing the compositions of invention comprising microspheres and substrates with discrete sites on a surface.", "Specifically, prior art sensors which can be adapted for use in the present invention include four broad classifications of microsphere sensors: 1) basic indicator chemistry sensors; 2) enzyme-based sensors; 3) immuno-based sensors (both of which are part of a broader general class of protein sensors); and 4) geno-sensors.", "In a preferred embodiment, the bioactive agents are used to detect chemical compounds.", "A large number of basic indicator sensors have been previously demonstrated.", "Examples include: TABLE III TARGET ANALYTE Bioactive agent NOTES (λAB/λEM) pH Sensors based on: seminaphthofluoresceins e.g., carboxyl-SNAFL seminaphthorhodafluors e.g., carboxyl-SNARF 8-hydroxypyrene-1,3,6- trisulfonic acid fluorescein CO2 Sensors based On: seminaphthofluoresceins e.g., carboxyl-SNAFL seminaphthorhodafluors e.g., carbody-SNARF 8-hydroxypyrene-1,3,6- trisulfonic acid Metal Ions Sensors based on: desferriozamine B e.g., Fe cyclen derivative e.g., Cu, Zn derivatized peptides e.g., FITC-Gly-Gly-His, and FITC-Gly His, Cu, Zn fluorexon (calcine) e.g., Ca, Mg, Cu, Pb, Ba calcine blue e.g., Ca, Mg, Cu methyl calcine blue e.g., Ca, Mg, Cu ortho-dianisidine tetracetic e.g., Zn acid (ODTA) bis-salicylidene e.g., Al ethylenediamine (SED) N-(6-methozy-8-quinolyl-p- e.g., Zn toluenesulfonamine (TSQ) Indo-1 e.g., Mn, Ni Fura-2 e.g., Mn, Ni Magesium Green e.g., Mg, Cd, Tb O2 Siphenylisobenzofuran 409/476 Methoxyvinyl pyrene 352/401 Nitrite diaminonaphthalene 340/377 NO luminol 355/411 dihydrohodamine 289/none Ca2+ Bis-fura 340/380 Calcium Green visible light/530 Fura-2 340/380 Indo-1 405/485 Fluo-3 visible light/525 Rhod-2 visible light/570 Mg2+ Mag-Fura-2 340/380 Mag-Fura-5 340/380 Mag-Indo-1 405/485 Magnesium Green 475/530 Magnesium Orange visible light/545 Zn2+ Newport Green 506/535 TSQ Methoxy-Quinobyl 334/385 Cu+ Phen Green 492/517 Na+ SBFI 339/565 SBFO 354/575 Sodium Green 506/535 K+ PBFI 336/557 Cl− SPQ 344/443 MQAE 350/460 Each of the chemicals listed in Table III directly produces an optically interrogatable signal or a change in the optical signature, as is more fully outlined below, in the presence of the targeted analyte.", "Enzyme-based microsphere sensors have also been demonstrated and could be manifest on microspheres.", "Examples include: TABLE IV SENSOR TARGET Bioactive agent Glucose Sensor glucose oxidase (enz.)", "+ O2-sensitive dye (see Table I) Penicillin Sensor penicillinase (enz.)", "+ pH-sensitive dye (see Table I) Urea Sensor urease (enz.)", "+ pH-sensitive dye (see Table I) Acetylcholine Sensor acetylcholinesterase (enz.)", "+ pH-sensitive dye (see Table I) Generally, as more fully outlined above, the induced change in the optical signal upon binding of the target analyte due to the presence of the enzyme-sensitive chemical analyte occurs indirectly in this class of chemical functionalities.", "The microsphere-bound enzyme, e.g., glucose oxidase, decomposes the target analyte, e.g., glucose, consume a co-substrate, e.g., oxygen, or produce some by-product, e.g., hydrogen peroxide.", "An oxygen sensitive dye is then used to trigger the signal change.", "Immuno-based microsphere sensors have been demonstrated for the detection for environmental pollutants such as pesticides, herbicides, PCB's and PAH's.", "Additionally, these sensors have also been used for diagnostics, such as bacterial (e.g., leprosy, cholera, lyme disease, and tuberculosis), viral (e.g., HIV, herpes simplex, cytomegalovirus), fungal (e.g., aspergillosis, candidiasis, cryptococcoses), Mycoplasmal (e.g., mycoplasmal pneumonia), Protozoal (e.g., amoebiasis, toxoplasmosis), Rickettsial (e.g., Rocky Mountain spotted fever), and pregnancy tests.", "Microsphere genosensors may also be made.", "These are typically constructed by attaching a probe sequence to the microsphere surface chemistry, typically via an NH2 group.", "A fluorescent dye molecule, e.g., fluorescein, is attached to the target sequence, which is in solution.", "The optically interrogatable signal change occurs with the binding of the target sequences to the microsphere.", "This produces a higher concentration of dye surrounding the microsphere than in the solution generally.", "A few demonstrated probe and target sequences, see Ferguson, J.", "A. et al.", "Nature Biotechnology, Vol.", "14, December 1996, are listed below in Table V. TABLE V PROBE SEQUENCES TARGET SEQUENCES B-glo(+) (segment of human B-globin)5′- B-glo(+)-CF NH2—(CH2)8—)TT TTT TTT TCA ACT TCA 5′-Fluorescein-TC AAC GTG GAT GAA GTT TCC ACG TTC ACC-3 C-3′ IFNG(interferon gamma 1)5′-NH2—(CH2)8-T12- IFNG-CF TGG CTT CTC TTG GCT GTT ACT-3′ 5′-Fluorescein-AG TAA CAG CCA AGA GAA CCC AAA-3′ IL2(interleukin-2)5′-NH2—(CH2)8-T12-TA ACC IL2-CF GAA TCC CAA ACT CAC CAG-3′ 5′-Fluorescein-CT GGT GAG TTT GGG ATT CTT GTA-3′ IL4(interleukin-4)5′NH2—(CH2)8-T12-CC AAC IL4-CF TGC TTC CCC CTC TGT-3′ 5′-Fluorescein-AC AGA GGG GGA AGC AGT TGG-3′ IL6(interleukin-6)5′NH2—(CH2)8-T12-GT TGG IL6-CF GTC AGG GGT GGT TAT T-3′ 5′-Fluorescein-AA TAA CCA CCC CTG ACC CAA C-3′ It should be further noted that the genosensors can be based on the use of hybridization indicators as the labels.", "Hybridization indicators preferentially associate with double stranded nucleic acid, usually reversibly.", "Hybridization indicators include intercalators and minor and/or major groove binding moieties.", "In a preferred embodiment, intercalators may be used; since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of target hybridization will the label light up.", "The present invention may be used with any or all of these types of sensors.", "As will be appreciated by those in the art, the type and composition of the sensor will vary widely, depending on the composition of the target analyte.", "That is, sensors may be made to detect nucleic acids, proteins (including enzyme sensors and immunosensors), lipids, carbohydrates, etc; similarly, these sensors may include bioactive agents that are nucleic acids, proteins, lipids, carbohydrates, etc.", "In addition, a single array sensor may contain different binding ligands for multiple types of analytes; for example, an array sensor for HIV may contain multiple nucleic acid probes for direct detection of the viral genome, protein binding ligands for direct detection of the viral particle, immuno-components for the detection of anti-HIV antibodies, etc.", "In addition to the beads and the substrate, the compositions of the invention may include other components, such as light sources, optical components such as lenses and filters, detectors, computer components for data analysis, etc.", "The arrays of the present invention are constructed such that information about the identity of the bioactive agent is built into the array, such that the random deposition of the beads on the surface of the substrate can be “decoded” to allow identification of the bioactive agent at all positions.", "This may be done in a variety of ways.", "In a preferred embodiment, the beads are loaded onto the substrate and then the array is decoded, prior to running the assay.", "This is done by detecting the optical response signature associated with the bead at each site on the array upon exposure to a reference analyte.", "This may be done all at once, if unique optical signatures are used, or sequentially, as is generally outlined above for the “reuse” of sets of optical signatures.", "Alternatively, full or partial decoding may occur after the assay is run.", "Once made and decoded if necessary, the compositions find use in a number of applications.", "As a preliminary matter, the invention finds use in methods for reducing the signal-to-noise ratio in the characteristic optical response signature of a sensor array having a subpopulations of array elements.", "The methods comprise a) decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array; b) measuring the characteristic optical response signature of each sensor element in the array; c) adjusting the baseline of the optical response signature for each sensor element in the array; d) summing the baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations; and e) reporting the characteristic optical response signature of each sensor subpopulation as a summation of the baseline-adjusted characteristic optical response signatures of all sensor elements within each of the subpopulations.", "This can result in an increase in the signal-to-noise ratio by a factor of at least about ten, with at least about 100 being preferred.", "Similarly, the invention provides methods for amplifying the characteristic optical response signature of a sensor array having subpopulations of array elements, comprising: a) decoding the array so as to identify the location of each sensor element within each sensor subpopulation within the array; b) measuring a characteristic optical response signature of each sensor element in the array; c) optionally adjusting the baseline of the optical response signature for each sensor element in the array; d) summing the baseline-adjusted characteristic optical response signature of all sensor elements within each of the sensor subpopulations; and e) reporting the characteristic optical response signature of each sensor subpopulation as a summation of the baseline-adjusted characteristic optical response signatures of all sensor elements within each of the subpopulations.", "In a preferred embodiment, the signal is amplified by a factor of at least about fifty and an analyte detection limit is reduced by a factor of at least about 100.Generally, a sample containing a target analyte (whether for detection of the target analyte or screening for binding partners of the target analyte) is added to the array, under conditions suitable for binding of the target analyte to at least one of the bioactive agents, i.e.", "generally physiological conditions.", "The presence or absence of the-target analyte is then detected.", "As will be appreciated by those in the art, this may be done in a variety of ways, generally through the use of a change in an optical signal.", "This change can occur via many different mechanisms.", "A few examples include the binding of a dye-tagged analyte to the bead, the production of a dye species on or near the beads, the destruction of an existing dye species, a change in the optical signature upon analyte interaction with dye on bead, or any other optical interrogatable event.", "In a preferred embodiment, the change in optical signal occurs as a result of the binding of a target analyte that is labeled, either directly or indirectly, with a detectable label, preferably an optical label such as a fluorochrome.", "Thus, for example, when a proteinaceous target analyte is used, it may be either directly labeled with a fluor, or indirectly, for example through the use of a labeled antibody.", "Similarly, nucleic acids are easily labeled with fluorochromes, for example during PCR amplification as is known in the art.", "Alternatively, upon binding of the target sequences, an intercalating dye (e.g., ethidium bromide) can be added subsequently to signal the presence of the bound target to the probe sequence.", "Upon binding of the target analyte to a bioactive agent, there is a new optical signal generated at that site, which then may be detected.", "Alternatively, in some cases, as discussed above, the target analyte such as an enzyme generates a species (for example, a fluorescent product) that is either directly or indirectly detectable optically.", "Furthermore, in some embodiments, a change in the optical response signature may be the basis of the optical signal.", "For example, the interaction of some chemical target analytes with some fluorescent dyes on the beads may alter the optical response signature, thus generating a different optical signal.", "For example, fluorophore derivatized receptors may be used in which the binding of the ligand alters the signal.", "As will be appreciated by those in the art, in some embodiments, the presence or absence of the target analyte may be done using changes in other optical or non-optical signals, including, but not limited to, surface enhanced Raman spectroscopy, surface plasmon resonance, radioactivity, etc.", "The assays may be run under a variety of experimental conditions, as will be appreciated by those in the art.", "A variety of other reagents may be included in the screening assays.", "These include reagents like salts, neutral proteins, e.g.", "albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.", "Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.", "The mixture of components may be added in any order that provides for the requisite binding.", "Various blocking and washing steps may be utilized as is known in the art.", "In a preferred embodiment, the compositions are used to probe a sample solution for the presence or absence of a target analyte.", "By “target analyte” or “analyte” or grammatical equivalents herein is meant any atom, molecule, ion, molecular ion, compound or particle to be either detected or evaluated for binding partners.", "As will be appreciated by those in the art, a large number of analytes may be used in the present invention; basically, any target analyte can be used which binds a bioactive agent or for which a binding partner (i.e.", "drug candidate) is sought.", "Suitable analytes include organic and inorganic molecules, including biomolecules.", "When detection of a target analyte is done, suitable target analytes include, but are not limited to, an environmental pollutant (including pesticides, insecticides, toxins, etc.", "); a chemical (including solvents, polymers, organic materials, etc.", "); therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.", "); biomolecules (including hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc); whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.", "); and spores; etc.", "Particularly preferred analytes are nucleic acids and proteins.", "In a preferred embodiment, the target analyte is a protein.", "As will be appreciated by those in the art, there are a large number of possible proteinaceous target analytes that may be detected or evaluated for binding partners using the present invention.", "Suitable protein target analytes include, but are not limited to, (1) immunoglobulins; (2) enzymes (and other proteins); (3) hormones and cytokines (many of which serve as ligands for cellular receptors); and (4) other proteins.", "In a preferred embodiment, the target analyte is a nucleic acid.", "These assays find use in a wide variety of applications.", "In a preferred embodiment, the probes are used in genetic diagnosis.", "For example, probes can be made using the techniques disclosed herein to detect target sequences-such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art.", "In an additional embodiment, viral and bacterial detection is done using the complexes of the invention.", "In this embodiment, probes are designed to detect target sequences from a variety of bacteria and viruses.", "For example, current blood-screening techniques rely on the detection of anti-HIV antibodies.", "The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences.", "In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies.", "Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may be detected in this way.", "Bacterial infections such as tuberculosis, clymidia and other sexually transmitted diseases, may also be detected.", "In a preferred embodiment, the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples.", "For example, samples may be treated to lyse the bacteria to release its nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E. coli, and Legionnaire's disease bacteria.", "Similarly, bioremediation strategies may be evaluated using the compositions of the invention.", "In a further embodiment, the probes are used for forensic “DNA fingerprinting” to match crime-scene DNA against samples taken from victims and suspects.", "In an additional embodiment, the probes in an array are used for sequencing by hybridization.", "The present invention also finds use as a methodology for the detection of mutations or mismatches in target nucleic acid sequences.", "For example, recent focus has been on the analysis of the relationship between genetic variation and phenotype by making use of polymorphic DNA markers.", "Previous work utilized short tandem repeats (STRs) as polymorphic positional markers; however, recent focus is on the use of single nucleotide polymorphisms (SNPs), which occur at an average frequency of more than 1 per kilobase in human genomic DNA.", "Some SNPs, particularly those in and around coding sequences, are likely to be the direct cause of therapeutically relevant phenotypic variants.", "There are a number of well known polymorphisms that cause clinically important phenotypes; for example, the apoE2/3/4 variants are associated with different relative risk of Alzheimer's and other diseases (see Cordor et al., Science 261(1993).", "Multiplex PCR amplification of SNP loci with subsequent hybridization to oligonucleotide arrays has been shown to be an accurate and reliable method of simultaneously genotyping at least hundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see also Schafer et al., Nature Biotechnology 16:33-39 (1998).", "The compositions of the present invention may easily be substituted for the arrays of the prior art.", "In a preferred embodiment, the compositions of the invention are used to screen bioactive agents to find an agent that will bind, and preferably modify the function of, a target molecule.", "As above, a wide variety of different assay formats may be run, as will be appreciated by those in the art.", "Generally, the target analyte for which a binding partner is desired is labeled; binding of the target analyte by the bioactive agent results in the recruitment of the label to the bead, with subsequent detection.", "In a preferred embodiment, the binding of the bioactive agent and the target analyte is specific; that is, the bioactive agent specifically binds to the target analyte.", "By “specifically bind” herein is meant that the agent binds the analyte, with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample.", "However, as will be appreciated by those in the art, it will be possible to detect analytes using binding which is not highly specific; for example, the systems may use different binding ligands, for example an array of different ligands, and detection of any particular analyte is via its “signature” of binding to a panel of binding ligands, similar to the manner in which “electronic noses” work.", "This finds particular utility in the detection of chemical analytes.", "The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding, although in some embodiments, wash steps are not desired; i.e.", "for detecting low affinity binding partners.", "In some embodiments, for example in the detection of certain biomolecules, the dissociation constants of the analyte to the binding ligand will be less than about 10−4-10−6 M−1, with less than about 10−5 to 10−9 M−1 being preferred and less than about 10−7-10−9 M−1 being particularly preferred.", "In a preferred embodiment, the compositions of the invention find use in training sensor arrays.", "That is, the methods include determining the response to a target analyte of a population of a first pool of sensors and transferring the knowledge of this response to subsequent arrays containing equivalent sensors.", "In some embodiments the method includes distributing a first population of a first pool of sensors on an array and detecting the response to a target analyte of the first pool.", "The method further includes distributing a second population of the first pool on a second array and exposing the second array to a sample solution.", "A signal on the second array that is similar to the signal obtained from the first array in response to the target analyte is indicative of the presence of the target analyte in the sample solution.", "The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention.", "It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.", "All references cited herein are incorporated by reference in their entireity.", "EXAMPLES Characteristic temporal optical response data measurements of sensor bead and sensor array response to specific vapor analytes and excitation light energy were made according to the established method and instrumentation disclosed by White, et al., Anal.", "Chem.", "68:2191-2202(1996).", "In FIG.", "8, a schematic diagram illustrates the experimental apparatus and instrumentation used for the data measurements reported in Examples 7 through 17.In a typical measurement, the proximal end 214 of a fiber optic bundle 202 was placed into a fiber chuck 300 and secured for viewing with an Olympus microscope-based imaging system.", "In other embodiments, a conventional Olympus microscope slide platform and slide clamp was used for positioning alternative sensor array substrates, such as glass cover slips.", "An Olympus microscope 320 equipped with an epi-illuminator was utilized for optical measurements.", "The microscope 320 was equipped with Olympus 20× and 40× and Zeiss 100× objectives.", "An Omega 560 DCRP dichroic mirror 330 was used to direct filtered exitation light energy from a 75 W Xenon arc lamp 340 to the sensor array 100 and to permit the emitted light energy, due to the characteristic optical response signature originating from each of the sensor beads 10 in the sensor array 100, to be recorded by a CCD frame transfer camera 310.The excitation light energy emanating from the arc lamp 340 was filtered by an Omega 535 BP40 integrated excitation light filter/shutter 350.The emission light energy which emitted from the sensor beads 10 of the sensor array 100 was filtered with an Omega 640 BP20 integrated emitted light filter/shutter 360 prior to the CCD frame transfer camera 310.Experiments generally consist of collecting video camera frames of fluorescence response images of the characteristic optical response signatures of individual sensor beads in the sensor array 100 conveyed by the fiber optic bundle 202 to its proximal end 214.The bead and array images are recorded with a CCD frame transfer camera 310 (Model TE512EFT from Princeton Instruments, Trenton, N.J.).", "A preselected number of image frames are captured and sent to a computer system 400, comprising a Princeton Instruments NUBus camera interface card installed in a 8100AV Macintosh Power PC.", "Camera frame rates can be set at any desired value and typically range between 80 to 250 ms/frame.", "The following is a list of frame rates (time between data points) used in acquiring the data shown in for FIGS.", "9-16.The specified frame rate corresponds to a specific time interval between data points.", "Figure Rate (ms/frame) Total No.", "of Frames 135 30 183.3 60 103.3 60 190.6 60 133 60 155 60 155 60 124 60 A conventional air dilution olfactometer and vacuum-controlled vapor delivery system 500, as commonly known and used in olfactory research and described in Kauer, et al., J. Physiol.", "272:495-516 (1977), was used to apply controlled pulses of analyte vapor and air carrier gas to either a sensor bead substrate or the distal end 212 of a fiber optic sensor array 100 containing an array of sensor beads 10 immobilized in microwells 250.To produce a saturated vapor sample, generally, a stream of air carrier gas is passed through a 5 ml cartridge containing filter paper saturated with the analyte.", "Analyte dilutions are produced by adjusting the relative flow rates of saturated vapor and clean carrier gas streams.", "Typically, a flow rate of 100 ml/min is used for the combined gas flow to the sensor array.", "At this flow rate, a 2 second pulse would deliver approximately 3.3 ml of analyte vapor with carrier gas.", "In generally, depending on the analyte vapor pressure and dilution factor, vapor pulses contain between 10−7 to 10−5 mol of analyte.", "The vapor pulse was typically delivered during the 11th through 30th frame, commencing on the 11th frame.", "The duration of the vapor pulse varied with the specific frame rate utilized and typically ranged between 2 to 3 seconds.", "Baseline control measurements were performed with high purity, Ultra Zero grade air.", "The air pulse measurements were performed to account for any bead responses due to the vapor carrier gas.", "Data processing: Following the collection of a temporal series of sensor bead or sensor array images, segments are drawn, using IPLab image processing software (Signal Analytics, Vienna, Va.), over each pixel which corresponds to an individual fiber where the fiber is coupled to one sensor bead at its distal end.", "The mean fluorescence intensity was measured for each one of these segments in each frame in the sequence.", "This is done for both the vapor pulse responses and the baseline air pulse responses.", "Averages of multiple runs of each may be performed to improve data quality where needed.", "The air pulse data is then subtracted from the vapor pulse data to subtract the background due to air alone.", "The resulting data can be plotted to yield temporal intensity responses for all beads of interest.", "In a preferred embodiment, the sensor array data are used in a neural network analysis according to the method disclosed in White, et al, Anal.", "Chem.", "68:2193-2202 (1996).", "All data manipulation is performed within the IPLab program environment using simple operator scripts that call standardized image or data processing routines included with the software.", "These scripts and routines consist of a data collection portion and a data analysis portion.", "In the data collection portion, there are three segments or loops as follows: Loop 1.This establishes the baseline fluorescence of each sensor.", "This loop can be shortened or extended to adjust to slower or faster response times of specific sensor beads or sensor arrays to certain analytes.", "For Examples 7 through 17, this loop was set between 5 to 10 frames.", "Loop 2.This is the vapor exposure loop.", "A vapor pulse is applied just before this loop starts by way of a script command that sends a 5 volt pulse to an attached solenoid valve which switches a vacuum line off, thereby allowing a vapor sample to emit from the end of a nozzle.", "Typically, this loop is 20 frames in duration.", "In Example 7, a 10 frame duration was utilized.", "Loop 3.This is a sensor recovery loop.", "Another 5 volt trigger pulse is sent to a solenoid which switches back to its initial position, causing the vacuum system to resume collection of the solvent vapor and carry it off to waste.", "Typically, this loop is of 30 frames duration.", "In Example 7, a 15 frame duration was utilized.", "Data analysis: In the data analysis portion, pre-selected segments taken from a previously collected “focus” image are transferred to the sequence of images collected.", "These segments, drawn by the user, allow the mean pixel intensity to be measured in particular regions throughout the image field.", "Typically, they are drawn over individual pixels of a fiber optic sensor array, each of which contains a bead.", "The script then enters a loop that steps through each frame, measuring the mean pixel intensity within each segment, and placing the values in data columns.", "The resulting columns can then be plotted to yield the temporal response of each bead of interest.", "Before plotting, however, responses are “standardized” by dividing the data for each bead response by its first point.", "Thus, all responses can be normalized to start at a value of 1.0.Redundancy: As shown in the Examples, the present invention shows that building redundancy into an array gives several significant advantages, including the ability to make quantitative estimates of confidence about the data and significant increases in sensitivity.", "Thus, preferred embodiments utilize array redundancy.", "As will be appreciated by those in the art, there are at least two types of redundancy that can be built into an array: the use of multiple identical sensor elements (termed herein “sensor redundancy”), and the use of multiple sensor elements directed to the same target analyte, but comprising different chemical functionalities (termed herein “target redundancy”).", "For example, for the detection of nucleic acids, sensor redundancy utilizes of a plurality of sensor elements such as beads comprising identical binding ligands such as probes.", "Target redundancy utilizes sensor elements with different probes to the same target: one probe may span the first 25 bases of the target, a second probe may span the second 25 bases of the target, etc.", "By building in either or both of these types of redundancy into an array, significant benefits are obtained.", "For example, a variety of statistical mathematical analyses may be done.", "In addition, while this is generally described herein for bead arrays, as will be appreciated by those in the art, this techniques can be used for any type of arrays designed to detect target analytes.", "Furthermore, while these techniques are generally described for nucleic acid systems, these techniques are useful in the detection of other binding ligand/target analyte systems as well.", "Bead response summing: In a preferred embodiment, sensor redundancy is used.", "In this embodiment, a plurality of sensor elements, e.g.", "beads, comprising identical bioactive agents are used.", "That is, each subpopulation comprises a plurality of beads comprising identical bioactive agents (e.g.", "binding ligands).", "By using a number of identical sensor elements for a given array, the optical signal from each sensor element can be combined and any number of statistical analyses run, as outlined below.", "This can be done for a variety of reasons.", "For example, in time varying measurements, redundancy can significantly reduce the noise in the system.", "For non-time based measurements, redundancy can significantly increase the confidence of the data.", "In a preferred embodiment, a plurality of identical sensor elements are used.", "As will be appreciated by those in the art, the number of identical sensor elements will vary with the application and use of the sensor array.", "In general, anywhere from 2 to thousands may be used, with from 2 to 100 being preferred, 2 to 50 being particularly preferred and from 5 to 20 being especially preferred.", "In general, preliminary results indicate that roughly 10 beads gives a sufficient advantage, although for some applications, more identical sensor elements can be used.", "Once obtained, the optical response signals from a plurality of sensor beads within each bead subpopulation can be manipulated and analyzed in a wide variety of ways, including baseline adjustment, averaging, standard deviation analysis, distribution and cluster analysis, confidence interval analysis, mean testing, etc.", "In a preferred embodiment, the first manipulation of the optical response signals is an optional baseline adjustment.", "In a typical procedure, the standardized optical responses are adjusted to start at a value of 0.0 by subtracting the integer 1.0 from all data points.", "Doing this allows the baseline-loop data to remain at zero even when summed together and the random-response signal noise is canceled out.", "When the sample is a vapor, the vapor pulse-loop temporal region, however, frequently exhibits a characteristic change in response, either positive, negative or neutral, prior to the vapor pulse and often requires a baseline adjustment to overcome noise associated with drift in the first few data points due to charge buildup in the CCD camera.", "If no drift is present, typically the baseline from the first data point for each bead sensor is subtracted from all the response data for the same bead.", "If drift is observed, the average baseline from the first ten data points for each bead sensor is substracted from the all the response data for the same bead.", "By applying this baseline adjustment, when multiple bead responses are added together they can be amplified while the baseline remains at zero.", "Since all beads respond at the same time to the sample (e.g.", "the vapor pulse), they all see the pulse at the exact same time and there is no registering or adjusting needed for overlaying their responses.", "In addition, other types of baseline adjustment may be done, depending on the requirements and output of the system used.", "Once the baseline has been adjusted, a number of possible statistical analyses may be run to generate known statistical parameters.", "Analyses based on redundancy are known and generally described in texts such as Freund and Walpole, Mathematical Statistics, Prentice Hall, Inc. New Jersey, 1980, hereby incorporated by reference in its entirety.", "In a preferred embodiment, signal summing is done by simply adding the intensity values of all responses at each time point, generating a new temporal response comprised of the sum of all bead responses.", "These values can be baseline-adjusted or raw.", "As for all the analyses described herein, signal summing can be performed in real time or during post-data acquisition data reduction and analysis.", "In one embodiment, signal summing is performed with a commercial spreadsheet program (Excel, Microsoft, Redmond, Wash.) after optical response data is collected.", "In a preferred embodiment, cummulative response data is generated by simply adding all data points in successive time intervals.", "This final column, comprised of the sum of all data points at a particular time interval, may then be compared or plotted with the individual bead responses to determine the extent of signal enhancement or improved signal-to-noise ratios as shown in FIGS.", "14 and 15.In a preferred embodiment, the mean of the subpopulation (i.e.", "the plurality of identical beads) is determined, using the well known Equation 1: μ = ∑ x i n Equation ⁢ ⁢ 1 In some embodiments, the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below).", "In a preferred embodiment, the standard deviation of the subpopulation can be determined, generally using Equation 2 (for the entire subpopulation) and Equation 3 (for less than the entire subpopulation): σ = ∑ ( x i - μ ) 2 n Equation ⁢ ⁢ 2 s = ∑ ( x i - x _ ) 2 n - 1 Equation ⁢ ⁢ 3 As for the mean, the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below).", "In a preferred embodiment, statistical analyses are done to evaluate whether a particular data point has statistical validity within a subpopulation by using techniques including, but not limited to, t distribution and cluster analysis.", "This may be done to statistically discard outliers that may otherwise skew the result and increase the signal-to-noise ratio of any particular experiment.", "This may be done using Equation 4: t = x _ - μ s / u Equation ⁢ ⁢ 4 In a preferred embodiment, the quality of the data is evaluated using confidence intervals, as is known in the art.", "Confidence intervals can be used to facilitate more comprehensive data processing to measure the statistical validity of a result.", "In a preferred embodiment, statistical parameters of a subpopulation of beads are used to do hypothesis testing.", "One application is tests concerning means, also called mean testing.", "In this application, statistical evaluation is done to determine whether two subpopulations are different.", "For example, one sample could be compared with another sample for each subpopulation within an array to determine if the variation is statistically significant.", "In addition, mean testing can also be used to differentiate two different assays that share the same code.", "If the two assays give results that are statistically distinct from each other, then the subpopulations that share a common code can be distinguished from each other on the basis of the assay and the mean test, shown below in Equation 5: z = x _ 1 - x _ 2 σ 1 2 n 1 + σ 2 2 n 2 Equation ⁢ ⁢ 5 Furthermore, analyzing the distribution of individual members of a subpopulation of sensor elements may be done.", "For example, a subpopulation distribution can be evaluated to determine whether the distribution is binomial, Poisson, hypergeometric, etc.", "Target redundancy: In addition to the sensor redundancy, a preferred embodiment utilizes a plurality of sensor elements that are directed to a single target analyte but yet are not identical.", "For example, a single target nucleic acid analyte may have two or more sensor elements each comprising a different probe.", "This adds a level of confidence as non-specific binding interactions can be statistically minimized.", "When nucleic acid target analytes are to be evaluated, the redundant nucleic acid probes may be overlapping, adjacent, or spatially separated.", "However, it is preferred that two probes do not compete for a single binding site, so adjacent or separated probes are preferred.", "Similarly, when proteinaceous target analytes are to be evaluated, preferred embodiments utilize bioactive agent binding agents that bind to different parts of the target.", "For example, when antibodies (or antibody fragments) are used as bioactive agents for the binding of target proteins, preferred embodiments utilize antibodies to different epitopes.", "In this embodiment, a plurality of different sensor elements may be used, with from about 2 to about 20 being preferred, and from about 2 to about 10 being especially preferred, and from 2 to about 5 being particularly preferred, including 2, 3, 4 or 5.However, as above, more may also be used, depending on the application.", "As above, any number of statistical analyses may be run on the data from target redundant sensors.", "One benefit of the sensor element summing (referred to herein as “bead summing” when beads are used), is the increase in sensitivity that can occur.", "As shown in Example 19, detection limits in the zeptomole range can be observed.", "EXAMPLES Example 1 Preparation of porous silica/Nile Red beads: Approximately 0.5 cm3 of nominally 3.2 micron diameter commercial porous silica beads were removed from a LUNA column (Phenomenex, Torrance, Calif.).", "Sample of beads were placed onto a filter paper and, using vacuum filtration, 0.5 mL of Nile Red (Eastman Kodak, Rochester, N.Y.) solution (1 mg/mL in toluene) was poured over beads.", "Nile Red was immediately taken up by silica beads, turning them a deep purple color.", "The beads were washed repeatedly with toluene to remove any excess, non-adsorbed Nile Red.", "The beads were dried on a watch glass overnight.", "Beads were then placed into microwells formed by etching a fiber optic bundle according to the method of the present invention.", "Example 2 Preparation of PDPO polymer coated porous silica beads: A silanizing solution was prepared from 20 uL N-octadecyl-triethyoxysilane in 980 uL of ethanol/water (95% ethanol, 5% ultrapure water with pH adjusted to 4.9 with acetic acid).", "The LUNA porous silica beads of Example 1 were dispersed in an excess of silanizing solution for approximately 10 minutes, vortexing continuously.", "The particles were rinsed three times with ethanol and dried in a 120° C. oven, overnight for approximately 12 hours.", "Stock solution of PDPO, poly(2,6-dimethyl-1,4-phenylene oxide),(Aldrich, Milwaukee, Wis.) and Nile Red was prepared from 0.09 g PDPO and 1.0 mL chloroform.", "After complete dissolution of the polymer, a 100 uL aliquot of 1 mg/mL nile red in chloroform was added.", "The resultant solution was vortexed continuously for uniform dispersion.", "Excess PDPO/Nile Red was added to a small fraction of the silanized porous beads, approximately 100 uL polymer/dye solution to approximately 1 mg of beads.", "The sample was vortexed for approximately 3 hours then washed.", "Excess polymer dye was removed and the beads were then washed repeatedly with methylene chloride, two to three times, followed by a washing with 0.01% polyoxyethylene-sorbitan monolaurate, Tween 20 (J.T.", "Baker, Cleveland, Ohio), in water.", "The washed beads were collected in a solution of 0.01% Tween 20/ultrapure water.", "A single, small drop was placed on a microscope coverslip and allowed to dry protected from light.", "Example 3 Preparation of non-porous silica/Nile Red beads coated with polysiloxane polymer: Commercially available non-porous 3.1 um silica beads (Bangs Laboratory, Fishers, Ind.)", "were first silanized in excess silanizing solution, a 10% solution by volume-of 3-(trimethoxysilyl)propyl methacrylate (Aldrich, Milwaukee, Wis.) in acetone, overnight.", "Excess silanizing solution was decanted and the beads were rinsed repeatedly, two to three times, with ultrapure acetone, vortexing and centrifuging between washes.", "The beads were soaked in excess Nile Red solution (1 mg/ml in toluene) for approximately 3 hours while vortexing so as to fully saturate the surface.", "The bead solution was centrifuged and excess dye solution was decanted.", "A mixture of 7.9 mg benzoin ethyl ether (Polysciences Inc., Warrington, Pa.), 250 microliters stock Nile Red in toluene and 250 microliters (15-20% acryloxypropyl-methylsiloxane) 80-85% dimethylsiloxane copolymer (Gelest Inc., Tullytown Pa.) were then added to the beads.", "The bead suspension was vortexed to uniformly coat the particles.", "The resultant suspension mixture was added dropwise to approximately 100 mL 0.1% Tween 20 in ultrapure water stirring at approximately 350 rpm.", "Polymerization was accomplished by ultraviolet excitation for 10 second durations for a total exposure of 30 seconds.", "The sample solution was stirred over night.", "The suspension was passed through a 230 micron sieve, followed by a 5 um sieve.", "The filtrate was centrifuged at 3000 rpm for approximately 5 minutes and the beads were collected into centrifuge tubes and washed with 0.01% Tween 20 in ultrapure water.", "A single small drop was placed on a microscope coverslip and allowed to dry protected from light.", "Example 4 Preparation of (15-20% acryloxypropylmethylsiloxane) 80-85% dimethylsiloxane copolymer beads with nile red: Approximately 25 mL of ultrapure water plus 25 mL ethanol were placed in a 100 mL round bottom flask and stirred with a stirbar at approximately 350 rpm.", "A mixture of 500 uL (15-20% acryloxypropylmethylsiloxane) 80-85% dimethylsiloxane copolymer, 200 uL Nile Red solution (1 mg/ml in chloroform) and 250 uL methylene chloride was made and added dropwise to the stirred water/ethanol solution.", "A solution of 5.5 mg AIBN, N,N′-azobis-isobutyl nitrile (2,2′-azobis-2-methylproprio-nitrile)(Phaltz & Bauer, Inc.), in methylene chloride was added to the stirring dispersion.", "The mixture was degassed with argon for approximately one hour and then heated to approximately 70 degrees celcius.", "After approximately three hours of heating, 20 mL of 0.01% Tween 20 in ultrapure water was added to the mixture.", "Heating and stirring was continued for approximately 12 hours.", "The mixture was passed through 230 micron sieve, then solids collected from centrifugation at up to 5000 rpm.", "The solids were washed twice with methanol and then washed with 0.01% Tween 20 in ultrapure water.", "The resultant beads were collected in a solution of 0.01% Tween 20 in ultrapure water.", "A single drop of the bead suspension was placed on a microscope coverslip and allowed to dry protected from light.", "Example 5 Nile red dyed poly(methylstyrene/divinyl benzene) beads: Approximately 1 mg of commercially available 3.15 um polymer beads, 87% methyl styrene, 13% divinyl benzene with amine functionalized surface (Bangs Laboratories, Fishers, Ind.", "), was washed in 1 ml of methanol by vortexing, centrifuging at approximately 3000 rpm and decanting the solvent.", "The beads were transferred to brown vial and approximately 100 uL of Nile Red solution (1 mg/ml in toluene) was added.", "The sample was vortexed and placed on a wrist shaker to agitate overnight.", "The suspension was transferred to a microcentrifuge tube and washed with methanol until the decanted solvent was clear.", "The beads were collected in approximately 0.5 mL of a solution of 0.01% Tween 20 in ultrapure water.", "A single drop placed on a microscope coverslip and allowed to dry protected from light.", "Example 6 Plasticizer modified poly(methylstyrene/divinyl benzene) beads with nile red incorporated: Approximately 1 mg of commercially available 3.15 um polymer beads, 87% methyl styrene, 13% divinyl benzene with amine functionalized surface (Bangs Laboratories, Fishers, Ind.", "), were rinsed with methanol according to Example 5 and transferred to a brown vial.", "Approximately 2-40% by wt plasticizer to polymer solutions of plasticizers, tritolyl phosphate (TTP), triphenyl phosphate (TPP),and dibutyl phthalate (DBP) (Aldrich, Milwaukee, Wis.), with nile red solution (1 mg/mL in toluene) were added to samples of beads, covered, vortexed then shaken on wrist shaker for approximately 12 hours.", "The beads were transferred to microcentifuge tubes and washed with Nile Red in methanol, then repeatedly with methanol until the decanted solvent was clear.", "The beads were collected in a solution of 0.01% Tween 20 in ultrapure water.", "A single drop of the suspension was placed on a microscope coverslip and allowed to dry protected from light.", "Example 7 The porous silica beads prepared by the method of Example 1 were evaluated to determine their characteristic optical response signature to toluene vapor following the experimental method described above.", "The results are presented in FIG.", "9 where the temporal optical response of 62 individual bead sensors to a pulse of toluene vapor is shown.", "Example 8 The poly(methylstyrene/divinyl benzene) beads prepared by the method of Example 5 were evaluated to determine their characteristic optical response signature to methanol vapor.", "The results are presented in FIG.", "10 where the temporal optical response of 39 individual bead sensors to a pulse of methanol vapor is shown.", "Example 9 The (15-20% acryloxypropylmethylsiloxane) 80-85% dimethylsiloxane copolymer beads prepared by the method of Example 4 were evaluated to determine their characteristic optical response signature to both toluene and methanol vapor.", "The results are presented in FIG.", "11 where the temporal optical responses of an individual bead sensor to a pulse of toluene and a pulse of methanol vapor is shown.", "Example 10 The PDPO polymer coated porous silica beads prepared by the method of Example 2 were evaluated to determine their characteristic optical response signature to both toluene and methanol vapor.", "The results are presented in FIG.", "12 where the temporal optical responses of an individual bead sensor to a pulse of toluene and a pulse of methanol vapor is shown.", "Example 11 Porous silica beads prepared by the method of Example 1 were incorporated into etched microwells on the distal end of a fiber optic bundle according to the method described above.", "The resultant sensor array was evaluated to determine the characteristic optical response signature of the bead subpopulation to ethyl acetate vapor.", "The results are presented in FIG.", "13 where the temporal optical response of 218 individual bead sensors to a pulse of ethyl acetate vapor is shown.", "Example 12 The signal summing method of the present invention was evaluated in analyzing the experimental measurements made on poly(methylstyrene /divinyl benzene) beads prepared by the method of Example 5 and tested by the method of Example 8.The results are shown in FIG.", "14 where the normalized temporal optical response for a single sensor bead, Bead #1, is compared with the summed responses of all 39 beads tested.", "As shown by FIG.", "14, the signal summing method of the present invention significantly reduces the experimental noise encountered in a single sensor bead measurement and provides a substantial improvement, ten-fold or greater, in the signal-to-noise ratio of analytical measurements.", "Example 13 The signal summing method of the present invention was evaluated in analyzing the 4 experimental measurements made on poly(methylstyrene /divinyl benzene) beads prepared by the method of Example 5 and tested by the method of Example 8.The results are shown in FIG.", "15 where the actual relative intensities of the temporal optical response for each of the 39 sensor beads is compared to relative intensity of the temporal optical response obtained from signal summing.", "As shown by FIG.", "15, substantial signal enhancement is obtained by signal summing with a correspondingly significant improvement, up to a hundred fold, in the detection limit for target analytes.", "Example 14 The polysiloxane coated porous silica beads prepared by the method of Example 3 were evaluated to determine their characteristic optical response signature to both toluene and methanol vapor.", "The results are presented in FIG.", "16 where the temporal optical responses of two bead sensors to both toluene and methanol are shown.", "The results shown in FIG.", "16 demonstrates the capability of this subpopulation of bead sensors to distinguish between two analytes of interest by utilizing the characteristic optical response signatures of the bead sensors to specific analytes.", "Example 15 A 50/50 mixture of porous silica beads prepared by the method of Example 1 and poly(methylstyrene/divinyl benzene) beads prepared by the method of Example 5 were randomly dispersed and incorporated into etched microwells on the distal end of a fiber optic bundle according to the method of the present invention as described above.", "The resultant sensor array was evaluated to determine the characteristic optical response signature of the bead subpopulation to methanol vapor.", "An 535 nm excitation filter and 600 nm emission filter was used in this experiment.", "The results are presented in FIG.", "17 where the normalized temporal optical response of 3 porous silica bead sensors and 6 PMS bead sensors to a pulse of methanol vapor is shown.", "In this example, the characteristic emitted light peak shapes of the bead subpopulations provide a distinguishable characteristic response signature for each subpopulation.", "FIG.", "17 demonstrates the innovative self-encoding feature of the present invention where the identity and location of the beads is determined in a single measurement of a reference vapor analyte.", "Example 16 The self-encoded fiber optic sensor array produced by the method of Example 15 was evaluated by measuring the characteristic temporal optical response signature of the porous silica and PMS sensor bead subpopulations of the array in response to a pulse of n-propanol vapor.", "The results are presented in FIG.", "18 where the temporal optical response of 3 porous silica bead sensors and 6 PMS bead sensors to a pulse of n-propanol vapor is shown.", "In this example, the characteristic emitted light intensities of the bead subpopulations provide a distinguishable characteristic response signature for each subpopulation.", "FIG.", "18 demonstrates the advantages of using the distinct characteristic temporal optical response signature of different bead subpopulations to detect a specific analyte of interest.", "Note that the identity and location of the bead sensors in the sensor array was decoded by the method of Example 15.By the combination of self-encoding the sensor array by the method of Example 15 and the sensor array measurement made by the method of the current Example 16, the sensor array was trained to detect and distinguish methanol from n-propanol.", "Example 17 The self-encoded fiber optic sensor array produced by the method of Example 15 was evaluated by measuring the characteristic temporal optical response signature of the porous silica and PMS sensor bead subpopulations of the array in response to a pulse of toluene vapor.", "The results are presented in FIG.", "19 where the temporal optical response of 3 porous silica bead sensors and 6 PMS bead sensors to a pulse of toluene vapor is shown.", "FIG.", "19 demonstrates the advantages of using the characteristic temporal optical response signature of different bead subpopulations to detect a specific analyte of interest.", "Note that the identity and location of the bead sensors in the sensor array was decoded by the method of Example 15.By the combination of decoding the self-encoding the sensor array by the method of Example 15, the sensor array measurement made by the method of Example 16, and the sensor array measurement made by the method of the current Example 17, the sensor array was trained to detect and distinguish between the group of target analytes comprising methanol, n-propanol, and toluene.", "Example 18 Samples of PS802 bead sensors produced by the method of Example 4, Poly methyl styrene/2% divinyl benzene bead sensors produced by the method of Example 5, and commercially available poly methyl styrene beads (Bangs Laboratory, Fishers, Ind.)", "were dispersed on a microscope coverslip substrate.", "Following equilibration of each bead subpopulation in air, each subpopulation was exposed to a pulse of saturated toluene vapor while illuminating the beads with excitation light energy.", "The changes in bead dimension due to the swelling response of each polymer type to toluene vapor was monitored using the apparatus of FIG.", "7.The response of the bead was recorded by filming the time varying fluorescence image of the beads and capturing changes in bead image dimensions with a CCD camera.", "FIG.", "20 illustrates the differences in swelling response of the three bead subpopulations by comparing the initial fluorescence image of each bead type in air with subsequent image of each bead type following exposure to toluene vapor.", "Such measurements of the swelling response characteristics of various polymer candidate materials is useful in prescreening bead sensor materials for use as bead sensor elements in the self-encoded sensor array of the present invention.", "Example 19 The use of bead summing for sensitive detection.", "An array containing 25 oligonucleotide probes attached to encoded microspheres was made.", "Before attaching oligonucleotides to the microspheres, a family of dye-encoded microspheres was created.", "Fluorescent dyes were used to encode the microspheres.", "Europium(III)thenoyltrifluoro-acetonate-3H2O (λex/λem=365/615) (Eu-dye), Cy5 (λex/λem=620/700) and 5-(and 6)-carboxytetramethyl-rhodamine, succinimidyl ester (λex/λem=535/580) (TAMRA, SE) were chosen for this demonstration.", "The dyes were incorporated by exploiting the chemical properties of the amino-modified polystyrene microspheres as follows.", "200-μL-aliquots of stock (1 mL of stock beads contains 5.8×109 beads in 0.01% merthiolate in water) 3.1 μm-diameter amine-modified poly(methylstyrene)divinylbenzene microspheres (Bangs Laboratories, Inc. Carmel, Ind.)", "were filtered and washed with dry THF then placed in a microcentrifuge tube.", "200 μL of europium(III)thenoyltrifluoroacetonate-3H2O [Eu-dye (Acros)] dye in THF was added to the beads.", "Eu-dye concentrations of 0, 0.001, 0.01, 0.025, 0.05, 0.1, 0.5, and 1 M were used.", "The microsphere/dye suspension was shaken (VWR Vortex Genie II) for 2 h. The suspensions were filtered separately (Millipore Type HVLP) and washed thoroughly with MeOH.", "The beads were stored in 0.01% Tween (essential for preparation and storage to prevent the beads from clumping together) in ultrapure water until use.", "Alternatively, external encoding was done.", "Ten μL of stock beads were rinsed (all rinsing procedures entailed placing the centrifuge tube containing the beads and solution into a microcentrifuge at 8000 rpm for 3 min, and liquid over the beads was removed using a pipette) with BT buffer (0.1 M boric acid, 0.1 M NaOH, 0.13 M NaHCO3, 0.01% Tween, pH 9).", "The beads were suspended in 100 μL BT buffer then 5 μL of dye solution [Cy5 (Amersham) or TAMRA(Molecular Probes)] in DMF was added.", "Cy5 concentrations of 0, 0.01, 0.05, 0.1, 0.3 mM and TAMRA concentrations of 0, 0.1, 0.4, and 3 mM were used.", "The beads were shaken for 2 h then rinsed three times with BT buffer then three times with PBST buffer (0.01 M phosphate buffer saline, 0.01% Tween, pH 7.4).", "The polystyrene microspheres swell in tetrahydrofuran (THF) enabling a dye to penetrate the microsphere and become entrapped when the microsphere contracts.", "The absorption and emission spectra of the dyes are not compromised within the microsphere's environment and their concentration remains constant over time.", "Eight distinguishable microsphere families were prepared by entrapping varying Eu-dye concentrations inside the microspheres.", "In addition to internal entrapment, the microspheres' amine-modified surface permitted coupling to amine-reactive dyes.", "Different concentrations of Cy5 and TAMRA were then attached to the surface amine groups of the eight Eu-dye beads.", "A library of 100 spectroscopically-distinguishable microsphere types was prepared using various combinations of the three dyes.", "Microsphere encoding was carried out prior to oligonucleotide attachment because reaction with the amine reactive dyes after probe attachment affected the hybridization reaction.", "On the other hand, the oligonucleotide probes on the surface of the microspheres are not affected by subsequent internal encoding with Eu-dye.", "DNA attachment.", "After the encoded microsphere library was in hand, we functionalized each encoded microsphere with a different single stranded DNA probe.", "Sequences of each probe are shown in Table 1.A protocol used previously to create a single core fiber optic DNA array was modified to prepare the DNA-microsphere sensors.", "DNA probes were synthesized with a 5′-amino-C6 modifier (Glen Research) in the Tufts Physiology Department using an ABI synthesizer.", "20 nmol of the 5′-amino-terminal oligonucleotide probe were dissolved in 180 μL of 0.1 M sodium borate buffer (SBB pH 8.3).", "Oligonucleotide activation was initiated by adding 40 nmol of cyanuric chloride in 40 μL of acetonitrile.", "After 1 h, unreacted cyanuric chloride was removed by three cycles of centrifugal ultrafiltration (Microcon 3, Amicon) and recovered in 200 μL of 0.1 M SBB.", "DNA functionalization.", "Five μL of stock beads were rinsed with 0.02 M phosphate buffer (pH 7).", "150 μL of 5% glutaraldehyde in phosphate buffer was added to the beads.", "The beads were shaken for 1 h then rinsed three times with phosphate buffer.", "150 μL of 5% polyethyleneimine (PEI) was then added to the beads.", "The beads were shaken for 1 h then rinsed three times with phosphate buffer then three times with 0.1 M SBB (sodium borate buffer, pH 8.3).", "100 μL of 150 μM cyanuric chloride-activated oligonucleotide probe in SBB buffer was added to the beads and shaken overnight.", "The probe solution was removed and saved for reuse.", "The beads were then rinsed three times with SBB buffer.", "Remaining amine groups were capped with succinic anhydride to prevent non-specific binding.", "100 μL of 0.1 M succinic anhydride in 90% DMSO, 10% SBB was added to the beads.", "The beads were shaken for 1 h then rinsed three times with SBB buffer then three times with TE buffer (10 mM Tris-HCL, pH 8.3, 1 mM EDTA, 0.1 M NaCl, 0.1% SDS).", "When the cyanuric chloride-activated probes were attached directly to the amine-modified polystyrene microspheres detectable fluorescent signals were generated by hybridized labeled targets.", "However, by first modifying the microspheres with polyethyleneimine (PEI) before DNA functionalization the signal increased ten-fold because the number of attachment sites available was amplified (data not shown).", "Non-specific binding of the target to the amine-functionalized microsphere surface was prevented by capping unreacted amines with succinic anhydride.", "The resulting encoded probe-functionalized microspheres can be stored for months and mixed in any desired combination to create or alter the DNA sensor array.", "Microsphere-based fiber-optic sensors.", "Recently, we reported an array consisting of randomly distributed independently addressable micron-bead-sensors using an imaging-optical-fiber substrate.", "This system employed imaging fibers consisting of six thousand individually clad fibers that were melted and drawn together to form a coherent, 500-μm diameter bundle.", "The compositional difference between the core and cladding of each fiber enables the cores to be etched selectively providing for the simultaneous formation of six thousand 3.5 μm-diameter wells in the surface of the fiber tip within seconds.", "See Michael et al., Anal.", "Chem 70:1242 (1998); Bronk et al., Anal.", "Chem.", "67:2750 (1995) and Pantano et al., Chem.", "Materials 8:2832 (1996), all of which are incorporated by reference.", "Microwell formation.", "500 μm-diameter imaging fiber bundles containing 6×104 individual fibers were chemically etched according to a previously detailed procedure; see Pantano et al.", "Chem.", "Materials 8:2832 (1996).", "Array formation.", "Five μL of probe-functionalized beads were stored in 40 μL of TE buffer.", "After selecting the desired probe-functionalized microspheres, 1 μL of each bead solution was placed in a microcentrifuge tube and vortexed.", "0.05 μL of this mixture was placed onto the distal face of the imaging fiber containing the microwells.", "After evaporation of the solvent (approximately 3 min), the distal tip of the fiber is wiped with an anti-static swab to remove excess beads.", "When a new sensor is desired, sonicating the fiber tip for 3 min will regenerate the substrate.", "Individual beads settle spontaneously into the wells as the water droplet evaporates to produce a randomly-distributed array of thousands of microsphere sensors.", "Excess microspheres are removed from the fiber tip while electrostatic interaction between the beads and the wells holds each microsphere in place.", "Controlling array formation.", "One of the primary advantages of this system is the ability to alter the types of microspheres contained in an array.", "Each milliliter of stock solution contains approximately 6×109 microspheres enabling functionalization of billions of beads at once.", "Even after a 20× dilution, a 1 μL volume of microsphere solution contains enough beads to produce hundreds of different arrays.", "The density of microspheres in solution can control the number of occupied wells.", "With dilute solutions, empty wells remain after the initial array production.", "Additional microspheres bearing different probes can be added to the unoccupied sites or to the original solution at any time to create a more diverse array.", "If a different selection of beads is desired, sonicating the fiber tip removes all of the beads from the wells, enabling a new sensor array to be made in the same substrate.", "Optical imaging and analysis system.", "Coupling the imaging fiber bundle to a detection system with a CCD camera enables us to resolve each fiber independently, and hence the microsphere residing in the well at each fiber tip, while simultaneously viewing the entire array.", "Hybridization was visualized using fluorescent-labeled complementary targets.", "The microspheres bearing a fluorescent signal due to a hybridized target are selected and the identity of the probe on each bead is determined by the microspheres' spectroscopic signature.", "Analysis set-up and protocol.", "The imaging system, described previously, consists of a light source, inverted microscope, and a modified Olympus epifluorescence microscope/charge coupled device camera (Photometrics PXL).", "A fiber chuck held the imaging fiber in a fixed position while electronically controlled filter wheels switch between the analytical wavelength and the encoding wavelengths, enabling complete analysis and identification of the microspheres within minutes.", "Excitation light was sent into the proximal tip of the imaging fiber and emission from the fluorescing molecules is captured and directed onto the CCD camera detector.", "Fluorescence measurements were acquired and analyzed using commercially available IPLab software (Signal Analytics).", "The fiber was not removed from the imaging system during testing, rinsing, or regeneration steps.", "The proximal tip of the fiber was secured in the fiber chuck of the imaging system and all solutions were brought to the fiber's distal tip which housed the microbead sensors.", "Images acquired immediately prior to each test while the fiber tip was in buffer were subtracted from the response images.", "Background signals from empty wells were then subtracted from signals generated during each test.", "Hybridization in real time.", "Each microsphere's fixed position made possible a hybridization study in real time.", "A DNA array containing identical beads was placed on the imaging system.", "The distal tip of the fiber bearing the microsphere sensors was placed in a labeled-target solution.", "Emission from hybridizing labeled-target was captured every minute for several minutes.", "In the small region of the imaging fiber selected for this study, 70 microspheres held the probe complementary to the target in solution.", "Each microsphere was monitored independently and simultaneously.", "Signals from 40 beads were averaged to provide kinetic data.", "At relatively high concentrations of target, hybridization could be detected immediately, as seen by the steep slope of the data.", "While the sensor remained on the imaging system it was regenerated by dipping the fiber tip into a room-temperature formamide solution.", "The same microspheres were assayed several times by placing the regenerated fiber into the target solution and repeating the experiment.", "Consecutive studies show that the same sensor can be used for multiple tests.", "A background fluorescence image was acquired at wavelengths specific to fluorescein (excitation 490 nm emission 530 nm) with the fiber's distal tip in buffer.", "The fiber's distal tip was then placed in 4 μL of fluorescein-labeled target solution and one image was acquired every minute for 10 min.", "Subsequently, the fiber was dipped in 90% formamide in TE buffer at room temperature (rt) to regenerate the sensor and a background image was taken with the fiber in buffer.", "The fiber was again placed in the target solution where images were acquired for another 10 min interval.", "Reproducibility and regenerabiliiy.", "The signal from the microspheres returns to background and the sensor can be used for multiple analyses with comparable results.", "100 assays of the same DNA array sensor were performed over several days.", "The average of fluorescent signals obtained after hybridization with an array containing two bead types was done.", "The low standard deviation exemplifies the robust nature of the DNA microspheres.", "At periodic intervals during the 100-assay test, microspheres carrying the second probe in the same array were tested to see if regeneration affected their response.", "Both probe types showed no compromise in response during the tests.", "Each array can be used for multiple tests since it is regenerated quickly and easily.", "The ability to reuse a single array hundreds of times significantly increases throughput and decreases the cost of each array.", "The fiber's distal tip was placed in 4 μL of labeled-target solution for 5 min, rinsed with TE buffer, and a fluorescence image was acquired for 5 s. The fiber tip was then dipped in 90% formamide in TE (rt) to remove any hybridized target and regenerate the sensor.", "This procedure was repeated 100 times using the IL2 target and 5 times (intermittently during the IL2 tests) using the IL6 target.", "Kinetic Study.", "The fiber tip was placed in the target solution for a given time, rinsed with TE and a fluorescence image was acquired with the sensor in buffer.", "After data acquisition, the fiber was placed back in the target solution for a given time, rinsed and analyzed in buffer.", "The sensor was monitored at elapsed times of 10 s, 20 s, 30 s, 1 min, 2 min, 3 min, 4 min, 5 min and 10 min.", "After a plateau was reached, the sensor was regenerated by dipping in a 90% formamide solution in TE (rt) and the test was repeated using a different concentration of target solution.", "Microsphere sensitivity.", "The fiber's distal tip was placed in 4 μL of target solution until the hybridization signal to noise ratio was three.", "The signal was monitored after rinsing the fiber tip with TE buffer and acquiring a fluorescence image for 5 s while the fiber tip was in buffer.", "For the hour-long assays, a 0.6 mL centrifuge tube was filled and capped.", "A hole was drilled in the cap to enable the fiber tip to be placed in the target solution while preventing evaporation.", "Sensitivity with an intensified CCD camera.", "The 21-mer cystic fibrosis oligonucleotide probe and complement with F508C mutation (5′-TAT CAT CTG TGG TGT TTC CTA-3′) were used for this study.", "The 5′-amino-terminal oligonucleotide probe was activated with 100 times excess of cyanuric chloride.", "The microspheres were incubated with 400 μM cyanuric chloride-activated oligonucleotide.", "The fluorescein-labeled target was dissolved in 6× saline sodium phosphate EDTA buffer (SSPE) containing 0.1% SDS.", "The fiber's distal tip was placed in 10 μL of target solution during hybridization with occasional stirring.", "The distal tip was then washed with 6×SSPE and a fluorescence image was acquired with a Pentamax ICCD camera (Princeton Instruments) for 1 s while the fiber tip was in 120 μL of 6×SSPE.", "Preparation of 10 to 125 bp ssDNA.", "One hundred μg of sperm DNA (587 to 831 base pairs) was incubated with nuclease S1 [3.96 U/μL (Gibco-BRL)] at 37° C. for 1 h in 30 mM sodium acetate buffer (pH 4.6) with 30 mM sodium chloride and 1 mM zinc acetate.", "After the reaction, the enzyme was removed from the DNA preparation by extraction with phenol:chloroform:isoamyl alcohol (25:24:1, equilibrated to pH 8.0).", "DNA between 10 and 125 base pairs was recovered by ultrafiltration with Microcon 3 (10 bp cut-off) and Microcon 50 (125 bp cut-off).", "The DNA was quantified with OligoGreen single-stranded DNA quantitation reagent (Molecular Probes).", "Multiplex Analysis.", "Images were acquired for 1 s and 0.5 s at wavelengths specific to each encoding dye.", "A 365 nm excitation filter and a 600 nm long pass emission filter were used for the Eu-dye.", "A 620 nm excitation filter and a 670 nm emission filter were used for the Cy5 dye.", "A 530 nm excitation filter and a 580 nm emission filter were used for the TAMRA.", "The images acquired at the three wavelength pairs were used to positionally register each microsphere sensor.", "The fiber's distal tip was placed in a target solution for 5 min, rinsed with TE buffer, and fluorescence images were acquired for 5 s while the fiber was in buffer.", "Overlay segments were drawn to select the beads bearing a hybridization signal using IPLab software.", "These overlay segments were copied and pasted onto each of the encoding images and the selected beads' identity was determined.", "The sensor was regenerated as described above and this procedure was repeated for each of the target solutions.", "Hybridization specificity in a multiplex assay.", "To demonstrate this microsphere array system, we first selected seven probes used in previous work (sequences 1-7 of Table 1, see FIG.", "22).", "The DNA sequences chosen for the array were designed to be completely specific at room temperature.", "The signals at two of the three encoding wavelengths is used to positionally register the microspheres.", "After registration at the encoding wavelengths, the array is ready for use.", "The fiber tip is dipped into a fluorescent-labeled target solution.", "After a specified time, the fiber tip is removed from the target solution, rinsed with buffer, and placed in buffer solution.", "Microspheres bearing a complementary probe display a fluorescent signal due to the hybridized labeled target.", "Completely specific hybridizations for seven different targets in an array were observed.", "Replicates of each bead type located randomly within the array yield redundant information which contributes to the array's reliability.", "Table 2 (FIG.", "23) shows the accuracy of the system to correctly identify the target.", "We have also demonstrated single-base-pair mismatch differentiation by conducting the hybridization at 53° C. (data not shown).", "Similar signals are generated from the hybridized complementary fluorescent-labeled target at room temperature and at the elevated temperature.", "The hybridized single base mismatch fluorescent-labeled target produced 50% less signal than the complementary target at room temperature.", "At the elevated temperature, the signal from the hybridized single base mismatch target was at the background level.", "After the initial demonstration, we selected 25 sequences from disease related genes (oncogenes and cystic fibrosis) and disease states (lymphocyte and cytokine expression) which are completely specific at room temperature (Table 1).", "An array sensor was created with the 25 different probes each attached to a different encoded microsphere.", "After registration, the array was interrogated with each of the 25 target solutions as described above with sensor regeneration between each test.", "Approximately 20% (1295 out of 6000) of the wells were occupied with an average of 50 replicates of each bead type in the array.", "The resulting data (Table 3) enabled the correct identification of each target solution.", "Hybridization at elevated temperature.", "An array was created using a 450 mm long fiber.", "The proximal end of the fiber was connected to the imaging system and the distal end was held in a vertical micropositioner.", "The buffer and target temperatures were controlled by a water bath.", "Testing was performed as described above.", "Sensitivity of the microspheres.", "There are three aspects to sensitivity: sample volume, target concentration, and absolute number of target molecules.", "The smaller the volume required, the less a sample needs to be amplified for detection since the same number of absolute target molecules in a smaller volume generates a higher local concentration.", "Sample volumes as small as 4 μL are required with this system since only the tip of the 500 μm-diameter fiber is dipped into the solution.", "Typically, we use 10 μL volumes for easier handling and to avoid evaporation.", "In order to evaluate the concentration sensitivity of the array, an intensified CCD (ICCD) camera was used.", "The camera is fitted with a microchannel plate image intensifier that is optically coupled to a CCD array.", "By employing the ICCD camera, the time needed to analyze the lowest concentrations was significantly reduced relative to an unintensified camera.", "For comparison, at 100 fM, an unintensified camera took 4 h to detect a signal.", "To determine the sensitivity for a given target, an array was prepared consisting of 500 identical beads.", "We reasoned that the sensitivity obtained by observing multiple beads in the array would provide us with a signal to background advantage.", "To our satisfaction, this advantage was borne out.", "Sensitivity experiments were carried out as follows: the array was hybridized in 10 μL solutions containing progressively decreasing concentrations of labeled target.", "The lowest concentration evaluated was 1 fM.", "At various times, the array was taken out of the hybridization solution, rinsed, and a fluorescence image was collected.", "The array was then placed back into the hybridization buffer.", "After hybridization, the array was dehybridized with formamide and five background measurements were taken in 6×SSPE.", "ROI's from 10 or 100 beads in the five images were averaged to provide the mean background.", "The mean background values were subtracted from the fluorescence intensities of the various numbers of beads.", "Individual beads exhibited significant variablilty such that it was not possible to ascertain whether or not a signal was present.", "On the other hand, summing signals from multiple beads provided detectable signals.", "The average signal of ten beads gave a 7% CV while 100 beads provided more precise average values with 3% CV.", "Results from three representative sets of ten beads for the complementary target and two non-complementary targets are presented in Table 5.The hybridization time was determined when the signal was over three times the standard deviation of the background signals (>3 sd).", "Using this criterion, the microsphere-fiber-optic system is able to detect a 1 fM target solution using a 10 μL volume in 1 hour.", "Both 10 and 100 beads from a total of 500 beads in the array were selected and monitored.", "In a 1 fM target solution, 10 μL contains ca.", "6000 DNA molecules.", "With 500 identical beads in the array giving a signal, each bead would be expected to contain, on average, ca.", "12 labeled target molecules on its surface.", "To confidently attest to the generation of signal, the average signal of at least ten beads was needed.", "Therefore, this system can give sufficient signal with only 120 molecules.", "Table 4 shows the specificity of the F508C oligonucleotide array to 1 fM target concentrations.", "Each target was tested three times.", "The non-specific binding signal was always less than three times the sd of the background.", "Hybridization of 1 fM target solution was also monitored using microspheres made with 4 times diluted cyanuric chloride activated oligonucleotide.", "In this case, no signal was obtained after 1 h demonstrating that the amount of probe on the surface of the microspheres plays an important role in the sensitivity.", "Analyses were performed in the presence of single-stranded salmon sperm DNA [587 to 831 base pairs (Sigma)].", "With up to 10 ng of sperm DNA, there was no observable inhibition in target hybridization.", "The analyses were also done with 10 ng of shorter lengths of sperm DNA (10 to 125 base pairs).", "In this case, hybridization of 1 and 10 fM target solution was inhibited.", "However, the same signal could be observed (>3 sd) with an additional 30 minutes of incubation time more than the values given in Table 3.Since fluorescein was used to label the DNA targets, we selected encoding dyes with spectral properties that would not overlap with the fluorescein spectrum.", "Covalently binding these dyes to the surface of the amine-functionalized microspheres yielded stable and reproducible signals.", "Unfortunately, such surface encoding reduces the number of amines available for the cyanuric chloride-activated oligonucleotide probe.", "Therefore, the concentrations of the dyes were optimized to enable sufficient signals from both the encoding dyes and the hybridized target.", "The finite number of surface amine groups reduces the range and number of dye combinations that can be generated with an external-labeling scheme.", "To increase the number of encoded microspheres, dyes also can be entrapped inside the bead.", "Lanthanide dyes are suitable for such internal encoding.", "The dyes' spectra are not compromised and their intensity remains constant once inside the microsphere.", "The DNA sequences employed in this work play important roles in the immune system.", "Not only is detection of these sequences important, but quantification of their expression can provide relevant clues in expression pattern during different disease states.", "Competition between labeled and unlabeled targets and kinetic rates of hybridization are methods used to quantitate analytes of interest.", "Single base mismatches are also an important area in genomic research.", "The microsphere fiber optic array completely discriminates between single base pair mismatches at elevated temperatures.", "This microarray has the shortest total assay time relative to other high density DNA analysis systems and can monitor hybridization directly in the target solution.", "The high density of probes on each bead and the small bead size contribute to the short analysis time and sensitivity of the system (Table 1).", "DNA samples presently require PCR amplification for analysis.", "Standard PCR starts with 102 to 105 copies of template.", "The DNA microbead array is capable of detecting 6000 target molecules.", "This result shows that DNA detection can be done without PCR amplification.", "At first, this result seems to defy logic; a standard white light source, camera, and optics are all employed.", "This level of sensitivity generally requires lasers, confocal optics and avalanche photodiodes.", "If we consider, however, that 12 molecules confined to a well volume (bead and liquid) of approximately 30 fL provides a local concentration of 1 nM, it becomes easy to understand why we are able to detect such small numbers of molecules.", "Nanomolar concentrations of fluorescence can be detected readily by the optical system.", "Thus, confining a small number of molecules to a small volume reduces the uncertainty of finding such low absolute molecule numbers by providing a relatively high local concentration.", "Bead replicates improve the confidence level even further.", "Longer strands of DNA that are typically in template DNA did not affect the sensitivity of the system.", "It can be concluded that the presence of a target sequence can be determined in a genomic DNA solution without PCR amplification.", "The DNA microarray presented here has smaller feature sizes and higher packing densities compared to other DNA arrays.", "We have demonstrated the fiber optic microarray using a 500 μm-diameter imaging fiber with well diameters of 3.5 82 m. Fibers have also been tapered to produce nanometer scale wells serving as host to nanometer-diameter beads.", "Using longer fibers, the microarray sensor tip can be brought to the sample and used to sequentially test multiple solutions.", "Utilizing the imaging fiber's remote sensing capabilities, arrays with nanometer dimensions potentially can be used for direct intracellular analysis.", "The advantages of this high-density randomly-distributed micrometer-sized high-resolution microsphere-based DNA array include cost effective production of the microbead array in seconds, high throughput analysis, easy replacement or addition with other microspheres when different testing is desired and facile regeneration of the sensor and substrate.", "In addition, the array can be brought to the sample solution rather than the solution being brought to the array.", "We are presently working on improving the sensitivity of the system to further reduce amplification requirements.", "With appropriate modifications, this general approach can be applied to the fabrication of libraries containing combinatorial peptides, antibodies, and other molecules.", "Example 20 Training Sensor Arrays Microsensor Preparation.", "Microsensors were prepared by soaking silica or polymer beads in a fluorescent indicator, Nile Red.", "Each sensor type was dyed in a batch process that produced a stock of ˜1.0×108 microspheres per 1 mg of microsensors.", "Once dyed, the sensors were placed on a vacuum filtration system and rinsed with toluene or chloroform to remove excess dye.", "The sensors were then dried in a 100° C. oven for approximately one hour, and stored in the dark until use.", "Table VI shows the bead material and dye concentration for each sensor type.", "While four of the sensor types were made directly from commercially available beads, the Lun802/BMA beads were fabricated by silanizing 45 mg of 3 μm Luna (OH) microspheres with a 10% solution of 3-(trimethyoxysilyl)propyl methacrylate in acetone for two hours.", "The excess silane solution was removed via vacuum filtration, and the beads were allowed to cure overnight.", "In a colorless 4 mL dram, the silanized beads were combined with 60 mg of BEE and 1 mL of a toluene solution that was 0.50% by volume in both PS802 and BMA.", "The mixture was purged with N2 for 15 minutes and allowed to photopolymerize for 1.5 hours under UV light with constant stirring.", "Excess monomer solution was removed via vacuum filtration.", "These beads were then dyed as described above.", "Sensor Array Fabrication.", "All tests employed a randomly distributed sensor array containing the five sensor types in Table VI.", "Three randomized sensor arrays were fabricated months apart, but all from the same sensor stock to yield three arrays with hundreds of copies of each of the five sensor types.", "Albert, K. J., Walt, D. R. Anal.", "Chem.", "2000, 72, 1947-1955.The mixed sensor stock was made by combining equal portions of each of the five sensor types to create a dry slurry.", "A small portion of this slurry was then placed on a glass coverslip, and a second coverslip was placed over the first to create a sandwich.", "The coverslips were then rubbed together to smear the sensors over the glass surface, creating two randomized sensor arrays.", "This randomized dispersion of sensors mimics the way odor receptors cells are randomly distributed in regions of the olfactory epithelium (Malnic, B., Hirono, J., Sato, T., Buck, L. Cell 1999, 96, 713-723; Sullivan, S. L., Resler, K. J., Buck, L. B. Curr.", "Opin.", "Genet Dev.", "1995, 5, 516-523), and simplifies array fabrication by eliminating the difficulty of positioning each sensor on a defined point within the array.", "In addition, a homogeneous array was made for each of the five sensor types, using an individual sensor type instead of the mixed sensor stock.", "TABLE VI Microsphere sensor materials.", "Bead Dye Solution Sensor size (mg dye/ml Name (mm) Bead Material solvent) IbsilC1 3 IB-Sil (Methyl group) 0.5 mg/ml toluene PhenosOH 3 Phenosphere (Hydroxyl 0.5 mg/ml toluene group) Selectosil 5 Selectosil (Stong cation 0.5 mg/ml chloroform exchange) Lun802/BMA 3 Luna (Hydroxyl group), 1.0 mg/ml toluene PS802, BMA 55% DVB 3 Polystyrene, 1.0 mg/ml toluene Divinylbenzene Instrumentation.", "A custom-built fluorescence imaging system described previously (Albert, K. J., Walt, D. R. Anal.", "Chem.", "2000, 72, 1947-1955; White, J., Kauer, J. S., Dickinson, T. A., Walt, D. R. Anal.", "Chem.", "1996, 68, 2191-2202)with an inverted microscope attached to a 640×480 pixel SensiCam high performance charge-coupled device (CCD) camera (Cooke Corporation, Auburn Hills, Mich.) was used to detect sensor array responses.", "The imaging system was computer controlled through IP LAB imaging software (Scanalytics, Fairfax, Va.).", "Vapor samples were delivered to the array in a pulsatile fashion via a previously described vacuum-controlled sparging apparatus.", "White, J., Kauer, J. S., Dickinson, T. A., Walt, D. R. Anal.", "Chem.", "1996, 68, 2191-2202.Binary mixtures were produced in conjunction with a Tedlar bag gas dilution vapor delivery system.", "Albert, K. J., Walt, D. R., Anal.", "Chem.", "2000, 72, 1947-1955.Sample Preparation.", "Volatile organic compounds (VOCs) and solid nitroaromatic compound (NAC) samples were placed in sealed Erlenmeyer flasks and delivered to the array in pure form at 25% and 50% saturated headspace vapor respectively (Table VII).", "The analytes used were acetone, air carrier gas (control), benzene, chloroform, ethanol, ethyl acetate, heptane, methanol, toluene, and NACs including 1,3-dinitrobenzene (1,3-DNB) and 4-nitrotoluene (4-NT).", "Binary mixtures of the VOCs and NACs were created using 50% saturated Tedlar bag samples of heptane, benzene, ethyl acetate and methanol in combination with NAC flask samples.", "The relative concentration of the VOC ranged from 100 to 75,000 times higher than that of the NAC in a mixture (See Table VIII).", "Tedlar bag samples were prepared as previously described (Albert, K. J., Walt, D. R. Anal.", "Chem.", "2000, 72, 1947-1955) by combining 3 L of ultra zero grade air carrier gas with the following volumes of organic solvent a) benzene (756 μL); b) ethyl acetate (826 μL); c) heptane (598 μL); and d) methanol (458 μL).", "TABLE VII The concentration of the pure analytes ± 15%.", "The concentrations were calculated based on the literature values (Lide, D. R., Ed.", "Handbook of Chemistry and Physics; CRC Press: Boca Raton, 1997; Howard, P. H., Meglan, W. M. Handbook of Physical Properties of Organic Chemicals; Lewis Publishers: New York, 1997) for the analyte vapor pressures listed.", "Vapor Pressure Sample Analyte @25° C. (mmHg) Concentration (ppm) Acetone 2.31E+02 7.6E+04 Benzene 9.53E+01 3.1E+04 Chloroform 1.97E+02 6.5E+04 Ethanol 5.90E+01 1.9E+04 Ethyl Acetate 9.45E+01 3.1E+04 Heptane 4.57E+01 1.5E+04 Methanol 1.27E+02 4.2E+04 Toluene 2.84E+01 9.4E+03 1,3-Dinitrobenzene 9.00E−04 6.0E−01 4-Nitrotoluene 1.64E−01 1.1E+02 TABLE VIII The concentration of the binary mixtures ± 15%.", "Concentration Concentration Analyte 1 Analyte 2 analyte1 (ppm) analyte2 (ppm) Benzene Methanol 3.1E+04 4.2E+04 Benzene 4-Nitrotoluene 3.1E+04 5.5E+01 Benzene 4-Nitrotoluene 3.1E+04 1.1E+02 Ethyl Heptane 3.1E+04 1.5E+04 Acetate Ethyl 1,3-Dinitrotoluene 3.1E+04 3.0E−01 Acetate Ethyl 1,3-Dinitrotoluene 3.1E+04 6.0E−01 Acetate Heptane 1,3-Dinitrotoluene 1.5E+04 6.0E−01 Heptane 4-Nitrotoluene 1.5E+04 1.1E+02 Methanol 4-Nitrotoluene 4.2E+04 5.5E+01 Methanol 4-Nitrotoluene 4.2E+04 1.1E+02 Data Acquisition.", "Fluorescence responses for all sensor elements in an array were recorded with a CCD camera by acquiring images before, during, and after vapor pulse delivery.", "For each vapor observation, a total of sixty images were collected in 4.12 seconds.", "First, registration pulses of 50% saturated ethanol and acetone were collected for each of five homogenous arrays, containing one of the five sensor types, as well as the three randomized arrays.", "Then NACs and VOCs were presented in both pure form and as components of binary mixtures to the three randomized arrays.", "The training array and first testing array were each exposed to the ten pure vapors and ten binary mixtures listed in Tables VII and VIII.", "Each sample was tested five times, and twelve air responses were collected intermittently during the data set, giving a total of 112 vapor responses collected for each array.", "The sampling order for the training array was not randomized, but samples were partially randomized for the first testing array.", "These two sensor arrays were tested one month apart using fresh samples and in a laboratory where temperature and humidity levels were not specifically controlled.", "A third sensor array was tested six months after the training array, with collection of three replicates of the ten pure analytes (Table VII) and four replicates of the binary mixtures (Table VIII), except for the three mixtures containing methanol.", "Seven air samples were collected intermittently to give a total of 65 vapor responses that were collected in a partially randomized order.", "Approximately 400 microsphere sensors were monitored on each of the three arrays.", "Sensor Registration.", "The sensors in the three randomized arrays had to be positionally registered as one of the five microsphere types.", "This task was accomplished by comparing the randomized sensor responses to the known response profiles of the five individual sensor types.", "Known response profiles were generated from the homogenous array sensor responses to ethanol and acetone registration pulses, by averaging the responses of 50 sensors over the sixty time points.", "These profiles were normalized to have the same amplitude and baseline.", "The ethanol and acetone responses from the randomized arrays were also normalized, and each sensor from the training and testing arrays was assigned to its closest bead type by comparing its response to the ethanol and acetone profile signals derived from the five homogeneous arrays.", "In each array, the twelve closest sensors to the known response profiles were chosen for each sensor type, where the distance was measured with respect to the L1 norm taken over sixty discrete frame observations.", "The L1 norm is defined as ∥(y1, y2, .", ".", ".", ", yn)−(x1, x2, .", ".", ".", ", xn)∥=|y1−x1|+|y2−x2|+ .", ".", ".", "+|yn−xn|.", "Data Analysis.", "First, air samples were identified by evaluating the raw fluorescence intensity of each sensor response.", "The difference between every sensor's raw maximum and minimum fluorescence intensity was measured for each vapor observation, and this intensity value was averaged over the approximately 400 analyzed sensors in an array.", "If the average change in intensity was less than 40 counts, the observation was classified as air, since air had less intense response features than the other analytes.", "Observations with an average value of 40 or higher counts were passed on to a classifier comprised of the simplest case of the Generalized-Mann-Whitney-Wilcoxon classifier (with k1=k2=2, henceforth called GWMW (2,2)) (Priebe, C. E., Cowen, L. Commun.", "Stat.-Theor.", "M. 1999, 28, 2871-2878) combined with an ordinary Mann-Whitney-Wilcoxon classifier (equivalent to the Generalized-Mann-Whitney-Wilcoxon classifier with k1=k2=1), for the region where GWMW (2,2) had low confidence in its answer.", "Data from the training array was used to define the classifier threshold values necessary to determine the presence or absence of NACs.", "Data from the testing arrays collected one and six months later were then analyzed with the same classifier.", "Prior to analysis with the classifier, the raw data from the training and testing arrays were pre-processed as follows.", "All sensor responses from the 12 registered beads of each of the five sensor types were normalized to have the same amplitude and baseline.", "Then each group of 12 responses was averaged to give one response per sensor type with 60 time points.", "Signal averaging eliminates differences between individual sensor responses and enhances the signal-to-noise ratio for each sensor type in the array.", "Dickinson, T. D., Michael, K. L., Kauer, J. S., Walt, D. R. Anal.", "Chem.", "1999, 71, 2192-2198; Albert, K. J., Walt, D. R. Anal.", "Chem.", "2000, 72, 1947-1955.The five averaged responses from the sensor types were then concatenated to yield a 300-dimensional discrete vector for each vapor.", "Thus the coordinates of the 300-dimensional vector would be expected to correspond for each array, even though the original arrays had different random mapping of sensor types.", "For each 300-dimensional test vector, the absolute value of its distance, coordinate-wise, to a training observation was computed, and summed over all 300-dimensional training vectors.", "The GWMW (1,1) classifier is similarly defined, where S (2,2) is replaced with S (1,1), and S (1,1) is defined as the collection of all 2-elements subsets of the training data, comprised exactly of one observation of class 1 and one observation of class 0.The larger the assigned weight, the more confidence that the observation is of class 1 and the lower the weight, the more confidence the observation is of class 0.Xie, J., Priebe, C. E. A Weighted Generalization of the Mann-Whitney-Wilcoxon Statistic; 579; John Hopkins Univ.", ":Baltimore, Md., 1999.A two-class problem was chosen to test if a classifier was transferable between sensor arrays.", "The problem was to determine if explosives-like nitroaromatic compound (NAC) vapors were present or absent in a series of organic vapor samples.", "While this is a straightforward yes/no question, determining whether NACs were present or absent in each observation was challenging due to the relatively low NAC levels and variable VOC backgrounds (Table VIII).", "The ability to sense NACs in variable high backgrounds was investigated because of its importance in explosives detection, especially buried land mines.", "George, V., Jenkins, T. F., Leggett, D. C., Cragin, J. H., Phelan, J., Oxley, J., Pennington, J. Proc.", "SPIE-Int.", "Opt.", "Eng., Orlando, Fla. 1999, 3710, 258-269.The microsensors response features result from the interaction between the analyte of interest, the sensor substrate and the indicator Nile Red, a solvatochromic dye.", "Solvatochromic dyes are sensitive to changes in the polarity of their environment, which is reported as shifts in the dye's excitation and/ or emission spectra.", "Reichardt, C. Chem.", "Rev.", "1994, 94, 2319-2358.Each sensor's temporal fluorescence change at a specific wavelength therefore gives rise to different response patterns based on how the vapor polarity and sensor surface functionality influenced the dye's emission properties.", "As can be seen in FIG.", "26, the data produced by these bead sensors is highly complex in shape, with some sensor types having non-linear response features.", "In developing a classifier, we sought to avoid feature selection, and instead developed a classifier that could deal directly with the raw high-dimensional data.", "We chose the GWMW family of classifiers, a non-parametric statistic that has the advantage of being distribution-free, that is, one does not need to make any restrictive assumptions about the data produced.", "Non-parametric statistics often have simple, closed mathematical formulations, and can be less computationally expensive than neural network methods.", "Another major advantage of non-parametric statistics is that they produce a mathematically rigorous notion of “confidence” associated with the class label assigned to each observation.", "This confidence level allows one to automatically tune the classifier and adjust the rate of false positives versus false negatives according to acceptable levels of accuracy.", "Priebe suggested specifically that the GWMW family of classifiers might be well suited for high-dimensional classification problems in general, and artificial nose data analysis in particular.", "Priebe, C. E. Olfactory Classification; 585; Johns Hopkins Univ.", ":Baltimore, Md.", "1999.In addition, because the GWMW family of classifier takes into account the relative distance-of all training observations, it seemed particularly suited to the task at hand, since a sensor-specific artifact in data that was array specific would not overly bias the classifier when tested on a new array.", "This insensitivity to artifacts is demonstrated in FIG.", "26 where the fourth sensor response to ethanol varies from array-to-array.", "However, this sensor variation does not overly influence the global ethanol response over all five sensor types, so that ethanol was always correctly classified as not containing a NAC.", "Also, instead of just looking at the k-nearest neighbors, the GWMW classifier family looks at inversions, counting when positive observations are closer than negative observations in the entire training data.", "If there were some consistent drift in signal from one array to another that led the same set of training observations to be close to all the testing observations, this drift should not completely erode the discriminatory power of the GWMW classifiers since they incorporate the relative distance order of all the training observations.", "In order for the two GWMW classifiers discussed above to be combined as one vapor discrimination model, thresholds were defined based on the training data to interpret the weights of each observation as class assignments.", "For a randomly ordered sequence of 50 class 1 observations and 62 class 0 observations, a score of 1,158,237.5 would be expected.", "The lowest scoring NAC-present example in the training data, based on a leave-one-out cross validation, received a score of 855,114 (one instance of 1,3-DNB).", "Excluding air, the highest scoring NAC-absent observation was 1,435,625 (one observation of ethyl acetate at 25%).", "This result is almost exactly a 25% error bar both above and below the mean value of the statistic.", "The GWMW (2,2) classifier was set to make 3 types of decisions: if GWMW (2,2) scores were under 750,000, the observation was classed NAC-absent, if GWMW (2,2) weight values were over 1,500,000, the class was assigned NAC-present, and for scores between 750,000 and 1,500,000, the decision was passed to the GWMW (1,1) classifier.", "For the GWMW (1,1) scores, excluding air, the highest-scoring NAC-absent example in a leave-one out cross validation of the training data had a score of 1325.Therefore scores above 1325 were classified as containing NAC vapor, and those below 1325 were classified as not containing NAC vapor.", "The classifier threshold values were defined to optimize the number of correctly assigned observations with the training data set.", "Score levels were calibrated across training and testing arrays to a base response from an initial air observation from each array.", "Thresholds for both classifiers were determined based on the training data, and then the GWMW classifiers were used to analyze the observations from the two test data sets collected one and six months after the training data.", "The GWMW (2,2) classifier misidentified two observations on the first test data set containing 1,3-DNB as being NAC-absent.", "The GWMW(1,1) classifier made no additional errors.", "FIG.", "27 shows a plot of the GWMW (1,1) vs. GWMW (2,2) scores for all 112 observations in the first test data set.", "The two false negatives obtained with the test data were both binary mixtures of 1,3-DNB and ethyl acetate.", "These errors are not surprising given that the concentration of ethyl acetate in this mixture was approximately 75,000 times higher than that of 1,3-DNB.", "A 98.2% correct classification rate was achieved on this data set without any additional training.", "The second test data set had two false positives and two false negatives (FIG.", "28).", "All four errors were made in the second step of the classifier, with two chloroform responses being classed as containing a NAC, and two 1,3-DNB responses classed as not containing a NAC.", "Despite these errors, the second test data set achieved a 93.8% correct classification rate.", "This data set was collected six months after the training data, demonstrating the longevity of the classifier.", "While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims." ] ]

[ [ "Whole cell engineering by mutagenizing a substantial portion of a starting genome combining mutations and optionally repeating", "This invention relates to the field of cellular and whole organism engineering.", "Specifically, this invention relates to a cellular transformation, directed evolution, and screening method for creating novel transgenic organisms having desirable properties.", "Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are diffenentially activatable." ], [ "1.A method for identifying proteins by differential labeling of peptides, the method comprising the following steps: (a) providing a sample comprising a polypeptide; (b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.", "2.The method of claim 1, wherein the sample of step (a) comprises a cell or a cell extract.", "3.The method of claim 1, further comprising providing two or more samples comprising a polypeptide.", "4.The method of claim 3, wherein one sample is derived from a wild type cell and one sample is derived from an abnormal or a modified cell.", "5.The method of claim 4, wherein the abnormal cell is a cancer cell.", "6.The method of claim 1, further comprising purifying or fractionating the polypeptide before the fragmenting of step (c).", "7.The method of claim 1, further comprising purifying or fractionating the polypeptide before the labeling of step (d).", "8.The method of claim 1, further comprising purifying or fractionating the labeled peptide before the chromatography of step (e).", "9.The method of claim 6, claim 8 or claim 8, wherein the purifying or fractionating comprises a method selected from the group consisting of size exclusion chromatography, size exclusion chromatography, HPLC, reverse phase HPLC and affinity purification.", "10.The method of claim 1, further comprising contacting the polypeptide with a labeling reagent of step (b) before the fragmenting of step (c).", "11.The method of claim 1, wherein the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: i. ZAOH and ZBOH, to esterify peptide C-terminals and/or Glu and Asp side chains; ii.", "ZANH2 and ZBNH2, to form amide bond with peptide C-terminals and/or Glu and Asp side chains; and iii.", "ZACO2H and ZBCO2H.", "to form amide bond with peptide N-terminals and/or Lys and Arg side chains; wherein ZA and ZB independently of one another comprise the general formula R-Z1-A1-Z2-A2-Z3-A3-Z4-A4-, Z1, Z2, Z3, and Z4 independently of one another, are selected from the group consisting of nothing, 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1, OBRR1, and OB(OR)(OR1), and R and R1 is an alkyl group, A1, A2, A3, and A4 independently of one another, are selected from the group consisting of nothing or (CRR1)n, wherein R, R1, independently from other R and R1 in Z1 to Z4 and independently from other R and R1 in A1 to A4, are selected from the group consisting of a hydrogen atom, a halogen atom and an alkyl group; n in Z1to Z4, independent of n in A1 to A4, is an integer having a value selected from the group consisting of 0 to about 51; 0 to about 41; 0 to about 31; 0 to about 21, 0 to about 11 and 0 to about 6.12.The method of claim 11, wherein the alkyl group is selected from the group consisting of an alkenyl, an alkynyl and an aryl group.", "13.The method of claim 11, wherein one or more C—C bonds from (CRR1)n are replaced with a double or a triple bond, 14.The method of claim 13, wherein an R or an R1 group is deleted.", "15.The method of claim 13, wherein (CRR1)n is selected from the group consisting of an o-arylene, an m-arylene and a p-arylene, wherein each group has none or up to 6 substituents.", "16.The method of claim 13, wherein (CRR1)n is selected from the group consisting of a carbocyclic, a bicyclic and a tricyclic fragment, wherein the fragment has up to 8 atoms in the cycle with or without a heteroatom selected from the group consisting of an O atom, a N atom and an S atom.", "17.The method of claim 1, wherein two or more labeling reagents have the same structure but a different isotope composition.", "18.The method of claim 11, wherein ZA has the same structure as ZB, but ZA has a different isotope composition than ZB.", "19.The method of claim 17, wherein the isotope is boron-10 and boron-11.20.The method of claim 17, wherein the isotope is carbon-12 and carbon-13.21.The method of claim 17, wherein the isotope is nitrogen-14 and nitrogen-15.22.The method of claim 17, wherein the isotope is sulfur-32 and sulfur-34.23.The method of claim 17, wherein, where the isotope with the lower mass is x and the isotope with the higher mass is y, and x and y are integers, x is greater than y.", "24.The method of claim 17, wherein x and y are between 1 and about 11, between 1 and about 21, between 1 and about 31, between 1 and about 41, or between 1 and about 51.25.The method of claim 1, wherein the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: i. CD3(CD2)nOH/CH3(CH2)nOH, to esterify peptide C-terminals, where n=0, 1, 2 or y; ii.", "CD3(CD2)nNH2 CH3(CH2)nNH2, to form amide bond with peptide C-terminals, where n=0, 1, 2 or y; and iii.", "D(CD2)nCO2H/H(CH2)nCO2H, to form amide bond with peptide N-terminals, where n=0, 1, 2 or y; wherein D is a deuteron atom, and y is an integer selected from the group consisting of about 51; about 41; about 31; about 21, about 11; about 6 and between about 5 and 51.26.The method of claim 1, wherein the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: i. ZAOH and ZBOH to esterify peptide C-terminals; ii.", "ZANH2/ZBNH2 to form an amide bond with peptide C-terminals; and iii.", "ZACO2H/ZBCO2H to form an amide bond with peptide N-terminals; wherein ZA and ZB have the general formula R-Z1-A1-Z2-A2-Z3-A3-Z4-A4-Z1, Z2, Z3, and Z4, independently of one another, are selected from the group consisting of nothing, 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1, and OB(OR)(OR1); A1, A2, A3, and A4, independently of one another, are selected from the group consisting of nothing and the general formulae (CRR1)n, and, R and R1 is an alkyl group.", "27.The method of claim 26, wherein a single C—C bond in a (CRR1)n group is replaced with a double or a triple bond.", "28.The method of claim 27, wherein R and R1 are absent.", "29.The method of claim 27, wherein (CRR1.", ")n comprises a moiety selected from the group consisting of an o-arylene, an m-arylene and ap-arylene, wherein the group has none or up to 6 substituents.", "30.The method of claim 27, wherein the group comprises a carbocyclic, a bicyclic, or a tricyclic fragments with up to 8 atoms in the cycle, with or without a heteroatom selected from the group consisting of an O atom, an N atom and an S atom.", "31.The method of claim 26, wherein R, R1, independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4, are selected from the group consisting of a hydrogen atom, a halogen and an alkyl group.", "32.The method of claim 31, wherein the alkyl group is selected from the group consisting of an alkenyl, an alkynyl and an aryl group.", "33.The method of claim 26, wherein n in Z1-Z4 is independent of n in A1-A4 and is an integer selected from the group consisting of about 51; about 41; about 31; about 21, about 11 and about 6.34.The method of claim 26, wherein ZA has the same structure a ZB but ZA further comprises x number of —CH2— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "35.The method of claim 26, wherein ZA has the same structure a ZB but ZA further comprises x number of —CF2— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "36.The method of claim 26, wherein ZA comprises x number of protons and ZB comprises y number of halogens in the place of protons, wherein x and y are integers.", "37.The method of claim 26, wherein ZA contains x number of protons and ZB contains y number of halogens, and there are x−y number of protons remaining in one or more A1-A4 fragments, wherein x and y are integers 38.The method of claim 26, wherein ZA further comprises x number of-O— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "39.The method of claim 26, wherein ZA further comprises x number of —S— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "40.The method of claim 26, wherein ZA further comprises x number of —O— fragment(s) and ZB further comprises y number of —S— fragment(s) in the place of —O— fragment(s), wherein x and y are integers.", "41.The method of claim 26, wherein ZA further comprises x−y number of —O— fragment(s) in one or more A1-A4 fragments, wherein x and y are integers.", "42.The method of claim 37, claim 40 or claim 41, wherein x and y are integers selected from the group consisting of between 1 about 51; between 1 about 41; between 1 about 31; between 1 about 21, between 1 about 11 and between 1 about 6, wherein x is greater than y.", "43.The method of claim 1, wherein the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: i. CH3(CH2)nOH/CH3(CH2)n+mOH, to esterify peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", "y; ii.", "CH3(CH2)n NH2/CH3(CH2)n+mNH2, to form amide bond with peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y; and, iii.", "H(CH2)nCO2H/H(CH2)n+mCO2H, to form amide bond with peptide N-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y; wherein n, m and y are integers.", "44.The method of claim 43, wherein n, m and y are integers selected from the group consisting of about 51; about 41; about 31; about 21, about 11; about 6 and between about 5 and 51.45.The method of claim 1, wherein the separating of step (e) comprises a liquid chromatography system.", "46.The method of claim 1, wherein the liquid chromatography system comprises a multidimensional liquid chromatography.", "47.The method of claim 1, wherein the mass spectrometer comprises a tandem mass spectrometry device.", "48.The method of claim 1, further comprising quantifying the amount of each polypeptide.", "49.The method of claim 1, further comprising quantifying the amount of each peptide.", "50.A method for defining the expressed proteins associated with a given cellular state, the method comprising the following steps: (a) providing a sample comprising a cell in the desired cellular state; (b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting polypeptides derived from the cell into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, thereby defining the expressed proteins associated with the cellular state.", "51.A method for quantifying changes in protein expression between at least two cellular states, the method comprising the following steps: state; (b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting polypeptides derived from the cells into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents, wherein the labels used in one same are different from the labels used in other samples; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which identifies from which sample each peptide was derived, compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, and compares the amount of each polypeptide in each sample, thereby quantifying changes in protein expression between at least two cellular states.", "52.A method for identifying proteins by differential labeling of peptides, the method comprising the following steps: (a) providing a sample comprising a polypeptide; (b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by multidimensional liquid chromatography to generate an eluate; (f) feeding the eluate of step (e) into a tandem mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.", "53.A chimeric labeling reagent comprising (a) a first domain comprising a biotin; and (b) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope.", "54.The chimeric labeling reagent of claim 53, wherein the isotope is in the first domain.", "55.The chimeric labeling reagent of claim 54, wherein the isotope is in the biotin.", "56.The chimeric labeling reagent of claim 53, wherein the isotope is in the second domain.", "57.The chimeric labeling reagent of claim 53, wherein the isotope is selected from the group consisting of a deuterium isotope, a boron-10 or boron-11 isotope, a carbon-12 or a carbon-13 isotope, a nitrogen-14 or a nitrogen-15 isotope and a sulfur-32 or a sulfur-34 isotope.", "58.The chimeric labeling reagent of claim 53 comprising two or more isotopes.", "59.The chimeric labeling reagent of claim 53, wherein the reactive group capable of covalently binding to an amino acid is selected from the group consisting of a succimide group, an isothiocyanate group and an isocyanate group.", "60.The chimeric labeling reagent of claim 53, wherein the reactive group capable of covalently binding to an amino acid binds to a lysine or a cysteine.", "61.The chimeric labeling reagent of claim 53, further comprising a linker moiety linking the biotin group and the reactive group.", "62.The chimeric labeling reagent of claim 53; wherein the linker moiety comprises at least one isotope.", "63.The chimeric labeling reagent of claim 53, wherein the linker is a cleavable moiety.", "64.The chimeric labeling reagent of claim 53, wherein the linker can be cleaved by enzymatic digest.", "65.The chimeric labeling reagent of claim 53, wherein the linker can be cleaved by reduction.", "66.A method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the small molecule tags are structurally identical but differ in their isotope composition, and the small molecules comprise reactive groups that covalently bind to cysteine or lysine residues or both; (b) providing at least two samples comprising polypeptides; (c) attaching covalently the differential small molecule tags to amino acids of the polypeptides; (d) determining the protein concentrations of each sample in a tandem mass spectrometer; and, (d) comparing relative protein concentrations of each sample.", "67.The method of claim 66, wherein the sample comprises a complete or a fractionated cellular sample.", "68.The method of claim 66, wherein differential small molecule tags comprise a chimeric labeling reagent comprising (a) a first domain comprising a biotin; and, (b) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope.", "69.The method of claim 68, wherein the isotope is selected from the group consisting of a deuterium isotope, a boron-10 or boron-lI 1 isotope, a carbon-12 or a carbon-13 isotope, a nitrogen-14 or a nitrogen-15 isotope and a sulfur-32 or a sulfur-34 isotope.", "70.The method of claim 68, wherein the chimeric labeling reagent comprises two or more isotopes.", "71.The method of claim 68, wherein the reactive group capable of covalently binding to an amino acid is selected from the group consisting of a succimide group, an isothiocyanate group and an isocyanate group.", "72.A method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the differential small molecule tags comprise a chimeric labeling reagent comprising (i) a first domain comprising a biotin; and, (ii) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope; (b) providing at least two samples comprising polypeptides; (c) attaching covalently the differential small molecule tags to amino acids of the polypeptides; (d) isolating the tagged polypeptides on a biotin-binding column by binding tagged polypeptides to the column, washing non-bound materials off the column, and eluting tagged polypeptides off the column; (e) determining the protein concentrations of each sample in a tandem mass spectrometer; and, (f) comparing relative protein concentrations of each sample.", "73.A method of producing an improved organism having a desirable trait comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence of said improved organism.", "74.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 15 different genes.", "75.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 50 different genes.", "76.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of a knocking out of at least 100 different genes.", "77.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 15 different genes.", "78.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 50 different genes.", "79.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an introduction of at least 100 different genes.", "80.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 15 different genes.", "81.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 50 different genes.", "82.The method of claim 73, wherein the set of substantial genetic mutations in step b) is comprised of an alteration in the expression of at least 100 different genes.", "83.A method of producing an improved organism having a desirable trait comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms each having at least one genetic mutation, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations c) detecting the manifestation of at least two genetic mutations, d) introducing at least two detected genetic mutations into one organism, and e) optionally repeating any of steps a), b), c), and d).", "84.The method of claim 83, wherein step d) is comprised of a knocking out of at least 15 different genes in one organism.", "85.The method of claim 83, wherein step d) is comprised of a knocking out of at least 50 different genes in one organism.", "86.The method of claim 83, wherein step d) is comprised of a knocking out of at least 100 different genes in one organism.", "87.The method of claim 83, wherein step d) is comprised of an introduction of at least 15 different genes into one organism.", "88.The method of claim 83, wherein step d) is comprised of an introduction of at least 50 different genes into one organism.", "89.The method of claim 83, wherein step d) is comprised of an introduction of at least 100 different genes into one organism.", "90.The method of claim 83, wherein step d) is comprised of an alteration in the expression of at least 15 different genes in one organism.", "91.The method of claim 83, wherein step d) is comprised of an alteration in the expression of at least 50 different genes in one organism.", "92.The method of claim 83, wherein step d) is comprised of an alteration in the expression of at least 100 different genes in one organism.", "93.A method for identifying a gene that alters a trait of an organism, comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence an organism having said altered trait, and d) determining the nucleotide sequence of a gene that has been mutagenized in the organism having the altered trait.", "94.A method for producing an organism with an improved trait, comprising: a) functionally knocking out an enogenous gene in a substantially clonal population of organisms; b) transferring a library of altered genes into the substantially clonal population of organisms, wherein each altered gene differs from the endogenous gene at only one codon; c) detecting a mutagenized organism having an improved trait; and d) determining the nucleotide sequence of an gene that has been transferred into the detected organism.", "95.A method of introducing differentially activatable stacked traits into a transgenic cell or organism, which method is comprised of the following steps: a) obtaining an initial cell or organism; b) introducing into the working cell or organism a plurality of traits (stacked traits), including selectively and differentially activatable traits, whereby serviceable traits for this purpose include traits conferred by genes and traits conferred by gene pathways; c) analyzing the information obtained from steps a) and b), and d) optionally repeating any number or all of the steps of a), b), c), and d); 96.The method of claim 95, wherein step a) also includes holistic monitoring of the strain or organism whereby holistic monitoring can include the detection and/or measurement of all detectable functions and physical parameters (such as but not limited to morphology, behavior, growth, responsiveness to stimuli [e.g., antibiotics, different environment, etc.", "], and profiles of all detectable molecules, including molecules that are chemically at least in part a nucleic acids, proteins, carbohydrates, proteoglycans, glycoproteins, or lipids) 97.The method of claim 95, wherein step d) also includes holistic monitoring of the strain or organism whereby holistic monitoring can include the detection and/or measurement of all detectable functions and physical parameters (such as but not limited to morphology, behavior, growth, responsiveness to stimuli [e.g., antibiotics, different environment, etc.", "], and profiles of all detectable molecules, including molecules that are chemically at least in part a nucleic acids, proteins, carbohydrates, proteoglycans, glycoproteins, or lipids) 98.The method of claim 95, wherein step a) and d) include holistic monitoring of the strain or organism whereby holistic monitoring can include the detection and/or measurement of all detectable functions and physical parameters (such as but not limited to morphology, behavior, growth, responsiveness to stimuli [e.g., antibiotics, different environment, etc.", "], and profiles of all detectable molecules, including molecules that are chemically at least in part a nucleic acids, proteins, carbohydrates, proteoglycans, glycoproteins, or lipids) 99.The method of claim 95, wherein step b) includes the introduction of at least 15 stacked traits 100.The method of claim 95, wherein step b) includes the introduction of at least 50 stacked traits 101.The method of claim 95, wherein step b) includes the introduction of at least 100 stacked traits 102.The method of claim 96, wherein step a) includes screening cellular characteristics by utilizing one or any combination of the following methods: a) genomics; b) transcriptome characterization or RNA profiling; c) proteomics; d) metabolomics or the analysis of metabolites; e) lipidomics or lipid profiling.", "103.A method of claim 102, wherein proteomics specifically includes the use of amino acid reactive tags 104.A method of claim 97, wherein step d) includes screening cellular characteristics by utilizing one or any combination of the following methods: f) genomics; g) transcriptome characterization or RNA profiling; h) proteomics; i) metabolomics or the analysis of metabolites; j) lipidomics or lipid profiling.", "105.A method of claim 104, wherein proteomics specifically includes the use of amino acid reactive tags 106.A method of claim 98, wherein steps a) and d) include screening cellular characteristics by utilizing one or any combination of the following methods: k) genomics; l) transcriptome characterization or RNA profiling; m) proteomics; n) metabolomics or the analysis of metabolites; o) lipidomics or lipid profiling.", "P) 107.A method of claim 106, wherein proteomics specifically includes the use of amino acid reactive tags 108.A method of claim 73, wherein step c) includes screening cellular characteristics by utilizing one or any combination of the following methods: q) genomics; r) transcriptome characterization or RNA profiling; s) proteomics; t) metabolomics or the analysis of metabolites; u) lipidomics or lipid profiling.", "109.A method of claim 108, wherein proteomics specifically includes the use of amino acid reactive tags 110.A method of claim 93, wherein step c) includes screening cellular characteristics by utilizing one or any combination of the following methods: v) genomics; w) transcriptome characterization or RNA profiling; x) proteomics; y) metabolomics or the analysis of metabolites; z) lipidomics or lipid profiling.", "111.A method of claim 110, wherein proteomics specifically includes the use of amino acid reactive tags 112.A method of claim 94, wherein step c) includes screening cellular characteristics by utilizing one or any combination of the following methods: aa) genomics; bb) transcriptome characterization or RNA profiling; cc) proteomics; dd) metabolomics or the analysis of metabolites; ee) lipidomics or lipid profiling.", "113.A method of claim 112, wherein proteomics specifically includes the use of amino acid reactive tags 114.A method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifyng an engineered phenotype in the cell using real-time metabolic flux analysis.", "115.The method of claim 114, wherein the genetic composition of the cell is modified by a method comprising addition of a nucleic acid to the cell.", "116.The method of claim 115, wherein the nucleic acid comprises a nucleic acid heterologous to the cell.", "117.The method of claim 115, wherein the nucleic acid comprises a nucleic acid homologous to the cell.", "118.The method of claim 117, wherein the homologous nucleic acid comprises a modified homologous nucleic acid.", "119.The method of claim 118, wherein the homologous nucleic acid comprises a modified homologous gene.", "120.The method of claim 114, wherein the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell.", "121.The method of claim 114, wherein the genetic composition of the cell is modified by a method comprising modifying or knocking out the expression of a gene.", "122.The method of claim 114, further comprising selecting a cell comprising a newly engineered phenotype.", "123.The method of claim 122, further comprising culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.", "124.The method of claim 122, wherein the newly engineered phenotype is selected from the group consisting of an increased or decreased expression or amount of a polypeptide, an increased or decreased amount of an mRNA transcript, an increased or decreased expression of a gene, an increased or decreased resistance or sensitivity to a toxin, an increased or decreased resistance use or production of a metabolite, an increased or decreased uptake of a compound by the cell, an increased or decreased rate of metabolism, and an increased or decreased growth rate.", "125.The method of claim 114, further comprising isolating a cell comprising a newly engineered phenotype.", "126.The method of claim 114, wherein the newly engineered phenotype is a stable phenotype.", "127.The method of claim 126, wherein modifying the genetic composition of a cell comprises insertion of a construct into the cell, wherein construct comprises a nucleic acid operably linked to a constitutively active promoter.", "128.The method of claim 114, wherein the newly engineered phenotype is an inducible phenotype.", "129.The method of claim 128, wherein modifying the genetic composition of a cell comprises insertion of a construct into the cell, wherein construct comprises a nucleic acid operably linked to an inducible promoter.", "130.The method of claim 115, wherein nucleic acid added to the cell in step (a) is stably inserted into the genome of the cell.", "131.The method of claim 115, wherein nucleic acid added to the cell in step (a) propagates as an episome in the cell.", "132.The method of claim 115, wherein nucleic acid added to the cell in step (a) encodes a polypeptide.", "133.The method of claim 132, wherein the polypeptide comprises a modified homologous polypeptide.", "134.The method of claim 132, wherein the polypeptide comprises a heterologous polypeptide.", "135.The method of claim 115, wherein the nucleic acid added to the cell in step (a) encodes a transcript comprising a sequence that is antisense to a homologous transcript.", "136.The method of claim 114, wherein modifying the genetic composition of the cell in step (a) comprises increasing or decreasing the expression of an mRNA transcript.", "137.The method of claim 114, wherein modifying the genetic composition of the cell in step (a) comprises increasing or decreasing the expression of a polypeptide.", "138.The method of claim 114, wherein modifying the homologous gene in step (a) comprises knocking out expression of the homologous gene.", "139.The method of claim 114, wherein modifying the homologous gene in step (a) comprises increasing the expression of the homologous gene.", "140.The method of claim 114, wherein the heterologous gene in step (a) comprises a sequence-modified homologous gene, wherein the sequence modification is made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises a homologous gene of the cell; (b) providing a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; (c) generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide of step (a) with the oligonucleotides of step (b), thereby generating polynucleotides comprising homologous gene sequence variations.", "141.The method of claim 114, wherein the heterologous gene in step (a) comprises a sequence-modified homologous gene, wherein the sequence modification is made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.", "142.The method of claim 114, wherein the cell is a prokaryotic cell.", "143.The method of claim 142, wherein the prokaryotic cell is a bacterial cell.", "144.The method of claim 114, wherein the cell is a selected from the group consisting of a fungal cell, a yeast cell, a plant cell and an insect cell.", "145.The method of claim 114, wherein the cell is a eukaryotic cell.", "146.The method of claim 145, wherein the cell is a mammalian cell.", "147.The method of claim 146, wherein the mammalian cell is a human cell.", "148.The method of claim 114, wherein the measured metabolic parameter comprises rate of cell growth.", "149.The method of claim 148, wherein the rate of cell growth is measured by a change in optical density of the culture.", "150.The method of claim 114, wherein the measured metabolic parameter comprises a change in the expression of a polypeptide.", "151.The method of claim 150, wherein the change in the expression of the polypeptide is measured by a method selected from the group consisting of a one-dimensional gel electrophoresis, a two-dimensional gel electrophoresis, a tandem mass spectography, an RIA, an ELISA, an immunoprecipitation and a Western blot.", "152.The method of claim 114, wherein the measured metabolic parameter comprises a change in expression of at least one transcript, or, the expression of a transcript of a newly introduced gene.", "153.The method of claim 152, wherein the change in expression of the transcript is measured by a method selected from the group consisting of a hybridization, a quantitative amplification and a Northern blot.", "154.The method of claim 153, wherein transcript expression is measured by hybridization of a sample comprising transcripts of a cell or nucleic acid representative of or complementary to transcripts of a cell by hybridization to immobilized nucleic acids on an array.", "155.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in a secondary metabolite.", "156.The method of claim 155, wherein secondary metabolite is selected from the group consisting of a glycerol and a methanol.", "157.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in an organic acid.", "158.The method of claim 157, wherein the organic acid is selected from the group consisting of an acetate, a butyrate, a succinate and an oxaloacetate.", "159.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in intracellular pH.", "160.The method of claim 159, wherein the increase or a decrease in intracellular pH is measured by intracellular application of a dye, and the change in fluorescence of the dye is measured over time.", "161.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in synthesis of DNA over time.", "162.The method of claim 161, wherein the increase or a decrease in synthesis of DNA over time is measured by intracellular application of a dye, and the change in fluorescence of the dye is measured over time.", "163.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in uptake of a composition.", "164.The method of claim 163, wherein the composition is a metabolite.", "165.The method of claim 164, wherein the metabolite is selected from the group consisting of a monosaccharide, a disaccharide, a polysaccharide, a lipid, a nucleic acid, an amino acid and a polypeptide.", "166.The method of claim 165, wherein the saccharide, disaccharide or polysaccharide comprises a glucose or a sucrose.", "167.The method of claim 163, wherein the composition is selected from the group consisting of an antibiotic, a metal, a steroid and an antibody.", "168.The method of claim 114, wherein the measured metabolic parameter comprises an increase or a decrease in the secretion of a byproduct or a secreted composition of a cell.", "169.The method of claim 168, wherein the byproduct or secreted composition is selected from the group consisting of a toxin, a lymphokine, a polysaccharide, a lipid, a nucleic acid, an amino acid, a polypeptide and an antibody.", "170.The method of claim 114, wherein the real time monitoring simultaneously measures a plurality of metabolic parameters.", "171.The method of claim 170, wherein real time monitoring of a plurality of metabolic parameters comprises use of a Cell Growth Monitor device.", "172.The method of claim 171, wherein the Cell Growth Monitor device is a Wedgewood Technology, Inc., Cell Growth Monitor model 652.173.The method of claim 171, wherein the real time simultaneous monitoring measures uptake of substrates, levels of intracellular organic acids and levels of intracellular amino acids.", "174.The method of claim 171, wherein the real time simultaneous monitoring measures: uptake of glucose; levels of acetate, butyrate, succinate or oxaloacetate; and, levels of intracellular natural amino acids.", "175.The method of claim 171, further comprising use of a computer-implemented program to real time monitor the change in measured metabolic parameters over time.", "176.The method of claim 175, wherein the computer-implemented program comprises a computer-implemented method as set forth in FIG.", "28.177.The method of claim 176, wherein the computer-implemented method comprises metabolic network equations.", "178.The method of claim 176, wherein the computer-implemented method comprises a pathway analysis.", "179.The method of claim 176, wherein the computer-implemented program comprises a preprocessing unit to filter out the errors for the measurement before the metabolic flux analysis." ], [ "<SOH> B—BACKGROUND <EOH>" ], [ "<SOH> C—SUMMARY OF THE INVENTION <EOH>This invention relates generally to the field of cellular and whole organism engineering.", "Specifically, this invention relates to a cellular transformation, directed evolution, and screening method for creating novel transgenic organisms having desirable properties.", "Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are differentially activatable.", "In one embodiment, this invention is directed to a method of producing an improved organism having a desirable trait to by: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence of said improved organism.", "This invention provides that any of steps a), b), and c) can be further repeated in any particular order and any number of times; accordingly, this invention specifically provides methods comprised of any iterative combination of steps a), b), and c), with a number of iterations.", "In another embodiment, this invention is directed to a method of producing an improved organism having a desirable trait to by: a) obtaining an initial population of organisms, which can be a clonal population or otherwise, b) generating a set of mutagenized organisms each having at least one genetic mutation, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations c) detecting the manifestation of at least two genetic mutations, and d) introducing at least two detected genetic mutations into one organism.", "Additionally, this invention provides that any of steps a), b), c), and d) can be further repeated in any particular order and any number of times; accordingly, this invention specifically provides methods comprised of any iterative combination of steps a), b), c), and d), with a total number of iterations can be from one up to one million, including specifically every integer value in between.", "In a preferred aspect of embodiments specified herein the step of b) generating a second set of mutagenized organisms is comprised of generating a plurality of organisms, each of which organisms has a particular transgenic mutation.", "As used herein, “generating a set of mutagenized organisms having genetic mutations” can be achieved by any means known in the art to mutagenized including any radiation known to mutagenized, such as ionizing and ultra violet.", "Further examples of serviceable mutagenizing methods include site-saturation mutagenesis, transposon-based methods, and homologous recombination.", "“Combining” means incorporating a plurality of different genetic mutations in the genetic makeup (e.g.", "the genome) of the same organism; and methods to achieve this combining” step including sexual recombination, homologous recombination, and transposon-based methods.", "As used herein, an “initial population of organisms” means a “Working population of organisms”, which refers simply to a population of organisms with which one is working, and which is comprised of at least one organism.", "An “initial population of organisms” which can be a clonal population or otherwise.", "Accordingly, in step 1) an “initial population of organisms” may be a population of multicellular organisms or of unicellular organisms or of both.", "An “initial population of organisms” may be comprised of unicellular organisms or multicellular organisms or both.", "An “initial population of organisms” may be comprised of prokaryotic organisms or eukaryotic organisms or both.", "This invention provides that an “initial population of organisms” is comprised of at least one organism, and preferred embodiments include at least that.", "By “organism” is meant any biological form or thing that is capable of self replication or replication in a host.", "Examples of “organisms” include the following kinds of organisms (which kinds are not necessarily mutually-exclusive): animals, plants, insects, cyanobacteria, microorganisms, fungi, bacteria, eukaryotes, prokaryotes, mycoplasma, viral organisms (including DNA viruses, RNA viruses), and prions.", "Non-limiting particularly preferred examples of kinds of “organisms” also include Archaea (archaebacteria) and Bacteria (eubacteria).", "Non-limiting examples of Archaea (archaebacteria) include Crenarchaeota, Euryarchaeota, and Korarchaeota.", "Non-limiting examples Bacteria (eubacteria) include Aquificales, CFB/Green sulfur bacteria group, Chlamydiales/Verrucomicrobia group, Chrysiogenes group, Coprothermobacter group, Cyanobacteria & chloroplasts, Cytophaga/Flexibacter/Bacteriods group, Dictyoglomus group, Fibrobacter/Acidobacteria group, Firmicutes, Flexistipes group, Fusobacteria, Green non-sulfur bacteria, Nitrospira group, Planctomycetales, Proteobacteria, Spirochaetales, Synergistes group, Thermodesulfobacterium group, Thermotogales, Thermus/Deinococcus group.", "As non-limiting examples, particularly preferred kinds of organisms include Aquifex, Aspergillus, Bacillus, Clostridium, E. coli, Lactobacillus, Mycobacterium, Pseudomonas, Streptomyces , and Thermotoga .", "As additional non-limiting examples, particularly preferred organisms include cultivated organisms such as CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.Particularly preferred non-limiting examples of organisms further include host organisms that are serviceable for the expression of recombinant molecules.", "Organisms further include primary cultures (e.g.", "cells from harvested mammalian tissues), immortalized cells, all cultivated and culturable cells and multicellular organisms, and all uncultivated and uculturable cells and multicellular organisms.", "In a preferred embodiment, knowledge of genomic information is useful for performing the claimed methods; thus, this invention provides the following as preferred but non-limiting examples of organisms that are particularly serviceable for this invention, because there is a significant amount of—if not complete—genomic sequence information (in terms of primary sequence &/or annotation) for these organisms: Human, Insect (e.g.", "Drosophila melanogaster ), Higher plants (e.g.", "Arabidopsis thaliana ), Protozoan (e.g.", "Plasmodium falciparum ), Nematode (e.g.", "Caenorhabditis elegans ), Fungi (e.g.", "Saccharomyces cerevisiae ), Proteobacteria gamma subdivision (e.g.", "Escherichia coli K-12, Haemophilus influenzae Rd, Xylella fastidiosa 9a5c, Vibrio cholerae E1 Tor N16961, Pseudomonas aeruginosa PA01, Buchnera sp.", "APS), Proteobacteria beta subdivision (e.g.", "Neisseria meningitidis MC58 (serogroup B), Neisseria meningitidis Z2491 (serogroup A)), Proteobacteria other subdivisions (e.g.", "Helicobacter pylori 26695, Helicobacter pylori J99, Campylobacter jejuni NCTCI 11168, Rickettsia prowazekii ), Gram-positive bacteria (e.g.", "Bacillus subtilis, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycobacterium tuberculosis H37Rv), Chlamydia (e.g.", "Chlamydia trachomatisserovar D, Chlamydia muridarum ( Chlamydia trachomatis MoPn), Chlamydia pneumoniae CWL029 , Chlamydia pneumoniae AR39 , Chlamydia pneumoniae J138), Spirochete (e.g.", "Borrelia burgdorferi B31, Treponema pallidum ), Cyanobacteria (e.g.", "Synechocystis sp.", "PCC6803), Radioresistant bacteria (e.g.", "Deinococcus radiodurans R1), Hyperthermophilic bacteria (e.g.", "Aquifex aeolicus VF5 , Thermotoga marilima MSB8), and Archaea (e.g.", "Methanococcus jannaschii, Methanobacterium thermoautotrophicum deltaH, Archaeoglobus fulgidus, Pyrococcus horikoshii OT3, Pyrococcus abyssi, Aeropyrum pernix K1).", "Non-limiting particularly preferred examples of kinds of plant “organisms” include those listed in Table 1.TABLE 1 Non-limiting examples of plant organisms and sources of transgenic molecules (e.g.", "nucleic acids & nucleic acid products) 1.Alfalfa 2.Amelanchier laevis 3.Apple 4.Arab.", "thaliana 5.Arabidopsis 6.Aspergillus flavus 7.Barley 8.Beet 9.Belladonna 10.Brassica oleracea 11.Carrot 12.Chrysanthemum 13.Cichorium intybus 14.Clavibacter 15.Clavibacter xyli 16.Coffee 17.Corn 18.Cotton 19.Cranberry 20.Creeping bentgrass 21.Cryphonectria parasitica 22.Eggplant 23.Festuca arundinacea 24.Fusarium graminearum 25.Fusarium moniliforme 26.Fusarium sporotrichioides 27.Gladiolus 28.Grape 29.Heterorhabditis bacteriophora 30.Kentucky bluegrass 31.Lettuce 32.Melon 33.Oat 34.Onion 35.Papaya 36.Pea 37.Peanut 38.Pelargonium 39.Pepper 40.Persimmon 41.Petunia 42.Pine 43.Pineapple 44.Pink bollworm 45.Plum 46.Poplar 47.Potato 48.Pseudomonas 49.Pseudomonas putida 50.Pseudomonas syringae 51.Rapeseed 52.Rhizobium 53.Rhizobium etli 54.Rhizobium fredii 55.Rhizobium leguminosarum 56.Rhizobium meliloti 57.Rice 58.Rubus idaeus 59.Spruce 60.Soybean 61.Squash 62.Squash-cucumber 63.Squash- cucurbita texana 64.Strawberry 65.Sugarcane 66.Sunflower 67.Sweet potato 68.Sweetgum 69.TMV 70.Tobacco 71.Tomato 72.Walnut 73.Watermelon 74.Wheat 75.Xanthomonas 76.Xanthomonas campestris As used herein, the meaning of “generating a set of mutagenized organisms having genetic mutations” includes the steps of substituting, deleting, as well as introducing a nucleotide sequence into organism; and this invention provides a nucleotide sequence that serviceable for this purpose may be a single-stranded or double-stranded and the fact that its length may be from one nucleotide up to 10,000,000,000 nucleotides in length including specifically every integer value in between.", "A mutation in an organism includes any alteration in the structure of one or more molecules that encode the organism.", "These molecules include nucleic acid, DNA, RNA, prionic molecules, and may be exemplified by a variety of molecules in an organism such as a DNA that is genomic, episomal, or nucleic, or by a nucleic acid that is vectoral (e.g.", "viral, cosmid, phage, phagemid).", "In one aspect, as used herein, a “set of substantial genetic mutations” is preferably a disruption (e.g.", "a functional knock-out) of at least about 15 to about 150,000 genomic locations or nucleotide sequences (e.g.", "genes, promoters, regulatory sequences, codons etc.", "), including specifically every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 15 to about 150,000 genes, including specifically every integer value in between.", "Corresponding to another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 15 to about 150,000 gene products &/or phenotypes &/or traits, including specifically every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” with respect to an organism (or type of organism) is preferably a disruption (e.g.", "a functional knock-out) of at least about 1% to about 100% of genomic locations or nucleotide sequences (e.g.", "genes, promoters, regulatory sequences, codons etc.)", "in the organism (or type of organism), including specifically percentages of every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 1% to about 100% of genes in an organism (or type of organism), including specifically percentages of every integer value in between.", "Corresponding to another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 1% to about 100% of the gene products &/or phenotypes &/or traits of an organism (or type of organism), including specifically every integer value in between.", "In yet another aspect, as used herein, a “set of substantial genetic mutations” is preferably an introduction or deletion of at least about 15 to 150,000 genes promoters or other nucleotide sequences (where each sequence is from 1 base to 10,000,000 bases), including specifically every integer value in between.", "For example, one can introduce a library of at least about 15 to 150,000 nucleotides (genes or promoters) produced by “site-saturation mutagenesis” &/or by “ligation reassembly” (including any specific aspect thereof provided herein) into an “initial population of organisms”.", "It is provided that wherever the manipulation of a plurality of “genes” is mentioned herein, gene pathways (e.g.", "that ultimately lead to the production of small molecules) are also included.", "It is appreciated herein that knocking-out, altering expression level, and altering expression pattern can be achieved, by non-limiting exemplification, by mutagenizing a nucleotide sequence corresponding gene as well as a corresponding promoter that affects the expression of the gene.", "As used herein, a “mutagenized organism” includes any organism that has been altered by a genetic mutation.", "A “genetic mutation” can be, by way of non-limiting and non-mutually exclusive exemplification, and change in the nucleotide sequence (DNA or RNA) with respect to genomic, extra-genomic, episomal, mitochondrial, and any nucleotide sequence associated with (e.g.", "contained within or considered part of) an organism.", "According to this invention, detecting the manifestation of a “genetic mutation” means “detecting the manifestation of a detectable parameter”, including but not limited to a change in the genomic sequence.", "Accordingly, this invention provides that a step of sequencing (&/or annotating) of and organism's genomic DNA is necessary for some methods of this invention, and exemplary but non-limiting aspects of this sequencing (&/or annotating) step are provided herein.", "A detectable “trait”, as used herein, is any detectable parameter associated with the organism.", "Accordingly, such a detectable “parameter” includes, by way of non-limiting exemplification, any detectable “nucleotide knock-in”, any detectable “nucleotide knock-outs”, any detectable “phenotype”, and any detectable “genotype”.", "By way of further illustration, a “trait” includes any substance produced or not produced by the organism.", "Accordingly, a “trait” includes viability or non-viability, behavior, growth rate, size, morphology.", "“Trait” includes increased (or alternatively decreased) expression of a gene product or gene pathway product.", "“Trait” also includes small molecule production (including vitamins, antibiotics), herbicide resistance, drought resistance, pest resistance, production of any recombinant biomolecule (ie.g.", "vaccines, enzymes, protein therapeutics, chiral enzymes).", "Additional examples of serviceable traits for this invention are shown in Table 2.TABLE 2 Non-limiting examples of serviceable genes, gene products, phenotypes, or traits according to the methods of this invention (e.g.", "knockouts, knockins, increased or decreased expression level, increased or decreased expression pattern) Table 2 - Part 1.Non-limiting examples of genes or gene products 1.17 kDa protein 2.3-hydroxy-3-methylglutaryl CoenzymeA reductase 3.4-Coumarate: CoA ligase knockout 4.60 kDa protein 5.Ac transposable element 6.ACC deaminase 7.ACC oxidase knockout 8.ACC synthase 9.ACC synthase knockout 10.Acetohydroxyacid synthase variant 11.Acetolactate synthase 12.Acetyl CoA carboxylase 13.ACP acyl-ACP thioesterase 14.ACP thioesterase 15.Acyl CoA reductase 16.Acyl-ACP knockout 17.Acyl-ACP desaturase 18.Acyl-ACP desaturase knockout 19.Acyl-ACP thioesterase 20.ADP glucose pyrophosphorylase 21.ADP glucose pyrophosphorylase knockout 22.Agglutinin 23.Aleurone 1 24.Alpha hordothinonin 25.Alpha-amylase 26.Alpha-hemoglobin 27.Aminoglycoside 3′-adenylytransferase 28.Amylase 29.Anionic peroxidase 30.Antibody 31.Antifungal protein 32.Antithrombin 33.Antitrypsin 34.Antiviral protein 35.Aspartokinase 36.Attacin E 37.B1 regulatory gene 38.B-1,3-glucanase knockout 39.B-1,4-endoglucanase knockout 40.Bacteropsin 41.Barnase 42.Barstar 43.Beta-hemoglobin 44.B-glucuronidase 45.C1 knockout 46.C1 regulatory gene 47.C2 knockout 48.C3 knockout 49.Caffeate O-methylthransferase 50.Caffeate O-methyltransferase knockout 51.Caffeoyl CoA O-methyltransferase knockout 52.Casein 53.Cecropin 54.Cecropin B 55.Cellulose binding protein 56.Chalcone synthase knockout 57.Chitinase 58.Chitobiosidase 59.Chloramphenicol acetyltransferase 60.Cholera toxin B 61.Choline oxidase 62.Cinnamate 4-hydroxylase 63.Cinnamate 4-hydroxylase knockout 64.Coat protein 65.Coat protein knockout 66.Conglycinin 67.CryIA 68.CryIAb 69.CryIAc 70.CryIB 71.CryIIA 72.CryIIIA 73.CryVIA 74.Cyclin dependent kinase 75.Cyclodexlrin glycosyltransferase 76.Cylindrical inclusion protein 77.Cystathionine synthase 78.Delta-12 desaturase 79.Delta-12 desaturase knockout 80.Delta-12 saturase 81.Delta-12 saturase knockout 82.Delta-15 desaturase 83.Delta-15 desaturase knockout 84.Delta-9 desaturase 85.Delta-9 desturase knockout 86.Deoxyhypusine synthase (DHS) 87.Deoxyhypusine synthase knockout 88.Diacylglycerol acetyl tansferase 89.Dihydrodipicolinate synthase 90.Dihydrofolate reductase 91.Diptheria toxin A 92.Disease resistance response gene 49 93.Double stranded ribonuclease 94.Ds transposable element 95.Elongase 96.EPSPS 97.Ethylene forming enzyme knockout 98.Ethylene receptor protein 99.Ethylene receptor protein knockout 100.Fatty acid elongase 101.Fluorescent protein 102.G glycoprotein 103.Galactanase 104.Galanthus nivalis agglutinin 105.Genome-linked protein 106.Glucanase 107.Glucanase knockout 108.Glucose oxidase 109.Glutamate dehydrogenase 110.Glutamine binding protein 111.Glutamine synthetase 112.Glutenin 113.Glycerol-3-phosphate acetyl transferase 114.Glyphosate exidoreductase 115.Glyphosate oxidoreductase 116.Green fluorescent protein 117.Helper component 118.Hemicellulase 119.Hup locus 120.Hygromycin phosphotransferase 121.Hyoscamine 6B-hydroxylase 122.IAA monooxygenase 123.Invertase 124.Invertase knockout 125.Isopentenyl transferase 126.Ketoacyl-ACP synthase 127.Ketoacyl-ACP synthase knockout 128.Larval serum protein 129.Leafy homeotic regulatory gene 130.Lectin 131.Lignin peroxidase 132.Luciferase 133.Lysine-2 gene 134.Lysophosphatidic acid acetyl transferase 135.Lysozyme 136.Mabinlin 137.Male sterility protein 138.Metallothionein 139.Modified ethylene receptor protein 140.Modified ethylene receptor protein knockout 141.Monooxygenase 142.Movement protein 143.Movement protein nonfunctional 144.N gene for TMV resistance 145.N-acetyl glucosidase 146.Nitrilase 147.Nopaline synthase 148.Notch 149.NptII 150.Nuclear inclusion protein a 151.Nuclear inclusion protein b 152.Nucleocapsid 153.Nucleoprotein 154.O-acyl transferase 155.Oleayl-ACP thioesterase 156.Omega 3 desaturase 157.Omega 3 desaturease knockout 158.Omega 6 desaturase 159.Omega 6 desaturase knockout 160.O-methyltransferase 161.Osmotin 162.Oxalate oxidase 163.Par locus 164.Pathogenesis protein 1a 165.Pectate lyase 166.Pectin esterase 167.Pectin esterase knockout 168.Pectin methylesterase 169.Pectin methylesterase knockout 170.Pentenlypyrophosphate isomerase 171.Phosphinothricin 172.Phosphinothricin acetyl transferase 173.Phytochrome A 174.Phytoene synthase 175.Phleomycin binding protein 176.Polygalacturonase 177.Polygalacturonase knockout 178.Polygalacturonase inhibitor protein 179.Prf regulatory gene 180.Prosystemin 181.Protease 182.Protein A 183.Protein kinase 184.Proteinase inhibitor 1 185.Pti5 transcription factor 186.R regulatory gene 187.Receptor kinase 188.Recombinase 189.Reductase 190.Replicase 191.Resveratrol synthase 192.Ribonuclease 193.ro1c 194.Rol hormone gene 195.S-adenosylmethione decarboxylase 196.S-adenosylmethione hydrolase 197.S-adenosylmethionine transferase 198.Salicylate hydroxylase 199.Satellite RNA 200.Seed storage protein 201.Serine-threonine protein kinase 202.Serum albumin 203.Shrunken 2 204.Sorbitol dehydrogenase 205.Sorbitol synthase 206.Stilbene synthase 207.Storage protein 208.Sucrose phosphate synthase 209.Systemic acquired resistance gene 8.2 210.Tetracycline binding protein 211.Thioesterase (×2) 212.Thiolase 213.TobRB7 214.Transcriptional activator 215.Transposon Tn5 216.Trehalase 217.Trehalase knockout 218.Trichodiene synthase 219.Trichosanthin 220.Trifolitoxin 221.Trypsin inhibitor 222.T-URF13 mitochondrial 223.UDP glucose glucosyltransferase 224.Violaxanthin de-epoxidase 225.Violaxanthin de-epoxidase knockout 226.Wheat germ agglutinin 227.Xanthosine-N7-methyltransferase knockout 228.Zein storage protein Table 2 - Part 2.Non-limiting examples of input traits/phenotypes 1.2,4-D tolerant 2.Alernaria resistant 3.Altered amino acid composition 4.Alternaria solani resistant 5.Ammonium assimilation increased 6.AMV resistant 7.Aphid resistant 8.Apple scab resistant 9.Aspergillus resistant 10.B-1,4-endoglucanase 11.Bacterial leaf blight resistant 12.Bacterial speck resistant 13.BCTV resistant 14.Blackspot bruise resistant 15.BLRV resistant 16.BNYVV Resistant 17.Botrytis cinerea resistant 18.Botrytis resistant 19.BPMV resistant 20.Bromoxynil tolerant 21.BYDV resistant 22.BYMV resistant 23.Carbohydrate metabolism altered 24.Cell wall altered 25.Chlorsulfuron tolerant 26.Clavibacter resistant 27.CLRV resistant 28.CMV resistant 29.Cold tolerant 30.Coleopteran resistant 31.Colletotrichum resistant 32.Colorado potato beetle resistant 33.Constitutive expression of glutamine synthetase 34.Corynebacterium sepedonicum resistant 35.Cottonwood leaf beetle resistant 36.Crown gall resistant 37.Crown rot resistant 38.Cucumovirus resistant 39.Cutting rootability increased 40.Downy mildew resistant 41.Drought tolerant 42.Erwinia carotovora resistant 43.Ethylene production reduced 44.European Corn Borer resistant 45.Female sterile 46.Fenthion susceptible 47.Fertility altered 48.Fire blight resistant 49.Flower and fruit abscission reduced 50.Flower and fruit set altered 51.Flowering altered 52.Flowering time altered 53.Frogeye leaf spot resistant 54.Fruit ripening altered 55.Fruit ripening delayed 56.Fruit rot resistant 57.Fruit solids increased 58.Fruit sweetness increased 59.Fungal post-harvest resistant 60.Fungal resistant 61.Fungal resistant general 62.Fusarium resistant 63.Glyphosate tolerant 64.Growth rate altered 65.Growth rate reduced 66.Heat stable glucanase produced 67.Hordothionin produced 68.Imidazolinone tolerant 69.Insect resistant general 70.Kanamycin resistant 71.Lepidopteran resistant 72.Lesser cornstalk borer resistant 73.LMV resistant 74.Loss of systemic resistance 75.Male sterile 76.Marssonina resistant 77.MCDV resistant 78.MCMV resistant 79.MDMV resistant 80.MDMV-B resistant 81.Mealybug wilt virus resistant 82.Melamtsora resistant 83.Melodgyne resistant 84.Methotrexate resistant 85.Mexican Rice Borer resistant 86.Nucleocapsid protein produced 87.Oblique banded leafroller resistant 88.PEMV resistant 89.PeSV resistant 90.Phoma resistant 91.Phosphinothricin tolerant 92.Phratora leaf beetle resistant 93.Phytophthora resistant 94.PLRV resistant 95.Polyamine metabolism altered 96.Potyvirus resistant 97.Powdery mildew resistant 98.PPV resistant 99.Pratylenchus vulnus resistant 100.Proteinase inhibitors level constitutive 101.PRSV resistant 102.PRV resistant 103.PSbMV resistant 104.Pseudomonas syringae resistant 105.PStV resistant 106.PVX resistant 107.PVY resistant 108.RBDV resistant 109.Rhizoctonia resistant 110.Rhizoctonia solani resistant 111.Ring rot resistance 112.Root-knot nematode resistant 113.SbMV resistant 114.Sclerotinia resistant 115.SCMV resistant 116.SCYLV resistant 117.Secondary metabolite increased 118.Seed set reduced 119.Selectable marker 120.Senescence altered 121.Septoria resistant 122.Shorter stems 123.Soft rot fungal resistant 124.Soft rot resistant 125.SqMV resistant 126.SrMV resistant 127.Storage protein altered 128.Streptomyces scabies resistant 129.Sulfonylurea tolerant 130.Tetracycline binding protein produced 131.TEV resistant 132.Thelaviopsis resistant 133.TMV resistant 134.Tobamovirus resistant 135.ToMoV resistant 136.ToMV resistant 137.Transposon activator 138.Transposon inserted 139.TRV resistant 140.TSWV resistant 141.TVMV resistant 142.TYLCV resistant 143.Tyrosine level increased 144.Venturia resistant 145.Verticillium dahliae resistant 146.Verticillium resistant 147.Visual marker 148.WMV2 resistant 149.WSMV resistant 150.Yield increased 151.ZYMV resistant Table 2 - Part 3.Non-limiting examples of output traits/phenotypes 1.ACC oxidase level decreased 2.Altered lignin biosynthesis 3.B-1,4-endoglucanase 4.Botrytis resistant 5.Carbohydrate metabolism altered 6.Carotenoid content altered 7.Cell wall altered 8.CMV resistant 9.Coleopteran resistant 10.Dry matter content increased 11.Ethylene production reduced 12.Ethylene synthesis reduced 13.Fatty acid metabolism altered 14.Fire blight resistant 15.Flower and fruit abscission reduced 16.Flower and fruit set altered 17.Flowering time altered 18.Fruit firmness increased 19.Fruit pectin esterase levels decreased 20.Fruit ripening altered 21.Fruit ripening delayed 22.Fruit solids increased 23.Fruit sugar profile altered 24.Fruit sweetness increased 25.Glucuronidase expressing 26.Heat stable glucanase produced 27.Heavy metals sequestered 28.Hordothionin produced 29.Improved fruit quality 30.Industrial enzyme produced 31.Lepidopteran resistant 32.Lysine level increased 33.Mealybug wilt virus resistant 34.Methionine level increased 35.Nucleocapsid protein produced 36.Oil profile altered 37.Pectin esterase level reduced 38.Pharmaceutical proteins produced 39.Phosphinothricin tolerant 40.Phytoene synthase activity increased 41.Pigment metabolism altered 42.Polygalacturonase level reduced 43.Processing characteristics altered 44.Prolonged shelf life 45.Protein altered 46.Protein quality altered 47.PRSV resistant 48.Root-knot nematode resistant 49.Sclerotinia resistant 50.Seed composition altered 51.Seed methionine storage increased 52.Seed set reduced 53.Seed storage protein 54.Senescence altered (e.g.", "Shelf life increased) 55.Shorter stems 56.Solids increased 57.SqMV resistant 58.Starch level increased 59.Starch metabolism altered 60.Starch reduced 61.Sterols increased 62.Storage protein altered 63.Sugar alcohol levels increased 64.Telracycline binding protein produced 65.Tyrosine level increased 66.Verticillium resistant 67.Visual marker 68.WMV2 resistant 69.Yield increased 70.ZYMV resistant Table 2 - Part 4.Non-limiting examples of traits/phenotypes with agronomic properties 1.ACC oxidase level decreased 2.Altered amino acid composition 3.Altered lignin biosynthesis 4.Altered maturing 5.Altered plant development 6.Aluminum tolerant 7.Ammonium assimilation increased 8.Anthocyanin produced in seed 9.B-1,4-endoglucanase 10.Calmodulin level altered 11.Carbohydrate metabolism altered 12.Carotenoid content altered 13.Cell wall altered 14.Cold tolerant 15.Constitutive expression of glutamine synthetase 16.Cutting root ability increased 17.Development altered 18.Drought tolerant 19.Dry matter content increased 20.Environmental stress reduced 21.Ethylene metabolism altered 22.Ethylene production reduced 23.Ethylene synthesis reduced 24.Fatty acid metabolism altered 25.Female sterile 26.Fenthion susceptible 27.Fertility altered 28.Fiber quality altered 29.Flower and fruit abscission reduced 30.Flower and fruit set altered 31.Flowering altered 32.Flower color altered 33.Flowering time altered 34.Fruit firmness increased 35.Fruit pectin esterase and levels decreased 36.Fruit polygalacturonase level decreased 37.Fruit ripening altered 38.Fruit ripening delayed 39.Fruit solids increased 40.Fruit sugar profile altered 41.Fruit sweetness increased 42.Glucuronidase expressing 43.Growth rate altered 44.Growth rate increased 45.Growth rate reduced 46.Heat stable glucanase produced 47.Heat tolerant 48.Heavy metals sequestered 49.Hordothionin produced 50.Improved fruit quality 51.Increased phosphorus 52.Increased stalk strength 53.Industrial enzyme produced 54.Lignin levels decreased 55.Lipase expressed in seeds 56.Lysine level increased 57.Male sterile 58.Male sterile reversible 59.Methionine level increased 60.Modified growth characteristics 61.Mycotoxin degradation 62.Nitrogen metabolism altered 63.Nucleocapsid protein produced 64.Oil profile altered 65.Oil quality altered 66.Oxidative stress tolerant 67.Pectin esterase level reduced 68.Pharmaceutical proteins produced 69.Photosynthesis enhanced 70.Phytoene synthase activity increased 71.Pigment metabolism altered 72.Polyamine metabolism altered 73.Polygalacturonase level reduced 74.Pratylenchus vulnus resistant 75.Processing characteristics altered 76.Prolonged shelf life 77.Protein altered 78.Protein lysine level increased 79.Protein quality altered 80.Proteinase inhibitors level constitutive 81.Salt tolerance increased 82.Seed composition altered 83.Seed methionine storage increased 84.Seed set reduced 85.Selectable marker 86.Senescence altered 87.Shorter stems 88.Solids increased 89.Starch level increased 90.Starch metabolism altered 91.Starch reduced 92.Sterols increased 93.Storage protein altered 94.Stress tolerant 95.Sugar alcohol levels increased 96.Tetracycline binding protein produced 97.Thermostable protein produced 98.Transposon activator 99.Transposon inserted 100.Tyrosine level increased 101.Visual marker 102.Vivipary increased 103.Yield increased Table 2 - Part 5.Non-limiting examples of traits/phenotypes with product quality properties 1.2,4-D tolerant 2.ACC oxidase level decreased 3.Altered amino acid composition 4.Altered lignin biosynthesis 5.Anthocyanin produced in seed 6.Antioxidant enzyme increased 7.Auxin metabolism and increased tuber solids 8.B-1,4-endoglucanase 9.Blackspot bruise resistant 10.Brown spot resistant 11.Bruising reduced 12.Caffeine levels reduced 13.Carbohydrate metabolism altered 14.Carotenoid content altered 15.Cell wall altered 16.Cold tolerant 17.Delayed softening 18.Disulfides reduced in endosperm 19.Dry matter content increased 20.Ear mold resistant 21.Ethylene production reduced 22.Ethylene synthesis reduced 23.Extended flower life 24.Fatty acid metabolism altered 25.Fiber quality altered 26.Fiber strength altered 27.Flavor enhancer 28.Flower and fruit abscission reduced 29.Fruit firmness increased 30.Fruit invertase level decreased 31.Fruit polygalacturonase level decreased 32.Fruit ripening altered 33.Fruit ripening delayed 34.Fruit solids increased 35.Fruit sugar profile altered 36.Fruit sweetness increased 37.Glyphosate tolerant 38.Heat stable glucanase produced 39.Improved fruit quality 40.Increased phosphorus 41.Increased protein levels 42.Lignin levels decreased 43.Lysine level increased 44.Male sterile 45.Melanin produced in cotton fibers 46.Metabolism altered 47.Methionine level increased 48.Mycotoxin degradation 49.Mycotoxin production inhibited 50.Nicotine levels reduced 51.Nitrogen metabolism altered 52.Novel protein produced 53.Nutritional quality altered 54.Oil profile altered 55.Oil quality altered 56.Pectin esterase level reduced 57.Photosynthesis enhanced 58.Phytoene synthase activity increased 59.Pigment metabolism altered 60.Polyamine metabolism altered 61.Polygalacturonase level reduced 62.Processing characteristics altered 63.Prolonged shelf life 64.Protein altered 65.Protein lysine level increased 66.Protein quality altered 67.Proteinase inhibitors level constitutive 68.Rust resistant 69.Seed composition altered 70.Seed methionine storage increased 71.Seed number increased 72.Seed quality altered 73.Seed set reduced 74.Seed weight increased 75.Senescence altered 76.Solids increased 77.Starch level increased 78.Starch metabolism altered 79.Starch reduced 80.Steroidal glycoalkaloids reduced 81.Sterols increased 82.Storage protein altered 83.Sugar alcohol levels increased 84.Thermostable protein produced 85.Tryptophan level increased 86.Tuber solids increased 87.Yield increased Table 2 - Part 6.Non-limiting examples of traits/phenotypes with herbicide tolerance properties 1.2,4-D tolerant 2.Chloroacetanilide tolerant 3.Fertility altered 4.Protein altered 5.Lignin levels decreased 6.Methionine level increased 7.Bromoxynil tolerant 8.Metabolism altered 9.Imidazole tolerant 10.Imidazolinone tolerant 11.Sulfonylurea tolerant 12.Northern corn leaf blight resistant 13.Herbicide tolerant 14.Isoxazole tolerant 15.Chlorsulfuron tolerant 16.Glyphosate tolerant 17.Lepidopteran resistant 18.Phosphinothricin tolerant 19.Sulfonylurea tolerant Table 2 - Part 7.Non-limiting examples of traits/phenotypes with pest resistance properties 1.Agrobacterium resistant - BR 2.Alternaria resistant - FR 3.Alternaria daucii resistant - FR 4.Alternaria solani resistant - FR 5.AMV resistant - VR 6.Anthracnose resistant - FR 7.Aphid resistant - IR 8.Apple scab resistant - FR 9.Aspergillus resistant - FR 10.Bacterial leaf blight resistant - BR 11.Bacterial resistant - BR 12.Bacterial soft rot resistant - BR 13.Bacterial soft rot resistant - VR 14.Bacterial speck resistant - BR 15.BCTV resistant - VR 16.Black shank resistant - FR 17.BLRV resistant - VR 18.BNYVV resistant - VR 19.Botrytis cinerea resistant - FR 20.Botrytis resistant - FR 21.BPMV resistant - VR 22.Brown spot resistant - FR 23.BYDV resistant - VR 24.BYMV resistant - VR 25.CaMV resistant - VR 26.Cercospora resistant - FR 27.Clavibacter resistant - BR 28.Closteroviurs resistant - BR 29.CLRV resistant - VR 30.CMV resistant - FR 31.Coleopteran resistant - IR 32.Colletotrichum resistant - FR 33.Colorado potato beetle resistant - IR 34.Corn earworm resistant - IR 35.Corynebacterium sepedonicum resistant - BR 36.Cottonwood leaf beetle resistant - IR 37.Criconnemella resistant - NR 38.Crown gall resistant - BR 39.Cucumovirus resistant - VR 40.Cylindrosporium resistant - FR 41.Disease resistant general - FR 42.Dollar spot resistant - FR 43.Downy mildew resistant - FR 44.Ear mold resistant - FR 45.Erwinia carotovora resistant - BR 46.European Corn Borer resistant - IR 47.Eyespot resistant - FR 48.Fall armyworm resistant - IR 49.Fire blight resistant - BR 50.Frogeye leaf spot resistanT - FR 51.Fruit rot resistant - FR 52.Fungal post-harvest resistant - FR 53.Fungal resistant - FR 54.Fungal resistant general - FR 55.Fusarium dehlae resistant - FR 56.Fusarium resistant - FR 57.Geminivirus resistant - VR 58.Gray lead spot resistant - FR 59.Helminthosporium resistant - FR 60.Hordothionin produced - BR 61.Insect predator resistant - IR 62.Insect resistant general - IR 63.Late blight resistant - FR 64.Leaf blight resistant - FR 65.Leaf spot resistant - FR 66.Lepidopteran resistant - IR 67.Lesser cornstalk borer resistant - IR 68.LMV resistant - VR 69.Loss of systemic resistance - VR 70.Marssonina resistant - FR 71.MCDV resistant - VR 72.MCMV resistant - VR 73.MDMV resistant - VR 74.MDMV-B resistant - VR 75.Mealybug wilt virus resistant - VR 76.Melamtsora resistant - FR 77.Melodgyne resistant - NR 78.Meloidogyne resistant - NR 79.Mexican Rice Borer resistant - IR 80.Mycotoxin degradation - FR 81.Nepovirus resistant - VR 82.Northern corn leaf blight resistant - IR 83.Nucleocapsid protein produced - VR 84.Oblique banded leafroller resistant - IR 85.Oomycete resistant - FR 86.Pathogenesis related proteins level increased - FR 87.PEMV resistant - VR 88.PeSV Resistant - VR 89.Phatora leaf beetle resistant - IR 90.Phoma resistant - FR 91.Phytophthora resistant - FR 92.PLRV resistant - VR 93.Potyvirus resistant - VR 94.Powdery mildew resistant - FR 95.PPV resistant - VR 96.Pralylenchus vulnus resistant - NR 97.PRSV resistant - VR 98.PRV resistant - VR 99.PSbMV resistant - VR 100.Pseudomonas syringae resistant - BR 101.PStV resistant - VR 102.PVX resistant - VR 103.PVY resistant - VR 104.RBDV resistant - VR 105.Rhizoctonia resistant - FR 106.Rhizoctonia solani resistant - FR 107.Ring rot resistance - BR 108.Root-knot nematode resistant - NR 109.Rust resistant - FR 110.SbMV resistant - VR 111.Sclerotinia resistant - FR 112.SCMV resistant - VR 113.SCYLV resistant - VR 114.Septoria resistant - FR 115.Smut resistant - FR 116.SMV resistant - VR 117.Sod web worm resistant - IR 118.Soft rot fungal resistant - FR 119.Soft rot resistant - BR 120.Southwestern corn borer resistant- IR 121.SPFMV resistant - VR 122.Sphaeropsis fruit rot resistant - FR 123.SqMV resistant - VR 124.SrMV resistant - VR 125.Streptomyces scabie s resistant - BR 126.Sugar cane borer resistant - IR 127.TEV resistant - VR 128.Thelaviopsis resistant - FR 129.TMV resistant - FR 130.Tobamovirus resistant - VR 131.ToMoV resistant - VR 132.ToMV resistant - VR 133.TRV resistant - VR 134.TSWV resistant - VR 135.TVMV resistant - VR 136.TYLCV resistant - VR 137.Venturia resistant - FR 138.Verticillium dahliae resistant - FR 139.Verticillium resistant - FR 140.Western corn root worm resistant - IR 141.WMV2 resistant - VR 142.WSMV resistant - VR 143.ZYMV resistant - VR Table 2 - Part 8.Non-limiting examples of miscellaneous traits/ phenotypes with properties 1.Antibiotic produced 2.Antiprotease producing 3.Capable of growth on defined synthetic media 4.Carbohydrate metabolism altered 5.Cell wall altered 6.Cold tolerant 7.Coleopteran resistant 8.Color altered 9.Color sectors in seeds 10.Colored sectors in leaves 11.Constitutive expression of glutaminc synthetase 12.Cre recombinase produced 13.Dalapon tolerant 14.Development altered 15.Disease resistant general 16.Ethylene metabolism altered 17.Expression optimization 18.Fenthion susceptible 19.Glucuronidase expressing 20.Glyphosate tolerant 21.Growth rate reduced 22.Heavy metals sequestered 23.Hygromycin tolerant 24.Inducible DNA modification 25.Industrial enzyme produced 26.Kanamycin resistant 27.Lipase expressed in seeds 28.Methotrexate resistant 29.Modified growth characteristics 30.Mycotoxin deficient 31.Mycotoxin production inhibited 32.Mycotoxin restored 33.Non-lesion forming mutant 34.Novel protein produced 35.Oil quality altered 36.Peroxidase levels increased 37.Pharmaceutical proteins produced 38.Phosphinothricin tolerant 39.Pigment metabolism altered 40.Pollen visual marker 41.Polyamine metablosim altered 42.Polymer produced 43.Recombinase produced 44.Secondary metabolite increased 45.Seed color altered 46.Seed weight increased 47.Selectable marker 48.Spectromycin resistant 49.Sterile 50.Sterols increased 51.Sulfonylurea susceptible 52.Syringomycin deficient 53.Transposon activator 54.Transposon elements inserted 55.Transposon inserted 56.Trifolitoxin producing 57.Trifolitoxin resistant 58.Virulence reduced 59.Visual marker 60.Visual marker inactive Legend BR—Bacterial Resistant FR—Fungal Resistant IR—Insent Resistant NR—Nematode Resistant VR—Viral Resistant In a particular examplification, “producing an organism having a desirable trait” includes an organism that is with respect to an organ or a part of an organ but not necessarily altered anywhere else.", "By “trait” is meant any detectable parameter associated with an organism under a set of conditions.", "Examples of “detectable parameters” include the ability to produce a substance, the ability to not produce a substance, an altered pattern of (such as an increased or a decreased) ability to produce a substance, viability, non-viability, behaviour, growth rate, size, morphology or morphological characteristic, In another embodiment, this invention is directed to a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining an initial population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one initial organism, c) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and d) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "A mutagenized organism having a desirable trait or a desirable improvement in a trait can be referred to as an “up-mutant”, and the associated mutation(s) contained in an up-mutant organism can be referred to as up-mutation(s).", "In one embodiment, step c) is comprised of selecting at least two different mutagenized organisms, each having a different mutagenized genome, and the method of producing an organism having a desirable trait or a desirable improvement in a trait is comprised of a) obtaining a starting population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one starting organism, c) selecting at least two mutagenized organism having a desirable trait or a desirable improvement in a trait, d) creating combinations of the mutations of the two or more mutagenized organisms, e) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and f) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "In one embodiment, the method is repeated.", "Thus, for example, an up-mutant organism can serve as a starting organism for the above method.", "Also, for example, an up mutant organism having a combination of two or more up-mutations in its genome can serve as a starting organism for the above method.", "Thus, in one embodiment, this invention is directed to a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining a starting population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one starting organism, c) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and d) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "A mutagenized organism having a desirable trait or a desirable improvement in a trait can be referred to as an “up-mutant”, and the associated mutation(s) contained in an up-mutant organism can be referred to as up-mutation(s).", "Mutagenizing a starting population such that mutations occur throughout a substantial part of the genome of at least one starting organism refers to mutagenizing at least approximately 1% of the genes of a genome, or at least approximately 10% of the genes of a genome, or at least approximately 20% of the genes of a genome, or at least approximately 30% of the genes of a genome, or at least approximately 40% of the genes of a genome, or at least approximately 50% of the genes of a genome, or at least approximately 60% of the genes of a genome, or at least approximately 70% of the genes of a genome, or at least approximately 80% of the genes of a genome, or at least approximately 90% of the genes of a genome, or at least approximately 95% of the genes of a genome, or at least approximately 98% of the genes of a genome.", "In a particular embodiment, this invention provides a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining sequence information of a genome; b) annotating the genomic sequence obtained; c) mutagenizing a substantial part of the genome the genome; d) selecting at least one mutagenized genome having a desirable trait or a desirable improvement in a trait; and e) optionally repeating the method by subjecting one or more mutagenized genomes to a repetition of the method.", "Thus in one aspect, this invention provides a process comprised of: 1.)", "Subjecting a working cell or organism to holistic monitoring (which can include the detection and/or measurement of all detectable functions and physical parameters).", "Examples of such parameters include morphology, behavior, growth, responsiveness to stimuli (e.g., antibiotics, different environment, etc.).", "Additional examples include all measurable molecules, including molecules that are chemically at least in part a nucleic acids, proteins, carbohydrates, proteoglycans, glycoproteins, or lipids.", "In a particular aspect, performing holistic monitoring is comprised of using a microarray-based method.", "In another aspect, performing holistic monitoring is comprised of sequencing a substantial portion of the genome, i.e.", "for example at least approximately 10% of the genome, or for example at least approximately 20% of the genome, or for example at least approximately 30% of the genome, or for example at least approximately 40% of the genome, or for example at least approximately 50% of the genome, or for example at least approximately 60% of the genome, or for example at least approximately 70% of the genome, or for example at least approximately 80% of the genome, or for example at least approximately 90% of the genome, or for example at least approximately 95% of the genome, or for example at least approximately 98% of the genome.", "2) Introducing into the working cell or organism a plurality of traits (stacked traits), including selectively and differentially activatable traits.", "Serviceable traits for this purpose include traits conferred by genes and traits conferred by gene pathways.", "3) Subjecting the working cell or organism to holistic monitoring.", "4) Compiling the information obtained from steps 1) and 3), and processing &/or analyzing it to better understand the changes introduced into the working cell or organisms.", "Such data processing includes identifying correlations between and/or among the measured parameters.", "5) Repeating any number or all of steps 2), 3), and 4).", "This invention provides that molecules serviceable for introducing transgenic traits into a plant include all known genes and nucleic acids.", "By way of non-limiting exemplification, this invention specifically names any number &/or combination of genes listed herein or listed in any reference incorporated herein by reference.", "Furthermore, by way of non-limiting exemplification, this invention specifically names any number &/or combination of genes & gene pathways listed herein as well as in any reference incorporated by reference herein.", "This invention provides that molecules serviceable as detectable parameters include molecule, any enzyme, substrate thereof, product thereof, and any gene or gene pathway listed herein including in any figure or table herein as well as in any reference incorporated by reference herein.", "This invention also relates generally to the field of nucleic acid engineering and correspondingly encoded recombinant protein engineering.", "More particularly, the invention relates to the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for resultant activity(ies) of interest, such nucleic acid activity(ies) &/or specified protein, particularly enzyme, activity(ies) of interest.", "Mutagenized molecules provided by this invention may have chimeric molecules and molecules with point mutations, including biological molecules that contain a carbohydrate, a lipid, a nucleic acid, &/or a protein component, and specific but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroidal and non-steroidal hormones.", "This invention relates generally to a method of: 1) preparing a progeny generation of molecule(s) (including a molecule that is comprised of a polynucleotide sequence, a molecule that is comprised of a polypeptide sequence, and a molecules that is comprised in part of a polynucleotide sequence and in part of a polypeptide sequence), that is mutagenized to achieve at least one point mutation, addition, deletion, &/or chimerization, from one or more ancestral or parental generation template(s); 2) screening the progeny generation molecule(s)—preferably using a high throughput method—for at least one property of interest (such as an improvement in an enzyme activity or an increase in stability or a novel chemotherapeutic effect); 3) optionally obtaining &/or cataloguing structural &/or and functional information regarding the parental &/or progeny generation molecules; and 4) optionally repeating any of steps 1) to 3).", "In a preferred embodiment, there is generated (e.g.", "from a parent polynucleotide template)—in what is termed “codon site-saturation mutagenesis”—a progeny generation of polynucleotides, each having at least one set of up to three contiguous point mutations (i.e.", "different bases comprising a new codon), such that every codon (or every family of degenerate codons encoding the same amino acid) is represented at each codon position.", "Corresponding to—and encoded by—this progeny generation of polynucleotides, there is also generated a set of progeny polypeptides, each having at least one single amino acid point mutation.", "In a preferred aspect, there is generated—in what is termed “amino acid site-saturation mutagenesis”—one such mutant polypeptide for each of the 19 naturally encoded polypeptide-forming alpha-amino acid substitutions at each and every amino acid position along the polypeptide.", "This yields—for each and every amino acid position along the parental polypeptide—a total of 20 distinct progeny polypeptides including the original amino acid, or potentially more than 21 distinct progeny polypeptides if additional amino acids are used either instead of or in addition to the 20 naturally encoded amino acids.", "Thus, in another aspect, this approach is also serviceable for generating mutants containing—in addition to &/or in combination with the 20 naturally encoded polypeptide-forming alpha-amino acids—other rare &/or not naturally-encoded amino acids and amino acid derivatives.", "In yet another aspect, this approach is also serviceable for generating mutants by the use of—in addition to &/or in combination with natural or unaltered codon recognition systems of suitable hosts—altered, mutagenized, &/or designer codon recognition systems (such as in a host cell with one or more altered tRNA molecules).", "In yet another aspect, this invention relates to recombination and more specifically to a method for preparing polynucleotides encoding a polypeptide by a method of in vivo re-assortment of polynucleotide sequences containing regions of partial homology, assembling the polynucleotides to form at least one polynucleotide and screening the polynucleotides for the production of polypeptide(s) having a useful property.", "In yet another preferred embodiment, this invention is serviceable for analyzing and cataloguing—with respect to any molecular property (e.g.", "an enzymatic activity) or combination of properties allowed by current technology—the effects of any mutational change achieved (including particularly saturation mutagenesis).", "Thus, a comprehensive method is provided for determining the effect of changing each amino acid in a parental polypeptide into each of at least 19 possible substitutions.", "This allows each amino acid in a parental polypeptide to be characterized and catalogued according to its spectrum of potential effects on a measurable property of the polypeptide.", "In another aspect, the method of the present invention utilizes the natural property of cells to recombine molecules and/or to mediate reductive processes that reduce the complexity of sequences and extent of repeated or consecutive sequences possessing regions of homology.", "It is an object of the present invention to provide a method for generating hybrid polynucleotides encoding biologically active hybrid polypeptides with enhanced activities.", "In accomplishing these and other objects, there has been provided, in accordance with one aspect of the invention, a method for introducing polynucleotides into a suitable host cell and growing the host cell under conditions that produce a hybrid polynucleotide.", "In another aspect of the invention, the invention provides a method for screening for biologically active hybrid polypeptides encoded by hybrid polynucleotides.", "The present method allows for the identification of biologically active hybrid polypeptides with enhanced biological activities.", "Other objects, features and advantages of the present invention will become apparent from the following detailed description.", "It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.", "In yet another aspect, this invention relates to a method of discovering which phenotype corresponds to a gene by disrupting every gene in the organism.", "Accordingly, this invention provides a method for determining a gene that alters a characteristic of an organism, comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence an organism having an altered trait, and d) determining the nucleotide sequence of a gene that has been mutagenized in the organism having the altered trait.", "In yet another aspect, this invention relates to a method of improving a trait in an organism by functionally knocking out a particular gene in the organism, and then transferring a library of genes, which only vary from the wild-type at one codon position, into the organism.", "Accordingly, this invention provides a method method for producing an organism with an improved trait, comprising: a) functionally knocking out an enogenous gene in a substantially clonal population of organisms; b) transferring the set of altered genes into the clonal population of organisms, wherein each altered gene differs from the endogenous gene at only one codon; and c) detecting a mutagenized organism having an improved trait; and d) determining the nucleotide sequence of a gene that has been transferred into the detected organism." ], [ "A—FIELD OF THE INVENTION This invention relates to the field of cellular and whole organism engineering.", "Specifically, this invention relates to a cellular transformation, directed evolution, and screening method for creating novel transgenic organisms having desirable properties.", "Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are differentially activatable.", "This invention also relates to the field of protein engineering.", "Specifically, this invention relates to a directed evolution method for preparing a polynucleotide encoding a polypeptide.", "More specifically, this invention relates to a method of using mutagenesis to generate a novel polynucleotide encoding a novel polypeptide, which novel polypeptide is itself an improved biological molecule &/or contributes to the generation of another improved biological molecule.", "More specifically still, this invention relates to a method of performing both non-stochastic polynucleotide chimerization and non-stochastic site-directed point mutagenesis.", "Thus, in one aspect, this invention relates to a method of generating a progeny set of chimeric polynucleotide(s) by means that are synthetic and non-stochastic, and where the design of the progeny polynucleotide(s) is derived by analysis of a parental set of polynucleotides &/or of the polypeptides correspondingly encoded by the parental polynucleotides.", "In another aspect this invention relates to a method of performing site-directed mutagenesis using means that are exhaustive, systematic, and non-stochastic.", "Furthermore this invention relates to a step of selecting from among a generated set of progeny molecules a subset comprised of particularly desirable species, including by a process termed end-selection, which subset may then be screened further.", "This invention also relates to the step of screening a set of polynucleotides for the production of a polypeptide &/or of another expressed biological molecule having a useful property.", "Novel biological molecules whose manufacture is taught by this invention include genes, gene pathways, and any molecules whose expression is affected thereby, including directly encoded polypetides &/or any molecules affected by such polypeptides.", "Said novel biological molecules include those that contain a carbohydrate, a lipid, a nucleic acid, &/or a protein component, and specific but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroidal and non-steroidal hormones.", "In a particular non-limiting aspect, the present invention relates to enzymes, particularly to thermostable enzymes, and to their generation by directed evolution.", "More particularly, the present invention relates to thermostable enzymes which are stable at high temperatures and which have improved activity at lower temperatures.", "B—BACKGROUND General Overview of the Problem to be Solved Brief Summary: It is instantly appreciated that the process of performing a genetic manipulation on a organism to achieve a genetic alteration, whether it is on a unicellular or on a multi-cellular organism, can lead to harmful, toxic, noxious, or even lethal effects on the manipulated organism.", "This is particularly true when the genetic manipulation becomes sizable.", "From a technical point of view, this problem is seen as one of the current obstacles that hinder the creation of genetically altered organisms having a large number of transgenic traits.", "On the marketing side, is instantly appreciated that the purchase price of a genetically altered organism is often dictated by, or proportional to, the number of transgenic traits that have been introduced into the organism.", "Consequently, a genetically altered organism having a large number of stacked transgenic traits can be quite costly to produce and purchase and economically in low demand.", "On the other hand, the generation of organism having but a single genetically introduced trait can also lead to the incurrence of undesirable costs, although for other reasons.", "It is thus appreciated that the separate production, marketing, & storage of genetically altered organisms each having a single transgenic traits can incur costs, including inventory costs, that are undesirable.", "For example, the storage of such organisms may require a separate bin to be used for each trait.", "Furthermore, the value of an organisms having a single particular trait is often intimately tied to the marketability of that particular trait, and when that marketability diminishes, inventories of such organisms cannot be sold in other markets.", "The instant invention solves these and other problems by providing a method of producing genetically altered organisms having a large number of stacked traits that are differentially activatable.", "Upon purchasing such a genetically altered organism (having a large number of differentially activatable stacked traits), the purchasing customer has the option of selecting and paying for particular traits among the total that can then be activated differentially.", "One economic advantage provided by this invention is that the storage of such genetically altered organisms is simplified since, for example, one bin could be used to store a large number of traits.", "Moreover, a single organism of this type can satisfy the demands for a variety of traits; consequently, such an organism can be sold in a variety of markets.", "To achieve the production of genetically altered organisms having a large number of stacked traits that are differentially activatable, this invention provides—in one specific aspect—a process comprising the step of monitoring a cell or organism at holistic level.", "This serves as a way of collecting holistic—rather than isolated—information about a working cell or organism that is being subjected to a substantial amount of genetic manipulation.", "This invention further provides that this type of holistic monitoring can include the detection of all morphological, behavioral, and physical parameters.", "Accordingly, the holistic monitoring provided by this invention can include the identification &/or quantification of all the genetic material contained in a working cell or organism (e.g.", "all nucleic acids including the entire genome, messenger RNA's, tRNA's, rRNA's, and mitochondrial nucleic acids, plasmids, phages, phagemids, viruses, as well as all episomal nucleic acids and endosymbiont nucleic acids).", "Furthermore this invention provides that this type of holistic monitoring can include all gene products produced by the working cell or organisms.", "Furthermore, the holistic monitoring provided by this invention can include the identification &/or quantification of all molecules that are chemically at least in part protein in a working cell or organism.", "The holistic monitoring provided by this invention can also include the identification &/or quantification of all molecules that are chemically at least in part carbohydrate in a working cell or organism.", "The holistic monitoring provided by this invention can also include the identification &/or quantification of all molecules that are chemically at least in part proteoglycan in a working cell or organism.", "The holistic monitoring provided by this invention can also include the identification &/or quantification of all molecules that are chemically at least in part glycoprotein in a working cell or organism.", "The holistic monitoring provided by this invention can also include the identification &/or quantification of all molecules that are chemically at least in part nucleic acids in a working cell or organism.", "The holistic monitoring provided by this invention can also include the identification &/or quantification of all molecules that are chemically at least in part lipids in a working cell or organism.", "In one aspect, this invention provides that the ability to differentially activate a trait from among many, such as a enzyme from among many enzymes, depends the enzyme(s) to be activated having a unique activity profile (or activity fingerprint).", "An enzyme's activity profile includes the reaction(s) it catalyzes and its specificity.", "Thus, an enzymes activity profile includes its: Catalyzed reaction(s) Reaction type Natural substrate(s) Substrate spectrum Product spectrum Inhibitor(s) Cofactor(s)/prostetic group(s) Metal compounds/salts that affect it Turnover number Specific activity Km value pH optimum pH range Temperature optimum Temperature range It is also instantly appreciated that enzymes are differentially affected by exposure to varying degrees of processing (e.g.", "upon extraction &/or purification) and exposure (e.g.", "to suboptimal storage conditions).", "Accordingly, enzyme differences may surface after exposure to: Isolation/Preparation Purification Crystallization Renaturation It is instantly appreciated that differences in molecular stability can also be used advantageously to differentially activate or inactivate selected enzymes, by exposing the enzymes for an appropriate time to variations in: pH Temperature Oxidation Organic solvent(s) Miscellaneous storage conditions It is thus appreciated that in order to be able to differentially activate selected traits among a plurality of stacked traits, it is desirable to introduce into a working cell or organism traits conferred by molecules (e.g.", "enzymes) having very unique profiles (e.g.", "unique enzyme fingerprints).", "Furthermore, it is appreciated that in order to obtain the molecules having a representation of a wide range of molecular fingerprints, it is advantageous to harvest molecules from the widest possible reaches nature's diversity.", "Thus, it is beneficial to harvest molecules not only from cultured mesophilic organisms, but also from extremophiles that are largely uncultured.", "In another aspect, it is instantly appreciated that harvesting the full potential of nature's diversity can include both the step of discovery and the step of optimizing what is discovered.", "For example, the step of discovery allows one to mine biological molecules that have commercial utility.", "It is instantly appreciated that the ability to harvest the full richness of biodiversity, i.e.", "to mine biological molecules from a wide range of environmental conditions, is critical to the ability to discover novel molecules adapted to function under a wide variety of conditions, including extremes of conditions, such as may be found in a commercial application.", "However, it is also instantly appreciated that only occassionally are there criteria for selection &/or survival in nature that point in the exact direction of particular commercial needs.", "Instead, it is often the case that a naturally occurring molecule will require a certain amount of change—from fine tuning to sweeping modification—in order to fulfill a particular unmet commercial need.", "Thus, to meet certain commercial needs (e.g., a need for a molecule that is fucntional under a specific set of commercial processing conditions) it is sometimes advantageous to experimentally modify a naturally expresed molecule to achieve properties beyond what natural evolution has provided &/or is likely to provide in the near future.", "The approach, termed directed evolution, of experimentally modifying a biological molecule towards a desirable property, can be achieved by mutagenizing one or more parental molecular templates and by idendifying any desirable molecules among the progeny molecules.", "Currently available technologies in directed evolution include methods for achieving stochastic (i.e.", "random) mutagenesis and methods for achieving non-stochastic (non-random) mutagenesis.", "However, critical shortfalls in both types of methods are identified in the instant disclosure.", "In prelude, it is noteworthy that it may be argued philosophically by some that all mutagenesis—if considered from an objective point of view—is non-stochastic; and furthermore that the entire universe is undergoing a process that—if considered from an objective point of view—is non-stochastic.", "Whether this is true is outside of the scope of the instant consideration.", "Accordingly, as used herein, the terms “randomness”, “uncertainty”, and “unpredictability” have subjective meanings, and the knowledge, particularly the predictive knowledge, of the designer of an experimental process is a determinant of whether the process is stochastic or non-stochastic.", "By way of illustration, stochastic or random mutagenesis is exemplified by a situation in which a progenitor molecular template is mutated (modified or changed) to yield a set of progeny molecules having mutation(s) that are not predetermined.", "Thus, in an in vitro stochastic mutagenesis reaction, for example, there is not a particular predetermined product whose production is intended; rather there is an uncertainty—hence randomness—regarding the exact nature of the mutations achieved, and thus also regarding the products generated.", "In contrast, non-stochastic or non-random mutagenesis is exemplified by a situation in which a progenitor molecular template is mutated (modified or changed) to yield a progeny molecule having one or more predetermined mutations.", "It is appreciated that the presence of background products in some quantity is a reality in many reactions where molecular processing occurs, and the presence of these background products does not detract from the non-stochastic nature of a mutagenesis process having a predetermined product.", "Thus, as used herein, stochastic mutagenesis is manifested in processes such as error-prone PCR and stochastic shuffling, where the mutation(s) achieved are random or not predetermined.", "In contrast, as used herein, non-stochastic mutagenesis is manifested in instantly disclosed processes such as gene site-saturation mutagenesis and synthetic ligation reassembly, where the exact chemical structure(s) of the intended product(s) are predetermined.", "In brief, existing mutagenesis methods that are non-stochastic have been serviceable in generating from one to only a very small number of predetermined mutations per method application, and thus produce per method application from one to only a few progeny molecules that have predetermined molecular structures.", "Moreover, the types of mutations currently available by the application of these non-stochastic methods are also limited, and thus so are the types of progeny mutant molecules.", "In contrast, existing methods for mutagenesis that are stochastic in nature have been serviceable for generating somewhat larger numbers of mutations per method application—though in a random fashion & usually with a large but unavoidable contingency of undesirable background products.", "Thus, these existing stochastic methods can produce per method application larger numbers of progeny molecules, but that have undetermined molecular structures.", "The types of mutations that can be achieved by application of these current stochastic methods are also limited, and thus so are the types of progeny mutant molecules.", "It is instantly appreciated that there is a need for the development of non-stochastic mutagenesis methods that: 1) Can be used to generate large numbers of progeny molecules that have predetermined molecular structures; 2) Can be used to readily generate more types of mutations; 3) Can produce a correspondingly larger variety of progeny mutant molecules; 4) Produce decreased unwanted background products; 5) Can be used in a manner that is exhaustive of all possibilities; and 6) Can produce progeny molecules in a systematic & non-repetitive way.", "The instant invention satisfies all of these needs.", "Directed Evolution Supplements Natural Evolution: Natural evolution has been a springboard for directed or experimental evolution, serving both as a reservoir of methods to be mimicked and of molecular templates to be mutagenized.", "It is appreciated that, despite its intrinsic process-related limitations (in the types of favored &/or allowed mutagenesis processes) and in its speed, natural evolution has had the advantage of having been in process for millions of years & and throughout a wide diversity of environments.", "Accordingly, natural evolution (molecular mutagenesis and selection in nature) has resulted in the generation of a wealth of biological compounds that have shown usefulness in certain commercial applications.", "However, it is instantly appreciated that many unmet commercial needs are discordant with any evolutionary pressure &/or direction that can be found in nature.", "Moreover, it is often the case that when commercially useful mutations would otherwise be favored at the molecular level in nature, natural evolution often overrides the positive selection of such mutations, e.g.", "when there is a concurrent detriment to an organism as a whole (such as when a favorable mutation is accompanied by a detrimental mutation).", "Additionally, natural evolution is often slow, and favors fidelity in many types of replication.", "Additionally still, natural evolution often favors a path paved mainly by consecutive beneficial mutations while tending to avoid a plurality of successive negative mutations, even though such negative mutations may prove beneficial when combined, or may lead—through a circuitous route—to final state that is beneficial.", "Moreover, natural evolution advances through specific steps (e.g.", "specific mutagenesis and selection processes), with avoidance of less favored steps.", "For example, many nucleic acids do not reach close enough proximity to each other in a operative environment to undergo chimerization or incorporation or other types of transfers from one species to another.", "Thus, e.g., when sexual intercourse between 2 particular species is avoided in nature, the chimerization of nucleic acids from these 2 species is likewise unlikely, with parasites common to the two species serving as an example of a very slow passageway for inter-molecular encounters and exchanges of DNA.", "For another example, the generation of a molecule causing self-toxicity or self-lethality or sexual sterility is avoided in nature.", "For yet another example, the propagation of a molecule having no particular immediate benefit to an organism is prone to vanish in subsequent generations of the organism.", "Furthermore, e.g., there is no selection pressure for improving the performance of molecule under conditions other than those to which it is exposed in its endogenous environment; e.g.", "a cytoplasmic molecule is not likely to acquire functional features extending beyond what is required of it in the cytoplasm.", "Furthermore still, the propagation of a biological molecule is susceptible to any global detrimental effects—whether caused by itself or not—on its ecosystem.", "These and other characteristics greatly limit the types of mutations that can be propagated in nature.", "On the other hand, directed (or experimental) evolution—particularly as provided herein—can be performed much more rapidly and can be directed in a more streamlined manner at evolving a predetermined molecular property that is commercially desirable where nature does not provide one &/or is not likely to provide.", "Moreover, the directed evolution invention provided herein can provide more wide-ranging possibilities in the types of steps that can be used in mutagenesis and selection processes.", "Accordingly, using templates harvested from nature, the instant directed evolution invention provides more wide-ranging possibilities in the types of progeny molecules that can be generated and in the speed at which they can be generated than often nature itself might be expected to in the same length of time.", "In a particular exemplification, the instantly disclosed directed evolution methods can be applied iteratively to produce a lineage of progeny molecules (e.g.", "comprising successive sets of progeny molecules) that would not likely be propagated (i.e., generated &/or selected for) in nature, but that could lead to the generation of a desirable downstream mutagenesis product that is not achievable by natural evolution.", "Previous Directed Evolution Methods are Suboptimal: Mutagenesis has been attempted in the past on many occasions, but by methods that are inadequate for the purpose of this invention.", "For example, previously described non-stochastic methods have been serviceable in the generation of only very small sets of progeny molecules (comprised often of merely a solitary progeny molecule).", "By way of illustration, a chimeric gene has been made by joining 2 polynucleotide fragments using compatible sticky ends generated by restriction enzyme(s), where each fragment is derived from a separate progenitor (or parental) molecule.", "Another example might be the mutagenesis of a single codon position (i.e.", "to achieve a codon substitution, addition, or deletion) in a parental polynucleotide to generate a single progeny polynucleotide encoding for a single site-mutagenized polypeptide.", "Previous non-stochastic approaches have only been serviceable in the generation of but one to a few mutations per method application.", "Thus, these previously described non-stochastic methods thus fail to address one of the central goals of this invention, namely the exhaustive and non-stochastic chimerization of nucleic acids.", "Accordingly previous non-stochastic methods leave untapped the vast majority of the possible point mutations, chimerizations, and combinations thereof, which may lead to the generation of highly desirable progeny molecules.", "In contrast, stochastic methods have been used to achieve larger numbers of point mutations and/or chimerizations than non-stochastic methods; for this reason, stochastic methods have comprised the predominant approach for generating a set of progeny molecules that can be subjected to screening, and amongst which a desirable molecular species might hopefully be found.", "However, a major drawback of these approaches is that—because of their stochastic nature—there is a randomness to the exact components in each set of progeny molecules that is produced.", "Accordingly, the experimentalist typically has little or no idea what exact progeny molecular species are represented in a particular reaction vessel prior to their generation.", "Thus, when a stochastic procedure is repeated (e.g.", "in a continuation of a search for a desirable progeny molecule), the re-generation and re-screening of previously discarded undesirable molecular species becomes a labor-intensive obstruction to progress, causing a circuitous—if not circular—path to be taken.", "The drawbacks of such a highly suboptimal path can be addressed by subjecting a stochastically generated set of progeny molecules to a labor-incurring process, such as sequencing, in order to identify their molecular structures, but even this is an incomplete remedy.", "Moreover, current stochastic approaches are highly unsuitable for comprehensively or exhaustively generating all the molecular species within a particular grouping of mutations, for attributing functionality to specific structural groups in a template molecule (e.g.", "a specific single amino acid position or a sequence comprised of two or more amino acids positions), and for categorizing and comparing specific grouping of mutations.", "Accordingly, current stochastic approaches do not inherently enable the systematic elimination of unwanted mutagenesis results, and are, in sum, burdened by too many inherently shortcomings to be optimal for directed evolution.", "In a non-limiting aspect, the instant invention addresses these problems by providing non-stochastic means for comprehensively and exhaustively generating all possible point mutations in a parental template.", "In another non-limiting aspect, the instant invention further provides means for exhaustively generating all possible chimerizations within a group of chimerizations.", "Thus, the aforementioned problems are solved by the instant invention.", "Specific shortfalls in the technological landscape addressed by this invention include: 1) Site-directed mutagenesis technologies, such as sloppy or low-fidelity PCR, are ineffective for systematically achieving at each position (site) along a polypeptide sequence the full (saturated) range of possible mutations (i.e.", "all possible amino acid substitutions).", "2) There is no relatively easy systematic means for rapidly analyzing the large amount of information that can be contained in a molecular sequence and in the potentially colossal number or progeny molecules that could be conceivably obtained by the directed evolution of one or more molecular templates.", "3) There is no relatively easy systematic means for providing comprehensive empirical information relating structure to function for molecular positions.", "4) There is no easy systematic means for incorporating internal controls, such as positive controls, for key steps in certain mutagenesis (e.g.", "chimerization) procedures.", "5) There is no easy systematic means to select for a specific group of progeny molecules, such as full-length chimeras, from among smaller partial sequences.", "An exceedingly large number of possibilities exist for the purposeful and random combination of amino acids within a protein to produce useful hybrid proteins and their corresponding biological molecules encoding for these hybrid proteins, i.e., DNA, RNA.", "Accordingly, there is a need to produce and screen a wide variety of such hybrid proteins for a desirable utility, particularly widely varying random proteins.", "The complexity of an active sequence of a biological macromolecule (e.g., polynucleotides, polypeptides, and molecules that are comprised of both polynucleotide and polypeptide sequences) has been called its information content (“IC”), which has been defined as the resistance of the active protein to amino acid sequence variation (calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function).", "Proteins that are more sensitive to random mutagenesis have a high information content.", "Molecular biology developments, such as molecular libraries, have allowed the identification of quite a large number of variable bases, and even provide ways to select functional sequences from random libraries.", "In such libraries, most residues can be varied (although typically not all at the same time) depending on compensating changes in the context.", "Thus, while a 100 amino acid protein can contain only 2,000 different mutations, 20100 sequence combinations are possible.", "Information density is the IC per unit length of a sequence.", "Active sites of enzymes tend to have a high information density.", "By contrast, flexible linkers of information in enzymes have a low information density.", "Current methods in widespread use for creating alternative proteins in a library format are error-prone polymerase chain reactions and cassette mutagenesis, in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide.", "In both cases, a substantial number of mutant sites are generated around certain sites in the original sequence.", "Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence.", "In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture.", "The published error-prone PCR protocols suffer from a low processivity of the polymerase.", "Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene.", "This inability limits the practical application of error-prone PCR.", "Some computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the large-scale block changes that are required for continued and dramatic sequence evolution.", "Further, the published error-prone PCR protocols do not allow for amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application.", "In addition, repeated cycles of error-prone PCR can lead to an accumulation of neutral mutations with undesired results, such as affecting a protein's immunogenicity but not its binding affinity.", "In oligonucleotide-directed mutagenesis, a short sequence is replaced with a synthetically mutagenized oligonucleotide.", "This approach does not generate combinations of distant mutations and is thus not combinatorial.", "The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization.", "Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping them into families, arbitrarily choosing a single family, and reducing it to a consensus motif.", "Such motif is re-synthesized and reinserted into a single gene followed by additional selection.", "This step process constitutes a statistical bottleneck, is labor intensive, and is not practical for many rounds of mutagenesis.", "Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine-tuning, but rapidly become too limiting when they are applied for multiple cycles.", "Another limitation of error-prone PCR is that the rate of down-mutations grows with the information content of the sequence.", "As the information content, library size, and mutagenesis rate increase, the balance of down-mutations to up-mutations will statistically prevent the selection of further improvements (statistical ceiling).", "In cassette mutagenesis, a sequence block of a single template is typically replaced by a (partially) randomized sequence.", "Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (i.e., library size).", "This eliminates other sequence families which are not currently best, but which may have greater long term potential.", "Also, mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round.", "Thus, such an approach is tedious and impractical for many rounds of mutagenesis.", "Thus, error-prone PCR and cassette mutagenesis are best suited, and have been widely used, for fine-tuning areas of comparatively low information content.", "One apparent exception is the selection of an RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection.", "In nature, the evolution of most organisms occurs by natural selection and sexual reproduction.", "Sexual reproduction ensures mixing and combining of the genes in the offspring of the selected individuals.", "During meiosis, homologous chromosomes from the parents line up with one another and cross-over part way along their length, thus randomly swapping genetic material.", "Such swapping or shuffling of the DNA allows organisms to evolve more rapidly.", "In recombination, because the inserted sequences were of proven utility in a homologous environment, the inserted sequences are likely to still have substantial information content once they are inserted into the new sequence.", "Theoretically there are 2,000 different single mutants of a 100 amino acid protein.", "However, a protein of 100 amino acids has 20100 possible sequence combinations, a number which is too large to exhaustively explore by conventional methods.", "It would be advantageous to develop a system which would allow generation and screening of all of these possible combination mutations.", "Some workers in the art have utilized an in vivo site specific recombination system to generate hybrids of combine light chain antibody genes with heavy chain antibody genes for expression in a phage system.", "However, their system relies on specific sites of recombination and is limited accordingly.", "Simultaneous mutagenesis of antibody CDR regions in single chain antibodies (scFv) by overlapping extension and PCR have been reported.", "Others have described a method for generating a large population of multiple hybrids using random in vivo recombination.", "This method requires the recombination of two different libraries of plasmids, each library having a different selectable marker.", "The method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).", "In vivo recombination between two homologous, but truncated, insect-toxin genes on a plasmid has been reported as a method of producing a hybrid gene.", "The in vivo recombination of substantially mismatched DNA sequences in a host cell having defective mismatch repair enzymes, resulting in hybrid molecule formation has been reported.", "C—SUMMARY OF THE INVENTION This invention relates generally to the field of cellular and whole organism engineering.", "Specifically, this invention relates to a cellular transformation, directed evolution, and screening method for creating novel transgenic organisms having desirable properties.", "Thus in one aspect, this invention relates to a method of generating a transgenic organism, such as a microbe or a plant, having a plurality of traits that are differentially activatable.", "In one embodiment, this invention is directed to a method of producing an improved organism having a desirable trait to by: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence of said improved organism.", "This invention provides that any of steps a), b), and c) can be further repeated in any particular order and any number of times; accordingly, this invention specifically provides methods comprised of any iterative combination of steps a), b), and c), with a number of iterations.", "In another embodiment, this invention is directed to a method of producing an improved organism having a desirable trait to by: a) obtaining an initial population of organisms, which can be a clonal population or otherwise, b) generating a set of mutagenized organisms each having at least one genetic mutation, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations c) detecting the manifestation of at least two genetic mutations, and d) introducing at least two detected genetic mutations into one organism.", "Additionally, this invention provides that any of steps a), b), c), and d) can be further repeated in any particular order and any number of times; accordingly, this invention specifically provides methods comprised of any iterative combination of steps a), b), c), and d), with a total number of iterations can be from one up to one million, including specifically every integer value in between.", "In a preferred aspect of embodiments specified herein the step of b) generating a second set of mutagenized organisms is comprised of generating a plurality of organisms, each of which organisms has a particular transgenic mutation.", "As used herein, “generating a set of mutagenized organisms having genetic mutations” can be achieved by any means known in the art to mutagenized including any radiation known to mutagenized, such as ionizing and ultra violet.", "Further examples of serviceable mutagenizing methods include site-saturation mutagenesis, transposon-based methods, and homologous recombination.", "“Combining” means incorporating a plurality of different genetic mutations in the genetic makeup (e.g.", "the genome) of the same organism; and methods to achieve this combining” step including sexual recombination, homologous recombination, and transposon-based methods.", "As used herein, an “initial population of organisms” means a “Working population of organisms”, which refers simply to a population of organisms with which one is working, and which is comprised of at least one organism.", "An “initial population of organisms” which can be a clonal population or otherwise.", "Accordingly, in step 1) an “initial population of organisms” may be a population of multicellular organisms or of unicellular organisms or of both.", "An “initial population of organisms” may be comprised of unicellular organisms or multicellular organisms or both.", "An “initial population of organisms” may be comprised of prokaryotic organisms or eukaryotic organisms or both.", "This invention provides that an “initial population of organisms” is comprised of at least one organism, and preferred embodiments include at least that.", "By “organism” is meant any biological form or thing that is capable of self replication or replication in a host.", "Examples of “organisms” include the following kinds of organisms (which kinds are not necessarily mutually-exclusive): animals, plants, insects, cyanobacteria, microorganisms, fungi, bacteria, eukaryotes, prokaryotes, mycoplasma, viral organisms (including DNA viruses, RNA viruses), and prions.", "Non-limiting particularly preferred examples of kinds of “organisms” also include Archaea (archaebacteria) and Bacteria (eubacteria).", "Non-limiting examples of Archaea (archaebacteria) include Crenarchaeota, Euryarchaeota, and Korarchaeota.", "Non-limiting examples Bacteria (eubacteria) include Aquificales, CFB/Green sulfur bacteria group, Chlamydiales/Verrucomicrobia group, Chrysiogenes group, Coprothermobacter group, Cyanobacteria & chloroplasts, Cytophaga/Flexibacter/Bacteriods group, Dictyoglomus group, Fibrobacter/Acidobacteria group, Firmicutes, Flexistipes group, Fusobacteria, Green non-sulfur bacteria, Nitrospira group, Planctomycetales, Proteobacteria, Spirochaetales, Synergistes group, Thermodesulfobacterium group, Thermotogales, Thermus/Deinococcus group.", "As non-limiting examples, particularly preferred kinds of organisms include Aquifex, Aspergillus, Bacillus, Clostridium, E. coli, Lactobacillus, Mycobacterium, Pseudomonas, Streptomyces, and Thermotoga.", "As additional non-limiting examples, particularly preferred organisms include cultivated organisms such as CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38.Particularly preferred non-limiting examples of organisms further include host organisms that are serviceable for the expression of recombinant molecules.", "Organisms further include primary cultures (e.g.", "cells from harvested mammalian tissues), immortalized cells, all cultivated and culturable cells and multicellular organisms, and all uncultivated and uculturable cells and multicellular organisms.", "In a preferred embodiment, knowledge of genomic information is useful for performing the claimed methods; thus, this invention provides the following as preferred but non-limiting examples of organisms that are particularly serviceable for this invention, because there is a significant amount of—if not complete—genomic sequence information (in terms of primary sequence &/or annotation) for these organisms: Human, Insect (e.g.", "Drosophila melanogaster), Higher plants (e.g.", "Arabidopsis thaliana), Protozoan (e.g.", "Plasmodium falciparum), Nematode (e.g.", "Caenorhabditis elegans), Fungi (e.g.", "Saccharomyces cerevisiae), Proteobacteria gamma subdivision (e.g.", "Escherichia coli K-12, Haemophilus influenzae Rd, Xylella fastidiosa 9a5c, Vibrio cholerae E1 Tor N16961, Pseudomonas aeruginosa PA01, Buchnera sp.", "APS), Proteobacteria beta subdivision (e.g.", "Neisseria meningitidis MC58 (serogroup B), Neisseria meningitidis Z2491 (serogroup A)), Proteobacteria other subdivisions (e.g.", "Helicobacter pylori 26695, Helicobacter pylori J99, Campylobacter jejuni NCTCI 11168, Rickettsia prowazekii), Gram-positive bacteria (e.g.", "Bacillus subtilis, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycobacterium tuberculosis H37Rv), Chlamydia (e.g.", "Chlamydia trachomatisserovar D, Chlamydia muridarum (Chlamydia trachomatis MoPn), Chlamydia pneumoniae CWL029, Chlamydia pneumoniae AR39, Chlamydia pneumoniae J138), Spirochete (e.g.", "Borrelia burgdorferi B31, Treponema pallidum), Cyanobacteria (e.g.", "Synechocystis sp.", "PCC6803), Radioresistant bacteria (e.g.", "Deinococcus radiodurans R1), Hyperthermophilic bacteria (e.g.", "Aquifex aeolicus VF5, Thermotoga marilima MSB8), and Archaea (e.g.", "Methanococcus jannaschii, Methanobacterium thermoautotrophicum deltaH, Archaeoglobus fulgidus, Pyrococcus horikoshii OT3, Pyrococcus abyssi, Aeropyrum pernix K1).", "Non-limiting particularly preferred examples of kinds of plant “organisms” include those listed in Table 1.TABLE 1 Non-limiting examples of plant organisms and sources of transgenic molecules (e.g.", "nucleic acids & nucleic acid products) 1.Alfalfa 2.Amelanchier laevis 3.Apple 4.Arab.", "thaliana 5.Arabidopsis 6.Aspergillus flavus 7.Barley 8.Beet 9.Belladonna 10.Brassica oleracea 11.Carrot 12.Chrysanthemum 13.Cichorium intybus 14.Clavibacter 15.Clavibacter xyli 16.Coffee 17.Corn 18.Cotton 19.Cranberry 20.Creeping bentgrass 21.Cryphonectria parasitica 22.Eggplant 23.Festuca arundinacea 24.Fusarium graminearum 25.Fusarium moniliforme 26.Fusarium sporotrichioides 27.Gladiolus 28.Grape 29.Heterorhabditis bacteriophora 30.Kentucky bluegrass 31.Lettuce 32.Melon 33.Oat 34.Onion 35.Papaya 36.Pea 37.Peanut 38.Pelargonium 39.Pepper 40.Persimmon 41.Petunia 42.Pine 43.Pineapple 44.Pink bollworm 45.Plum 46.Poplar 47.Potato 48.Pseudomonas 49.Pseudomonas putida 50.Pseudomonas syringae 51.Rapeseed 52.Rhizobium 53.Rhizobium etli 54.Rhizobium fredii 55.Rhizobium leguminosarum 56.Rhizobium meliloti 57.Rice 58.Rubus idaeus 59.Spruce 60.Soybean 61.Squash 62.Squash-cucumber 63.Squash-cucurbita texana 64.Strawberry 65.Sugarcane 66.Sunflower 67.Sweet potato 68.Sweetgum 69.TMV 70.Tobacco 71.Tomato 72.Walnut 73.Watermelon 74.Wheat 75.Xanthomonas 76.Xanthomonas campestris As used herein, the meaning of “generating a set of mutagenized organisms having genetic mutations” includes the steps of substituting, deleting, as well as introducing a nucleotide sequence into organism; and this invention provides a nucleotide sequence that serviceable for this purpose may be a single-stranded or double-stranded and the fact that its length may be from one nucleotide up to 10,000,000,000 nucleotides in length including specifically every integer value in between.", "A mutation in an organism includes any alteration in the structure of one or more molecules that encode the organism.", "These molecules include nucleic acid, DNA, RNA, prionic molecules, and may be exemplified by a variety of molecules in an organism such as a DNA that is genomic, episomal, or nucleic, or by a nucleic acid that is vectoral (e.g.", "viral, cosmid, phage, phagemid).", "In one aspect, as used herein, a “set of substantial genetic mutations” is preferably a disruption (e.g.", "a functional knock-out) of at least about 15 to about 150,000 genomic locations or nucleotide sequences (e.g.", "genes, promoters, regulatory sequences, codons etc.", "), including specifically every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 15 to about 150,000 genes, including specifically every integer value in between.", "Corresponding to another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 15 to about 150,000 gene products &/or phenotypes &/or traits, including specifically every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” with respect to an organism (or type of organism) is preferably a disruption (e.g.", "a functional knock-out) of at least about 1% to about 100% of genomic locations or nucleotide sequences (e.g.", "genes, promoters, regulatory sequences, codons etc.)", "in the organism (or type of organism), including specifically percentages of every integer value in between.", "In another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 1% to about 100% of genes in an organism (or type of organism), including specifically percentages of every integer value in between.", "Corresponding to another aspect, as used herein, a “set of substantial genetic mutations” is preferably an alteration in an expression level (e.g.", "decreased or increased expression level) or an alteration in the expression pattern (e.g.", "throughout a period of time) of at least about 1% to about 100% of the gene products &/or phenotypes &/or traits of an organism (or type of organism), including specifically every integer value in between.", "In yet another aspect, as used herein, a “set of substantial genetic mutations” is preferably an introduction or deletion of at least about 15 to 150,000 genes promoters or other nucleotide sequences (where each sequence is from 1 base to 10,000,000 bases), including specifically every integer value in between.", "For example, one can introduce a library of at least about 15 to 150,000 nucleotides (genes or promoters) produced by “site-saturation mutagenesis” &/or by “ligation reassembly” (including any specific aspect thereof provided herein) into an “initial population of organisms”.", "It is provided that wherever the manipulation of a plurality of “genes” is mentioned herein, gene pathways (e.g.", "that ultimately lead to the production of small molecules) are also included.", "It is appreciated herein that knocking-out, altering expression level, and altering expression pattern can be achieved, by non-limiting exemplification, by mutagenizing a nucleotide sequence corresponding gene as well as a corresponding promoter that affects the expression of the gene.", "As used herein, a “mutagenized organism” includes any organism that has been altered by a genetic mutation.", "A “genetic mutation” can be, by way of non-limiting and non-mutually exclusive exemplification, and change in the nucleotide sequence (DNA or RNA) with respect to genomic, extra-genomic, episomal, mitochondrial, and any nucleotide sequence associated with (e.g.", "contained within or considered part of) an organism.", "According to this invention, detecting the manifestation of a “genetic mutation” means “detecting the manifestation of a detectable parameter”, including but not limited to a change in the genomic sequence.", "Accordingly, this invention provides that a step of sequencing (&/or annotating) of and organism's genomic DNA is necessary for some methods of this invention, and exemplary but non-limiting aspects of this sequencing (&/or annotating) step are provided herein.", "A detectable “trait”, as used herein, is any detectable parameter associated with the organism.", "Accordingly, such a detectable “parameter” includes, by way of non-limiting exemplification, any detectable “nucleotide knock-in”, any detectable “nucleotide knock-outs”, any detectable “phenotype”, and any detectable “genotype”.", "By way of further illustration, a “trait” includes any substance produced or not produced by the organism.", "Accordingly, a “trait” includes viability or non-viability, behavior, growth rate, size, morphology.", "“Trait” includes increased (or alternatively decreased) expression of a gene product or gene pathway product.", "“Trait” also includes small molecule production (including vitamins, antibiotics), herbicide resistance, drought resistance, pest resistance, production of any recombinant biomolecule (ie.g.", "vaccines, enzymes, protein therapeutics, chiral enzymes).", "Additional examples of serviceable traits for this invention are shown in Table 2.TABLE 2 Non-limiting examples of serviceable genes, gene products, phenotypes, or traits according to the methods of this invention (e.g.", "knockouts, knockins, increased or decreased expression level, increased or decreased expression pattern) Table 2 - Part 1.Non-limiting examples of genes or gene products 1.17 kDa protein 2.3-hydroxy-3-methylglutaryl CoenzymeA reductase 3.4-Coumarate: CoA ligase knockout 4.60 kDa protein 5.Ac transposable element 6.ACC deaminase 7.ACC oxidase knockout 8.ACC synthase 9.ACC synthase knockout 10.Acetohydroxyacid synthase variant 11.Acetolactate synthase 12.Acetyl CoA carboxylase 13.ACP acyl-ACP thioesterase 14.ACP thioesterase 15.Acyl CoA reductase 16.Acyl-ACP knockout 17.Acyl-ACP desaturase 18.Acyl-ACP desaturase knockout 19.Acyl-ACP thioesterase 20.ADP glucose pyrophosphorylase 21.ADP glucose pyrophosphorylase knockout 22.Agglutinin 23.Aleurone 1 24.Alpha hordothinonin 25.Alpha-amylase 26.Alpha-hemoglobin 27.Aminoglycoside 3′-adenylytransferase 28.Amylase 29.Anionic peroxidase 30.Antibody 31.Antifungal protein 32.Antithrombin 33.Antitrypsin 34.Antiviral protein 35.Aspartokinase 36.Attacin E 37.B1 regulatory gene 38.B-1,3-glucanase knockout 39.B-1,4-endoglucanase knockout 40.Bacteropsin 41.Barnase 42.Barstar 43.Beta-hemoglobin 44.B-glucuronidase 45.C1 knockout 46.C1 regulatory gene 47.C2 knockout 48.C3 knockout 49.Caffeate O-methylthransferase 50.Caffeate O-methyltransferase knockout 51.Caffeoyl CoA O-methyltransferase knockout 52.Casein 53.Cecropin 54.Cecropin B 55.Cellulose binding protein 56.Chalcone synthase knockout 57.Chitinase 58.Chitobiosidase 59.Chloramphenicol acetyltransferase 60.Cholera toxin B 61.Choline oxidase 62.Cinnamate 4-hydroxylase 63.Cinnamate 4-hydroxylase knockout 64.Coat protein 65.Coat protein knockout 66.Conglycinin 67.CryIA 68.CryIAb 69.CryIAc 70.CryIB 71.CryIIA 72.CryIIIA 73.CryVIA 74.Cyclin dependent kinase 75.Cyclodexlrin glycosyltransferase 76.Cylindrical inclusion protein 77.Cystathionine synthase 78.Delta-12 desaturase 79.Delta-12 desaturase knockout 80.Delta-12 saturase 81.Delta-12 saturase knockout 82.Delta-15 desaturase 83.Delta-15 desaturase knockout 84.Delta-9 desaturase 85.Delta-9 desturase knockout 86.Deoxyhypusine synthase (DHS) 87.Deoxyhypusine synthase knockout 88.Diacylglycerol acetyl tansferase 89.Dihydrodipicolinate synthase 90.Dihydrofolate reductase 91.Diptheria toxin A 92.Disease resistance response gene 49 93.Double stranded ribonuclease 94.Ds transposable element 95.Elongase 96.EPSPS 97.Ethylene forming enzyme knockout 98.Ethylene receptor protein 99.Ethylene receptor protein knockout 100.Fatty acid elongase 101.Fluorescent protein 102.G glycoprotein 103.Galactanase 104.Galanthus nivalis agglutinin 105.Genome-linked protein 106.Glucanase 107.Glucanase knockout 108.Glucose oxidase 109.Glutamate dehydrogenase 110.Glutamine binding protein 111.Glutamine synthetase 112.Glutenin 113.Glycerol-3-phosphate acetyl transferase 114.Glyphosate exidoreductase 115.Glyphosate oxidoreductase 116.Green fluorescent protein 117.Helper component 118.Hemicellulase 119.Hup locus 120.Hygromycin phosphotransferase 121.Hyoscamine 6B-hydroxylase 122.IAA monooxygenase 123.Invertase 124.Invertase knockout 125.Isopentenyl transferase 126.Ketoacyl-ACP synthase 127.Ketoacyl-ACP synthase knockout 128.Larval serum protein 129.Leafy homeotic regulatory gene 130.Lectin 131.Lignin peroxidase 132.Luciferase 133.Lysine-2 gene 134.Lysophosphatidic acid acetyl transferase 135.Lysozyme 136.Mabinlin 137.Male sterility protein 138.Metallothionein 139.Modified ethylene receptor protein 140.Modified ethylene receptor protein knockout 141.Monooxygenase 142.Movement protein 143.Movement protein nonfunctional 144.N gene for TMV resistance 145.N-acetyl glucosidase 146.Nitrilase 147.Nopaline synthase 148.Notch 149.NptII 150.Nuclear inclusion protein a 151.Nuclear inclusion protein b 152.Nucleocapsid 153.Nucleoprotein 154.O-acyl transferase 155.Oleayl-ACP thioesterase 156.Omega 3 desaturase 157.Omega 3 desaturease knockout 158.Omega 6 desaturase 159.Omega 6 desaturase knockout 160.O-methyltransferase 161.Osmotin 162.Oxalate oxidase 163.Par locus 164.Pathogenesis protein 1a 165.Pectate lyase 166.Pectin esterase 167.Pectin esterase knockout 168.Pectin methylesterase 169.Pectin methylesterase knockout 170.Pentenlypyrophosphate isomerase 171.Phosphinothricin 172.Phosphinothricin acetyl transferase 173.Phytochrome A 174.Phytoene synthase 175.Phleomycin binding protein 176.Polygalacturonase 177.Polygalacturonase knockout 178.Polygalacturonase inhibitor protein 179.Prf regulatory gene 180.Prosystemin 181.Protease 182.Protein A 183.Protein kinase 184.Proteinase inhibitor 1 185.Pti5 transcription factor 186.R regulatory gene 187.Receptor kinase 188.Recombinase 189.Reductase 190.Replicase 191.Resveratrol synthase 192.Ribonuclease 193.ro1c 194.Rol hormone gene 195.S-adenosylmethione decarboxylase 196.S-adenosylmethione hydrolase 197.S-adenosylmethionine transferase 198.Salicylate hydroxylase 199.Satellite RNA 200.Seed storage protein 201.Serine-threonine protein kinase 202.Serum albumin 203.Shrunken 2 204.Sorbitol dehydrogenase 205.Sorbitol synthase 206.Stilbene synthase 207.Storage protein 208.Sucrose phosphate synthase 209.Systemic acquired resistance gene 8.2 210.Tetracycline binding protein 211.Thioesterase (×2) 212.Thiolase 213.TobRB7 214.Transcriptional activator 215.Transposon Tn5 216.Trehalase 217.Trehalase knockout 218.Trichodiene synthase 219.Trichosanthin 220.Trifolitoxin 221.Trypsin inhibitor 222.T-URF13 mitochondrial 223.UDP glucose glucosyltransferase 224.Violaxanthin de-epoxidase 225.Violaxanthin de-epoxidase knockout 226.Wheat germ agglutinin 227.Xanthosine-N7-methyltransferase knockout 228.Zein storage protein Table 2 - Part 2.Non-limiting examples of input traits/phenotypes 1.2,4-D tolerant 2.Alernaria resistant 3.Altered amino acid composition 4.Alternaria solani resistant 5.Ammonium assimilation increased 6.AMV resistant 7.Aphid resistant 8.Apple scab resistant 9.Aspergillus resistant 10.B-1,4-endoglucanase 11.Bacterial leaf blight resistant 12.Bacterial speck resistant 13.BCTV resistant 14.Blackspot bruise resistant 15.BLRV resistant 16.BNYVV Resistant 17.Botrytis cinerea resistant 18.Botrytis resistant 19.BPMV resistant 20.Bromoxynil tolerant 21.BYDV resistant 22.BYMV resistant 23.Carbohydrate metabolism altered 24.Cell wall altered 25.Chlorsulfuron tolerant 26.Clavibacter resistant 27.CLRV resistant 28.CMV resistant 29.Cold tolerant 30.Coleopteran resistant 31.Colletotrichum resistant 32.Colorado potato beetle resistant 33.Constitutive expression of glutamine synthetase 34.Corynebacterium sepedonicum resistant 35.Cottonwood leaf beetle resistant 36.Crown gall resistant 37.Crown rot resistant 38.Cucumovirus resistant 39.Cutting rootability increased 40.Downy mildew resistant 41.Drought tolerant 42.Erwinia carotovora resistant 43.Ethylene production reduced 44.European Corn Borer resistant 45.Female sterile 46.Fenthion susceptible 47.Fertility altered 48.Fire blight resistant 49.Flower and fruit abscission reduced 50.Flower and fruit set altered 51.Flowering altered 52.Flowering time altered 53.Frogeye leaf spot resistant 54.Fruit ripening altered 55.Fruit ripening delayed 56.Fruit rot resistant 57.Fruit solids increased 58.Fruit sweetness increased 59.Fungal post-harvest resistant 60.Fungal resistant 61.Fungal resistant general 62.Fusarium resistant 63.Glyphosate tolerant 64.Growth rate altered 65.Growth rate reduced 66.Heat stable glucanase produced 67.Hordothionin produced 68.Imidazolinone tolerant 69.Insect resistant general 70.Kanamycin resistant 71.Lepidopteran resistant 72.Lesser cornstalk borer resistant 73.LMV resistant 74.Loss of systemic resistance 75.Male sterile 76.Marssonina resistant 77.MCDV resistant 78.MCMV resistant 79.MDMV resistant 80.MDMV-B resistant 81.Mealybug wilt virus resistant 82.Melamtsora resistant 83.Melodgyne resistant 84.Methotrexate resistant 85.Mexican Rice Borer resistant 86.Nucleocapsid protein produced 87.Oblique banded leafroller resistant 88.PEMV resistant 89.PeSV resistant 90.Phoma resistant 91.Phosphinothricin tolerant 92.Phratora leaf beetle resistant 93.Phytophthora resistant 94.PLRV resistant 95.Polyamine metabolism altered 96.Potyvirus resistant 97.Powdery mildew resistant 98.PPV resistant 99.Pratylenchus vulnus resistant 100.Proteinase inhibitors level constitutive 101.PRSV resistant 102.PRV resistant 103.PSbMV resistant 104.Pseudomonas syringae resistant 105.PStV resistant 106.PVX resistant 107.PVY resistant 108.RBDV resistant 109.Rhizoctonia resistant 110.Rhizoctonia solani resistant 111.Ring rot resistance 112.Root-knot nematode resistant 113.SbMV resistant 114.Sclerotinia resistant 115.SCMV resistant 116.SCYLV resistant 117.Secondary metabolite increased 118.Seed set reduced 119.Selectable marker 120.Senescence altered 121.Septoria resistant 122.Shorter stems 123.Soft rot fungal resistant 124.Soft rot resistant 125.SqMV resistant 126.SrMV resistant 127.Storage protein altered 128.Streptomyces scabies resistant 129.Sulfonylurea tolerant 130.Tetracycline binding protein produced 131.TEV resistant 132.Thelaviopsis resistant 133.TMV resistant 134.Tobamovirus resistant 135.ToMoV resistant 136.ToMV resistant 137.Transposon activator 138.Transposon inserted 139.TRV resistant 140.TSWV resistant 141.TVMV resistant 142.TYLCV resistant 143.Tyrosine level increased 144.Venturia resistant 145.Verticillium dahliae resistant 146.Verticillium resistant 147.Visual marker 148.WMV2 resistant 149.WSMV resistant 150.Yield increased 151.ZYMV resistant Table 2 - Part 3.Non-limiting examples of output traits/phenotypes 1.ACC oxidase level decreased 2.Altered lignin biosynthesis 3.B-1,4-endoglucanase 4.Botrytis resistant 5.Carbohydrate metabolism altered 6.Carotenoid content altered 7.Cell wall altered 8.CMV resistant 9.Coleopteran resistant 10.Dry matter content increased 11.Ethylene production reduced 12.Ethylene synthesis reduced 13.Fatty acid metabolism altered 14.Fire blight resistant 15.Flower and fruit abscission reduced 16.Flower and fruit set altered 17.Flowering time altered 18.Fruit firmness increased 19.Fruit pectin esterase levels decreased 20.Fruit ripening altered 21.Fruit ripening delayed 22.Fruit solids increased 23.Fruit sugar profile altered 24.Fruit sweetness increased 25.Glucuronidase expressing 26.Heat stable glucanase produced 27.Heavy metals sequestered 28.Hordothionin produced 29.Improved fruit quality 30.Industrial enzyme produced 31.Lepidopteran resistant 32.Lysine level increased 33.Mealybug wilt virus resistant 34.Methionine level increased 35.Nucleocapsid protein produced 36.Oil profile altered 37.Pectin esterase level reduced 38.Pharmaceutical proteins produced 39.Phosphinothricin tolerant 40.Phytoene synthase activity increased 41.Pigment metabolism altered 42.Polygalacturonase level reduced 43.Processing characteristics altered 44.Prolonged shelf life 45.Protein altered 46.Protein quality altered 47.PRSV resistant 48.Root-knot nematode resistant 49.Sclerotinia resistant 50.Seed composition altered 51.Seed methionine storage increased 52.Seed set reduced 53.Seed storage protein 54.Senescence altered (e.g.", "Shelf life increased) 55.Shorter stems 56.Solids increased 57.SqMV resistant 58.Starch level increased 59.Starch metabolism altered 60.Starch reduced 61.Sterols increased 62.Storage protein altered 63.Sugar alcohol levels increased 64.Telracycline binding protein produced 65.Tyrosine level increased 66.Verticillium resistant 67.Visual marker 68.WMV2 resistant 69.Yield increased 70.ZYMV resistant Table 2 - Part 4.Non-limiting examples of traits/phenotypes with agronomic properties 1.ACC oxidase level decreased 2.Altered amino acid composition 3.Altered lignin biosynthesis 4.Altered maturing 5.Altered plant development 6.Aluminum tolerant 7.Ammonium assimilation increased 8.Anthocyanin produced in seed 9.B-1,4-endoglucanase 10.Calmodulin level altered 11.Carbohydrate metabolism altered 12.Carotenoid content altered 13.Cell wall altered 14.Cold tolerant 15.Constitutive expression of glutamine synthetase 16.Cutting root ability increased 17.Development altered 18.Drought tolerant 19.Dry matter content increased 20.Environmental stress reduced 21.Ethylene metabolism altered 22.Ethylene production reduced 23.Ethylene synthesis reduced 24.Fatty acid metabolism altered 25.Female sterile 26.Fenthion susceptible 27.Fertility altered 28.Fiber quality altered 29.Flower and fruit abscission reduced 30.Flower and fruit set altered 31.Flowering altered 32.Flower color altered 33.Flowering time altered 34.Fruit firmness increased 35.Fruit pectin esterase and levels decreased 36.Fruit polygalacturonase level decreased 37.Fruit ripening altered 38.Fruit ripening delayed 39.Fruit solids increased 40.Fruit sugar profile altered 41.Fruit sweetness increased 42.Glucuronidase expressing 43.Growth rate altered 44.Growth rate increased 45.Growth rate reduced 46.Heat stable glucanase produced 47.Heat tolerant 48.Heavy metals sequestered 49.Hordothionin produced 50.Improved fruit quality 51.Increased phosphorus 52.Increased stalk strength 53.Industrial enzyme produced 54.Lignin levels decreased 55.Lipase expressed in seeds 56.Lysine level increased 57.Male sterile 58.Male sterile reversible 59.Methionine level increased 60.Modified growth characteristics 61.Mycotoxin degradation 62.Nitrogen metabolism altered 63.Nucleocapsid protein produced 64.Oil profile altered 65.Oil quality altered 66.Oxidative stress tolerant 67.Pectin esterase level reduced 68.Pharmaceutical proteins produced 69.Photosynthesis enhanced 70.Phytoene synthase activity increased 71.Pigment metabolism altered 72.Polyamine metabolism altered 73.Polygalacturonase level reduced 74.Pratylenchus vulnus resistant 75.Processing characteristics altered 76.Prolonged shelf life 77.Protein altered 78.Protein lysine level increased 79.Protein quality altered 80.Proteinase inhibitors level constitutive 81.Salt tolerance increased 82.Seed composition altered 83.Seed methionine storage increased 84.Seed set reduced 85.Selectable marker 86.Senescence altered 87.Shorter stems 88.Solids increased 89.Starch level increased 90.Starch metabolism altered 91.Starch reduced 92.Sterols increased 93.Storage protein altered 94.Stress tolerant 95.Sugar alcohol levels increased 96.Tetracycline binding protein produced 97.Thermostable protein produced 98.Transposon activator 99.Transposon inserted 100.Tyrosine level increased 101.Visual marker 102.Vivipary increased 103.Yield increased Table 2 - Part 5.Non-limiting examples of traits/phenotypes with product quality properties 1.2,4-D tolerant 2.ACC oxidase level decreased 3.Altered amino acid composition 4.Altered lignin biosynthesis 5.Anthocyanin produced in seed 6.Antioxidant enzyme increased 7.Auxin metabolism and increased tuber solids 8.B-1,4-endoglucanase 9.Blackspot bruise resistant 10.Brown spot resistant 11.Bruising reduced 12.Caffeine levels reduced 13.Carbohydrate metabolism altered 14.Carotenoid content altered 15.Cell wall altered 16.Cold tolerant 17.Delayed softening 18.Disulfides reduced in endosperm 19.Dry matter content increased 20.Ear mold resistant 21.Ethylene production reduced 22.Ethylene synthesis reduced 23.Extended flower life 24.Fatty acid metabolism altered 25.Fiber quality altered 26.Fiber strength altered 27.Flavor enhancer 28.Flower and fruit abscission reduced 29.Fruit firmness increased 30.Fruit invertase level decreased 31.Fruit polygalacturonase level decreased 32.Fruit ripening altered 33.Fruit ripening delayed 34.Fruit solids increased 35.Fruit sugar profile altered 36.Fruit sweetness increased 37.Glyphosate tolerant 38.Heat stable glucanase produced 39.Improved fruit quality 40.Increased phosphorus 41.Increased protein levels 42.Lignin levels decreased 43.Lysine level increased 44.Male sterile 45.Melanin produced in cotton fibers 46.Metabolism altered 47.Methionine level increased 48.Mycotoxin degradation 49.Mycotoxin production inhibited 50.Nicotine levels reduced 51.Nitrogen metabolism altered 52.Novel protein produced 53.Nutritional quality altered 54.Oil profile altered 55.Oil quality altered 56.Pectin esterase level reduced 57.Photosynthesis enhanced 58.Phytoene synthase activity increased 59.Pigment metabolism altered 60.Polyamine metabolism altered 61.Polygalacturonase level reduced 62.Processing characteristics altered 63.Prolonged shelf life 64.Protein altered 65.Protein lysine level increased 66.Protein quality altered 67.Proteinase inhibitors level constitutive 68.Rust resistant 69.Seed composition altered 70.Seed methionine storage increased 71.Seed number increased 72.Seed quality altered 73.Seed set reduced 74.Seed weight increased 75.Senescence altered 76.Solids increased 77.Starch level increased 78.Starch metabolism altered 79.Starch reduced 80.Steroidal glycoalkaloids reduced 81.Sterols increased 82.Storage protein altered 83.Sugar alcohol levels increased 84.Thermostable protein produced 85.Tryptophan level increased 86.Tuber solids increased 87.Yield increased Table 2 - Part 6.Non-limiting examples of traits/phenotypes with herbicide tolerance properties 1.2,4-D tolerant 2.Chloroacetanilide tolerant 3.Fertility altered 4.Protein altered 5.Lignin levels decreased 6.Methionine level increased 7.Bromoxynil tolerant 8.Metabolism altered 9.Imidazole tolerant 10.Imidazolinone tolerant 11.Sulfonylurea tolerant 12.Northern corn leaf blight resistant 13.Herbicide tolerant 14.Isoxazole tolerant 15.Chlorsulfuron tolerant 16.Glyphosate tolerant 17.Lepidopteran resistant 18.Phosphinothricin tolerant 19.Sulfonylurea tolerant Table 2 - Part 7.Non-limiting examples of traits/phenotypes with pest resistance properties 1.Agrobacterium resistant - BR 2.Alternaria resistant - FR 3.Alternaria daucii resistant - FR 4.Alternaria solani resistant - FR 5.AMV resistant - VR 6.Anthracnose resistant - FR 7.Aphid resistant - IR 8.Apple scab resistant - FR 9.Aspergillus resistant - FR 10.Bacterial leaf blight resistant - BR 11.Bacterial resistant - BR 12.Bacterial soft rot resistant - BR 13.Bacterial soft rot resistant - VR 14.Bacterial speck resistant - BR 15.BCTV resistant - VR 16.Black shank resistant - FR 17.BLRV resistant - VR 18.BNYVV resistant - VR 19.Botrytis cinerea resistant - FR 20.Botrytis resistant - FR 21.BPMV resistant - VR 22.Brown spot resistant - FR 23.BYDV resistant - VR 24.BYMV resistant - VR 25.CaMV resistant - VR 26.Cercospora resistant - FR 27.Clavibacter resistant - BR 28.Closteroviurs resistant - BR 29.CLRV resistant - VR 30.CMV resistant - FR 31.Coleopteran resistant - IR 32.Colletotrichum resistant - FR 33.Colorado potato beetle resistant - IR 34.Corn earworm resistant - IR 35.Corynebacterium sepedonicum resistant - BR 36.Cottonwood leaf beetle resistant - IR 37.Criconnemella resistant - NR 38.Crown gall resistant - BR 39.Cucumovirus resistant - VR 40.Cylindrosporium resistant - FR 41.Disease resistant general - FR 42.Dollar spot resistant - FR 43.Downy mildew resistant - FR 44.Ear mold resistant - FR 45.Erwinia carotovora resistant - BR 46.European Corn Borer resistant - IR 47.Eyespot resistant - FR 48.Fall armyworm resistant - IR 49.Fire blight resistant - BR 50.Frogeye leaf spot resistanT - FR 51.Fruit rot resistant - FR 52.Fungal post-harvest resistant - FR 53.Fungal resistant - FR 54.Fungal resistant general - FR 55.Fusarium dehlae resistant - FR 56.Fusarium resistant - FR 57.Geminivirus resistant - VR 58.Gray lead spot resistant - FR 59.Helminthosporium resistant - FR 60.Hordothionin produced - BR 61.Insect predator resistant - IR 62.Insect resistant general - IR 63.Late blight resistant - FR 64.Leaf blight resistant - FR 65.Leaf spot resistant - FR 66.Lepidopteran resistant - IR 67.Lesser cornstalk borer resistant - IR 68.LMV resistant - VR 69.Loss of systemic resistance - VR 70.Marssonina resistant - FR 71.MCDV resistant - VR 72.MCMV resistant - VR 73.MDMV resistant - VR 74.MDMV-B resistant - VR 75.Mealybug wilt virus resistant - VR 76.Melamtsora resistant - FR 77.Melodgyne resistant - NR 78.Meloidogyne resistant - NR 79.Mexican Rice Borer resistant - IR 80.Mycotoxin degradation - FR 81.Nepovirus resistant - VR 82.Northern corn leaf blight resistant - IR 83.Nucleocapsid protein produced - VR 84.Oblique banded leafroller resistant - IR 85.Oomycete resistant - FR 86.Pathogenesis related proteins level increased - FR 87.PEMV resistant - VR 88.PeSV Resistant - VR 89.Phatora leaf beetle resistant - IR 90.Phoma resistant - FR 91.Phytophthora resistant - FR 92.PLRV resistant - VR 93.Potyvirus resistant - VR 94.Powdery mildew resistant - FR 95.PPV resistant - VR 96.Pralylenchus vulnus resistant - NR 97.PRSV resistant - VR 98.PRV resistant - VR 99.PSbMV resistant - VR 100.Pseudomonas syringae resistant - BR 101.PStV resistant - VR 102.PVX resistant - VR 103.PVY resistant - VR 104.RBDV resistant - VR 105.Rhizoctonia resistant - FR 106.Rhizoctonia solani resistant - FR 107.Ring rot resistance - BR 108.Root-knot nematode resistant - NR 109.Rust resistant - FR 110.SbMV resistant - VR 111.Sclerotinia resistant - FR 112.SCMV resistant - VR 113.SCYLV resistant - VR 114.Septoria resistant - FR 115.Smut resistant - FR 116.SMV resistant - VR 117.Sod web worm resistant - IR 118.Soft rot fungal resistant - FR 119.Soft rot resistant - BR 120.Southwestern corn borer resistant- IR 121.SPFMV resistant - VR 122.Sphaeropsis fruit rot resistant - FR 123.SqMV resistant - VR 124.SrMV resistant - VR 125.Streptomyces scabies resistant - BR 126.Sugar cane borer resistant - IR 127.TEV resistant - VR 128.Thelaviopsis resistant - FR 129.TMV resistant - FR 130.Tobamovirus resistant - VR 131.ToMoV resistant - VR 132.ToMV resistant - VR 133.TRV resistant - VR 134.TSWV resistant - VR 135.TVMV resistant - VR 136.TYLCV resistant - VR 137.Venturia resistant - FR 138.Verticillium dahliae resistant - FR 139.Verticillium resistant - FR 140.Western corn root worm resistant - IR 141.WMV2 resistant - VR 142.WSMV resistant - VR 143.ZYMV resistant - VR Table 2 - Part 8.Non-limiting examples of miscellaneous traits/ phenotypes with properties 1.Antibiotic produced 2.Antiprotease producing 3.Capable of growth on defined synthetic media 4.Carbohydrate metabolism altered 5.Cell wall altered 6.Cold tolerant 7.Coleopteran resistant 8.Color altered 9.Color sectors in seeds 10.Colored sectors in leaves 11.Constitutive expression of glutaminc synthetase 12.Cre recombinase produced 13.Dalapon tolerant 14.Development altered 15.Disease resistant general 16.Ethylene metabolism altered 17.Expression optimization 18.Fenthion susceptible 19.Glucuronidase expressing 20.Glyphosate tolerant 21.Growth rate reduced 22.Heavy metals sequestered 23.Hygromycin tolerant 24.Inducible DNA modification 25.Industrial enzyme produced 26.Kanamycin resistant 27.Lipase expressed in seeds 28.Methotrexate resistant 29.Modified growth characteristics 30.Mycotoxin deficient 31.Mycotoxin production inhibited 32.Mycotoxin restored 33.Non-lesion forming mutant 34.Novel protein produced 35.Oil quality altered 36.Peroxidase levels increased 37.Pharmaceutical proteins produced 38.Phosphinothricin tolerant 39.Pigment metabolism altered 40.Pollen visual marker 41.Polyamine metablosim altered 42.Polymer produced 43.Recombinase produced 44.Secondary metabolite increased 45.Seed color altered 46.Seed weight increased 47.Selectable marker 48.Spectromycin resistant 49.Sterile 50.Sterols increased 51.Sulfonylurea susceptible 52.Syringomycin deficient 53.Transposon activator 54.Transposon elements inserted 55.Transposon inserted 56.Trifolitoxin producing 57.Trifolitoxin resistant 58.Virulence reduced 59.Visual marker 60.Visual marker inactive Legend BR—Bacterial Resistant FR—Fungal Resistant IR—Insent Resistant NR—Nematode Resistant VR—Viral Resistant In a particular examplification, “producing an organism having a desirable trait” includes an organism that is with respect to an organ or a part of an organ but not necessarily altered anywhere else.", "By “trait” is meant any detectable parameter associated with an organism under a set of conditions.", "Examples of “detectable parameters” include the ability to produce a substance, the ability to not produce a substance, an altered pattern of (such as an increased or a decreased) ability to produce a substance, viability, non-viability, behaviour, growth rate, size, morphology or morphological characteristic, In another embodiment, this invention is directed to a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining an initial population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one initial organism, c) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and d) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "A mutagenized organism having a desirable trait or a desirable improvement in a trait can be referred to as an “up-mutant”, and the associated mutation(s) contained in an up-mutant organism can be referred to as up-mutation(s).", "In one embodiment, step c) is comprised of selecting at least two different mutagenized organisms, each having a different mutagenized genome, and the method of producing an organism having a desirable trait or a desirable improvement in a trait is comprised of a) obtaining a starting population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one starting organism, c) selecting at least two mutagenized organism having a desirable trait or a desirable improvement in a trait, d) creating combinations of the mutations of the two or more mutagenized organisms, e) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and f) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "In one embodiment, the method is repeated.", "Thus, for example, an up-mutant organism can serve as a starting organism for the above method.", "Also, for example, an up mutant organism having a combination of two or more up-mutations in its genome can serve as a starting organism for the above method.", "Thus, in one embodiment, this invention is directed to a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining a starting population of organisms comprised of at least one starting organism, b) mutagenizing the population such that mutations occur throughout a substantial part of the genome of at least one starting organism, c) selecting at least one mutagenized organism having a desirable trait or a desirable improvement in a trait, and d) optionally repeating the method by subjecting one or more mutagenized organisms to a repetition of the method.", "A mutagenized organism having a desirable trait or a desirable improvement in a trait can be referred to as an “up-mutant”, and the associated mutation(s) contained in an up-mutant organism can be referred to as up-mutation(s).", "Mutagenizing a starting population such that mutations occur throughout a substantial part of the genome of at least one starting organism refers to mutagenizing at least approximately 1% of the genes of a genome, or at least approximately 10% of the genes of a genome, or at least approximately 20% of the genes of a genome, or at least approximately 30% of the genes of a genome, or at least approximately 40% of the genes of a genome, or at least approximately 50% of the genes of a genome, or at least approximately 60% of the genes of a genome, or at least approximately 70% of the genes of a genome, or at least approximately 80% of the genes of a genome, or at least approximately 90% of the genes of a genome, or at least approximately 95% of the genes of a genome, or at least approximately 98% of the genes of a genome.", "In a particular embodiment, this invention provides a method of producing an organism having a desirable trait or a desirable improvement in a trait by: a) obtaining sequence information of a genome; b) annotating the genomic sequence obtained; c) mutagenizing a substantial part of the genome the genome; d) selecting at least one mutagenized genome having a desirable trait or a desirable improvement in a trait; and e) optionally repeating the method by subjecting one or more mutagenized genomes to a repetition of the method.", "Thus in one aspect, this invention provides a process comprised of: 1.)", "Subjecting a working cell or organism to holistic monitoring (which can include the detection and/or measurement of all detectable functions and physical parameters).", "Examples of such parameters include morphology, behavior, growth, responsiveness to stimuli (e.g., antibiotics, different environment, etc.).", "Additional examples include all measurable molecules, including molecules that are chemically at least in part a nucleic acids, proteins, carbohydrates, proteoglycans, glycoproteins, or lipids.", "In a particular aspect, performing holistic monitoring is comprised of using a microarray-based method.", "In another aspect, performing holistic monitoring is comprised of sequencing a substantial portion of the genome, i.e.", "for example at least approximately 10% of the genome, or for example at least approximately 20% of the genome, or for example at least approximately 30% of the genome, or for example at least approximately 40% of the genome, or for example at least approximately 50% of the genome, or for example at least approximately 60% of the genome, or for example at least approximately 70% of the genome, or for example at least approximately 80% of the genome, or for example at least approximately 90% of the genome, or for example at least approximately 95% of the genome, or for example at least approximately 98% of the genome.", "2) Introducing into the working cell or organism a plurality of traits (stacked traits), including selectively and differentially activatable traits.", "Serviceable traits for this purpose include traits conferred by genes and traits conferred by gene pathways.", "3) Subjecting the working cell or organism to holistic monitoring.", "4) Compiling the information obtained from steps 1) and 3), and processing &/or analyzing it to better understand the changes introduced into the working cell or organisms.", "Such data processing includes identifying correlations between and/or among the measured parameters.", "5) Repeating any number or all of steps 2), 3), and 4).", "This invention provides that molecules serviceable for introducing transgenic traits into a plant include all known genes and nucleic acids.", "By way of non-limiting exemplification, this invention specifically names any number &/or combination of genes listed herein or listed in any reference incorporated herein by reference.", "Furthermore, by way of non-limiting exemplification, this invention specifically names any number &/or combination of genes & gene pathways listed herein as well as in any reference incorporated by reference herein.", "This invention provides that molecules serviceable as detectable parameters include molecule, any enzyme, substrate thereof, product thereof, and any gene or gene pathway listed herein including in any figure or table herein as well as in any reference incorporated by reference herein.", "This invention also relates generally to the field of nucleic acid engineering and correspondingly encoded recombinant protein engineering.", "More particularly, the invention relates to the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for resultant activity(ies) of interest, such nucleic acid activity(ies) &/or specified protein, particularly enzyme, activity(ies) of interest.", "Mutagenized molecules provided by this invention may have chimeric molecules and molecules with point mutations, including biological molecules that contain a carbohydrate, a lipid, a nucleic acid, &/or a protein component, and specific but non-limiting examples of these include antibiotics, antibodies, enzymes, and steroidal and non-steroidal hormones.", "This invention relates generally to a method of: 1) preparing a progeny generation of molecule(s) (including a molecule that is comprised of a polynucleotide sequence, a molecule that is comprised of a polypeptide sequence, and a molecules that is comprised in part of a polynucleotide sequence and in part of a polypeptide sequence), that is mutagenized to achieve at least one point mutation, addition, deletion, &/or chimerization, from one or more ancestral or parental generation template(s); 2) screening the progeny generation molecule(s)—preferably using a high throughput method—for at least one property of interest (such as an improvement in an enzyme activity or an increase in stability or a novel chemotherapeutic effect); 3) optionally obtaining &/or cataloguing structural &/or and functional information regarding the parental &/or progeny generation molecules; and 4) optionally repeating any of steps 1) to 3).", "In a preferred embodiment, there is generated (e.g.", "from a parent polynucleotide template)—in what is termed “codon site-saturation mutagenesis”—a progeny generation of polynucleotides, each having at least one set of up to three contiguous point mutations (i.e.", "different bases comprising a new codon), such that every codon (or every family of degenerate codons encoding the same amino acid) is represented at each codon position.", "Corresponding to—and encoded by—this progeny generation of polynucleotides, there is also generated a set of progeny polypeptides, each having at least one single amino acid point mutation.", "In a preferred aspect, there is generated—in what is termed “amino acid site-saturation mutagenesis”—one such mutant polypeptide for each of the 19 naturally encoded polypeptide-forming alpha-amino acid substitutions at each and every amino acid position along the polypeptide.", "This yields—for each and every amino acid position along the parental polypeptide—a total of 20 distinct progeny polypeptides including the original amino acid, or potentially more than 21 distinct progeny polypeptides if additional amino acids are used either instead of or in addition to the 20 naturally encoded amino acids.", "Thus, in another aspect, this approach is also serviceable for generating mutants containing—in addition to &/or in combination with the 20 naturally encoded polypeptide-forming alpha-amino acids—other rare &/or not naturally-encoded amino acids and amino acid derivatives.", "In yet another aspect, this approach is also serviceable for generating mutants by the use of—in addition to &/or in combination with natural or unaltered codon recognition systems of suitable hosts—altered, mutagenized, &/or designer codon recognition systems (such as in a host cell with one or more altered tRNA molecules).", "In yet another aspect, this invention relates to recombination and more specifically to a method for preparing polynucleotides encoding a polypeptide by a method of in vivo re-assortment of polynucleotide sequences containing regions of partial homology, assembling the polynucleotides to form at least one polynucleotide and screening the polynucleotides for the production of polypeptide(s) having a useful property.", "In yet another preferred embodiment, this invention is serviceable for analyzing and cataloguing—with respect to any molecular property (e.g.", "an enzymatic activity) or combination of properties allowed by current technology—the effects of any mutational change achieved (including particularly saturation mutagenesis).", "Thus, a comprehensive method is provided for determining the effect of changing each amino acid in a parental polypeptide into each of at least 19 possible substitutions.", "This allows each amino acid in a parental polypeptide to be characterized and catalogued according to its spectrum of potential effects on a measurable property of the polypeptide.", "In another aspect, the method of the present invention utilizes the natural property of cells to recombine molecules and/or to mediate reductive processes that reduce the complexity of sequences and extent of repeated or consecutive sequences possessing regions of homology.", "It is an object of the present invention to provide a method for generating hybrid polynucleotides encoding biologically active hybrid polypeptides with enhanced activities.", "In accomplishing these and other objects, there has been provided, in accordance with one aspect of the invention, a method for introducing polynucleotides into a suitable host cell and growing the host cell under conditions that produce a hybrid polynucleotide.", "In another aspect of the invention, the invention provides a method for screening for biologically active hybrid polypeptides encoded by hybrid polynucleotides.", "The present method allows for the identification of biologically active hybrid polypeptides with enhanced biological activities.", "Other objects, features and advantages of the present invention will become apparent from the following detailed description.", "It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.", "In yet another aspect, this invention relates to a method of discovering which phenotype corresponds to a gene by disrupting every gene in the organism.", "Accordingly, this invention provides a method for determining a gene that alters a characteristic of an organism, comprising: a) obtaining an initial population of organisms, b) generating a set of mutagenized organisms, such that when all the genetic mutations in the set of mutagenized organisms are taken as a whole, there is represented a set of substantial genetic mutations, and c) detecting the presence an organism having an altered trait, and d) determining the nucleotide sequence of a gene that has been mutagenized in the organism having the altered trait.", "In yet another aspect, this invention relates to a method of improving a trait in an organism by functionally knocking out a particular gene in the organism, and then transferring a library of genes, which only vary from the wild-type at one codon position, into the organism.", "Accordingly, this invention provides a method method for producing an organism with an improved trait, comprising: a) functionally knocking out an enogenous gene in a substantially clonal population of organisms; b) transferring the set of altered genes into the clonal population of organisms, wherein each altered gene differs from the endogenous gene at only one codon; and c) detecting a mutagenized organism having an improved trait; and d) determining the nucleotide sequence of a gene that has been transferred into the detected organism.", "D. BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1.Exonuclease Activity.", "FIG.", "1 shows the activity of the enzyme exonuclease III.", "This is an exemplary enzyme that can be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks.", "The asterisk indicates that the enzyme acts from the 3′ direction towards the 5′ direction of the polynucleotide substrate.", "FIG.", "2.Generation of A Nucleic Acid Building Block by Polymerase-Based Amplification.", "FIG.", "2 illustrates a method of generating a double-stranded nucleic acid building block with two overhangs using a polymerase-based amplification reaction (e.g., PCR).", "As illustrated, a first polymerase-based amplification reaction using a first set of primers, F2 and R1, is used to generate a blunt-ended product (labeled Reaction 1, Product 1), which is essentially identical to Product A.", "A second polymerase-based amplification reaction using a second set of primers, F1 and R2, is used to generate a blunt-ended product (labeled Reaction 2, Product 2), which is essentially identical to Product B.", "These two products are then mixed and allowed to melt and anneal, generating a potentially useful double-stranded nucleic acid building block with two overhangs.", "In the example of FIG.", "1, the product with the 3′ overhangs (Product C) is selected for by nuclease-based degradation of the other 3 products using a 3′ acting exonuclease, such as exonuclease III.", "Alternate primers are shown in parenthesis to illustrate serviceable primers may overlap, and additionally that serviceable primers may be of different lengths, as shown.", "FIG.", "3.Unique Overhangs And Unique Couplings.", "FIG.", "3 illustrates the point that the number of unique overhangs of each size (e.g.", "the total number of unique overhangs composed of 1 or 2 or 3, etc.", "nucleotides) exceeds the number of unique couplings that can result from the use of all the unique overhangs of that size.", "For example, there are 4 unique 3′ overhangs composed of a single nucleotide, and 4 unique 5′ overhangs composed of a single nucleotide.", "Yet the total number of unique couplings that can be made using all the 8 unique single-nucleotide 3′ overhangs and single-nucleotide 5′ overhangs is 4.FIG.", "4.Unique Overall Assembly Order Achieved by Sequentially Coupling the Building Blocks.", "FIG.", "4 illustrates the fact that in order to assemble a total of “n” nucleic acid building blocks, “n-1” couplings are needed.", "Yet it is sometimes the case that the number of unique couplings available for use is fewer that the “n-1” value.", "Under these, and other, circumstances a stringent non-stochastic overall assembly order can still be achieved by performing the assembly process in sequential steps.", "In this example, 2 sequential steps are used to achieve a designed overall assembly order for five nucleic acid building blocks.", "In this illustration the designed overall assembly order for the five nucleic acid building blocks is: 5′-(#1-#2-#3-#4-#5)-3′, where #1 represents building block number 1, etc.", "FIG.", "5.Unique Couplings Available Using a Two-Nucleotide 3′ Overhang.", "FIG.", "5 further illustrates the point that the number of unique overhangs of each size (here, e.g.", "the total number of unique overhangs composed of 2 nucleotides) exceeds the number of unique couplings that can result from the use of all the unique overhangs of that size.", "For example, there are 16 unique 3′ overhangs composed of two nucleotides, and another 16 unique 5′ overhangs composed of two nucleotides, for a total of 32 as shown.", "Yet the total number of couplings that are unique and not self-binding that can be made using all the 32 unique double-nucleotide 3′ overhangs and double-nucleotide 5′ overhangs is 12.Some apparently unique couplings have “identical twins” (marked in the same shading), which are visually obvious in this illustration.", "Still other overhangs contain nucleotide sequences that can self-bind in a palindromic fashion, as shown and labeled in this figure; thus they not contribute the high stringency to the overall assembly order.", "FIG.", "6.Generation of an Exhaustive Set of Chimeric Combinations by Synthetic Ligation Reassembly.", "FIG.", "6 showcases the power of this invention in its ability to generate exhaustively and systematically all possible combinations of the nucleic acid building blocks designed in this example.", "Particularly large sets (or libraries) of progeny chimeric molecules can be generated.", "Because this method can be performed exhaustively and systematically, the method application can be repeated by choosing new demarcation points and with correspondingly newly designed nucleic acid building blocks, bypassing the burden of re-generating and re-screening previously examined and rejected molecular species.", "It is appreciated that, codon wobble can be used to advantage to increase the frequency of a demarcation point.", "In other words, a particular base can often be substituted into a nucleic acid building block without altering the amino acid encoded by progenitor codon (that is now altered codon) because of codon degeneracy.", "As illustrated, demarcation points are chosen upon alignment of 8 progenitor templates.", "Nucleic acid building blocks including their overhangs (which are serviceable for the formation of ordered couplings) are then designed and synthesized.", "In this instance, 18 nucleic acid building blocks are generated based on the sequence of each of the 8 progenitor templates, for a total of 144 nucleic acid building blocks (or double-stranded oligos).", "Performing the ligation synthesis procedure will then produce a library of progeny molecules comprised of yield of 818 (or over 1.8×1016) chimeras.", "FIG.", "7.Synthetic genes from oligos: According to one embodiment of this invention, double-stranded nucleic acid building blocks are designed by aligning a plurality of progenitor nucleic acid templates.", "Preferably these templates contain some homology and some heterology.", "The nucleic acids may encode related proteins, such as related enzymes, which relationship may be based on function or structure or both.", "FIG.", "7 shows the alignment of three polynucleotide progenitor templates and the selection of demarcation points (boxed) shared by all the progenitor molecules.", "In this particular example, the nucleic acid building blocks derived from each of the progenitor templates were chosen to be approximately 30 to 50 nucleotides in length.", "FIG.", "8.Nucleic acid building blocks for synthetic ligation gene reassembly.", "FIG.", "8 shows the nucleic acid building blocks from the example in FIG.", "7.The nucleic acid building blocks are shown here in generic cartoon form, with their compatible overhangs, including both 5′ and 3′ overhangs.", "There are 22 total nucleic acid building blocks derived from each of the 3 progenitor templates.", "Thus, the ligation synthesis procedure can produce a library of progeny molecules comprised of yield of 322 (or over 3.1×1010) chimeras.", "FIG.", "9.Addition of Introns by Synthetic Ligation Reassembly.", "FIG.", "9 shows in generic cartoon form that an intron may be introduced into a chimeric progeny molecule by way of a nucleic acid building block.", "It is appreciated that introns often have consensus sequences at both termini in order to render them operational.", "It is also appreciated that, in addition to enabling gene splicing, introns may serve an additional purpose by providing sites of homology to other nucleic acids to enable homologous recombination.", "For this purpose, and potentially others, it may be sometimes desirable to generate a large nucleic acid building block for introducing an intron.", "If the size is overly large easily genrating by direct chemical synthesis of two single stranded oligos, such a specialized nucleic acid building block may also be generated by direct chemical synthesis of more than two single stranded oligos or by using a polymerase-based amplification reaction as shown in FIG.", "2.FIG.", "10.Ligation Reassembly Using Fewer Than All The Nucleotides Of An Overhang.", "FIG.", "10 shows that coupling can occur in a manner that does not make use of every nucleotide in a participating overhang.", "The coupling is particularly lively to survive (e.g.", "in a transformed host) if the coupling reinforced by treatment with a ligase enzyme to form what may be referred to as a “gap ligation” or a “gapped ligation”.", "It is appreciated that, as shown, this type of coupling can contribute to generation of unwanted background product(s), but it can also be used advantageously increase the diversity of the progeny library generated by the designed ligation reassembly.", "FIG.", "11.Avoidance of unwanted self-ligation in palindromic couplings.", "As mentioned before and shown in FIG.", "5, certain overhangs are able to undergo self-coupling to form a palindromic coupling.", "A coupling is strengthened substantially if it is reinforced by treatment with a ligase enzyme.", "Accordingly, it is appreciated that the lack of 5′ phosphates on these overhangs, as shown, can be used advantageously to prevent this type of palindromic self-ligation.", "Accordingly, this invention provides that nucleic acid building blocks can be chemically made (or ordered) that lack a 5′ phosphate group (or alternatively they can be remove—e.g.", "by treatment with a phosphatase enzyme such as a calf intestinal alkaline phosphatase (CIAP)— in order to prevent palindromic self-ligations in ligation reassembly processes.", "FIG.", "12.Pathway Engineering.", "It is a goal of this invention to provide ways of making new gene pathways using ligation reassembly, optionally with other directed evolution methods such as saturation mutagenesis.", "FIG.", "12 illustrates a preferred approach that may be taken to achieve this goal.", "It is appreciated that naturally-occurring microbial gene pathways are linked more often than naturally-occurring eukaryotic (e.g.", "plant) gene pathways, which are sometime only partially linked.", "In a particular embodiment, this invention provides that regulatory gene sequences (including promoters) can be introduced in the form of nucleic acid building blocks into progeny gene pathways generated by ligation reassembly processes.", "Thus, originally linked microbial gene pathways, as well as originally unlinked genes and gene pathways, can be thus converted to acquire operability in plants and other eukaryotes.", "FIG.", "13.Avoidance of unwanted self-ligation in palindromic couplings.", "FIG.", "13 illustrates that another goal of this invention, in addition to the generation of novel gene pathways, is the subjection of gene pathways—both naturally occurring and man-made—to mutagenesis and selection in order to achieve improved progeny molecules using the instantly disclosed methods of directed evolution (including saturation mutagenesis and synthetic ligation reassembly).", "In a particular embodiment, as provided by the instant invention, both microbial and plant pathways can be improved by directed evolution, and as shown, the directed evolution process can be performed both on genes prior to linking them into pathways, and on gene pathways themselves.", "FIG.", "14.Conversion of Microbial Pathways to Eukaryotic Pathways.", "In a particular embodiment, this invention provides that microbial pathways can be converted to pathways operable in plants and other eukaryotic species by the introduction of regulatory sequences that function in those species.", "Preferred regulatory sequences include promoters, operators, and activator binding sites.", "As shown, a preferred method of achieving the introduction of such serviceable regulatory sequences is in the form of nucleic acid building blocks, particularly through the use of couplings in ligation reassembly processes.", "These couplings in FIG.", "14 are marked with the letters A, B, C, D and F. FIG.", "15.Engineering of differentially activatable stacked traits in novel transgenic plants using directed evolution and holistic whole cell monitoring.", "It is a goal of this invention to provide ways of introducing differentially activatable stacked traits into a transgenic cell or organism, the effects of which is holistically monitored.", "FIG.", "15 illustrates an approach that may be taken to introduce a plurality of stacked traits into an organism, such as but not limited to a plant, and to carry out holistic whole cell or organism monitoring.", "Holistic monitoring can include methods pertaining to genomics, RNA profiling, proteomics, metabolomics, and lipid profiling.", "FIG.", "16.Differential Activation of Selected Traits Can Be Achieved by Adjusting and Controlling the Environment of the Traits.", "In a particular embodiment, this invention provides that stacked traits can be introduced into an organism that are differentially activatable, allowing screening under various conditions.", "FIG.", "16 illustrates an example in which the stacked traits comprise genetically introduced enzymes.", "In this example, the enzymes can be selectively and differentially activated by adjusting the environment to which they are exposed.", "FIG.", "17.Desired or improved traits for harvesting, processing, and storage conditions.", "One of the goals of this invention is to provide a method that allows the generation of recombinant proteins with desired or improved activities.", "In a particular embodiment, as illustrated in this figure, a potential application of this method is screening transgenic cells for various responses to harvesting, processing, and storage conditions of biological reagents and strains.", "The transgenic cells have had stacked traits that are differentially activatable introduced.", "Screening methods that pertain to methods of genomics, proteomics, RNA profiling, metabolomics, and lipid profiling can be utilized and assessed under various specific conditions that include but are not limited to variations in pH, temperature, and other environmental conditions.", "FIG.", "18.Mutagenesis and production of a transgenic organism.", "In another embodiment of this invention, it provides a general method to introduce a library of mutagenized nucleotide sequences (e.g., saturation mutagenesis and/or ligation reassembly) into an organism, and to screen the transgenic organisms for various holistic phenotypes (preferably using a high throughput method).", "Optionally, mutations can be combined and the organisms rescreened and/or a second library can be introduced into the transgenic organisms and the process repeated.", "In a preferred embodiment, the starting population is comprised of an organism strain to be subjected to improvement or evolution in order to produce a resultant population comprised of an improved organism strain that has a desired trait.", "FIG.", "19 Gene Product Processing.", "FIG.", "19 illustrates that various processing or decorating steps occur to a gene product prior to it being active.", "This is a schematic of various processing steps that render a product active or inactive.", "Once a gene product is active it can be differentially expressed and in certain cases modifications in its activities or properties can be screened.", "FIG.", "20.Differential Activation of Selected Precursor (Inactive) Gene Products.", "FIG.", "20 is a schematic that illustrates post-translational modifications as a potential process that differentially activates gene products.", "Differential activation of gene products should be considered when designing screening assays.", "In screening assays, a transgenic organism may not be selected if the gene product has been inactivated due to post-translational effects such as proteolytic cleavage.", "FIG.", "21.Production of an improved organism or strain that has a desired trait.", "In another embodiment of this invention, it provides a general method to introduce a library of mutagenized nucleotide sequences into an organism, and to screen the transgenic organisms or strain for various phenotypes (preferably using a high throughput method).", "Screening methods that pertain to methods of genomics, proteomics, RNA profiling, metabolomics, and lipid profiling can be utilized to identify a subset of desired mutants, such as “up-mutants”.", "Optionally, mutations can be combined and the organisms rescreened and/or a second library can be introduced into the transgenic organisms and the process repeated.", "In a preferred embodiment, the starting population is comprised of an organism strain to be subjected to improvement or evolution in order to produce a resultant population comprised of an improved organism strain that has a desired trait.", "FIG.", "22.Reassortment of polynucleotide sequences to produce an improved sequence that has a desired trait.", "Another goal of this invention is to provide a method to prepare mutagenized polynucleotides, to screen the polynucleotide products, and thereby produce an improved sequence with a desired trait.", "For example, as illustrated in FIG.", "22, mutagenized polynucleotides can be generated by in vivo based reassortment methods such as transposon-based or homologous recombination-based methods.", "Subsequently, the transgenic organisms can be screened to select a desirable subset of mutants (such as those with an enhanced trait or “up mutant”).", "The subset of organisms can be selected and various mutations can be combined.", "The resultant strain can undergo further rounds of selection for an “up mutant” and/or the improved genomic sequence can be selected and determined.", "FIG.", "23.Strain Improvement.", "FIG.", "23 further illustrates the utility of this invention for the generation of improved strains or organisms.", "This schematic illustratively compares classical and modified classical genetic methods with a method provided in this invention.", "This invention provides for the generation of strains that harbor more mutations than are typically harbored by strains generated by classical genetic approaches.", "The generation of strains with numerous mutations and subsequent screening of such strains will allow for the selection of improved strains.", "As illustrated in this figure, an embodiment of this invention is to generate random clones (e.g., that are a result of three levels of mutagenesis), create transgenic organisms upon the transfer of these clones in a high throughput process, allow in vivo recombination due to homologous recombination, transposon insertion, or suicide plasmids, and identify strains with improved characteristics by screening.", "Subsequently, the clones that rendered improved characteristics could be identified and combined into one strain with the goal of generating an improved strain due to multiple genetic mutations.", "FIG.", "24.Iterative Strain Improvement.", "This figure illustrates how this invention provides a method for iterative strain improvement by allowing multiple rounds of mutagenesis, recombination, and selection.", "In this schematic, a library from an organism is subjected to mutagenesis and then transformed into a parent organism.", "Once in the cell, additional variation is introduced by in vivo recombination (e.g., homologous recombination).", "Resultant strains are screened for a desired or enhanced trait (an “up mutant”) and the mutations are identified and sequenced.", "Subsequently, various set or subsets of identified clones can be recombined to create further strain improvements.", "FIG.", "25.Illustrative diagram for the introduction of mutations for genome site saturated mutagenesis.", "In one sense, this method permits the targeted construction of markerless deletions, insertions, and point mutations into a genome (such as a bacterial chromosome) for genome site saturation mutagenesis.", "Libraries of genomes can be mutagenized (and multiply mutagenized) and introduced into cells, allowing recombination with genomic alleles.", "For example as illustrated in this diagram, a suicide plasmid that carries a mutant allele and the recognition site of the yeast meganuclease I-SceI, can be inserted into a genome by homologous recombination between the mutant and the wild-type alleles.", "Further recombination results in either a mutant or a wildtype chromosome.", "Pools of mutants generated from the same genome fragment can be combined and stored in one position of an array such that every fragment of the genome can be mutated to saturation.", "FIG.", "26.Producing polynucleotides via interrupted synthesis methods.", "An embodiment of this invention provides for the production of chimeric/mutagenized polynucleotides (including coding and noncoding regions) generated by incomplete extension.", "Incomplete extension can be used to generate intermediate products of varying length that ultimately may be utilized to generate pools of chimeric/mutagenized polynucleotides.", "Various methods can be utilized to interrupt synthesis of nucleic acids: abbreviated annealing times (as exemplified in FIG.", "27), decreased dNTP concentrations, multiple monobinders priming one polybinder template, template chemistry (such as using a template with chemically modified bases), a DNA polymerase with decreased activity, and/or the use of modified nucleotides during synthesis (such as ddCTP).", "FIG.", "27.Utilizing PCR cycles with abbreviated annealing times for interrupted synthesis.", "An embodiment of this invention provides for the production of chimeric/mutagenized polynucleotides (including coding and noncoding regions) generated by interrupted synthesis methods.", "Variations of standard PCR cycles that utilize abbreviated annealing times is one method that can lead to incomplete extension.", "As illustrated, there are numerous possible variations (such as, but not limited to, variations 1-5) that could be utilized.", "FIG.", "28.Example of a flow chart that is serviceable for performing computer-aided analysis according to this invention.", "E. DEFINITIONS OF TERMS In order to facilitate understanding of the examples provided herein, certain frequently occurring methods and/or terms will be described.", "The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds (e.g., a VLSIPS peptide array, polynucleotide array, and/or combinatorial small molecule array), biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made form biological materials such as bacteria, plants, fungi, or animal (particular mammalian) cells or tissues.", "Agents are evaluated for potential activity as anti-neoplastics, anti-inflammatories or apoptosis modulators by inclusion in screening assays described hereinbelow.", "Agents are evaluated for potential activity as specific protein interaction inhibitors (i.e., an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which doe snot substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.", "An “ambiguous base requirement” in a restriction site refers to a nucleotide base requirement that is not specified to the fullest extent, i.e.", "that is not a specific base (such as, in a non-limiting exemplification, a specific base selected from A, C, G, and T), but that are used in the art as well as herein to represent ambiguity in bases include the following: R=G or A; Y=C or T; M=A or C; K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C; V=G or C or A; D=G or A or T; N=A or C or G or T. The term “amino acid” as used herein refers to any organic compound that contains an amino group (—NH2) and a carboxyl group (—COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds.", "The “twenty naturally encoded polypeptide-forming alpha-amino acids” are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C), gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or 1), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (trp or W), tyrosine (tyr or Y), and valine (val or V).", "The term “amplification” means that the number of copies of a polynucleotide is increased.", "The term “antibody”, as used herein, refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab′, (Fab′)2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen.", "These antibody fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., Harlow and Lane, supra), and are described further, as follows.", "(1) An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.", "(2) An Fab′ fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule fragments are obtained per antibody molecule treated in this manner.", "(3) An (Fab′)2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.", "A (Fab′)2 fragment is a dimer of two Fab′ fragments, held together by two disulfide bonds.", "(4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.", "(5) An single chain antibody (“SCA”) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.", "The term “Applied Molecular Evolution” (“AME”) means the application of an evolutionary design algorithm to a specific, useful goal.", "While many different library formats for AME have been reported for polynucleotides, peptides and proteins (phage, lad and polysomes), none of these formats have provided for recombination by random cross-overs to deliberately create a combinatorial library.", "A molecule that has a “chimeric property” is a molecule that is: 1) in part homologous and in part heterologous to a first reference molecule; while 2) at the same time being in part homologous and in part heterologous to a second reference molecule; without 3) precluding the possibility of being at the same time in part homologous and in part heterologous to still one or more additional reference molecules.", "In a non-limiting embodiment, a chimeric molecule may be prepared by assemblying a reassortment of partial molecular sequences.", "In a non-limiting aspect, a chimeric polynucleotide molecule may be prepared by synthesizing the chimeric polynucleotide using plurality of molecular templates, such that the resultant chimeric polynucleotide has properties of a plurality of templates.", "The term “cognate” as used herein refers to a gene sequence that is evolutionarily and functionally related between species.", "For example, but not limitation, in the human genome the human CD4 gene is the cognate gene to the mouse 3d4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.", "A “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.", "Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.", "As used herein, the term “complementarity-determining region” and “CDR” refer to the art-recognized term as exemplified by the Kabat and Chothia CDR definitions also generally known as supervariable regions or hypervariable loops (Chothia and Lesk, 1987; Clothia et al, 1989; Kabat et al, 1987; and Tramontano et al, 1990).", "Variable region domains typically comprise the amino-terminal approximately 105-115 amino acids of a naturally-occurring immunoglobulin chain (e.g., amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.", "“Conservative amino acid substitutions” refer to the interchangeability of residues having similar side chains.", "For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.", "Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.", "The term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.", "In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.", "For illustration, the nucleotide sequence “TATAC” corresponds to a reference “TATAC” and is complementary to a reference sequence “GTATA.” The term “degrading effective” amount refers to the amount of enzyme which is required to process at least 50% of the substrate, as compared to substrate not contacted with the enzyme.", "Preferably, at least 80% of the substrate is degraded.", "As used herein, the term “defined sequence framework” refers to a set of defined sequences that are selected on a non-random basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a β-sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations.", "A “defined sequence kernal” is a set of sequences which encompass a limited scope of variability.", "Whereas (1) a completely random 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences but will exhibit a bias for certain residues at certain positions and/or overall, (3) a defined sequence kernal is a subset of sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids).", "A defined sequence kernal generally comprises variant and invariant residue positions and/or comprises variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence.", "Defined sequence kernels can refer to either amino acid sequences or polynucleotide sequences.", "Of illustration and not limitation, the sequences (NNK)10 and (NNM)10, wherein N represents A, T, G, or C; K represents G or T; and M represents A or C, are defined sequence kernels.", "“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.", "The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.", "For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution.", "For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume.", "Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer.", "Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions.", "After digestion the reaction is electrophoresed directly on a gel to isolate the desired fragment.", "“Directional ligation” refers to a ligation in which a 5′ end and a 3′ end of a polynuclotide are different enough to specify a preferred ligation orientation.", "For example, an otherwise untreated and undigested PCR product that has two blunt ends will typically not have a preferred ligation orientation when ligated into a cloning vector digested to produce blunt ends in its multiple cloning site; thus, directional ligation will typically not be displayed under these circumstances.", "In contrast, directional ligation will typically displayed when a digested PCR product having a 5′ EcoR I-treated end and a 3′ BamH I-is ligated into a cloning vector that has a multiple cloning site digested with EcoR I and BamH I.", "The term “DNA shuffling” is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like.", "As used in this invention, the term “epitope” refers to an antigenic determinant on an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as an phytase-specific antibody, binds.", "Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.", "As used herein “epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding body of an antibody.", "Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.", "The terms “fragment”, “derivative” and “analog” when referring to a reference polypeptide comprise a polypeptide which retains at least one biological function or activity that is at least essentially same as that of the reference polypeptide.", "Furthermore, the terms “fragment”, “derivative” or “analog” are exemplified by a “pro-form” molecule, such as a low activity proprotein that can be modified by cleavage to produce a mature enzyme with significantly higher activity.", "A method is provided herein for producing from a template polypeptide a set of progeny polypeptides in which a “full range of single amino acid substitutions” is represented at each amino acid position.", "As used herein, “full range of single amino acid substitutions” is in reference to the naturally encoded 20 naturally encoded polypeptide-forming alpha-amino acids, as described herein.", "The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).", "“Genetic instability”, as used herein, refers to the natural tendency of highly repetitive sequences to be lost through a process of reductive events generally involving sequence simplification through the loss of repeated sequences.", "Deletions tend to involve the loss of one copy of a repeat and everything between the repeats.", "The term “heterologous” means that one single-stranded nucleic acid sequence is unable to hybridize to another single-stranded nucleic acid sequence or its complement.", "Thus areas of heterology means that areas of polynucleotides or polynucleotides have areas or regions within their sequence which are unable to hybridize to another nucleic acid or polynucleotide.", "Such regions or areas are for example areas of mutations.", "The term “homologous” or “homeologous” means that one single-stranded nucleic acid nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence.", "The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentrations as discussed later.", "Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.", "An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, also called CDR's.", "The extent of the framework region and CDR's have been precisely defined; see “Sequences of Proteins of Immunological Interest” (Kabat et al, 1987).", "The sequences of the framework regions of different light or heavy chains are relatively conserved within a specie.", "As used herein, a “human framework region” is a framework region that is substantially identical (about 85 or more, usually 90-95 or more) to the framework region of a naturally occurring human immunoglobulin.", "the framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's.", "The CDR's are primarily responsible for binding to an epitope of an antigen.", "The benefits of this invention extend to “commercial applications” (or commercial processes), which term is used to include applications in commercial industry proper (or simply industry) as well as non-commercial commercial applications (e.g.", "biomedical research at a non-profit institution).", "Relevant applications include those in areas of diagnosis, medicine, agriculture, manufacturing, and academia.", "The term “identical” or “identity” means that two nucleic acid sequences have the same sequence or a complementary sequence.", "Thus, “areas of identity” means that regions or areas of a polynucleotide or the overall polynucleotide are identical or complementary to areas of another polynucleotide or the polynucleotide.", "The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).", "For example, a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated.", "Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.", "By “isolated nucleic acid” is meant a nucleic acid, e.g., a DNA or RNA molecule, that is not immediately contiguous with the 5′ and 3′ flanking sequences with which it normally is immediately contiguous when present in the naturally occurring genome of the organism from which it is derived.", "The term thus describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs); and a nucleic acid that exists as a separate molecule, e.g., a DNA fragment produced by PCR amplification or restriction enzyme digestion, or an RNA molecule produced by in vitro transcription.", "The term also describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein.", "As used herein “ligand” refers to a molecule, such as a random peptide or variable segment sequence, that is recognized by a particular receptor.", "As one of skill in the art will recognize, a molecule (or macromolecular complex) can be both a receptor and a ligand.", "In general, the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor.", "“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Sambrook et al, 1982, p. 146; Sambrook, 1989).", "Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.", "As used herein, “linker” or “spacer” refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in a preferred configuration, e.g., so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.", "As used herein, a “molecular property to be evolved” includes reference to molecules comprised of a polynucleotide sequence, molecules comprised of a polypeptide sequence, and molecules comprised in part of a polynucleotide sequence and in part of a polypeptide sequence.", "Particularly relevant—but by no means limiting—examples of molecular properties to be evolved include enzymatic activities at specified conditions, such as related to temperature; salinity; pressure; pH; and concentration of glycerol, DMSO, detergent, &/or any other molecular species with which contact is made in a reaction environment.", "Additional particularly relevant—but by no means limiting—examples of molecular properties to be evolved include stabilities—e.g.", "the amount of a residual molecular property that is present after a specified exposure time to a specified environment, such as may be encountered during storage.", "The term “mutations” includes changes in the sequence of a wild-type or parental nucleic acid sequence or changes in the sequence of a peptide.", "Such mutations may be point mutations such as transitions or transversions.", "The mutations may be deletions, insertions or duplications.", "A mutation can also be a “chimerization”, which is exemplified in a progeny molecule that is generated to contain part or all of a sequence of one parental molecule as well as part or all of a sequence of at least one other parental molecule.", "This invention provides for both chimeric polynucleotides and chimeric polypeptides.", "As used herein, the degenerate “N,N,G/T” nucleotide sequence represents 32 possible triplets, where “N” can be A, C, G or T. The term “naturally-occurring” as used herein as applied to the object refers to the fact that an object can be found in nature.", "For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.", "Generally, the term naturally occurring refers to an object as present in a non-pathological (un-diseased) individual, such as would be typical for the species.", "As used herein, a “nucleic acid molecule” is comprised of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively.", "Furthermore, a nucleic acid molecule may belong exclusively or chimerically to any group of nucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occurring and not naturally occurring nucleic acids, and synthetic nucleic acids.", "This includes, by way of non-limiting example, nucleic acids associated with any organelle, such as the mitochondria, ribosomal RNA, and nucleic acid molecules comprised chimerically of one or more components that are not naturally occurring along with naturally occurring components.", "Additionally, a “nucleic acid molecule” may contain in part one or more non-nucleotide-based components as exemplified by, but not limited to, amino acids and sugars.", "Thus, by way of example, but not limitation, a ribozyme that is in part nucleotide-based and in part protein-based is considered a “nucleic acid molecule”.", "In addition, by way of example, but not limitation, a nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or alternatively a non-radioactive label, is likewise considered a “nucleic acid molecule”.", "The terms “nucleic acid sequence coding for” or a “DNA coding sequence of” or a “nucleotide sequence encoding” a particular enzyme—as well as other synonymous terms—refer to a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences.", "A “promotor sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.", "The promoter is part of the DNA sequence.", "This sequence region has a start codon at its 3′ terminus.", "The promoter sequence does include the minimum number of bases where elements necessary to initiate transcription at levels detectable above background.", "However, after the RNA polymerase binds the sequence and transcription is initiated at the start codon (3′ terminus with a promoter), transcription proceeds downstream in the 3′ direction.", "Within the promotor sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1) as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.", "The terms “nucleic acid encoding an enzyme (protein)” or “DNA encoding an enzyme (protein)” or “polynucleotide encoding an enzyme (protein)” and other synonymous terms encompasses a polynucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.", "In one preferred embodiment, a “specific nucleic acid molecule species” is defined by its chemical structure, as exemplified by, but not limited to, its primary sequence.", "In another preferred embodiment, a specific “nucleic acid molecule species” is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species.", "Thus, by way of non-limiting example, a “specific nucleic acid molecule species” may be defined by one or more activities or properties attributable to it, including activities or properties attributable its expressed product.", "The instant definition of “assembling a working nucleic acid sample into a nucleic acid library” includes the process of incorporating a nucleic acid sample into a vector-based collection, such as by ligation into a vector and transformation of a host.", "A description of relevant vectors, hosts, and other reagents as well as specific non-limiting examples thereof are provided hereinafter.", "The instant definition of “assembling a working nucleic acid sample into a nucleic acid library” also includes the process of incorporating a nucleic acid sample into a non-vector-based collection, such as by ligation to adaptors.", "Preferably the adaptors can anneal to PCR primers to facilitate amplification by PCR.", "Accordingly, in a non-limiting embodiment, a “nucleic acid library” is comprised of a vector-based collection of one or more nucleic acid molecules.", "In another preferred embodiment a “nucleic acid library” is comprised of a non-vector-based collection of nucleic acid molecules.", "In yet another preferred embodiment a “nucleic acid library” is comprised of a combined collection of nucleic acid molecules that is in part vector-based and in part non-vector-based.", "Preferably, the collection of molecules comprising a library is searchable and separable according to individual nucleic acid molecule species.", "The present invention provides a “nucleic acid construct” or alternatively a “nucleotide construct” or alternatively a “DNA construct”.", "The term “construct” is used herein to describe a molecule, such as a polynucleotide (e.g., a phytase polynucleotide) may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector.", "In a specific—but by no means limiting—aspect, a nucleotide construct is exemplified by a DNA expression DNA expression constructs suitable for the transformation of a host cell.", "An “oligonucleotide” (or synonymously an “oligo”) refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized.", "Such synthetic oligonucleotides may or may not have a 5′ phosphate.", "Those that do not will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase.", "A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.", "To achieve polymerase-based amplification (such as with PCR), a “32—fold degenerate oligonucleotide that is comprised of, in series, at least a first homologous sequence, a degenerate N,N,G/T sequence, and a second homologous sequence” is mentioned.", "As used in this context, “homologous” is in reference to homology between the oligo and the parental polynucleotide that is subjected to the polymerase-based amplification.", "As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship.", "A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.", "For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.", "Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.", "A coding sequence is “operably linked to” another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences.", "The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein.", "As used herein the term “parental polynucleotide set” is a set comprised of one or more distinct polynucleotide species.", "Usually this term fis used in reference to a progeny polynucleotide set which is preferably obtained by mutagenization of the parental set, in which case the terms “parental”, “starting” and “template” are used interchangeably.", "As used herein the term “physiological conditions” refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.", "For example, the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.", "Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions.", "In general, in vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45 C and 0.001-10 mM divalent cation (e.g., Mg++, Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein (e.g., BSA).", "A non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).", "Particular aqueous conditions may be selected by the practitioner according to conventional methods.", "For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents and/or membrane fractions and/or anti-foam agents and/or scintillants.", "Standard convention (5′ to 3′) is used herein to describe the sequence of double standed polynucleotides.", "The term “population” as used herein means a collection of components such as polynucleotides, portions or polynucleotides or proteins.", "A “mixed population: means a collection of components which belong to the same family of nucleic acids or proteins (i.e., are related) but which differ in their sequence (i.e., are not identical) and hence in their biological activity.", "A molecule having a “pro-form” refers to a molecule that undergoes any combination of one or more covalent and noncovalent chemical modifications (e.g.", "glycosylation, proteolytic cleavage, dimerization or oligomerization, temperature-induced or pH-induced conformational change, association with a co-factor, etc.)", "en route to attain a more mature molecular form having a property difference (e.g.", "an increase in activity) in comparison with the reference pro-form molecule.", "When two or more chemical modification (e.g.", "two proteolytic cleavages, or a proteolytic cleavage and a deglycosylation) can be distinguished en route to the production of a mature molecule, the referemce precursor molecule may be termed a “pre-pro-form” molecule.", "As used herein, the term “pseudorandom” refers to a set of sequences that have limited variability, such that, for example, the degree of residue variability at another position, but any pseudorandom position is allowed some degree of residue variation, however circumscribed.", "“Quasi-repeated units”, as used herein, refers to the repeats to be re-assorted and are by definition not identical.", "Indeed the method is proposed not only for practically identical encoding units produced by mutagenesis of the identical starting sequence, but also the reassortment of similar or related sequences which may diverge significantly in some regions.", "Nevertheless, if the sequences contain sufficient homologies to be reasserted by this approach, they can be referred to as “quasi-repeated” units.", "As used herein “random peptide library” refers to a set of polynucleotide sequences that encodes a set of random peptides, and to the set of random peptides encoded by those polynucleotide sequences, as well as the fusion proteins contain those random peptides.", "As used herein, “random peptide sequence” refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a stochastic or random process.", "A random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.", "As used herein, “receptor” refers to a molecule that has an affinity for a given ligand.", "Receptors can be naturally occurring or synthetic molecules.", "Receptors can be employed in an unaltered state or as aggregates with other species.", "Receptors can be attached, covalently or non-covalently, to a binding member, either directly or via a specific binding substance.", "Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.", "“Recombinant” enzymes refer to enzymes produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired enzyme.", "“Synthetic” enzymes are those prepared by chemical synthesis.", "The term “related polynucleotides” means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.", "“Reductive reassortment”, as used herein, refers to the increase in molecular diversity that is accrued through deletion (and/or insertion) events that are mediated by repeated sequences.", "The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence.", "Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.", "Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.", "“Repetitive Index (RI)”, as used herein, is the average number of copies of the quasi-repeated units contained in the cloning vector.", "The term “restriction site” refers to a recognition sequence that is necessary for the manifestation of the action of a restriction enzyme, and includes a site of catalytic cleavage.", "It is appreciated that a site of cleavage may or may not be contained within a portion of a restriction site that comprises a low ambiguity sequence (i.e.", "a sequence containing the principal determinant of the frequency of occurrence of the restriction site).", "Thus, in many cases, relevant restriction sites contain only a low ambiguity sequence with an internal cleavage site (e.g.", "G/AATTC in the EcoR I site) or an immediately adjacent cleavage site (e.g.", "/CCWGG in the EcoR II site).", "In other cases, relevant restriction enzymes [e.g.", "the Eco57 I site or CTGAAG(16/14)] contain a low ambiguity sequence (e.g.", "the CTGAAG sequence in the Eco57 I site) with an external cleavage site (e.g.", "in the N16 portion of the Eco57 I site).", "When an enzyme (e.g.", "a restriction enzyme) is said to “cleave” a polynucleotide, it is understood to mean that the restriction enzyme catalyzes or facilitates a cleavage of a polynucleotide.", "In a non-limiting aspect, a “selectable polynucleotide” is comprised of a 5′ terminal region (or end region), an intermediate region (i.e.", "an internal or central region), and a 3′ terminal region (or end region).", "As used in this aspect, a 5′ terminal region is a region that is located towards a 5′ polynucleotide terminus (or a 5′ polynucleotide end); thus it is either partially or entirely in a 5′ half of a polynucleotide.", "Likewise, a 3′ terminal region is a region that is located towards a 3′ polynucleotide terminus (or a 3′ polynucleotide end); thus it is either partially or entirely in a 3′ half of a polynucleotide.", "As used in this non-limiting exemplification, there may be sequence overlap between any two regions or even among all three regions.", "The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.", "The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or 1) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.", "This “substantial identity”, as used herein, denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 80 percent sequence identity, preferably at least 85 percent identity, often 90 to 95 percent sequence identity, and most commonly at least 99 percent sequence identity as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.", "As known in the art “similarity” between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme.", "Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).", "As used herein, the term “single-chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally liked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]x), and which may comprise additional amino acid sequences at the amino- and/or carboxy-termini.", "For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.", "As an example, a scFv is a single-chain antibody.", "Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino substantially encoded by genes of the immunoglobulin superfamily (e.g., see Williams and Barclay, 1989, pp.", "361-368, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human heavy chain or light chain gene sequence.", "A functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).", "The members of a pair of molecules (e.g., an antibody-antigen pair or a nucleic acid pair) are said to “specifically bind” to each other if they bind to each other with greater affinity than to other, non-specific molecules.", "For example, an antibody raised against an antigen to which it binds more efficiently than to a non-specific protein can be described as specifically binding to the antigen.", "(Similarly, a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions (see above).)", "“Specific hybridization” is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.", "The term “specific polynucleotide” means a polynucleotide having certain end points and having a certain nucleic acid sequence.", "Two polynucleotides wherein one polynucleotide has the identical sequence as a portion of the second polynucleotide but different ends comprises two different specific polynucleotides.", "“Stringent hybridization conditions” means hybridization will occur only if there is at least 90% identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences.", "See Sambrook et al, 1989, which is hereby incorporated by reference in its entirety.", "Also included in the invention are polypeptides having sequences that are “substantially identical” to the sequence of a phytase polypeptide, such as one of SEQ ID 1.A “substantially identical” amino acid sequence is a sequence that differs from a reference sequence only by conservative amino acid substitutions, for example, substitutions of one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).", "Additionally a “substantially identical” amino acid sequence is a sequence that differs from a reference sequence or by one or more non-conservative substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site the molecule, and provided that the polypeptide essentially retains its behavioural properties.", "For example, one or more amino acids can be deleted from a phytase polypeptide, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity.", "For example, amino- or carboxyl-terminal amino acids that are not required for phytase biological activity can be removed.", "Such modifications can result in the development of smaller active phytase polypeptides.", "The present invention provides a “substantially pure enzyme”.", "The term “substantially pure enzyme” is used herein to describe a molecule, such as a polypeptide (e.g., a phytase polypeptide, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated.", "For example, a substantially pure molecule, such as a polypeptide, can be at least 60%, by dry weight, the molecule of interest.", "The purity of the polypeptides can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis.", "As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.", "Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition.", "Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.", "Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.", "As used herein, the term “variable segment” refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence.", "A variable segment” refers to a portion of a nascent peptide which comprises a random pseudorandom, or defined kernal sequence.", "A variable segment can comprise both variant and invariant residue positions, and the degree of residue variation at a variant residue position may be limited: both options are selected at the discretion of the practitioner.", "Typically, variable segments are about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable segments may be longer and may comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.", "The term “wild-type” means that the polynucleotide does not comprise any mutations.", "A “wild type” protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.", "The term “working”, as in “working sample”, for example, is simply a sample with which one is working.", "Likewise, a “working molecule”, for example is a molecule with which one is working.", "1.SCREENING AND SELECTION 1.1.Overview of Screening and Selection Screening is, in general, a two-step process in which one first determines which cells do and do not express a screening marker and then physically separates the cells having the desired property.", "Screening markers include, for example, luciferase, beta-galactosidase, and green fluorescent protein.", "Screening can also be done by observing a cell holistically including but not limited to utilizing methods pertaining to genomics, RNA profiling, proteomics, metabolomics, and lipidomics as well as observing such aspects of growth as colony size, halo formation, etc.", "Additionally, screening for production of a desired compound, such as a therapeutic drug or “designer chemical” can be accomplished by observing binding of cell products to a receptor or ligand, such as on a solid support or on a column.", "Such screening can additionally be accomplished by binding to antibodies, as in an ELISA.", "In some instances the screening process is preferably automated so as to allow screening of suitable numbers of colonies or cells.", "Some examples of automated screening devices include fluorescence activated cell sorting (FACS), especially in conjunction with cells immobilized in agarose (see Powell et. al.", "Bio/Technology 8: 333-337 (1990); Weaver et. al.", "Methods 2: 234-247 (1991)), automated ELISA assays, scintillation proximity assays (Hart, H. E. et al., Molecular Immunol.", "16: 265-267 (1979)) and the formation of fluorescent, colored or UV absorbing compounds on agar plates or in microtitre wells (Krawiec, S., Devel.", "Indust.", "Microbiology 31: 103-114 (1990)).", "Selection is a form of screening in which identification and physical separation are achieved simultaneously, for example, by expression of a selectable marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa).", "Selectable markers can include, for example, drug, toxin resistance, or nutrient synthesis genes.", "Selection is also done by such techniques as growth on a toxic substrate to select for hosts having the ability to detoxify a substrate, growth on a new nutrient source to select for hosts having the ability to utilize that nutrient source, competitive growth in culture based on ability to utilize a nutrient source, etc.", "In particular, uncloned but differentially expressed proteins (e.g., those induced in response to new compounds, such as biodegradable pollutants in the medium) can be screened by differential display (Appleyard et al.", "Mol.", "Gen. Gent.", "247: 338-342 (1995)).", "Hopwood (Phil Trans R. Soc.", "Lond B 324: 549-562) provides a review of screens for antibiotic production.", "Omura (Microbio.", "Rev.", "50: 259-279 (1986) and Nisbet (Ann Rev.", "Med.", "Chem.", "21: 149-157 (1986)) disclose screens for antimicrobial agents, including supersensitive bacteria, detection of beta-lactamase and D,D-carboxypeptidase inhibition, beta-lactamase induction, chromogenic substrates and monoclonal antibody screens.", "Antibiotic targets can also be used as screening targets in high throughput screening.", "Antifungals are typically screened by inhibition of fungal growth.", "Pharmacological agents can be identified as enzyme inhibitors using plates containing the enzyme and a chromogenic substrate, or by automated receptor assays.", "Hydrolytic enzymes (e.g., proteases, amylases) can be screened by including the substrate in an agar plate and scoring for a hydrolytic clear zone or by using a colorimetric indicator (Steele et al.", "Ann.", "Rev.", "Microbiol.", "45: 89-106 (1991)).", "This can be coupled with the use of stains to detect the effects of enzyme action (such as congo red to detect the extent of degradation of celluloses and hemicelluloses).", "Tagged substrates can also be used.", "For example, lipases and esterases can be screened using different lengths of fatty acids linked to umbelliferyl.", "The action of lipases or esterases removes this tag from the fatty acid, resulting in a quenching or enhancement of umbelliferyl fluorescence.", "These enzymes can be screened in microtiter plates by a robotic device.", "1.2.High-Throughput Cellular Screening: Utilizing Various Types of “Omics” Functional genomics seeks to discover gene function once nucleotide sequence information is available.", "Proteomics (the study of protein properties such as expression, post-translational modifications, interactions, etc.)", "and metabolomics (analysis of metabolite pools) are fast-emerging fields complementing functional genomics, that provide a global, integrated view of cellular processes.", "The variety of techniques and methods used in this effort include the use of bioinformatics, gene-array chips, mRNA differential display, disease models, protein discovery and expression, and target validation.", "The ultimate goal of many of these efforts has been to develop high-throughput screens for genes of unknown function.", "For review see Greenbaum D. et al.", "Genome Res, 11(9): 1463-8 (2001).", "1.2.1 Genomics An embodiment of this invention provides for cellular screening; in a particular embodiment, cellular screening may include genomics.", "“High throughput genomics” refers to application of genomic or genetic data or analysis techniques that use microarrays or other genomic technologies to rapidly identify large numbers of genes or proteins, or distinguish their structure, expression or function from normal or abnormal cells or tissues.", "An observer can be a person viewing a slide with a microscope or an observer who views digital images.", "Alternatively, an observer can be a computer-based image analysis system, which automatically observes, analyses and quantitates biological arrayed samples with or without user interaction.", "Genomics can refer to various investigative techniques that are broad in scope but often refers to measuring gene expression for multitudes of genes simultaneously.", "For a review see Lockhart, D. J. and Winzeler, E. A.", "2000.Genomics, gene expression and DNA arrays.", "Nature, 405(6788): 827-36.1.2.1.1.Biological Chips 1.2.1.1.1.General Considerations In one aspect the present invention provides for the use of arrays of oligonucleotide probes immobilized in microfabricated patterns on silica chips for analyzing molecular interactions of biological interest.", "In some assay formats, the oligonucleotide probe is tethered, i.e., by covalent attachment, to a solid support, and arrays of oligonucleotide probes immobilized on solid supports have been used to detect specific nucleic acid sequences in a target nucleic acid.", "See, e.g., PCT patent publication Nos.", "WO 89/10977 and 89/11548.Others have proposed the use of large numbers of oligonucleotide probes to provide the complete nucleic acid sequence of a target nucleic acid but failed to provide an enabling method for using arrays of immobilized probes for this purpose.", "See U.S. Pat.", "Nos.", "5,202,231 and 5,002,867 and PCT patent publication No.", "WO 93/17126.See U.S. Pat.", "No.", "5,143,854 and PCT patent publication Nos.", "WO 90/15070 and 92/10092, each of which is incorporated herein by reference.", "Microfabricated arrays of large numbers of oligonucleotide probes, called “DNA chips” offer great promise for a wide variety of applications.", "New methods and reagents are required to realize this promise, and the present invention helps meet that need.", "1.2.1.1.2.General Strategies for Utilizing Nucleic Acid Arrays The invention provides several strategies employing immobilized arrays of probes for comparing a reference sequence of known sequence with a target sequence showing substantial similarity with the reference sequence, but differing in the presence of, e.g., mutations.", "In a first embodiment, the invention provides a tiling strategy employing an array of immobilized oligonucleotide probes comprising at least two sets of probes.", "A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence.", "A second probe set comprises a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets.", "The probes in the first probe set have at least two interrogation positions corresponding to two contiguous nucleotides in the reference sequence.", "One interrogation position corresponds to one of the contiguous nucleotides, and the other interrogation position to the other.", "In a second embodiment, the invention provides a tiling strategy employing an array comprising four probe sets.", "A first probe set comprises a plurality of probes, each probe comprising a segment of at least three nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence.", "Second, third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set.", "The probes in the second, third and fourth probe sets are identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.", "The first probe set often has at least 100 interrogation positions corresponding to 100 contiguous nucleotides in the reference sequence.", "Sometimes the first probe set has an interrogation position corresponding to every nucleotide in the reference sequence.", "The segment of complementarity within the probe set is usually about 9-21 nucleotides.", "Although probes may contain leading or trailing sequences in addition to the 9-21 sequences, many probes consist exclusively of a 9-21 segment of complementarity.", "In a third embodiment, the invention provides immobilized arrays of probes tiled for multiple reference sequences.", "one such array comprises at least one pair of first and second probe groups, each group comprising first and second sets of probes as defined in the first embodiment.", "Each probe in the first probe set from the first group is exactly complementary to a subsequence of a first reference sequence, and each probe in the first probe set from the second group is exactly complementary to a subsequence of a second reference sequence.", "Thus, the first group of probes are tiled with respect to a first reference sequence and the second group of probes with respect to a second reference sequence.", "Each group of probes can also include third and fourth sets of probes as defined in the second embodiment.", "In some arrays of this type, the second reference sequence is a mutated form of the first reference sequence.", "In a fourth embodiment, the invention provides arrays for block tiling.", "Block tiling is a species of the general tiling strategies described above.", "The usual unit of a block tiling array is a group of probes comprising a wildtype probe, a first set of three mutant probes and a second set of three mutant probes.", "The wildtype probe comprises a segment of at least three nucleotides exactly complementary to a subsequence of a reference sequence.", "The segment has at least first and second interrogation positions corresponding to first and second nucleotides in the reference sequence.", "The probes in the first set of three mutant probes are each identical to a sequence comprising the wildtype probe or a subsequence of at least three nucleotides thereof including the first and second interrogation positions, except in the first interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "The probes in the second set of three mutant probes are each identical to a sequence comprising the wildtype probes or a subsequence of at least three nucleotides thereof including the first and second interrogation positions, except in the second interrogation position, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "In a fifth embodiment, the invention provides methods of comparing a target sequence with a reference sequence using arrays of immobilized pooled probes.", "The arrays employed in these methods represent a further species of the general tiling arrays noted above.", "In these methods, variants of a reference sequence differing from the reference sequence in at least one nucleotide are identified and each is assigned a designation.", "An array of pooled probes is provided, with each pool occupying a separate cell of the array.", "Each pool comprises a probe comprising a segment exactly complementary to each variant sequence assigned a particular designation.", "The array is then contacted with a target sequence comprising a variant of the reference sequence.", "The relative hybridization intensities of the pools in the array to the target sequence are determined.", "The identity of the target sequence is deduced from the pattern of hybridization intensities.", "Often, each variant is assigned a designation having at least one digit and at least one value for the digit.", "In this case, each pool comprises a probe comprising a segment exactly complementary to each variant sequence assigned a particular value in a particular digit.", "When variants are assigned successive numbers in a numbering system of base m having n digits, n×(m−1) pooled probes are used are used to assign each variant a designation.", "In a sixth embodiment, the invention provides a pooled probe for trellis tiling, a further species of the general tiling strategy.", "In trellis tiling, the identity of a nucleotide in a target sequence is determined from a comparison of hybridization intensities of three pooled trellis probes.", "A pooled trellis probe comprises a segment exactly complementary to a subsequence of a reference sequence except at a first interrogation position occupied by a pooled nucleotide N, a second interrogation position occupied by a pooled nucleotide selected from the group of three consisting of (1) M or K, (2) R or Y and (3) S or W, and a third interrogation position occupied by a second pooled nucleotide selected from the group.", "The pooled nucleotide occupying the second interrogation position comprises a nucleotide complementary to a corresponding nucleotide from the reference sequence when the second pooled probe and reference sequence are maximally aligned, and the pooled nucleotide occupying the third interrogation position comprises a nucleotide complementary to a corresponding nucleotide from the reference sequence when the third pooled probe and the reference sequence are maximally aligned.", "Standard IUPAC nomenclature is used for describing pooled nucleotides.", "In trellis tiling, an array comprises at least first, second and third cells, respectively occupied by first, second and third pooled probes, each according to the generic description above.", "However, the segment of complementarity, location of interrogation positions, and selection of pooled nucleotide at each interrogation position may or may not differ between the three pooled probes subject to the following constraint.", "One of the three interrogation positions in each of the three pooled probes must align with the same corresponding nucleotide in the reference sequence.", "This interrogation position must be occupied by a N in one of the pooled probes, and a different pooled nucleotide in each of the other two pooled probes.", "In a seventh embodiment, the invention provides arrays for bridge tiling.", "Bridge tiling is a species of the general tiling strategies noted above, in which probes from the first probe set contain more than one segment of complementarity.", "In bridge tiling, a nucleotide in a reference sequence is usually determined from a comparison of four probes.", "A first probe comprises at least first and second segments, each of at least three nucleotides and each exactly complementary to first and second subsequences of a reference sequences.", "The segments including at least one interrogation position corresponding to a nucleotide in the reference sequence.", "Either (1) the first and second subsequences are noncontiguous in the reference sequence, or (2) the first and second subsequences are contiguous and the first and second segments are inverted relative to the first and second subsequences.", "The arrays further comprises second, third and fourth probes, which are identical to a sequence comprising the first probe or a subsequence thereof comprising at least three nucleotides from each of the first and second segments, except in the at least one interrogation position, which differs in each of the probes.", "In a species of bridge tiling, referred to as deletion tiling, the first and second subsequences are separated by one or two nucleotides in the reference sequence.", "In an eighth embodiment, the invention provides arrays of probes for multiplex tiling.", "Multiplex tiling is a strategy, in which the identity of two nucleotides in a target sequence is determined from a comparison of the hybridization intensities of four probes, each having two interrogation positions.", "Each of the probes comprising a segment of at least 7 nucleotides that is exactly complementary to a subsequence from a reference sequence, except that the segment may or may not be exactly complementary at two interrogation positions.", "The nucleotides occupying the interrogation positions are selected by the following rules: (1) the first interrogation position is occupied by a different nucleotide in each of the four probes, (2) the second interrogation position is occupied by a different nucleotide in each of the four probes, (3) in first and second probes, the segment is exactly complementary to the subsequence, except at no more than one of the interrogation positions, (4) in third and fourth probes, the segment is exactly complementary to the subsequence, except at both of the interrogation positions.", "In a ninth embodiment, the invention provides arrays of immobilized probes including helper mutations.", "Helper mutations are useful for, e.g., preventing self-annealing of probes having inverted repeats.", "In this strategy, the identity of a nucleotide in a target sequence is usually determined from a comparison of four probes.", "A first probe comprises a segment of at least 7 nucleotides exactly complementary to a subsequence of a reference sequence except at one or two positions, the segment including an interrogation position not at the one or two positions.", "The one or two positions are occupied by helper mutations.", "Second, third and fourth mutant probes are each identical to a sequence comprising the wildtype probe or a subsequence thereof including the interrogation position and the one or two positions, except in the interrogation position, which is occupied by a different nucleotide in each of the four probes.", "In a tenth embodiment, the invention provides arrays of probes comprising at least two probe sets, but lacking a probe set comprising probes that are perfectly matched to a reference sequence.", "Such arrays are usually employed in methods in which both reference and target sequence are hybridized to the array.", "The first probe set comprising a plurality of probes, each probe comprising a segment exactly complementary to a subsequence of at least 3 nucleotides of a reference sequence except at an interrogation position.", "The second probe set comprises a corresponding probe for each probe in the first probe set, the corresponding probe in the second probe set being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least three nucleotides thereof that includes the interrogation position, except that the interrogation position is occupied by a different nucleotide in each of the two corresponding probes and the complement to the reference sequence.", "In an eleventh embodiment, the invention provides methods of comparing a target sequence with a reference sequence comprising a predetermined sequence of nucleotides using any of the arrays described above.", "The methods comprise hybridizing the target nucleic acid to an array and determining which probes, relative to one another, in the array bind specifically to the target nucleic acid.", "The relative specific binding of the probes indicates whether the target sequence is the same or different from the reference sequence.", "In some such methods, the target sequence has a substituted nucleotide relative to the reference sequence in at least one undetermined position, and the relative specific binding of the probes indicates the location of the position and the nucleotide occupying the position in the target sequence.", "In some methods, a second target nucleic acid is also hybridized to the array.", "The relative specific binding of the probes then indicates both whether the target sequence is the same or different from the reference sequence, and whether the second target sequence is the same or different from the reference sequence.", "In some methods, when the array comprises two groups of probes tiled for first and second reference sequences, respectively, the relative specific binding of probes in the first group indicates whether the target sequence is the same or different from the first reference sequence.", "The relative specific binding of probes in the second group indicates whether the target sequence is the same or different from the second reference sequence.", "Such methods are particularly useful for analyzing heterologous alleles of a gene.", "Some methods entail hybridizing both a reference sequence and a target sequence to any of the arrays of probes described above.", "Comparison of the relative specific binding of the probes to the reference and target sequences indicates whether the target sequence is the same or different from the reference sequence.", "In a twelfth embodiment, the invention provides arrays of immobilized probes in which the probes are designed to tile a reference sequence from a human immunodeficiency virus.", "Reference sequences from either the reverse transcriptase gene or protease gene of HIV are of particular interest.", "Some chips further comprise arrays of probes tiling a reference sequence from a 16S RNA or DNA encoding the 16S RNA from a pathogenic microorganism.", "The invention further provides methods of using such arrays in analyzing a HIV target sequence.", "The methods are particularly useful where the target sequence has a substituted nucleotide relative to the reference sequence in at least one position, the substitution conferring resistance to a drug use in treating a patient infected with a HIV virus.", "The methods reveal the existence of the substituted nucleotide.", "The methods are also particularly useful for analyzing a mixture of undetermined proportions of first and second target sequences from different HIV variants.", "The relative specific binding of probes indicates the proportions of the first and second target sequences.", "In a thirteenth embodiment, the invention provides arrays of probes tiled based on reference sequence from a CFTR gene.", "A preferred array comprises at least a group of probes comprising a wildtype probe, and five sets of three mutant probes.", "The wildtype probe is exactly complementary to a subsequence of a reference sequence from a cystic fibrosis gene, the segment having at least five interrogation positions corresponding to five contiguous nucleotides in the reference sequence.", "The probes in the first set of three mutant probes are each identical to the wildtype probe, except in a first of the five interrogation positions, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "The probes in the second set of three mutant probes are each identical to the wildtype probe, except in a second of the five interrogation positions, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "The probes in the third set of three mutant probes are each identical to the wildtype probe, except in a third of the five interrogation positions, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "The probes in the fourth set of three mutant probes are each identical to the wildtype probe, except in a fourth of the five interrogation positions, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "The probes in the fifth set of three mutant probes are each identical to the wildtype probe, except in a fifth of the five interrogation positions, which is occupied by a different nucleotide in each of the three mutant probes and the wildtype probe.", "Preferably, a chip comprises two such groups of probes.", "The first group comprises a wildtype probe exactly complementary to a first reference sequence, and the second group comprises a wildtype probe exactly complementary to a second reference sequence that is a mutated form of the first reference sequence.", "The invention further provides methods of using the arrays of the invention for analyzing target sequences from a CFTR gene.", "The methods are capable of simultaneously analyzing first and second target sequences representing heterozygous alleles of a CFTR gene.", "In a fourteenth embodiment, the invention provides arrays of probes tiling a reference sequence from a p53 gene, an hMLHI gene and/or an MSH2 gene.", "The invention further provides methods of using the arrays described above to analyze these genes.", "The method are useful, e.g., for diagnosing patients susceptible to developing cancer.", "In a fifteenth embodiment, the invention provides arrays of probes tiling a reference sequence from a mitochondrial genome.", "The reference sequence may comprise part or all of the D-loop region, or all, or substantially all, of the mitochondrial genome.", "The invention further provides method of using the arrays described above to analyze target sequences from a mitochondrial genome.", "The methods are useful for identifying mutations associated with disease, and for forensic, epidemiological and evolutionary studies.", "1.2.1.1.3.Specific Strategies for Utilizing Nucleic Acid Arrays The invention provides a number of strategies for comparing a polynucleotide of known sequence (a reference sequence) with variants of that sequence (target sequences).", "The comparison can be performed at the level of entire genomes, chromosomes, genes, exons or introns, or can focus on individual mutant sites and immediately adjacent bases.", "The strategies allow detection of variations, such as mutations or polymorphisms, in the target sequence irrespective whether a particular variant has previously been characterized.", "The strategies both define the nature of a variant and identify its location in a target sequence.", "The strategies employ arrays of oligonucleotide probes immobilized to a solid support.", "Target sequences are analyzed by determining the extent of hybridization at particular probes in the array.", "The strategy in selection of probes facilitates distinction between perfectly matched probes and probes showing single-base or other degrees of mismatches.", "The strategy usually entails sampling each nucleotide of interest in a target sequence several times, thereby achieving a high degree of confidence in its identity.", "This level of confidence is further increased by sampling of adjacent nucleotides in the target sequence to nucleotides of interest.", "The number of probes on the chip can be quite large (e.g., 105-106).", "However, usually only a small proportion of the total number of probes of a given length are represented.", "Some advantage of the use of only a small proportion of all possible probes of a given length include: (i) each position in the array is highly informative, whether or not hybridization occurs; (ii) nonspecific hybridization is minimized; (iii) it is straightforward to correlate hybridization differences with sequence differences, particularly with reference to the hybridization pattern of a known standard; and (iv) the ability to address each probe independently during synthesis, using high resolution photolithography, allows the array to be designed and optimized for any sequence.", "For example the length of any probe can be varied independently of the others.", "The present tiling strategies result in sequencing and comparison methods suitable for routine large-scale practice with a high degree of confidence in the sequence output.", "1.2.1.1.4.General Tiling Strategies 1.1.1.1.4.1.Selection of Reference Sequence The chips are designed to contain probes exhibiting complementarity to one or more selected reference sequence whose sequence is known.", "The chips are used to read a target sequence comprising either the reference sequence itself or variants of that sequence.", "Target sequences may differ from the reference sequence at one or more positions but show a high overall degree of sequence identity with the reference sequence (e.g., at least 75, 90, 95, 99, 99.9 or 99-99%).", "Any polynucleotide of known sequence can be selected as a reference sequence.", "Reference sequences of interest include sequences known to include mutations or polymorphisms associated with phenotypic changes having clinical significance in human patients.", "For example, the CFTR gene and P53 gene in humans have been identified as the location of several mutations resulting in cystic fibrosis or cancer respectively.", "Other reference sequences of interest include those that serve to identify pathogenic microorganisms and/or are the site of mutations by which such microorganisms acquire drug resistance (e.g., the HIV reverse transcriptase gene).", "Other reference sequences of interest include regions where polymorphic variations are known to occur (e.g., the D-loop region of mitochondrial DNA).", "These reference sequences have utility for, e.g., forensic or epidemiological studies.", "Other reference sequences of interest include p34 (related to p53), p65 (implicated in breast, prostate and liver cancer), and DNA segments encoding cytochromes P450 (see Meyer et al., Pharmac.", "Ther.", "46, 349-355 (1990)).", "Other reference sequences of interest include those from the genome of pathogenic viruses (e.g., hepatitis J, B, or Q, herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.", "Other reference sequences of interest are from genomes or episomes of pathogenic bacteria, particularly regions that confer drug resistance or allow phylogenic characterization of the host (e.g., 16S rRNA or corresponding DNA).", "For example, such bacteria include chlanydia, rickettsial bacteria, mycobacteria, staphylococci, treptocci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.", "Other reference sequences of interest include those in which mutations result in the following autosomal recessive disorders: sickle cell anemia, beta-thalassemia, phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe combined immunodeficiency, alpha-1-antitrypsin deficiency, albinism, alkaptonuria, lysosomal storage diseases and Ehlers-Danlos syndrome.", "Other reference sequences of interest include those in which mutations result in X-linked recessive disorders: hemophilia, glucose-6-phosphate dehydrogenase, agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease and fragile X-syndrome.", "Other reference sequences of interest includes those in which mutations result in the following autosomal dominant disorders: familial hypercholesterolemia, polycystic kidney disease, Huntingdon's disease, hereditary spherocytosis, Marfan's syndrome, von Willebrand's disease, neurofibromatosis, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, myotonic dystrophy, muscular dystrophy, osteogenesis imperfecta, acute intermittent porphyria, and von Hippel-Lindau disease.", "The length of a reference sequence can vary widely from a full-length genome, to an individual chromosome, episome, gene, component of a gene, such as an exon, intron or regulatory sequences, to a few nucleotides.", "A reference sequence of between about 2, 5, 10, 20, 50, 100, 5000, 1000, 5,000 or 10,000, 20,000 or 100,000 nucleotides is common.", "Sometimes only particular regions of a sequence (e.g., exons of a gene) are of interest.", "In such situations, the particular regions can be considered as separate reference sequences or can be considered as components of a single reference sequence, as matter of arbitrary choice.", "A reference sequence can be any naturally occurring, mutant, consensus or purely hypothetical sequence of nucleotides, RNA or DNA.", "For example, sequences can be obtained from computer data bases, publications or can be determined or conceived de novo.", "Usually, a reference sequence is selected to show a high degree of sequence identity to envisaged target sequences.", "Often, particularly, where a significant degree of divergence is anticipated between target sequences, more than one reference sequence is selected.", "Combinations of wildtype and mutant reference sequences are employed in several applications of the tiling strategy.", "1.2.1.1.5.Chip Design 1.2.1.1.5.1.Basic Tiling Strategy The basic tiling strategy provides an array of immobilized probes for analysis of target sequences showing a high degree of sequence identity to one or more selected reference sequences.", "The strategy is first illustrated for an array that is subdivided into four probe sets, although it will be apparent that in some situations, satisfactory results are obtained from only two probe sets.", "A first probe set comprises a plurality of probes exhibiting perfect complementarity with a selected reference sequence.", "The perfect complementarity usually exists throughout the length of the probe.", "However, probes having a segment or segments of perfect complementarity that is/are flanked by leading or trailing sequences lacking complementarity to the reference sequence can also be used.", "Within a segment of complementarity, each probe in the first probe set has at least one interrogation position that corresponds to a nucleotide in the reference sequence.", "That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarity between the two.", "If a probe has more than one interrogation position, each corresponds with a respective nucleotide in the reference sequence.", "The identity of an interrogation position and corresponding nucleotide in a particular probe in the first probe set cannot be determined simply by inspection of the probe in the first set.", "As will become apparent, an interrogation position and corresponding nucleotide is defined by the comparative structures of probes in the first probe set and corresponding probes from additional probe sets.", "In principle, a probe could have an interrogation position at each position in the segment complementary to the reference sequence.", "Sometimes, interrogation positions provide more accurate data when located away from the ends of a segment of complementarity.", "Thus, typically a probe having a segment of complementarity of length x does not contain more than x-2 interrogation positions.", "Since probes are typically 9-21 nucleotides, and usually all of a probe is complementary, a probe typically has 1-19 interrogation positions.", "Often the probes contain a single interrogation position, at or near the center of probe.", "For each probe in the first set, there are, for purposes of the present illustration, three corresponding probes from three additional probe sets.", "Thus, there are four probes corresponding to each nucleotide of interest in the reference sequence.", "Each of the four corresponding probes has an interrogation position aligned with that nucleotide of interest.", "Usually, the probes from the three additional probe sets are identical to the corresponding probe from the first probe set with one exception.", "The exception is that at least one (and often only one) interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, is occupied by a different nucleotide in the four probe sets.", "For example, for an A nucleotide in the reference sequence, the corresponding probe from the first probe set has its interrogation position occupied by a T, and the corresponding probes from the additional three probe sets have their respective interrogation positions occupied by A, C, or G, a different nucleotide in each probe.", "Of course, if a probe from the first probe set comprises trailing or flanking sequences lacking complementarity to the reference sequences, these sequences need not be present in corresponding probes from the three additional sets.", "Likewise corresponding probes from the three additional sets can contain leading or trailing sequences outside the segment of complementarity that are not present in the corresponding probe from the first probe set.", "Occasionally, the probes from the additional three probe set are identical (with the exception of interrogation position(s)) to a contiguous subsequence of the full complementary segment of the corresponding probe from the first probe set.", "In this case, the subsequence includes the interrogation position and usually differs from the full-length probe only in the omission of one or both terminal nucleotides from the termini of a segment of complementarity.", "That is, if a probe from the first probe set has a segment of complementarity of length n, corresponding probes from the other sets will usually include a subsequence of the segment of at least length n-2.Thus, the subsequence is usually at least 3, 4, 7, 9, 15, 21, or 25 nucleotides long, most typically, in the range of 9-21 nucleotides.", "The subsequence should be sufficiently long to allow a probe to hybridize detectably more strongly to a variant of the reference sequence mutated at the interrogation position than to the reference sequence.", "The probes can be oligodeoxyribonucleotides or oligoribonucleotides, or any modified forms of these polymers that are capable of hybridizing with a target nucleic sequence by complementary base-pairing.", "Complementary base pairing means sequence-specific base pairing which includes e.g., Watson-Crick base pairing as well as other forms of base pairing such as Hoogsteen base pairing.", "Modified forms include 2□-0-methyl oligoribonucleotides and so-called PNAs, in which oligodeoxyribonucleotides are linked via peptide bonds rather than phophodiester bonds.", "The probes can be attached by any linkage to a support (e.g., 3□, 5□ or via the base).", "3 □ attachment is more usual as this orientation is compatible with the preferred chemistry for solid phase synthesis of oligonucleotides.", "The number of probes in the first probe set (and as a consequence the number of probes in additional probe sets) depends on the length of the reference sequence, the number of nucleotides of interest in the reference sequence and the number of interrogation positions per probe.", "In general, each nucleotide of interest in the reference sequence requires the same interrogation position in the four sets of probes.", "Consider, as an example, a reference sequence of 100 nucleotides, 50 of which are of interest, and probes each having a single interrogation position.", "In this situation, the first probe set requires fifty probes, each having one interrogation position corresponding to a nucleotide of interest in the reference sequence.", "The second, third and fourth probe sets each have a corresponding probe for each probe in the first probe set, and so each also contains a total of fifty probes.", "The identity of each nucleotide of interest in the reference sequence is determined by comparing the relative hybridization signals at four probes having interrogation positions corresponding to that nucleotide from the four probe sets.", "In some reference sequences, every nucleotide is of interest.", "In other reference sequences, only certain portions in which variants (e.g., mutations or polymorphisms) are concentrated are of interest.", "In other reference sequences, only particular mutations or polymorphisms and immediately adjacent nucleotides are of interest.", "Usually, the first probe set has interrogation positions selected to correspond to at least a nucleotide (e.g., representing a point mutation) and one immediately adjacent nucleotide.", "Usually, the probes in the first set have interrogation positions corresponding to at least 3, 1.0, 50, 100, 1000, or 20,000 contiguous nucleotides.", "The probes usually have interrogation positions corresponding to at least 5, 10, 30, 50, 75, 90, 99 or sometimes 100% of the nucleotides in a reference sequence.", "Frequently, the probes in the first probe set completely span the reference sequence and overlap with one another relative to the reference sequence.", "For example, in one common arrangement each probe in the first probe set differs from another probe in that set by the omission of a 3□ base complementary to the reference sequence and the acquisition of a 5□ base complementary to the reference sequence.", "For conceptual simplicity, the probes in a set are usually arranged in order of the sequence in a lane across the chip.", "A lane contains a series of overlapping probes, which represent or tile across, the selected reference sequence.", "The components of the four sets of probes are usually laid down in four parallel lanes, collectively constituting a row in the horizontal direction and a series of 4-member columns in the vertical direction.", "Corresponding probes from the four probe sets (i.e., complementary to the same subsequence of the reference sequence) occupy a column.", "Each probe in a lane usually differs from its predecessor in the lane by the omission of a base at one end and the inclusion of additional base at the other end.", "However, this orderly progression of probes can be interrupted by the inclusion of control probes or omission of probes in certain columns of the array.", "Such columns serve as controls to orient the chip, or gauge the background, which can include target sequence nonspecifically bound to the chip.", "The probes sets are usually laid down in lanes such that all probes having an interrogation position occupied by an A form an-A-lane, all probes having an interrogation position occupied by a C form a C-lane, all probes having an interrogation position occupied by a G form a G-lane, and all probes having an interrogation position occupied by a T (or U) form a T lane (or a U lane).", "Note that in this arrangement there is not a unique correspondence between probe sets and lanes.", "Thus, the probe from the first probe set is laid down in the A-lane, C-lane, A-lane, A-lane and T-lane for the five columns.", "The interrogation position on a column of probes corresponds to the position in the target sequence whose identity is determined from analysis of hybridization to the probes in that column.", "The interrogation position can be anywhere in a probe but is usually at or near the central position of the probe to maximize differential hybridization signals between a perfect match and a single-base mismatch.", "For example, for an 11 mer probe, the central position is the sixth nucleotide.", "Although the array of probes is usually laid down in rows and columns as described above, such a physical arrangement of probes on the chip is not essential.", "Provided that the spatial location of each probe in an array is known, the data from the probes can be collected apd processed to yield the sequence of a target irrespective of the physical arrangement of the probes on a chip.", "In processing the data, the hybridization signals from the respective probes can be reassorted into any conceptual array desired for subsequent data reduction whatever the physical arrangement of probes on the chip.", "A range of lengths of probes can be employed in the chips.", "As noted above, a probe may consist exclusively of a complementary segments, or may have one or more complementary segments juxtaposed by flanking, trailing and/or intervening segments.", "In the latter situation, the total length of complementary segment(s) is more important than the length of the probe.", "In functional terms, the complementarity segment(s) of the first probe sets should be sufficiently long to allow the probe to hybridize detectably more strongly to a reference sequence compared with a variant of the reference including a single base mutation at the nucleotide corresponding to the interrogation position of the probe.", "Similarly, the complementarity segment(s) in corresponding probes from additional probe sets should be sufficiently long to allow a probe to hybridize detectably more strongly to a variant of the reference sequence having a single nucleotide substitution at the interrogation position relative to the reference sequence.", "A probe usually has a single complementary segment having a length of at least 3 nucleotides, and more usually at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or bases exhibiting perfect complementarity (other than possibly at the interrogation position(s) depending on the probe set) to the reference sequence.", "In bridging strategies, where more than one segment of complementarity is present, each segment provides at least three complementary nucleotides to the reference sequence and the combined segments provide at least two segments of three or a total of six complementary nucleotides.", "As in the other strategies, the combined length of complementary segments is typically from 6-30 nucleotides, and preferably from about 9-21 nucleotides.", "The two segments are often approximately the same length.", "Often, the probes (or segment of complementarity within probes) have an odd number of bases, so that an interrogation position can occur in the exact center of the probe.", "In some chips, all probes are the same length.", "Other chips employ different groups of probe sets, in which case the probes are of the same size within a group, but differ between different groups.", "For example, some chips have one group comprising four sets of probes as described above in which all the probes are 11 mers, together with a second group comprising four sets of probes in which all of the probes are 13 mers.", "Of course, additional groups of probes can be added.", "Thus, some chips contain, e.g., four groups of probes having sizes of 11 mers, 13 mers, 15 mers and 17 mers.", "Other chips have different size probes within the same group of four probe sets.", "In these chips, the probes in the first set can vary in length independently of each other.", "Probes in the other sets are usually the same length as the probe occupying the same column from the first set.", "However, occasionally different lengths of probes can be included at the same column position in the four lanes.", "The different length probes are included to equalize hybridization signals from probes irrespective of whether A-T or C-G bonds are formed at the interrogation position.", "The length of probe can be important in distinguishing between a perfectly matched probe and probes showing a single-base mismatch with the target sequence.", "The discrimination is usually greater for short probes.", "Shorter probes are usually also less susceptible to formation of secondary structures.", "However, the absolute amount of target sequence bound, and hence the signal, is greater for larger probes.", "The probe length representing the optimum compromise between these competing considerations may vary depending on inter alia the GC content of a particular region of the target DNA sequence, secondary structure, synthesis efficiency and cross-hybridization.", "In some regions of the target, depending on hybridization conditions, short probes (e.g., 11 mers) may provide information that is inaccessible from longer probes (e.g., 19 mers) and vice versa.", "Maximum sequence information can be read by including several groups of different sized probes on the chip as noted above.", "However, for many regions of the target sequence, such a strategy provides redundant information in that the same sequence is read multiple times from the different groups of probes.", "Equivalent information can be obtained from a single group of different sized probes in which the sizes are selected to maximize readable sequence at particular regions of the target sequence.", "The strategy of customizing probe length within a single group of probe sets minimizes the total number of probes required to read a particular target sequence.", "This leaves ample capacity for the chip to include probes to other reference sequences.", "The invention provides an optimization block which allows systematic variation of probe length and interrogation position to optimize the selection of probes for analyzing a particular nucleotide in a reference sequence.", "The block comprises alternating columns of probes complementary to the wildtype target and probes complementary to a specific mutation.", "The interrogation position is varied between columns and probe length is varied down a column.", "Hybridization of the chip to the reference sequence or the mutant form of the reference sequence identifies the probe length and interrogation position providing the greatest differential hybridization signal.", "The probes are designed to be complementary to either strand of the reference sequence (e.g., coding or non-coding).", "some chips contain separate groups of probes, one complementary to the coding strand, the other complementary to the noncoding strand.", "Independent analysis of coding and noncoding strands provides largely redundant information.", "However, the regions of ambiguity in reading the coding strand are not always the same as those in reading the noncoding strand.", "Thus, combination of the information from coding and noncoding strands increases the overall accuracy of sequencing.", "Some chips contain additional probes or groups of probes designed to be complementary to a second reference sequence.", "The second reference sequence is often a subsequence of the first reference sequence bearing one or more commonly occurring mutations or interstrain variations.", "The second group of probes is designed by the same principles as described above except that the probes exhibit complementarity to the second reference sequence.", "The inclusion of a second group is particular useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (i.e., two or more mutations within 9 to 21 bases).", "Of course, the same principle can be extended to provide chips containing groups of probes for any number of reference sequences.", "Alternatively, the chips may contain additional probe(s) that do not form part of a tiled array as noted above, but rather serves as probe(s) for a conventional reverse dot blot.", "For example, the presence of mutation can be detected from binding of a target sequence to a single oligomeric probe harboring the mutation.", "Preferably, an additional probe containing the equivalent region of the wildtype sequence is included as a control.", "The chips are read by comparing the intensities of labelled target bound to the probes in an array.", "Specifically, a comparison is performed between each lane of probes (e.g., A, C, G and T lanes) at each columnar position (physical or conceptual).", "For a particular columnar position, the lane showing the greatest hybridization signal is called as the nucleotide present at the position in the target sequence corresponding to the interrogation position in the probes.", "The corresponding position in the target sequence is that aligned with the interrogation position in corresponding probes when the probes and target are aligned to maximize complementarity.", "Of the four probes in a column, only one can exhibit a perfect match to the target sequence whereas the others usually exhibit at least a one base pair mismatch.", "The probe exhibiting a perfect match usually produces a substantially greater hybridization signal than the other three probes in the column and is thereby easily identified.", "However, in some regions of the target sequence, the distinction between a perfect match and a one-base mismatch is less clear.", "Thus, a call ratio is established to define the ratio of signal from the best hybridizing probes to the second best hybridizing probe that must be exceeded for a particular target position to be read from the probes.", "A high call ratio ensures that few if any errors are made in calling target nucleotides, but can result in some nucleotides being scored as ambiguous, which could in fact be accurately read.", "A lower call ratio results in fewer ambiguous calls, but can result in more erroneous calls.", "It has been found that at a call ratio of 1.2 virtually all calls are accurate.", "However, a small but significant number of bases (e.g., up to about %) may have to be scored as ambiguous.", "Although small regions of the target sequence can sometimes be ambiguous, these regions usually occur at the same or similar segments in different target sequences.", "Thus, for precharacterized mutations, it is known in advance whether that mutation is likely to occur within a region of unambiguously determinable sequence.", "An array of probes is most useful for analyzing the reference sequence from which the probes were designed and variants of that sequence exhibiting substantial sequence similarity with the reference sequence (e.g., several single-base mutants spaced over the reference sequence).", "When an array is used to analyze the exact reference sequence from which it was designed, one probe exhibits a perfect match to the reference sequence, and the other three probes in the same column exhibits single-base mismatches.", "Thus, discrimination between hybridization signals is usually high and accurate sequence is obtained.", "High accuracy is also obtained when an array is used for analyzing a target sequence comprising a variant of the reference sequence that has a single mutation relative to the reference sequence, or several widely spaced mutations relative to the reference sequence.", "At different mutant loci, one probe exhibits a perfect match to the target, and the other three probes occupying the same column exhibit single-base mismatches, the difference (with respect to analysis of the reference sequence) being the lane in which the perfect match occurs.", "For target sequences showing a high degree of divergence from the reference strain or incorporating several closely spaced mutations from the reference strain, a single group of probes (i.e., designed with respect to a single reference sequence) will not always provide accurate sequence for the highly variant region of this sequence.", "At some particular columnar positions, it may be that no single probe exhibits perfect complementarity to the target and that any comparison must be based on different degrees of mismatch between the four probes.", "Such a comparison does not always allow the target nucleotide corresponding to that columnar position to be called.", "Deletions in target sequences can be detected by loss of signal from probes having interrogation positions encompassed by the deletion.", "However, signal may also be lost from probes having interrogation positions closely proximal to the deletion resulting in some regions of the target sequence that cannot be read.", "Target sequence bearing insertions will also exhibit short regions including and proximal to the insertion that usually cannot be read.", "The presence of short regions of difficult-to-read target because of closely spaced mutations, insertions or deletion, does not prevent determination of the remaining sequence of the target as different regions of a target sequence are determined independently.", "Moreover, such ambiguities as might result from analysis of diverse variants with a single group of probes can be avoided by including multiple groups of probe sets on a chip.", "For example, one group of probes can be designed based on a full-length reference sequence, and the other groups on subsequences of the reference sequence incorporating frequently occurring mutations or strain variations.", "A particular advantage of the present sequencing strategy over conventional sequencing methods is the capacity simultaneously to detect and quantify proportions of multiple target sequences.", "Such capacity is valuable, e.g., for diagnosis of patients who are heterozygous with respect to a gene or who are infected with a virus, such as HIV, which is usually present in several polymorphic forms.", "Such capacity is also useful in analyzing targets from biopsies of tumor cells and surrounding tissues.", "The presence of multiple target sequences is detected from the relative signals of the four probes at the array columns corresponding to the target nucleotides at which diversity occurs.", "The relative signals at the four probes for the mixture under test are compared with the corresponding signals from a homogeneous reference sequence.", "An increase in a signal from a probe that is mismatched with respect to the reference sequence, and a corresponding decrease in the signal from the probe which is matched with the reference sequence signal the presence of a mutant strain in the mixture.", "The extent in shift in hybridization signals of the probes is related to the proportion of a target sequence in the mixture.", "Shifts in relative hybridization signals can be quantitatively related to proportions of reference and mutant sequence by prior calibration of the chip with seeded mixtures of the mutant and reference sequences.", "By this means, a chip can be used to detect variant or mutant strains constituting as little as 1, 5, 20, or 25% of a mixture of stains.", "Similar principles allow the simultaneous analysis of multiple target sequences even when none is identical to the reference sequence.", "For example, with a mixture of two target sequences bearing first and second mutations, there would be a variation in the hybridization patterns of probes having interrogation positions corresponding to the first and second mutations relative to the hybridization pattern with the reference sequence.", "At each position, one of the probes having a mismatched interrogation position relative to the reference sequence would show an increase in hybridization signal, and the probe having a matched interrogation position relative to the reference sequence would show a decrease in hybridization signal.", "Analysis of the hybridization pattern of the mixture of mutant target sequences, preferably in comparison with the hybridization pattern of the reference sequence, indicates the presence of two mutant target sequences, the position and nature of the mutation in each strain, and the relative proportions of each strain.", "In a variation of the above method, the different components in a mixture of target sequences are differentially labelled before being applied to the array.", "For example, a variety of fluorescent labels emitting at different wavelength are available.", "The use of differential labels allows independent analysis of different targets bound simultaneously to the array.", "For example, the methods permit comparison of target sequences obtained from a patient at different stages of a disease.", "1.2.1.1.5.2.Omission of Probes The general strategy outlined above employs four probes to read each nucleotide of interest in a target sequence.", "One probe (from the first probe set) shows a perfect match to the reference sequence and the other three probes (from the second, third and fourth probe sets) exhibit a mismatch with the reference sequence and a perfect match with a target sequence bearing a mutation at the nucleotide of interest.", "The provision of three probes from the second, third and fourth probe sets allows detection of each of the three possible nucleotide substitutions of any nucleotide of interest.", "However, in some reference sequences or regions of reference sequences, it is known in advance that only certain mutations are likely to occur.", "Thus, for example, at one site it might be known that an A nucleotide in the reference sequence may exist as a T mutant in some target sequences but is unlikely to exist as a C or G mutant.", "Accordingly, for analysis of this region of the reference sequence, one might include only the first and second probe sets, the first probe set exhibiting perfect complementarity to the reference sequence, and the second probe set having an interrogation position occupied by an invariant A residue (for detecting the T mutant).", "In other situations, one might include the first, second and third probes sets (but not the fourth) for detection of a wildtype nucleotide in the reference sequence and two mutant variants thereof in target sequences.", "In some chips, probes that would detect silent mutations (i.e., not affecting amino acid sequence) are omitted.", "In some chips, the probes from the first probe set are omitted corresponding to some or all positions of the reference sequences.", "Such chips comprise at least two probe sets.", "The first probe set has a plurality of probes.", "Each probe comprises a segment exactly complementary to a subsequence of a reference sequence except in at least one interrogation position.", "A second probe set has a corresponding probe for each probe in the first probe set.", "The corresponding probe in the second probe set is identical to a sequence comprising the corresponding probe form the first probe set or a subsequence thereof that includes the at least one (and usually only one) interrogation position except that the at least one interrogation position is occupied by a different nucleotide in each of the two corresponding probes from the first and second probe sets.", "A third probe set, if present, also comprises a corresponding probe for each probe in the first probe set except at the at least one interrogation position, which differs in the corresponding probes from the three sets.", "Omission of probes having a segment exhibiting perfect complementarity to the reference sequence results in loss of control information, i.e., the detection of nucleotides in a target sequence that are the same As those in a reference sequence.", "However, similar information can be obtained by hybridizing a chip lacking probes from the first probe set to both target and reference sequences.", "The hybridization can be performed sequentially, or concurrently, if the target and reference are differentially labelled.", "In this situation, the presence of a mutation is detected by a shift in the background hybridization intensity of the reference sequence to a perfectly matched hybridization signal of the target sequence, rather than by a comparison of the hybridization intensities of probes from the first set with corresponding probes from the second, third and fourth sets.", "1.2.1.1.5.3.Wildtype Probe Lane When the chips comprise four probe sets, as discussed supra, and the probe sets are laid down in four lanes, an A-lane, a C-lane, a G-lane and a T or U-lane, the probe having a segment exhibiting perfect complementarity to a reference sequence varies between the four lanes from one column to another.", "This does not present any significant difficulty in computer analysis of the data from the chip.", "However, visual inspection of the hybridization pattern of the chip is sometimes facilitated by provision of an extra lane of probes, in which each probe has a segment exhibiting perfect complementarity to the reference sequence.", "This segment-is identical to a segment from one of the probes in the other four lanes (which lane depending on the column position).", "The extra lane of probes (designated the wildtype lane) hybridizes to a target sequence at all nucleotide positions except those in which deviations from the reference sequence occurs.", "The hybridization pattern of the wildtype lane thereby provides a simple visual indication of mutations.", "1.2.1.1.5.4.Deletion, Insertion and Multiple-Mutation Probes Some chips provide an additional probe set specifically designed for analyzing deletion mutations.", "The additional probe set comprises a probe corresponding to each probe in the first probe set as described above.", "However, a probe from the additional probe set differs from the corresponding probe in the first probe set in that the nucleotide occupying the interrogation position is deleted in the probe from the additional probe set.", "Optionally, the probe from the additional probe set bears an additional nucleotide at one of its termini relative to the corresponding probe from the first probe set.", "The probe from the additional probe set will hybridize more strongly than the corresponding probe from the first probe set to a target sequence having a single base deletion at the nucleotide corresponding to the interrogation position.", "Additional probe sets are provided in which not only the interrogation position, but also an adjacent nucleotide is detected.", "Similarly, other chips provide additional probe sets for analyzing insertions.", "For example, one additional probe set has a probe corresponding to each probe in the first probe set as described above.", "However, the probe in the additional probe set has an extra T nucleotide inserted adjacent to the interrogation position.", "Optionally, the probe has one fewer nucleotide at one of its termini relative to the corresponding probe from the first probe set.", "The probe from the additional probe set hybridizes more strongly than the corresponding probe from the first probe set to a target sequence having an A nucleotide inserted in a position adjacent to that corresponding to the interrogation position.", "Similar additional probe sets are constructed having C, G or T/U nucleotides inserted adjacent to the interrogation position.", "Usually, four such probe sets, one for each nucleotide, are used in combination.", "Other chips provide additional probes (multiple-mutation probes) for analyzing target sequences having multiple closely spaced mutations.", "A multiple-mutation probe is usually identical to a corresponding probe from the first set as described above, except in the base occupying the interrogation position, and except at one or more additional positions, corresponding to nucleotides in which substitution may occur in the reference sequence.", "The one or more additional positions in the multiple mutation probe are occupied by nucleotides complementary to the nucleotides occupying corresponding positions in the reference sequence when the possible substitutions have occurred.", "1.2.1.1.5.5.Block Tiling As noted in the discussion of the general tiling strategy, a probe in the first probe set sometimes has more than one interrogation position.", "In this situation, a probe in the first probe set is sometimes matched with multiple groups of at least one, and usually, three additional probe sets.", "Three additional probe sets are used to allow detection of the three possible nucleotide substitutions at any one position.", "If only certain types of substitution are likely to occur (e.g., transitions), only one or two additional probe sets are required (analogous to the use of probes in the basic tiling strategy).", "To illustrate for the situation where a group comprises three additional probe sets, a first such group comprises second, third and fourth probe sets, each of which has a probe corresponding to each probe in the first probe set.", "The corresponding probes from the second, third and fourth probes sets differ from the corresponding probe in the first set at a first of the interrogation positions.", "Thus, the relative hybridization signals from corresponding probes from the first, second, third and fourth probe sets indicate the identity of the nucleotide in a target sequence corresponding to the first interrogation position.", "A second group of three probe sets (designated fifth, sixth and seventh probe sets), each also have a probe corresponding to each probe in the first probe set.", "These corresponding probes differ from that in the first probe set at a second interrogation position.", "The relative hybridization signals from corresponding probes from the first, fifth, sixth, and seventh probe sets indicate the identity of the nucleotide in the target sequence corresponding to the second interrogation position.", "As noted above, the probes in the first probe set often have seven or more interrogation positions.", "If there are seven interrogation positions, there are seven groups of three additional probe sets, each group of three probe sets serving to identify the nucleotide corresponding to one of the seven interrogation positions.", "Each block of probes allows short regions of a target sequence to be read.", "For example, for a block of probes having seven interrogation positions, seven nucleotides in the target sequence can be read.", "Of course, a chip can contain any number of blocks depending on how many nucleotides of the target are of interest.", "The hybridization signals for each block can be analyzed independently of any other block.", "The block tiling strategy can also be combined with other tiling strategies, with different parts of the same reference sequence being tiled by different strategies.", "The block tiling strategy offers two advantages over the basic strategy in which each probe in the first set has a single interrogation position.", "One advantage is that the same sequence information can be obtained from fewer probes.", "A second advantage is that each of the probes constituting a block (i.e., a probe from the first probe set and a corresponding probe from each of the other probe sets) can have identical 3 □ and 5 □ sequences, with the variation confined to a central segment containing the interrogation positions.", "The identity of 3 □ sequence between different probes simplifies the strategy for solid phase synthesis of the probes on the chip and results in more uniform deposition of the different probes on the chip, thereby in turn increasing the uniformity of signal to noise ratio for different regions of the chip.", "A third advantage is that greater signal uniformity is achieved within a block.", "1.2.1.1.5.6.Multiplex Tiling In the block tiling strategy discussed above, the identity of a nucleotide in a target or reference sequence is determined by comparison of hybridization patterns of one probe having a segment showing a perfect match with that of other probes (usually three other probes) showing a single base mismatch.", "In multiplex tiling, the identity of at least two nucleotides in a reference or target sequence is determined by comparison of hybridization signal intensities of four probes, two of which have a segment showing perfect complementarity or a single base mismatch to the reference sequence, and two of which have a segment showing perfect complementarity or a double-base mismatch to a segment.", "The four probes whose hybridization patterns are to be compared each have a segment that is exactly complementary to a reference sequence except at two interrogation positions, in which the segment may or may not be complementary to the reference sequence.", "The interrogation positions correspond to the nucleotides in a reference or target sequence which are determined by the comparison of intensities.", "The nucleotides occupying the interrogation positions in the four probes are selected according to the following rule.", "The first interrogation position is occupied by a different nucleotide in each of the four probes.", "The second interrogation position is also occupied by a different nucleotide in each of the four probes.", "In two of the four probes, designated the first and second probes, the segment is exactly complementary to the reference sequence except at not more than one of the two interrogation positions.", "In other words, one of the interrogation positions is occupied by a nucleotide that is complementary to the corresponding nucleotide from the reference sequence and the other interrogation position may or may not be so occupied.", "In the other two of the four probes, designated the third and fourth probes, the segment is exactly complementary to the reference sequence except that both interrogation positions are occupied by nucleotides which are noncomplementary to the respective corresponding nucleotides in the reference sequence.", "There are number of ways of satisfying these conditions depending on whether the two nucleotides in the reference sequence corresponding to the two interrogation positions are the same or different.", "If these two nucleotides are different in the reference sequence (probability ¾), the conditions are satisfied by each of the two interrogation positions being occupied by the same nucleotide in any given probe.", "For example, in the first probe, the two interrogation positions would both be A, in the second probe, both would be C, in the third probe, each would be G, and in the fourth probe each would be T or U.", "If the two nucleotides in the reference sequence corresponding to the two interrogation positions are different, the conditions noted above are satisfied by each of the interrogation positions in any one of the four probes being occupied by complementary nucleotides.", "For example, in the first probe, the interrogation positions could be occupied by A and T, in the second probe by C and G, in the third probe by G and C and in the four probe, by T and A.", "When the four probes are hybridized to a target that is the same as the reference sequence or differs from the reference sequence at one (but not both) of the interrogation positions, two of the four probes show a double-mismatch with the target and two probes show a single mismatch.", "The identity of probes showing these different degrees of mismatch can be determined from the different hybridization signals.", "From the identity of the probes showing the different degrees of mismatch, the nucleotides occupying both of the interrogation positions in the target sequence can be deduced.", "For ease of illustration, the multiplex strategy has been initially described for the situation where there are two nucleotides of interest in a reference sequence and only four probes in an array.", "Of course, the strategy can be extended to analyze any number of nucleotides in a target sequence by using additional probes.", "In one variation, each pair of interrogation positions is read from a unique group of four probes.", "In a block variation, different groups of four probes exhibit the same segment of complementarity with the reference sequence, but the interrogation positions move within a block.", "The block and standard multiplex tiling variants can of course be used in combination for different regions of a reference sequence.", "Either or both variants can also be used in combination with any of the other tiling strategies described.", "1.2.1.1.5.7.Helper Mutations Occasionally small regions of a reference sequence give a low hybridization signal as a result of annealing of probes.", "The self-annealing reduces the amount of probe effectively available for hybridizing to the target.", "Although such regions of the target are generally small and the reduction of hybridization signal is usually not so substantial as to obscure the sequence of this region, this concern can be avoided by the use of probes incorporating helper mutations.", "The helper mutation(s) serve to break-up regions of internal complementarity within a probe and thereby prevent annealing.", "Usually, one or two helper mutations are quite sufficient for this purpose.", "The inclusion of helper mutations can be beneficial in any of the tiling strategies noted above.", "In general each probe having a particular interrogation position has the same helper mutation(s).", "Thus, such probes have a segment in common which shows perfect complementarity with a reference sequence, except that the segment contains at least one helper mutation (the same in each of the probes) and at least one interrogation position (different in all of the probes).", "For example, in the basic tiling strategy, a probe from the first probe set comprises a segment containing an interrogation position and showing perfect complementarity with a reference sequence except for one or two helper mutations.", "The corresponding probes from the second, third and fourth probe sets usually comprise the same segment (or sometimes a subsequence thereof including the helper mutation(s) and interrogation position), except that the base occupying the interrogation position varies in each probe.", "Usually, the helper mutation tiling strategy is used in conjunction with one of the tiling strategies described above.", "The probes containing helper mutations are used to tile regions of a reference sequence otherwise giving low hybridization signal (e.g., because of self-complementarity), and the alternative tiling strategy is used to tile intervening regions.", "1.2.1.1.5.8.Pooling Strategies Pooling strategies also employ arrays of immobilized probes.", "Probes are immobilized in cells of an array, and the hybridization signal of each cell can be determined independently of any other cell.", "A particular cell may be occupied by pooled mixture of probes.", "Although the identity of each probe in the mixture is known, the individual probes in the pool are not separately addressable.", "Thus, the hybridization signal from a cell is the aggregate of that of the different probes occupying the cell.", "In general, a cell is scored as hybridizing to a target sequence if at least one probe occupying the cell comprises a segment exhibiting perfect complementarity to the target sequence.", "A simple strategy to show the increased power of pooled strategies over a standard tiling is to create three cells each containing a pooled probe having a single pooled position, the pooled position being the same in each of the pooled probes.", "At the pooled position, there are two possible nucleotides, allowing the pooled probe to hybridize to two target sequences.", "In tiling terminology, the pooled position of each probe is an interrogation position.", "As will become apparent, comparison of the hybridization intensities of the pooled probes from the three cells reveals the identity of the nucleotide in the target sequence corresponding to the interrogation position (i.e., that is matched with the interrogation position when the target sequence and pooled probes are maximally aligned for complementarity).", "The three cells are assigned probe pools that are perfectly complementary to the target except at the pooled position, which is occupied by a different pooled nucleotide in each probe.", "With 3 pooled probes, all 4 possible single base pair states (wild and 3 mutants) are detected.", "A pool hybridizes with a target if some probe contained within that pool is complementary to that target.", "A cell containing a pair (or more) of oligonucleotides lights up when a target complementary to any of the oligonucleotide in the cell is present.", "Using the simple strategy, each of the four possible targets (wild and three mutants) yields a unique hybridization pattern among the three cells.", "Since a different pattern of hybridizing pools is obtained for each possible nucleotide in the target sequence corresponding to the pooled interrogation position in the probes, the identity of the nucleotide can be determined from the hybridization pattern of the pools.", "Whereas, a standard tiling requires four cells to detect and identify the possible single-base substitutions at one location, this simple pooled 45 strategy only requires three cells.", "A more efficient pooling strategy for sequence analysis is the ‘Trellis’ strategy.", "In this strategy, each pooled probe has a segment of perfect complementarity to a reference sequence except at three pooled positions.", "One pooled position is an N pool.", "The three pooled positions may or may not be contiguous in a probe.", "The other two pooled positions are selected from the group of three pools consisting of (1) M or K, (2) R or Y and (3) W or S, where the single letters are IUPAC standard ambiguity codes.", "The sequence of a pooled probe is thus, of the form XXXN[(M/K) or (R/Y) or (W/S)][(M/K) or (R/Y) or (W/S)]XXXXX, where XXX represents bases complementary to the reference sequence.", "The three pooled positions may be in any order, and may be contiguous or separated by intervening nucleotides.", "For, the two positions occupied by [(M/K) or (RN) or (W/S)], two choices must be made.", "First, one must select one of the following three pairs of pooled nucleotides (1) M/K, (2) R/Y and (3) W/S.", "The one of three pooled nucleotides selected may be the same or different at the two pooled positions.", "Second, supposing, for example, one selects M/K at one position, one must then chose between M or K. This choice should result in selection of a pooled nucleotide comprising a nucleotide that complements the corresponding nucleotide in a reference sequence, when the probe and reference sequence are maximally aligned.", "The same principle governs the selection between R and Y, and between W and S. A trellis pool probe has one pooled position with four possibilities, and two pooled positions, each with two possibilities.", "Thus, a trellis pool probe comprises a mixture of 16 (4×2×2) probes.", "Since each pooled position includes one nucleotide that complements the corresponding nucleotide from the reference sequence, one of these 16 probes has a segment that is the exact complement of the reference sequence.", "A target sequence that is the same as the reference sequence (i.e., a wildtype target) gives a hybridization signal to each probe cell.", "Here, as in other tiling methods, the segment of complementarity should be sufficiently long to permit specific hybridization of a pooled probe to a reference sequence be detected relative to a variant of that reference sequence.", "Typically, the segment of complementarity is about 9-21 nucleotides.", "A target sequence is analyzed by comparing hybridization intensities at three pooled probes, each having the structure described above.", "The segments complementary to the reference sequence present in the three pooled probes show some overlap.", "Sometimes the segments are identical (other than at the interrogation positions).", "However, this need not be the case.", "For example, the segments can tile across a reference sequence in increments of one nucleotide (i.e., one pooled probe differs from the next by the acquisition of one nucleotide at the 5 □ end and loss of a nucleotide at the 3 □ end).", "The three interrogation positions may or may not occur at the same relative positions within each pooled probe (i.e., spacing from a probe terminus).", "All that is required is that one of the three interrogation positions from each of the three pooled probes aligns with the same nucleotide in the reference sequence, and that this interrogation position is occupied by a different pooled nucleotide in each of the three probes.", "In one of the three probes, the interrogation position is occupied by an N. In the other two pooled probes the interrogation position is occupied by one of (M/K) or (R/Y) or (W/S).", "In the simplest form of the trellis strategy, three pooled probes are used to analyze a single nucleotide in the reference sequence.", "Much greater economy of probes is achieved when more pooled probes are included in an array.", "For example, consider an array of five pooled probes each having the general structure outlined above.", "Three of these pooled probes have an interrogation position that aligns with the same nucleotide in the reference sequence and are used to read that nucleotide.", "A different combination of three probes have an interrogation position that aligns with a different nucleotide in the reference sequence.", "Comparison of these three probe intensities allows analysis of this second nucleotide.", "Still another combination of three pooled probes from the set of five have an interrogation position that aligns with a third nucleotide in the reference sequence and these probes are used to analyze that nucleotide.", "Thus, three nucleotides in the reference sequence are fully analyzed from only five pooled probes.", "By comparison, the basic tiling strategy would require 12 probes for a similar analysis.", "The trellis strategy employs an array of probes having at least three cells, each of which is occupied by a pooled probe as described above.", "Consider the use of three such pooled probes for analyzing a target sequence, of which one position may contain any single base substitution to the reference sequence (i.e, there are four possible target sequences to be distinguished).", "Three cells are occupied by pooled probes having a pooled interrogation position corresponding to the position of possible substitution in the target sequence, one cell with an □N□, one cell with one of □M□ or □K□, and one cell with □R□ or □Y□.", "An interrogation position corresponds to a nucleotide in the target sequence if it aligns adjacent with that nucleotide when the probe and target sequence are aligned to maximize 45 complementarity.", "Note that although each of the pooled probes has two other pooled positions, these positions are not relevant for the present illustration.", "The positions are only relevant when more than one position in the target sequence is to be read, a circumstance that will be considered later.", "For present purposes, the cell with the □N□ in the interrogation position lights up for the wildtype sequence and any of the three single base substitutions of the target sequence.", "A further class of strategies involving pooled probes are termed coding strategies.", "These strategies assign code words from some set of numbers to variants of a reference sequence.", "Any number of variants can be coded.", "The variants can include multiple closely spaced substitutions, deletions or insertions.", "The designation letters or other symbols assigned to each variant may be any arbitrary set of numbers, in any order.", "For example, a binary code is often used, but codes to other bases are entirely feasible.", "The numbers are often assigned such that each variant has a designation having at least one digit and at least one nonzero value for that digit.", "For example, in a binary system, a variant assigned the number 101, has a designation of three digits, with one possible nonzero value for each digit.", "The designation of the variants are coded into an array of pooled probes comprising a pooled probe for each nonzero value of each digit in the numbers assigned to the variants.", "For example, if the variants are assigned successive number in a numbering system of base m, and the highest number assigned to a variant has n digits, the array would have about n×(m−1) pooled probes.", "In general, logm(3N+1) probes are required to analyze all variants of N locations in a reference sequence, each having three possible mutant substitutions.", "For example, 10 base pairs of sequence may be analyzed with only 5 pooled probes using a binary coding system.", "Each pooled probe has a segment exactly complementary to the reference sequence except that certain positions are pooled.", "The segment should be sufficiently long to allow specific hybridization of the pooled probe to the reference sequence relative to a mutated form of the reference sequence.", "As in other tiling strategies, segments lengths of 9-21 nucleotides are typical.", "Often the probe has no nucleotides other than the 9-21 nucleotide segment.", "The pooled positions comprise nucleotides that allow the pooled probe to hybridize to every variant assigned a particular nonzero value in a particular digit.", "Usually, the pooled positions further comprises a nucleotide that allows the pooled probe to hybridize to the reference sequence.", "Thus, a wildtype target (or reference sequence) is immediately recognizable from all the pooled probes being lit.", "When a target is hybridized to the pools, only those pools comprising a component probe having a segment that is exactly complementary to the target light up.", "The identity of the target is then decoded from the pattern of hybridizing pools.", "Each pool that lights up is correlated with a particular value in a particular digit.", "Thus, the aggregate hybridization patterns of each lighting pool reveal the value of each digit in the code defining the identity of the target hybridized to the array.", "1.2.1.1.5.9.Bridging Strategy Probes that contain partial matches to two separate (i.e., non contiguous) subsequences of a target sequence sometimes hybridize strongly to the target sequence.", "In certain instances, such probes have generated stronger signals than probes of the same length which are perfect matches to the target sequence.", "It is believed (but not necessary to the invention) that this observation results from interactions of a single target sequence with two or more probes simultaneously.", "This invention exploits this observation to provide arrays of probes having at least first and second segments, which are respectively complementary to first and second subsequences of a reference sequence.", "Optionally, the probes may have a third or more complementary segments.", "These probes can be employed in any of the strategies noted above.", "The two segments of such a probe can be complementary to disjoint subsequences of the reference sequences or contiguous subsequences.", "* If the latter, the two segments in the probe are inverted relative to the order of the complement of the reference sequence.", "The two subsequences of the reference sequence each typically comprises about 3 to 30 contiguous nucleotides.", "The subsequences of the reference sequence are sometimes separated by 0, 1, 2 or 3 bases.", "Often the sequences, are adjacent and nonoverlapping.", "The bridging strategy offers the following advantages: (1) Higher discrimination between matched and mismatched probes, (2) The possibility of using longer probes in a bridging tiling, thereby increasing the specificity of the hybridization, without sacrificing discrimination, (3) The use of probes in which an interrogation position is located very off-center relative to the regions of target complementarity.", "This may be of particular advantage when, for example, when a probe centered about one region of the target gives low hybridization signal.", "The low signal is overcome by using a probe centered about an adjoining region giving a higher hybridization signal.", "(4) Disruption of secondary structure that might result in annealing of certain probes (see previous discussion of helper mutations).", "1.2.1.1.5.10.Deletion Tiling Deletion tiling is related to both the bridging and helper mutant strategies described above.", "In the deletion strategy, comparisons are performed between probes sharing a common deletion but differing from each other at an interrogation position located outside the deletion.", "For example, a first probe comprises first and second segments, each exactly complementary to respective first and second subsequences of a reference sequence, wherein the first and second subsequences of the reference sequence are separated by a short distance (e.g., 1 or 2 nucleotides).", "The order of the first and second segments in the probe is usually the same as that of the complement to the first and second subsequences in the reference sequence.", "Such tilings sometimes offer superior discrimination in hybridization intensities between the probe having an interrogation position complementary to the target and other probes.", "Thermodynamically, the difference between the hybridizations to matched and mismatched targets for the probe set shown above is the difference between a single-base bulge, and a large asymmetric loop (e.g., two bases of target, one of probe).", "This often results in a larger difference in stability than the comparison of a perfectly matched probe with a probe showing a single base mismatch in the basic tiling strategy.", "The use of deletion or bridging probes is quite general.", "These probes can be used in any of the tiling strategies of the invention.", "As well as offering superior discrimination, the use of deletion or bridging strategies is advantageous for certain probes to avoid self-hybridization (either within a probe or between two probes of the same sequence).", "1.2.1.1.6.Preparation of Target Samples The target polynucleotide, whose sequence is to be determined, is usually isolated from a tissue sample.", "If the target is genomic, the sample may be from any tissue (except exclusively red blood cells).", "For example, whole blood, peripheral blood lymphocytes or PBMC, skin, hair or semen are convenient sources of clinical samples.", "These sources are also suitable if the target is RNA.", "Blood and other body fluids are also a convenient source for isolating viral nucleic acids.", "If the target is mRNA, the sample is obtained from a tissue in which the mRNA is expressed.", "If the polynucleotide in the sample is RNA, it is usually reverse transcribed to DNA.", "DNA samples or cDNA resulting from reverse transcription are usually amplified, e.g., by PCR.", "Depending on the selection of primers and amplifying enzyme(s), the amplification product can be RNA or DNA.", "Paired primers are selected to flank the borders of a target polynucleotide of interest.", "More than one target can be simultaneously amplified by multiplex PCR in which multiple paired primers are employed.", "The target can be labelled at one or more nucleotides during or after amplification.", "For some target polynucleotides (depending on size of sample), e.g., episomal DNA, sufficient DNA is present in the tissue sample to dispense with the amplification step.", "When the target strand is prepared in single-stranded form as in preparation of target RNA, the sense of the strand should of course be complementary to that of the probes on the chip.", "This is achieved by appropriate selection of primers.", "The target is preferably fragmented before application to the chip to reduce or eliminate the formation of secondary structures in the target.", "The average size of targets segments following hybridization is usually larger than the size of probe on the chip.", "1.2.1.2.Sequencing This invention provides that the method of performing whole cell engineering may comprise the step of cell screening.", "In a preferred embodiment, this invention provides that the step of cell screening may comprise the step of genomic sequencing.", "In one exemplification, genome sequencing can be accomplished according to the enzymatic/Sanger method (described in F. Sanger, S. Nicklen, and A. R. Coulson, Proc.", "Natl.", "Acad.", "Sci, USA, 74: 5463-5467 (1977)) and involve cloning and subcloning (described in U.S. Pat.", "No.", "4,725,677; Chen and Seeburg, DNA 4, 165-170 (1985); Lim et al., Gene Anal., Techn.", "5, 32-39 (1988); PCR Protocols—A Guide to Methods and Applications.", "Innis et al., editors, Academic Press, San Diego (1990); Innis et al., Proc.", "Nat.", "Acad.", "Sci.", "USA 85, 9436-9440 (1988)).", "In another exemplification, sequencing can be accomplished according to the chemical/Maxam and Gilbert method which is described in references: A. M. Maxam, and W. Gilbert, Proc.", "Nat.", "Acad.", "of Sci., USA, 74: 560-564 (1977) and Church et al., Proc.", "Natl.", "Acad.", "Sci., 81: 1991 (1984).", "In additional exemplifications, genome sequencing can be accomplished by methodology described by Guo and Wu (Guo and Wu, Nucleic Acids Res., 10: 2065 (1982); and Meth.", "Enz., 100: 60 (1983)) or those methods that utilize 3′hydroxy-protected and labeled nucleotides as exemplified in the following references: Churchich, J. E., Eur.", "J.", "Biochem., 231: 736 (1995); Metzket, M. L. et al., Nucleic Acids Research, 22: 4259 (1994); Beabealashvilli, R. S. et al, Biochimica et Biophysica Acta, 868: 136 (1986); Chidgeavadze, Z. G.; Kukhanova, M. K. et al.", "Biochimica et Biophysica Acta, 868: 145 (1986); Hiratsuka, T et Biophysica Acta, 742: 496 (1983); Jeng, S. J. and Guillory, R. J. J., Supramolecular Structure, 3: 448 (1975).", "The invention also provides that sequencing may be read by autoradiography using radioisotopes (as described in Ornstein et al., Biotechniques 2, 476 (1985)) or by using non-radioactively labeling strategies that have been integrated into partly automated DNA sequencing procedures (Smith et al., Nature M, 674-679 (1986) and EPO Patent No.", "873 00998.9; Du Pont De Nemours EPO Application No.", "03 59225; Ansorge et al., L Biochem.", "Biophys.", "Method 13, 325-32 (19860; Prober et al.", "Science M, 33641 (1987); Applied Biosystems, PCT Application WO 91/05060; Smith et al., Science 235, G89 (1987); U.S. Pat.", "Nos.", "570,973 and 689,013), Du Pont De Nemours, U.S. Pat.", "Nos.", "881,372 and 57,566, Ansorge et al.", "Nucleic Acids Res.", "15-, 4593-4602 (1987) and EMBL Patent Application DE P3724442 and P3805808.1) and Hitachi (JP 1-90844 and DE 4011991 A1; U.S. Pat.", "No.", "4,729,947; PCT Application WO92/02635; U.S. Pat.", "No.", "594,676; Beck, O'Keefe, Coull and Köster, Nucleic Acids Res.", "7, 5115-5123 (1989).L7 and Beck and Köster, Anal.", "Chem.", "62 2258-2270 (1990); Church et al., Science 240, 185-188 (1988); Köster et al., Nucleic Acids Res.", "Symposium Ser.", "No.", "24, 318-321 (1991), University of Utah, PCT Application No.", "WO 90/15883; Smith et al., Nature (1986) 321: 674-679; Orion-Yhtyma Oy, U.S. Pat.", "No.", "277,643; M. Uhlen et al.", "Nucleic Acids Res.", "16, 3025-38 (1988); Cemu Bioteknik, PCT Application No.", "WO 89/09282 and Medical Research Council, GB, PCT Application No.", "WO 92/03575; Du Pont De Nemours, PCT Application WO 91/11533).", "In addition, this invention provides for various methods of reading sequencing data such as capillary zone electrophoresis (described in Jorgenson et al., J. Chromatography 352, 337 (1986); Gesteland et al., Nucleic Acids Res.", "18, 1415-1419 (1990)), mass spectrometry (including ES [described in Fenn et al.", "J. Phys.", "Chem.", "18, 4451-59 (1984); PCT Application No.", "WO 90/14148; R. D. Smith et al., Anal.", "Chem.", "62, 882-89 (1990) and B. Ardrey, Electrospray Mass Spectrometry, Spectroscopy Europe 4, 10-18 (1992)] and MALDI [Hillenkamp et al.", "Matrix Assisted UV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry of Large Biomolecules, Biological Mass Spectrometry (Burlingame and McCloskey, editors), Elsevier Science Publishers, Amsterdam, pp.", "49-60, (1990); Williams et al., Science, 246, 1585-87 (1989); Williams et al., Rapid Communications in Mass Spectrometry, 4, 348-351 (1990)]), tube gel electrophoresis and a mass analyzer to sequence (described in EPO Patent Applications No.", "0360676 A1 and 0360677).", "In order to analyze the sequencing data, this invention provides for the use of probes in large arrays (as described in PCT patent Publication No.", "92/10588; U.S. Pat.", "No.", "5,143,854; U.S. application Ser.", "No.", "07/805,727; U.S. Pat.", "No.", "5,202,231; PCT patent Publication No.", "89/10977).", "This invention provides that the method of performing whole cell engineering may comprise the step of cell screening which in a particular embodiment may include the method of DNA amplification.", "In a particular embodiment, this invention provides that DNA amplification.", "DNA can be amplified by a variety of procedures including cloning (Sambrook et at., Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, 1989), polymerase chain reaction (PCR) (C. R. Newton and A. Graham, PCF, BIOS Publishers, 1994; Bevan et al., “Sequencing of PCR-Amplified DNA” PCR Meth.", "App.", "4: 222 (1992)), ligase chain reaction (LCR) (F. Barany Proc.", "Natl.", "Acad Sci USA 88, 189-93 (1991), strand displacement amplification (SDA) (G. Terrance Walker et al., Nucleic Acids Res.", "22, 2670-77 (1994)) and variations such as RT-PCR (Arens, M. Clin Microbiol Rev, 12(4): 612-26 (1999)), allele-specific amplification (ASA) (Nichols, W. C. et al.", "Genomics.", "October; 5(3): 53540(1989); Giffard, P. M. et al.", "Anal Biochem, 292(2): 207-15 (2001)).", "In additional embodiments of this invention, it provides for additional sequencing methods (as described in Labeit et al., MA 5, 173-177 (1986); Amersham, PCT-Application GB86/00349; Eckstein et al., Nucleic Acids Res.", "1˜, 9947 (1988); Max-Planck-Geselischaft, DE 3930312 A1; Saiki, R. et al., Science 239: 487-491 (1998); Sarkat, G. and Bolander Mark E., Semi Exponential Cycle Sequencing Nucleic Acids Research, 1995, Vol.", "23, No.", "7, p. 1269-1270).", "This invention also provides for the following sequencing strategies: shotgun sequencing, transposon-mediated directed sequencing (Strathmann, M. et al.", "Proc Natl Acad Sci USA (1991) 88: 1247-1250), and large scale variations thereof (as exemplified in K. B. Mullis et al., U.S. Pat.", "No.", "4,683,202; July/1987; 435/91; and U.S. Pat.", "No.", "4,683,195, July/1987; 435/6).", "According to alternative embodiments of this invention, the step of genomic sequencing may include constructing ordered clone maps of DNA sequencing (as described in sections of U.S. Pat.", "No.", "5,604,100 and PCT Patent Publication No.", "WO9627025).", "This invention provides that the method of genome sequencing be achieved by various steps that may utilize modifications of certain methods mentioned above (described in the following patents: PCT Publication Nos.", "WO9737041, WO9742348, WO9627025, WO9831834, WO9500530, and WO9831833; U.S. Pat.", "No.", "5,604,100, U.S. Pat.", "No.", "5,670,321, U.S. Pat.", "No.", "5,453,247, U.S. Pat.", "No.", "5,994,058, and U.S. Pat.", "No.", "5,354,656).", "1.2.1.3.Annotating In one aspect this invention discloses the use of a relational database system for storing and manipulating biomolecular sequence information and storing and displaying genetic information, the database including genomic libraries for a plurality of types of organisms, the libraries having multiple genomic sequences, at least some of which represent open reading frames located along a contiguous sequence on each the plurality of organisms' genomes, and a user interface capable of receiving a selection of two or more of the genomic libraries for comparison and displaying the results of the comparison.", "Associated with the database is a software system that allows a user to determine the relative position of a selected gene sequence within a genome.", "The system allows execution of a method of displaying the genetic locus of a biomolecular sequence.", "The method involves providing a database including multiple biomolecular sequences, at least some of which represent open reading frames located along a contiguous sequence on an organism's genome.", "The system also provides a user interface capable of receiving a selection of one or more probe open reading frames for use in determining homologous matches between such probe open reading frame(s) and the open reading frames in the genomic libraries, and displaying the results of the determination.", "An open reading frame for the sequence is selected and displayed together with adjacent open reading frames located upstream and downstream in the relative positions in which they occur on the contiguous sequence.", "Also disclosed is a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein function hierarchies.", "The hierarchies allow searches for sequences based upon a protein's biological function or molecular function.", "Also disclosed is a mechanism for automatically grouping new sequences into protein function hierarchies.", "This mechanism uses descriptive information obtained from “external hits” which are matches of stored sequences against gene sequences stored in an external database such as GenBank.", "The descriptive information provided with the external database is evaluated according to a specific algorithm and used to automatically group the external hits (or the sequences associated with the hits) in the categories.", "Ultimately, the biomolecular sequences stored in databases of this invention are provided with both descriptive information from the external hit and category information from a relevant hierarchy or hierarchies.", "Disclosed is a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to association with one or more projects for obtaining full-length biomolecular sequences from shorter sequences.", "The relational database has sequence records containing information identifying one or more projects to which each of the sequence records belong.", "Each project groups together one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The computer system has a user interface allowing a user to selectively view information regarding one or more projects.", "The relational database also provides interfaces and methods for accessing and manipulating and analyzing project-based information.", "Polymer sequences are assembled into bins.", "A first number of bins are populated with polymer sequences.", "The polymer sequences in each bin are assembled into one or more consensus sequences representative of the polymer sequences of the bin.", "The consensus sequences of the bins are compared to determine relationships, if any, between the consensus sequences of the bins.", "The bins are modified based on the relationships between the consensus sequences of the bins.", "The polymer sequences are reassembled in the modified bins to generate one or more modified consensus sequences for each bin representative of the modified bins.", "In another aspect of the invention, sequence similarities and dissimilarities are analyzed in a set of polymer sequences.", "Pairwise alignment data is generated for pairs of the polymer sequences.", "The pairwise alignment data defines regions of similarity between the pairs of polymer sequences with boundaries.", "Additional boundaries in particular polymer sequences are determined by applying at least one boundary from at least one pairwise alignment for one pair of polymer sequences to at least one other pairwise alignment for another pair of polymer sequences including one of the particular polymer sequences.", "Additional regions of similarity are generated based on the boundaries.", "1.2.1.3.1.Annotating—General Methodology In one aspect this present invention relates generally to relational databases for storing and retrieving biological information.", "More particularly the invention relates to systems and methods for providing sequences of biological molecules in a relational format allowing retrieval in a client-server environment and for providing full-length cDNA sequences in a relational format allowing retrieval in a client-server environment.", "Informatics is the study and application of computer and statistical techniques to the management of information.", "In genome projects, bioinformatics includes the development of methods to search databases quickly, to analyze nucleic acid sequence information, and to predict protein sequence, structure and function from DNA sequence data.", "Increasingly, molecular biology is shifting from the laboratory bench to the computer desktop.", "Today's researchers require advanced quantitative analyses, database comparisons, and computational algorithms to explore the relationships between sequence and phenotype.", "Thus, by all accounts, researchers can not and will not be able to avoid using computer resources to explore gene expression, gene sequencing and molecular structure.", "One use of bioinformatics involves studying an organism's genome to determine the sequence and placement of its genes and their relationship to other sequences and genes within the genome or to genes in other organisms.", "Another use of bioinformatics involves studying genes differentially or commonly expressed in different tissues or cell lines (e.g.", "normal and cancerous tissue).", "Such information is of significant interest in biomedical and pharmaceutical research, for instance to assist in the evaluation of drug efficacy and resistance.", "The sequence tag method involves generation of a large number (e.g., thousands) of Expressed Sequence Tags (“ESTs”) from cDNA libraries (each produced from a different tissue or sample).", "ESTs are partial transcript sequences that may cover different parts of the cDNA(s) of a gene, depending on cloning and sequencing strategy.", "Each EST includes about 50 to 300 nucleotides.", "If it is assumed that the number of tags is proportional to the abundance of transcripts in the tissue or cell type used to make the cDNA library, then any variation in the relative frequency of those tags, stored in computer databases, can be used to detect the differential abundance and potentially the expression of the corresponding genes.", "To make genomic and EST information manipulation easy to perform and understand, sophisticated computer database systems have been developed.", "In one database system, developed by Incyte Pharmaceuticals, Inc. of Palo Alto, Calif., genomic sequence data and the abundance levels of mRNA species represented in a given sample is electronically recorded and annotated with information available from public sequence databases such as GenBank.", "Examples of such databases include GenBank (NCBI) and TIGR.", "The resulting information is stored in a relational database that may be employed to determine relationships between sequences and genes within and among genomes and establish a cDNA profile for a given tissue and to evaluate changes in gene expression caused by disease progression, pharmacological treatment, aging, etc.", "In one database system, developed by Incyte Pharmaceuticals, Inc. of Palo Alto, Calif., abundance levels of mRNA species represented in a given sample are electronically recorded and annotated with information available from public sequence databases such as GenBank.", "The resulting information is stored in a relational database that may be employed to establish a cDNA profile for a given tissue and to evaluate changes in gene expression caused by disease progression, pharmacological treatment, aging, etc.", "Genetic information for a number of organisms has been catalogued in computer databases.", "Genetic databases for organisms such as Eschericia coli, Haemophilus influenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae, among others, are publicly available.", "At present, however, complete sequence data is available for relatively few species, and the ability to manipulate sequence data within and between species and databases is limited.", "While genetic data processing and relational database systems such as those developed by Incyte Pharmaceuticals, Inc. provide great power and flexibility in analyzing genetic information and gene expression information, this area of technology is still in its infancy and further improvements in genetic data processing and relational database systems and their content will help accelerate biological research for numerous applications.", "In genome projects, bioinformatics includes the development of methods to search databases quickly, to analyze nucleic acid sequence information, and to predict protein sequence and structure from DNA sequence data.", "Increasingly, molecular biology is shifting from the laboratory bench to the computer desktop.", "Advanced quantitative analyses, database comparisons, and computational algorithms are needed to explore the relationships between sequence and phenotype.", "1.2.1.3.2.Annotating—Exemplary Aspects The annotation methods of this invention include those described in PCT patent publication Nos.", "98/26407, 98/26408, and 99/49403 and U.S. Pat.", "Nos.", "6,023,659 and 5,953,727 and are herein incorporated by reference in their entirety to the same extent as if each individual patent or patent application were specifically and individually indicated to be incorporated by reference in its entirety.", "Thus, in one aspect, this present invention provides relational database systems for storing and analyzing biomolecular sequence information together with biological annotations detailing the source and interpretation the sequence data.", "The present invention provides a powerful database tool for drug development and other research and development purposes.", "The present invention provides relational database systems for storing and analyzing biomolecular sequence information together with biological detailing the source and interpretation the sequence data.", "Disclosed is a relational database systems for storing and displaying genetic information.", "Associated with the database is a software system the allows a user to determine the relative position of a selected gene sequence within a genome.", "The system allows execution of a method of displaying the genetic locus of a biomolecular sequence.", "The method involves providing a database including multiple biomolecular sequences, at least some of which represent open reading frames located along a contiguous sequence on an organism's genome.", "An open reading frame for the sequence is selected and displayed together with adjacent open reading frames located upstream and downstream in the relative positions in which they occur on the contiguous sequence.", "The invention provides a method of displaying the genetic locus of a biomolecular sequence.", "The method involve providing a database including multiple biomolecular sequences, at least some of which represent open reading frames located along a contiguous sequence on an organism's genome.", "The method further involves identifying a selected open reading frame, and displaying the selected open reading frame together with adjacent open reading frames located upstream and downstream from the selected open reading frame.", "The adjacent open reading frames and the selected open reading frame are displayed in the relative positions in which they occur on the contiguous sequence, textually and/or graphically.", "The method of the invention may be practiced with sequences from microbial organisms, and the sequences may include nucleic acid or protein sequences.", "The invention also provides a computer system including a database having multiple biomolecular sequences, at least some of which represent open reading frames located along a contiguous sequence on an organism□s genome.", "The computer system also includes a user interface capable of identifying a selected open reading frame, and displaying the selected open reading frame together with adjacent open reading frames located upstream and downstream from the selected open reading frame.", "The adjacent the open reading frames and the selected open reading frame are displayed in the relative positions in which they occur on the contiguous sequence.", "The user interface may also capable of detecting a scrolling command, and based upon the direction and magnitude of the scrolling command, identifying a new selected open reading frame from the contiguous sequence.", "The invention further provides a computer program product comprising a computer-usable medium having computer-readable program code embodied thereon relating to a database including multiple biomolecular sequences, at least some of which represent open reading frames located along a contiguous sequence on an organism's genome.", "The computer program product includes computer-readable program code for identifying a selected open reading frame, and displaying the selected open reading frame together with adjacent open reading frames located upstream and downstream from the selected open reading frame.", "The adjacent open reading frames and the selected open reading frame are displayed in the relative positions in which they occur on the contiguous sequence.", "Comparative Genomics is a feature of the database system of the present invention which allows a user to compare the sequence data of sets of different organism types.", "Comparative searches may be formulated in a number of ways using the Comparative Genomics feature.", "For example, genes common to a set of organisms may be identified through a “commonality” query, and genes unique to one of a set of organisms may be identified through a “subtraction” query.", "Electronic Southern is a feature of the present database system which is useful for identifying genomic libraries in which a given gene or ORF exists.", "A Southern analysis is a conventional molecular biology technique in which a nucleic acid of known sequence is used to identify matching (complementary) sequences in a sample of nucleic acid to be analyzed.", "Like their laboratory counterparts, Electronic Southerns according to the present invention may be used to locate homologous matches between a “probe” DNA sequence and a large number of DNA sequences in one or more libraries.", "The present invention provides a method of comparing genetic complements of different types of organisms.", "The method involves providing a database having sequence libraries with multiple biomolecular sequences for different types of organisms, where at least some of the sequences represent open reading frames located along one or more contiguous sequences on each of the organisms' genomes.", "The method further involves receiving a selection of two or more of the sequence libraries for comparison, determining open reading frames common or unique to the selected sequence libraries, and displaying the results of the determination.", "The invention also provides a method of comparing genomic complements of different types of organisms.", "The method involves providing a database having genomic sequence libraries with multiple biomolecular sequences for different types of organisms, where at least some of the sequences represent open reading frames located along one or more contiguous sequences on each of the organisms' genomes.", "The method further involves receiving a selection of two or more of the sequence libraries for comparison, determining sequences common or unique to the selected sequence libraries, and displaying the results of the determination.", "The invention further provides a computer system including a database containing genomic libraries for different types of organisms, which libraries have multiple genomic sequences, at least some of which representing open reading frames located along one or more contiguous sequences on each the organisms' genomes.", "The system also includes a user interface capable of receiving a selection of two or more genomic libraries for comparison and displaying the results of the comparison.", "Another aspect of the present invention provides a method of identifying libraries in which a given gene exists.", "The method involves providing a database including genomic libraries for one or more types of organisms.", "The libraries have multiple genomic sequences, at least some of which represent open reading frames located along one or more contiguous sequences on each the organisms' genomes.", "The method further involves receiving a selection of one or more probe sequences, determining homologous matches between the selected probe sequences and the sequences in the genomic libraries, and displaying the results of the determination.", "The invention also provides a computer system including a database including genomic libraries for one or more types of organisms, which libraries have multiple genomic sequences, at least some of which represent open reading frames located along one or more contiguous sequences on each the organisms' genomes.", "The system also includes a user interface capable of receiving a selection of one or more probe sequences for use in determining homologous matches between one or more probe sequences and the sequences in the genomic libraries, and displaying the results of the determination.", "Also provided is a computer program product including a computer-usable medium having computer-readable program code embodied thereon relating to a database including genomic libraries for one or more types of organisms.", "The libraries have multiple genomic sequences, at least some of which represent open reading frames located along one or more contiguous sequences on each the organisms' genomes.", "The computer program product includes computer-readable program code for providing, within a computing system, an interface for receiving a selection of two or more genomic libraries for comparison, determining sequences common or unique to the selected genomic libraries, and displaying the results of the determination.", "Additionally provided is a computer program product including a computer-usable medium having computer-readable program code embodied thereon relating to a database including genomic libraries for one or more types of organisms.", "The libraries have multiple genomic sequences, at least some of which represent open reading frames located along one or more contiguous sequences on each the organisms' genomes.", "The computer program product includes computer-readable program code for providing, within a computing system, an interface for receiving a selection of one or more probe open reading frames, determining homologous matches between the probe sequences and the sequences in the genomic libraries, and displaying the results of the determination.", "The invention further provides a method of presenting the genetic complement of an organism.", "The method involves providing a database including sequence libraries for a plurality of types of organisms, where the libraries have multiple biomolecular sequences, at least some of which represent open reading frames located along one or more contiguous sequences on each of the organisms' genomes.", "The method further involves receiving a selection of one of the sequence libraries, determining open reading frames within the selected sequence library, and displaying the results as one or more unique identifiers for groups of related opening reading frames.", "The present invention provides relational database systems for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein function hierarchies.", "The hierarchies are provided to allow carefully tailored searches for sequences based upon a protein's biological function or molecular function.", "To make this capability available in large sequence databases, the invention provides a mechanism for automatically grouping new sequences into protein function hierarchies.", "This mechanism takes advantage of descriptive information obtained from “external hits” which are matches of stored sequences against gene sequences stored in an external database such as GenBank.", "The descriptive information provided with GenBank is evaluated according to a specific algorithm and used to automatically group the external hits (or the sequences associated with the hits) in the categories.", "Ultimately, the biomolecular sequences stored in databases of this invention are provided with both descriptive information from the external hit and category information from a relevant hierarchy or hierarchies.", "The invention provides a computer system having a database containing records pertaining to a plurality of biomolecular sequences.", "At least some of the biomolecular sequences are grouped into a first hierarchy of protein function categories, the protein function categories specifying biological functions of proteins corresponding to the biomolecular sequences and the first hierarchy.", "The hierarchy includes a first set of protein function categories specifying biological functions at a cellular level, and a second set of protein function categories specifying biological functions at a level above the cellular level.", "The computer system of the invention also includes a user interface allowing a user to selectively view information regarding the plurality of biomolecular sequences as it relates to the first hierarchy.", "The computer system may also include additional protein function categories based, for example, on molecular or enzymatic function of proteins.", "The biomolecular sequences may include nucleic acid or amino acid sequences.", "Some of said biomolecular sequences may be provided as part of one or more projects for obtaining full-length gene sequences from shorter sequences, and the database records may contain information about such projects.", "The invention also provides a method of using a computer system to present information pertaining to a plurality of biomolecular sequence records stored in a database.", "The method involves displaying a list of the records or a field for entering information identifying one or more of the records, identifying one or more of the records that a user has selected from the list or field, matching the one or more selected records with one or more protein function categories from a first hierarchy of protein function categories into which at least some of the biomolecular sequence records are grouped, and displaying the one or more categories matching the one or more selected records.", "The protein function categories specify biological functions of proteins corresponding to the biomolecular sequences and the first hierarchy includes a first set of protein function categories specifying biological functions at a cellular level, and a second set of protein function categories specifying biological functions at a tissue level.", "The method may also involve matching the records against other protein function hierarchies, such as hierarchies based on molecular and/or enzymatic function, and displaying the results.", "At least some of the biomolecular sequences may be provided as part of one or more projects for obtaining full-length gene sequences from shorter sequences, and the database records may contain information about those projects.", "Additionally, the invention provides a method of using a computer system to present information pertaining to a plurality of biomolecular sequence records stored in a database.", "The method involves displaying a list of one or more protein biological function categories from a first hierarchy of protein biological function categories into which at least some of the biomolecular sequence records are grouped, identifying one or more of the protein biological function categories that a user has selected from the list, matching the one or more selected protein biological function categories with one or more biomolecular sequence records which are grouped in the selected protein biological function categories, and displaying the one or more sequence records matching the one or more selected protein biological function categories.", "The protein biological function categories specify biological functions of proteins corresponding to the biomolecular sequences and the first hierarchy includes a first set of protein biological function categories specifying biological functions at a cellular level, and a second set of protein biological function categories specifying biological functions at a tissue level.", "The method may also involve matching the records against other protein function hierarchies, such as hierarchies based on molecular and/or enzymatic function, and displaying the results.", "At least some of the biomolecular sequences may be provided as part of one or more projects for obtaining full-length gene sequences from shorter sequences, and the database records may contain information about those projects.", "Another aspect of the invention provides a database system having a plurality of internal records.", "The database includes a plurality of sequence records specifying biomolecular sequences, at least some of which records reference hits to an external database, which hits specify genes having sequences that at least partially match those of the biomolecular sequences.", "The database also includes a plurality of external hit records specifying the hits to the external database, and at least some of the records reference protein function hierarchy categories which specify at least one of biological functions of proteins or molecular functions of proteins.", "At least some of the biomolecular sequences may be provided as part of one or more projects for obtaining full-length gene sequences from shorter sequences, and the database records may contain information about those projects.", "Further aspects of the present invention provide a method of using a computer system and a computer readable medium having program instructions to automatically categorize biomolecular sequence records into protein function categories in an internal database.", "The method and program involve receiving descriptive information about a biomolecular sequence in the internal database from a record in an external database pertaining to a gene having a sequence that at least partially matches that of the biomolecular sequence.", "Next, a determination is made whether the descriptive information contains one or more terms matching one or more keywords associated with a first protein function category, the keywords being terms consistent with a classification in the first protein function category.", "When at least one keyword is found to match a term in the descriptive information, a determination is made whether the descriptive information contains a term matching one or more anti-keywords associated with the first protein function category, the anti-keywords being terms inconsistent with a classification in the first protein function category.", "Then, the biomolecular sequence is grouped in the first protein function category when the descriptive information contains a term matching a keyword but contains no term matching an anti-keyword.", "with reference to the drawings, The present invention provides relational database systems for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more characteristics.", "The sequence information of the database is generated by one or more “projects” which are concerned with identifying the full-length coding sequence of a gene (i.e., mRNA).", "The projects involve the extension of an initial sequenced portion of a clone of a gene of interest (e.g., an EST) by a variety of methods which use conventional molecular biological techniques, recently developed adaptations of these techniques, and certain novel database applications.", "Data accumulated in these projects may be provided to the database of the present invention throughout the course of the projects and may be available to database users (subscribers) throughout the course of these projects for research, product (i.e., drug) development, and other purposes.", "In a preferred embodiment, the database of the present invention and its associated projects may provide sequence and related data in amounts and forms not previously available.", "The present invention preferably makes partial and full-length sequence information for a given gene available to a user both during the course of the data acquisition and once the full-length sequence of the gene has been elucidated.", "The database also preferably provides a variety of tools for analysis and manipulation of the data, including Northern analysis and Expression summaries.", "The present invention should permit more complete and accurate annotation of sequence data, as well as the study of relationships between genes of different tissues, systems or organisms, and ultimately detailed expression studies of full-length gene sequences.", "The invention provides a computer system including a database having sequence records containing information identifying one or more projects to which each of the sequence records belong.", "Each project groups together one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The computer system also has a user interface allowing a user to selectively view information regarding one or more projects.", "The biomolecular sequences may include nucleic acid or amino acid sequences.", "The user interface may allow users to view at least three levels of project information including a project information results level listing at least some of the projects in said database, a sequence information results level listing at least some of the sequences associated with a given project, and a sequence retrieval results level sequentially listing monomers which comprise a given sequence.", "A method of using a computer system and a computer program product to present information pertaining to a plurality of sequence records stored in a database are also provided by the present invention.", "The sequence records contain information identifying one or more projects to which each of the sequence records belong.", "Each of the projects groups one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The method and program involve providing an interface for entering query information relating to one or more projects, locating data corresponding to the entered query information, and displaying the data corresponding to the entered query information.", "Additionally, the invention provides a method of using a computer system to present information pertaining to a plurality of sequence records stored in a database.", "The sequence records contains information identifying one or more projects to which each of the sequence records belong.", "Each of the projects groups one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The method involves displaying a list of one or more project identifiers, determining which project identifier or identifiers from the list is selected by a user, then displaying a second list of one or more biomolecular sequence identifiers associated with the selected project identifier or identifiers, determining which sequence identifier or identifiers from the second list has been selected by a user, and displaying a third list of one or more sequences corresponding to the selected sequence identifier or identifiers.", "Following the display of the third list, a determination may be made whether and which sequence from the third list has been selected by a user.", "If a sequence is selected, a sequence alignment search of the selected sequence against other databased sequences may be initiated, and the results of the alignment search displayed.", "For Electronic Northern analysis, the invention further provides a computer system including a database having sequence records containing information identifying one or more projects to which each of the sequence records belong, each of said projects grouping one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The system also has a user interface capable of allowing a user to select one or more project identifiers or project member identifiers specifying one or more sequences to be compared with one or more cDNA sequence libraries, and displaying matches resulting from that comparison.", "A method of using a computer system to present comparative information pertaining to a plurality of sequence records stored in a database is also provided by the present invention.", "The sequence records contain information identifying one or more projects to which each of the sequence records belong, each of the projects grouping one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The method involves providing an interface capable of allowing a user to select one or more project identifiers or project member identifiers specifying one or more sequences, comparing the one or more specified sequences with one or more cDNA sequence libraries, and displaying matches resulting from the comparison.", "In addition, for Expression analysis, the invention provides a computer system including a database having sequence records containing information identifying one or more projects to which each of the sequence records belong, each of the projects grouping one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "The system also has a user interface allowing a user to view expression information pertaining to the projects by selecting one or more expression categories for a query, and displaying the result of the query.", "A method of using a computer system to view expression information pertaining to one or more projects, each of the projects grouping one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence, is also provided in accordance with the present invention.", "The computer system includes a database storing a plurality of sequence records, the sequence records containing information identifying one or more projects to which each of the sequence records belong.", "The method involves providing an interface which allows a user to select one or more expression categories as a query, locating projects belonging to the selected one or more expression categories, and displaying a list of located projects.", "Finally, the present invention provides a computer system including a database having sequence records containing information identifying one or more projects to which each of the sequence records belong, each of the projects grouping one or more biomolecular sequences generated during work to obtain a full-length gene sequence from a shorter sequence.", "This computer system has a user interface allowing a user to selectively view information regarding said one or more projects and which displays information to a user in a format common to one or more other sequence databases.", "These and other features and advantages of the invention will be described in more detail below with reference to the drawings.", "Polymer sequences are assembled into bins.", "A first number of bins are populated with polymer sequences.", "The polymer sequences in each bin are assembled into one or more consensus sequences representative of the polymer sequences of the bin.", "The consensus sequences of the bins are compared to determine relationships, if any, between the consensus sequences.", "The bins are modified based on the relationships between the consensus sequences.", "The polymer sequences are reassembled in the modified bins to generate one or more modified consensus sequences for each bin representative of the modified bins.", "In another aspect of the invention, sequence similarities and dissimilarities are analyzed in a set of polymer sequences.", "Pairwise alignment data is generated for pairs of the polymer sequences.", "The pairwise alignment data defines regions of similarity between the pairs of polymer sequences with boundaries.", "Additional boundaries in particular polymer sequences are determined by applying at least one boundary from at least one pairwise alignment for one pair of polymer sequences to at least one other pairwise alignment for another pair of polymer sequences including one of the particular polymer sequences.", "Additional regions of similarity are generated based on the boundaries.", "1.2.1.33.Annotating—Preferred Embodiments Generally, the present invention provides an improved relational database for storing and manipulating genomic sequence information.", "While the invention is described in terms of a database optimized for microbial data, it is by no means so limited.", "The invention may be employed to investigate data from various sources.", "For example, the invention covers databases optimized for other sources of sequence data, such as animal sequences (e.g., human, primate, rodent, amphibian, insect, etc.", "), plant sequences and microbial sequences.", "In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention.", "It will be apparent, however, that the present invention may be practiced without limitation to some of the specific details presented herein.", "Generally, the present invention provides an improved relational database for storing sequence information.", "The invention may be employed to investigate data from various sources.", "For example, it may catalogue animal sequences (e.g., human, primate, rodent, amphibian, insect, etc.", "), plant sequences, and microbial sequences.", "1.3.Transcriptome Analysis or RNA Profiling The characterization of RNA expression and transcript populations (the transcriptome) can be referred to as RNA profiling and/or expression profiling, utilizing high throughput techniques such as RNA differential displays and DNA microarrays.", "One potential method to characterize gene expression, SAGE (Serial Analysis of Gene Expression) utilizes combinatorial chemistry technology and short sequence tags in the screening of compound libraries.", "For further information see references: Burge, C. B.", "2001.Chipping away at the transcriptome.", "Nat Genet, 27(3): 2324; Hughes, T. R. and Shoemaker, D. D. 2001.DNA microarrays for expression profiling.", "Curr Opin Chem Biol, 5(1): 21-5; Yamamoto, M. et al.", "2001.Use of serial analysis of gene expression (SAGE) technology.", "J Immunol Methods 250(1-2): 45-66.1.3.1 Screening and Selecting Nucleotides for Protein Binding An embodiment of this invention provides for screening methods that include the user of recombinant and in vitro chemical synthesis methods.", "In these hybrid methods, cell-free enzymatic machinery is employed to accomplish the in vitro synthesis of the library members (i.e., peptides or polynucleotides).", "In one type of method, RNA molecules with the ability to bind a predetermined protein or a predetermined dye molecule were selected by alternate rounds of selection and PCR amplification (Tuerk and Gold, 1990; Ellington and Szostak, 1990).", "A similar technique was used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach, 1990; Beaudry and Joyce, 1992; PCT patent publications WO 92/05258 and WO 92/14843).", "1.4.Proteomics In another embodiment of this invention, this invention relates to the emerging field of proteomics, Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantitating expression at the protein level, rather than at the messenger RNA level.", "Proteomics also involves the study of non-genome encoded events, including the post-translational modification of proteins (including glycosylation or other modifications), interactions between proteins, and the location of proteins within a cell.", "The structure, function, and/or level of activity of the proteins expressed by the cell are also of interest.", "Essentially, proteomics involves the study of part or all of the status of the total protein contained within or secreted by a cell.", "Proteomics requires means of separating proteins in complex mixtures and identifying both low-and high-abundance species.", "Examples of powerful methods currently used to resolve complex protein mixtures are 2D gel electrophoresis, reverse phase HPLC, capillary electrophoresis, isoelectric focusing and related hybrid techniques.", "Commonly used protein identification techniques include N-terminal Edman and mass spectrometry (electrospray [ESI] or matrix-assisted laser desorption ionization [MALDI] MS) and sophisticated database search programs, such as SEQUEST, to identify proteins in World Wide Web protein and nucleic acid databases from the MS-MS spectra of their peptides.", "Using a computer, the output of the mass spectrometry can be analyzed so as to link a gene and the particular protein for which it codes.", "This overall process is sometimes referred to as “functional genomics”.", "For general information on proteome research, see, for example, J. S. Fruton; 1999, Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology, Yale Univ.", "Pr.", "; Wilkins et al., 1997, Proteome Research: New Frontiers in Functional Genomics (Principles and Practice), Springer Verlag; A. J.", "Link, 1999, 2-D Proteome Analysis Protocols (Methods in Molecular Biology, 1112, Humana Pr.", "); and Kamp et al., 1999, Proteome and Protein Analysis, Springer Verlag.", "Signal Transduction See also, James, Peter, “Protein identification in the post-genome era: the rapid rise of proteomics”, Q. Rev.", "Biophysics, Vol.", "30, No.", "4, pp.", "279-331 (1997), which is incorporated by reference, herein.", "1.4.1 Screening Peptides: Peptide Display Methods The present invention is further directed to a method for generating a selected mutant polynucleotide sequence (or a population of selected polynucleotide sequences) typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequences(s) possess at least one desired phenotypic characteristic (e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like) which can be selected for.", "One method for identifying hybrid polypeptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor), involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.", "One method of screening peptides involves the display of a peptide sequence, antibody, or other protein on the surface of a bacteriophage particle or cell.", "Generally, in these methods each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences.", "Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.", "A well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein.", "The bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule.", "The bacteriophage particles (i.e., library members) which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage sub-population for a subsequent round of affinity enrichment and phage replication.", "After several rounds of affinity enrichment and phage replication, the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor).", "Such methods are further described in PCT patent publications WO 91/17271, WO 91/18980, WO 91/19818 and WO 93/08278.The latter PCT publication describes a recombinant DNA method for the display of peptide ligands that involves the production of a library of fusion proteins with each fusion protein composed of a first polypeptide portion, typically comprising a variable sequence, that is available for potential binding to a predetermined macromolecule, and a second polypeptide portion that binds to DNA, such as the DNA vector encoding the individual fusion protein.", "When transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the DNA vector encoding it.", "Upon lysis of the host cell, the fusion protein/vector DNA complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).", "The displayed peptide sequences can be of varying lengths, typically from 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and often from about 8-15 amino acids long.", "A library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence.", "Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kernal, fixed, or the like.", "The present display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scfv displayed on phage, which enable large-scale screening of scfv libraries having broad diversity of variable region sequences and binding specificities.", "The present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion.", "The random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.", "The mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or incorporation in a cell.", "1.4.2.Screening that Utilizes In Vitro Translation Systems An embodiment of this invention provides for the use of in vitro translation during the step of screening.", "In vitro translation has been used to synthesize proteins of interest and has been proposed as a method for generating large libraries of peptides.", "These methods, generally comprising stabilized polysome complexes, are described further in WO 91/05058, and WO 92/02536.Applicants have described methods in which library members comprise a fusion protein having a first polypeptide portion with DNA binding activity and a second polypeptide portion having the library member unique peptide sequence; such methods are suitable for use in cell-free in vitro selection formats, among others.", "1.4.3.Affinity Enrichment An aspect of this invention provides for the use of affinity enrichment which allows a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected.", "The polynucleotide can then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just VHI, VLI or CDR portions thereof).", "Using these methods, one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of shuffling to converge rapidly to a desired high-affinity peptide or scfv.", "The peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic or diagnostic agent).", "A significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest.", "The peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.", "The present invention also provides a method for shuffling a pool of polynucleotide sequences selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).", "The invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).", "1.4.4.Antibody Display The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g., for affinity for binding a predetermined antigen (ligand).", "Various prokaryotic expression systems have been developed that can be manipulated to produce combinatorial antibody libraries which may be screened for high-affinity antibodies to specific antigens.", "Recent advances in the expression of antibodies in Escherichia coli and bacteriophage systems (see “alternative peptide display methods”, infra) have raised the possibility that virtually any specificity can be obtained by either cloning antibody genes from characterized hybridomas or by de novo selection using antibody gene libraries (e.g., from Ig cDNA).", "Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al, 1989); Caton and Koprowski, 1990; Mullinax et al, 1990; Persson et al, 1991).", "Various embodiments of bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang et al, 1991; Clackson et al, 1991; McCafferty et al, 1990; Burton et al, 1991; Hoogenboom et al, 1991; Chang et al, 1991; Breitling et al, 1991; Marks et al, 1991, p. 581; Barbas et al, 1992; Hawkins and Winter, 1992; Marks et al, 1992, p. 779; Marks et al, 1992, p. 16007; and Lowman et al, 1991; Lerner et al, 1992; all incorporated herein by reference).", "Typically, a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).", "One particularly advantageous approach has been the use of so-called single-chain fragment variable (scfv) libraries (Marks et al, 1992, p. 779; Winter and Milstein, 1991; Clackson et al, 1991; Marks et al, 1991, p. 581; Chaudhary et al, 1990; Chiswell et al, 1992; McCafferty et al, 1990; and Huston et al, 1988).", "Various embodiments of scfv libraries displayed on bacteriophage coat proteins have been described.", "Bacteriophage display of scfv have already yielded a variety of useful antibodies and antibody fusion proteins.", "A bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al, 1994).", "Intracellular expression of an anti-Rev scfv has been shown to inhibit HIV-1 virus replication in vitro (Duan et al, 1994), and intracellular expression of an anti-p21rar, scfv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al, 1993).", "Recombinant scfv which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility of scfv (Lilley et al, 1994).", "Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al, 1992; Nicholls et al, 1993).", "Various methods have been reported for increasing the combinatorial diversity of a scfv library to broaden the repertoire of binding species (idiotype spectrum).", "Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scfv site-directed hybrids (Stemmer et al, 1993), as has error-prone PCR and chemical mutagenesis (Deng et al, 1994).", "Riechmann (Riechmann et al, 1993) showed semi-rational design of an antibody scfv fragment using site-directed randomization by degenerate oligonucleotide PCR and subsequent phage display of the resultant scfv hybrids.", "Barbas (Barbas et al, 1992) attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.", "Displayed peptide/polynucleotide complexes (library members) which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique.", "This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species.", "Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.", "The library members without the desired specificity are removed by washing.", "The degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope.", "A certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing.", "The temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule.", "Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route.", "This may be done either by continued incubation in the presence of a saturating amount of free predetermined macromolecule, or by increasing the volume, number, and length of the washes.", "In each case, the rebinding of dissociated nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.", "Additional modifications of the binding and washing procedures may be applied to find peptides with special characteristics.", "The affinities of some peptides are dependent on ionic strength or cation concentration.", "This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.", "One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scfv library can be simultaneously screened for a multiplicity of scfv which have different binding specificities.", "Given that the size of a scfv library often limits the diversity of potential scfv sequences, it is typically desirable to us scfv libraries of as large a size as possible.", "The time and economic considerations of generating a number of very large polysome scFv-display libraries can become prohibitive.", "To avoid this substantial problem, multiple predetermined epitope species (receptor species) can be concomitantly screened in a single library, or sequential screening against a number of epitope species can be used.", "In one variation, multiple target epitope species, each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scfv library under suitable binding conditions.", "The collection of beads, comprising multiple epitope species, can then be used to isolate, by affinity selection, scfv library members.", "Generally, subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species.", "This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.", "1.4.5.Expression Systems The DNA expression constructs will typically include an expression control DNA sequence operably linked to the coding sequences, including naturally-associated or heterologous promoter regions.", "Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.", "Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the mutant' “engineered” antibodies.", "The DNA sequences will be expressed in hosts after the sequences have been operably linked to an expression control sequence (i.e., positioned to ensure the transcription and translation of the structural gene).", "These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.", "Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat.", "No.", "4,704,362, which is incorporated herein by reference).", "In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell culture may also be used to produce the polypeptides of the present invention (see Winnacker, 1987), which is incorporated herein by reference).", "Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, and myeloma cell lines, but preferably transformed Bcells or hybridomas.", "Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.", "Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.", "Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the vector.", "Enhancers are cis-acting sequences of between 10 to 300 bp that increase transcription by a promoter.", "Enhancers can effectively increase transcription when either 5′ or 3′ to the transcription unit.", "They are also effective if located within an intron or within the coding sequence itself.", "Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers.", "Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.", "Mammalian expression vector systems will also typically include a selectable marker gene.", "Examples of suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance.", "The first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium.", "Transformed cells can then be identified by their ability to grow on non-supplemented media.", "Examples of prokaryotic drug resistance genes useful as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.", "The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.", "For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment.", "lipofection, or electroporation may be used for other cellular hosts.", "Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation, and micro-injection (see, generally, Sambrook et al, 1982 and 19891.Once expressed, the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, 1982).", "Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Lefkovits and Pernis, 1979 and 1981; Lefkovits, 1997).", "1.4.6 Two-Hybrid Based Screening Assays This invention provides for screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence.", "The selected library members are pooled and shuffled by in vitro and/or in vivo recombination.", "The shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetemnined polypeptide sequence (e.g., an SH2 domain from another protein species).", "An approach to identifying polypeptide sequences which bind to a predetermined polypeptide sequence has been to use a so-called “two-hybrid” system wherein the predetermined polypeptide sequence is present in a fusion protein (Chien et al, 1991).", "This approach identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator (Fields and Song, 1989), the yeast Gal4 transcription protein.", "Typically, the method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation.", "Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell.", "Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacz, HIS3) which is operably linked to a Gal4 binding site.", "Typically, the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver and Hunt, 1993; Durfee et al, 1993; Yang et al, 1992; Luban et al, 1993; Hardy et al, 1992; Bartel et al, 1993; and Vojtek et al, 1993).", "However, variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li and Fields, 1993; Lalo et al, 1993; Jackson et al, 1993; and Madura et al, 1993).", "Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al, 1993; Chakrabarty et al, 1992; Staudinger et al, 1993; and Milne and Weaver 1993) or domains responsible for oligomerization of a single protein (Iwabuchi et al, 1993; Bogerd et al, 1993).", "Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al, 1992).", "Alternatively, an E. coli/BCCP interactive screening system (Germino et al, 1993; Guarente, 1993) can be used to identify interacting protein sequences (i.e., protein sequences which heterodimerize or form higher order heteromultimers).", "Sequences selected by a two-hybrid system can be pooled and shuffled and introduced into a two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence.", "The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kernals.", "1.4.7.Improved Methods for Cellular Engineering, Protein Expression Profiling, Differential Labeling of Peptides, and Novel Reagents Therefore In one embodiment, this invention relates to peptide chemistry, proteomics, and mass spectrometry technology.", "In particular, the invention provides novel methods for determining polypeptide profiles and protein expression variations, as with proteome analyses.", "The present invention provides methods of simultaneously identifying and quantifying individual proteins in complex protein mixtures by selective differential labeling of amino acid residues followed by chromatographic and mass spectrographic analysis.", "The diagnosis and treatment, as well as the predisposition of, a variety of diseases and disorders may often be accomplished through identification and quantitative measurement of polypeptide expression variations between different cell types and cell states.", "Biochemical pathways and metabolic networks can also be analyzed by globally and quantitatively measuring protein expression in various cell types and biological states (see, e.g., Ideker (2001) Science 292: 929-934).", "State-of-the-art techniques such as liquid-chromatography-electrospray-ionization tandem mass spectrometry have, in conjunction with database-searching computer algorithms, revolutionized the analysis of biochemical species from complex biological mixtures.", "With these techniques, it is now possible to perform high-throughput protein identification at picomolar to subpicomolar levels from complex mixtures of biological molecules (see, e.g., Dongre (1997) Trends Biotechnol.", "15: 418-425).", "One such method is based on a class of chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry.", "The method labels multiple cysteinyl residues and uses stable isotope dilution techniques.", "For example, Gygi (1999) Nat.", "Biotechnol.", "10: 994-999, compared protein expression in a yeast using ethanol or galactose as a carbon source.", "The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions.", "In another technique, two different protein mixtures for quantitative comparison are digested to peptide mixtures, the peptides mixtures are separately methylated using either d0- or d3-methanol, the mixtures of methylated peptide combined and subjected to microcapillary HPLC-MS/MS (see, e.g., Goodlett, D. R., et al., (2000) “Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation,” 49th ASMS; Zhou, H; Watts, J D; Aebersold, R. A systematic approach to the analysis of protein phosphorylation.", "Comment In: Nat Biotechnol.", "2001 April; 19(4): 317-8; Nature Biotechnology 2001 April, 19(4): 375-8).", "Parent proteins of methylated peptides are identified by correlative database searching of fragment ion spectra using a computer program assisted paradigms or automated de novo sequencing that compares all tandem mass spectra of d0- and d3-methylated peptide ion pairs.", "In Goodlett (2000) supra, ratios of proteins in two different mixtures were calculated for d0- to d3-methylated peptide pairs.", "However, there are several limitations to this approach, including: use of differential labeling reagents, which relied on stable isotopes, which are expensive, and not flexible to differential labeling of more than two mixtures of peptides; labeling methods limited only to methylation of carboxy-termini; protein expression profiling limited to duplex comparison; one dimensional capillary HPLC chromatography was employed to separate peptides, which doesn't has enough capacity and resolving power for complex mixtures of peptides.", "In one embodiment this invention provides a method for identifying proteins by differential labeling of peptides, the method comprising the following steps: (a) providing a sample comprising a polypeptide; (b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.", "In one aspect, the sample of step (a) comprises a cell or a cell extract.", "The method can further comprise providing two or more samples comprising a polypeptide.", "One or more of the samples can be derived from a wild type cell and one sample can be derived from an abnormal or a modified cell.", "The abnormal cell can be a cancer cell.", "The modified cell can be a cell that is mutagenized &/or treated with a chemical, a physiological factor, or the presence of another organism (including, e.g.", "a eukaryotic organism, prokaryotic organism, virus, vector, prion, or part thereof), &/or exposed to an environmental factor or change or physical force (including, e.g., sound, light, heat, sonication, and radiation).", "The modification can be genetic change (including, for example, a change in DNA or RNA sequence or content) or otherwise.", "In one aspect, the method further comprises purifying or fractionating the polypeptide before the fragmenting of step (c).", "The method can further comprise purifying or fractionating the polypeptide before the labeling of step (d).", "The method can further comprise purifying or fractionating the labeled peptide before the chromatography of step (e).", "In alternative aspects, the purifying or fractionating comprises a method selected from the group consisting of size exclusion chromatography, size exclusion chromatography, HPLC, reverse phase HPLC and affinity purification.", "In one aspect, the method further comprises contacting the polypeptide with a labeling reagent of step (b) before the fragmenting of step (c).", "In one aspect, the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: ZAOH and ZBOH, to esterify peptide C-terminals and/or Glu and Asp side chains; ZANH2 and ZBNH2, to form amide bond with peptide C-terminals and/or Glu and Asp side chains; and ZACO2H and ZBCO2H.", "to form amide bond with peptide N-terminals and/or Lys and Arg side chains; wherein ZA and ZB independently of one another comprise the general formula R-Z1-A1-Z2-A2-Z3-A3-Z4-A4-, Z1, Z2, Z3, and Z4 independently of one another, are selected from the group consisting of nothing, 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1, and OB(OR)(OR1), and R and R1 is an alkyl group, A1, A2, A3, and A4 independently of one another, are selected from the group consisting of nothing or (CRR1)n, wherein R, R1, independently from other R and R1 in Z1 to Z4 and independently from other R and R1 in A1 to A4, are selected from the group consisting of a hydrogen atom, a halogen atom and an alkyl group; “n” in Z1 to Z4, independent of n in A1 to A4, is an integer having a value selected from the group consisting of 0 to about 51; 0 to about 41; 0 to about 31; 0 to about 21, 0 to about 11 and 0 to about 6.In one aspect, the alkyl group (see definition below) is selected from the group consisting of an alkenyl, an alkynyl and an aryl group.", "One or more C—C bonds from (CRR1)n can be replaced with a double or a triple bond; thus, in alternative aspects, an R or an R1 group is deleted.", "The (CRR1)n can be selected from the group consisting of an o-arylene, an m-arylene and ap-arylene, wherein each group has none or up to 6 substituents.", "The (CRR1)n can be selected from the group consisting of a carbocyclic, a bicyclic and a tricyclic fragment, wherein the fragment has up to 8 atoms in the cycle with or without a heteroatom selected from the group consisting of an O atom, a N atom and an S atom.", "In one aspect, two or more labeling reagents have the same structure but a different isotope composition.", "For example, in one aspect, ZA has the same structure as ZB, while ZA has a different isotope composition than ZB.", "In alternative aspects, the isotope is boron-10 and boron-11; carbon-12 and carbon-13; nitrogen-14 and nitrogen-15; and, sulfur-32 and sulfur-34.In one aspect, where the isotope with the lower mass is x and the isotope with the higher mass is y, and x and y are integers, x is greater than y.", "In alternative aspects, x and y are between 1 and about 11, between 1 and about 21, between 1 and about 31, between 1 and about 41, or between 1 and about 51.In one aspect, the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: CD3(CD2)nOH/CH3(CH2)nOH, to esterify peptide C-terminals, where n=0, 1, 2 or y; CD3(CD2)nNH2/CH3(CH2)nNH2, to form amide bond with peptide C-terminals, where n=0, 1, 2 or y; and, D(CD2)nCO2H/H(CH2)nCO2H, to form amide bond with peptide N-terminals, where n=0, 1, 2 or y; wherein D is a deuteron atom, and y is an integer selected from the group consisting of about 51; about 41; about 31; about 21, about 11; about 6 and between about 5 and 51.In one aspect, the labeling reagent of step (b) can comprise the general formulae selected from the group consisting of ZAOH and ZBOH to esterify peptide C-terminals; ZANH2/ZBNH2 to form an amide bond with peptide C-terminals; and, ZACO2H/ZBCO2H to form an amide bond with peptide N-terminals; wherein ZA and ZB have the general formula R-Z1-A1-Z2-A2-Z3-A3-Z4-A4-; Z1, Z2, Z3, and Z4, independently of one another, are selected from the group consisting of nothing, 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1, and OB(OR)(OR1); A1, A2, A3, and A4, independently of one another, are selected from the group consisting of nothing and the general formulae (CRR1)n, and, R and R1 is an alkyl group.", "In one aspect, a single C—C bond in a (CRR1)n group is replaced with a double or a triple bond; thus, the R and R1 can be absent.", "The (CRR1)n can comprise a moiety selected from the group consisting of an o-arylene, an m-arylene and a p-arylene, wherein the group has none or up to 6 substituents.", "The group can comprise a carbocyclic, a bicyclic, or a tricyclic fragments with up to 8 atoms in the cycle, with or without a heteroatom selected from the group consisting of an O atom, an N atom and an S atom.", "In one aspect, R, R1, independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4, are selected from the group consisting of a hydrogen atom, a halogen and an alkyl group The alkyl group (see definition below) can be an alkenyl, an alkynyl or an aryl group.", "In one aspect, the “n” in Z1-Z4 is independent of n in A1-A4 and is an integer selected from the group consisting of about 51; about 41; about 31; about 21, about 11 and about 6.In one aspect, ZA has the same structure a ZB but ZA further comprises x number of —CH2— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "In one aspect, ZA has the same structure a ZB but ZA further comprises x number of —CF2— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "In one aspect, ZA comprises x number of protons and ZB comprises y number of halogens in the place of protons, wherein x and y are integers.", "In one aspect, ZA contains x number of protons and ZB contains y number of halogens, and there are x−y number of protons remaining in one or more A1-A4 fragments, wherein x and y are integers.", "In one aspect, ZA further comprises x number of —O— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "In one aspect, ZA further comprises x number of —S— fragment(s) in one or more A1-A4 fragments, wherein x is an integer.", "In one aspect, ZA further comprises x number of —O— fragment(s) and ZB further comprises y number of —S— fragment(s) in the place of —O— fragment(s), wherein x and y are integers.", "In one aspect, ZA further comprises x−y number of —O— fragment(s) in one or more A1-A4 fragments, wherein x and y are integers.", "In alternative aspects, x and y are integers selected from the group consisting of between 1 about 51; between 1 about 41; between 1 about 31; between 1 about 21, between 1 about 11 and between 1 about 6, wherein x is greater than y.", "In one aspect, the labeling reagent of step (b) comprises the general formulae selected from the group consisting of: CH3(CH2)nOH/CH3(CH2)n+mOH, to esterify peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y; CH3(CH2)nNH2/CH3(CH2)n+mNH2, to form amide bond with peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y; and, H(CH2)nCO2H/H(CH2)n+mCO2H, to form amide bond with peptide N-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y; wherein n, m and y are integers.", "In one aspect, n, m and y are integers selected from the group consisting of about 51; about 41; about 31; about 21, about 11; about 6 and between about 5 and 51.In one aspect, the separating of step (e) comprises a liquid chromatography system, such as a multidimensional liquid chromatography or a capillary chromatography system.", "In one aspect, the mass spectrometer comprises a tandem mass spectrometry device.", "In one aspect, the method further comprises quantifying the amount of each polypeptide or each peptide.", "The invention provides a method for defining the expressed proteins associated with a given cellular state, the method comprising the following steps: (a) providing a sample comprising a cell in the desired cellular state; (b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting polypeptides derived from the cell into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, thereby defining the expressed proteins associated with the cellular state.", "The invention provides a method for quantifying changes in protein expression between at least two cellular states, the method comprising the following steps: (a) providing at least two samples comprising cells in a desired cellular state; (b) providing a plurality of labeling reagents which differ in molecular mass that can generate differential labeled peptides that do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting polypeptides derived from the cells into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents, wherein the labels used in one same are different from the labels used in other samples; (e) separating the peptides by chromatography to generate an eluate; (f) feeding the eluate of step (e) into a mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which identifies from which sample each peptide was derived, compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated, and compares the amount of each polypeptide in each sample, thereby quantifying changes in protein expression between at least two cellular states.", "The invention provides a method for identifying proteins by differential labeling of peptides, the method comprising the following steps: (a) providing a sample comprising a polypeptide; (b) providing a plurality of labeling reagents which differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, wherein the differences in molecular mass are distinguishable by mass spectrographic analysis; (c) fragmenting the polypeptide into peptide fragments by enzymatic digestion or by non-enzymatic fragmentation; (d) contacting the labeling reagents of step (b) with the peptide fragments of step (c), thereby labeling the peptides with the differential labeling reagents; (e) separating the peptides by multidimensional liquid chromatography to generate an eluate; (f) feeding the eluate of step (e) into a tandem mass spectrometer and quantifying the amount of each peptide and generating the sequence of each peptide by use of the mass spectrometer; (g) inputting the sequence to a computer program product which compares the inputted sequence to a database of polypeptide sequences to identify the polypeptide from which the sequenced peptide originated.", "The invention provides a chimeric labeling reagent comprising (a) a first domain comprising a biotin; and (b) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope.", "The isotope(s) can be in the first domain or the second domain.", "For example, the isotope(s) can be in the biotin.", "In alternative aspects, the isotope can be a deuterium isotope, a boron-10 or boron-11 isotope, a carbon-12 or a carbon-13 isotope, a nitrogen-14 or a nitrogen-15 isotope, or, a sulfur-32 or a sulfur-34 isotope.", "The chimeric labeling reagent can comprise two or more isotopes.", "The chimeric labeling reagent reactive group capable of covalently binding to an amino acid can be a succimide group, an isothiocyanate group or an isocyanate group.", "The reactive group can be capable of covalently binding to an amino acid binds to a lysine or a cysteine.", "The chimeric labeling reagent can further comprising a linker moiety linking the biotin group and the reactive group.", "The linker moiety can comprise at least one isotope.", "In one aspect, the linker is a cleavable moiety that can be cleaved by, e.g., enzymatic digest or by reduction.", "The invention provides a method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the small molecule tags are structurally identical but differ in their isotope composition, and the small molecules comprise reactive groups that covalently bind to cysteine or lysine residues or both; (b) providing at least two samples comprising polypeptides; (c) attaching covalently the differential small molecule tags to amino acids of the polypeptides; (d) determining the protein concentrations of each sample in a tandem mass spectrometer; and, (d) comparing relative protein concentrations of each sample.", "In one aspect, the sample comprises a complete or a fractionated cellular sample.", "In one aspect of the method, the differential small molecule tags comprise a chimeric labeling reagent comprising (a) a first domain comprising a biotin; and, (b) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope.", "The isotope can be a deuterium isotope, a boron-10 or boron-11 isotope, a carbon-12 or a carbon-13 isotope, a nitrogen-14 or a nitrogen-15 isotope, or, a sulfur-32 or a sulfur-34 isotope.", "The chimeric labeling reagent can comprise two or more isotopes.", "The reactive group can be capable of covalently binding to an amino acid is selected from the group consisting of a succimide group, an isothiocyanate group and an isocyanate group.", "The invention provides a method of comparing relative protein concentrations in a sample comprising (a) providing a plurality of differential small molecule tags, wherein the differential small molecule tags comprise a chimeric labeling reagent comprising (i) a first domain comprising a biotin; and, (ii) a second domain comprising a reactive group capable of covalently binding to an amino acid, wherein the chimeric labeling reagent comprises at least one isotope; (b) providing at least two samples comprising polypeptides; (c) attaching covalently the differential small molecule tags to amino acids of the polypeptides; (d) isolating the tagged polypeptides on a biotin-binding column by binding tagged polypeptides to the column, washing non-bound materials off the column, and eluting tagged polypeptides off the column; (e) determining the protein concentrations of each sample in a tandem mass spectrometer; and, (f) comparing relative protein concentrations of each sample.", "The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below.", "Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.", "All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.", "The invention provides methods for simultaneously identifying individual proteins in complex mixtures of biological molecules and quantifying the expression levels of those proteins, e.g., proteome analyses.", "The methods compare two or more samples of proteins, one of which can be considered as the standard sample and all others can be considered as samples under investigation.", "The proteins in the standard and investigated samples are subjected separately to a series of chemical modifications, i.e., differential chemical labeling, and fragmentation, e.g., by proteolytic digestion and/or other enzymatic reactions or physical fragmenting methodologies.", "The chemical modifications can be done before, or after, or before and after fragmentation/digestion of the polypeptide into peptides.", "Peptides derived from the standard and the investigated samples are labeled with chemical residues of different mass, but of similar properties, such that peptides with the same sequence from both samples are eluted together in the separation procedure and their ionization and detection properties regarding the mass spectrometry are very similar.", "Differential chemical labeling can be performed on reactive functional groups on some or all of the carboxy- and/or amino-termini of proteins and peptides and/or on selected amino acid side chains.", "A combination of chemical labeling, proteolytic digestion and other enzymatic reaction steps, physical fragmentation and/or fractionation can provide access to a variety of residues to general different specifically labeled peptides to enhance the overall selectivity of the procedure.", "The standard and the investigated samples are combined, subjected to multidimensional chromatographic separation, and analyzed by mass spectrometry methods.", "Mass spectrometry data is processed by special software, which allows for identification and quantification of peptides and proteins.", "Depending on the complexity and composition of the protein samples, it may be desirable, or be necessary, to perform protein fractionation using such methods as size exclusion, ion exchange, reverse phase, or other methods of affinity purifications prior to one or more chemical modification steps, proteolytic digestion or other enzymatic reaction steps, or physical fragmentation steps.", "The combined mixtures of peptides are first separated by a chromatography method, such as a multidimensional liquid chromatography, system, before being fed into a coupled mass spectrometry device, such as a tandem mass spectrometry device.", "The combination of multidimensional liquid chromatography and tandem mass spectrometry can be called “LC-LC-MS/MS.” LC-LC-MS/MS was first developed by Link A. and Yates J. R., as described, e.g., by Link (1999) Nature Biotechnology 17: 676-682; Link (1999) Electrophoresis 18: 1314-1334; Washburn, M P; Wolters, D; Yates, J R, Nature Biotechnology 2001 March, 19(3): 242-7.In practicing the methods of the invention, proteins can be first substantially or partially isolated from the biological samples of interest.", "The polypeptides can be treated before selective differential labeling; for example, they can be denatured, reduced, preparations can be desalted, and the like.", "Conversion of samples of proteins into mixtures of differentially labeled peptides can include preliminary chemical and/or enzymatic modification of side groups and/or termini; proteolytic digestion or fragmentation; post-digestion or post-fragmentation chemical and/or enzymatic modification of side groups and/or termini.", "The differentially modified polypeptides and peptides are then combined into one or more peptide mixtures.", "Solvent or other reagents can be removed, neutralized or diluted, if desired or necessary.", "The buffer can be modified, or, the peptides can be redissolved in one or more different buffers, such as a “MudPIT” (see below) loading buffer.", "The peptide mixture is then loaded onto chromatography column, such as a liquid chromatography column, a 2D capillary column or a multidimensional chromatography column, to generate an eluate.", "The eluate is fed into a mass spectrometer, such as a tandem mass spectrometer.", "In one aspect, an LC ESI MS and MS/MS analysis is complete.", "Finally, data output is processed by appropriate software using database searching and data analysis.", "In practicing the methods of the invention, high yields of peptides can generated for mass spectrograph analysis.", "Two or more samples can be differentially labeled by selective labeling of each sample.", "Peptide modifications, i.e., labeling, are stable.", "Reagents having differing masses or reactive groups can be chosen to maximize the number of reactive groups and differentially labeled samples, thus allowing for a multiplex analysis of sample, polypeptides and peptides.", "In one aspect, a “MudPIT” protocol is used for peptide analysis, as described herein.", "The methods of the invention can be fully automated and can essentially analyze every protein in a sample.", "Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.", "As used herein, the following terms have the meanings ascribed to them unless specified otherwise.", "As used herein, the term “alkyl” is used to refer to a genus of compounds including branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radicals, including substituted derivatives and equivalents thereof.", "In one aspect, the hydrocarbons have from about 1 to about 100 carbons, about 1 to about 50 carbons or about 1 to about 30 carbons, about 1 to about 20 carbons, about 1 to about 10 carbons.", "When the alkyl group has from about 1 to 6 carbon atoms, it is referred to as a “lower alkyl.” Suitable alkyl radicals include, e.g., structures containing one or more methylene, methine and/or methyne groups arranged in acyclic and/or cyclic forms.", "Branched structures have a branching motif similar to isopropyl, tert-butyl isobutyl, 2-ethylpropyl, etc.", "As used herein, the term encompasses “substituted alkyls.” “Substituted alkyl” refers to alkyl as just described including one or more functional groups such as lower alkyl, aryl, acyl, halogen (i.e., alkylhalos, e.g., CF3), hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, arylamino, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like.", "These groups may be attached to any carbon of the alkyl moiety.", "Additionally, these groups may be pendent from, or integral to, the alkyl chain.", "The term “alkoxy” is used herein to refer to the to a COR group, where R is a lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or substituted arylalkyl wherein the alkyl, aryl, substituted aryl, arylalkyl and substituted arylalkyl groups are as described herein.", "Suitable alkoxy radicals include, for example, methoxy, ethoxy, phenoxy, substituted phenoxy, benzyloxy phenethyloxy, tert.-butoxy, etc.", "The term “aryl” is used herein to refer to an aromatic substituent that may be a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.", "The common linking group may also be a carbonyl as in benzophenone.", "The aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.", "The term “aryl” encompasses “arylalkyl.” “Substituted aryl” refers to aryl as just described including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF3), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto and both saturated and unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety.", "The linking group may also be a carbonyl such as in cyclohexyl phenyl ketone.", "The term “substituted aryl” encompasses “substituted arylalkyl.” The term “arylalkyl” is used herein to refer to a subset of “aryl” in which the aryl group is further attached to an alkyl group, as defined herein.", "The term “biotin” as used herein refers to any natural or synthetic biotin or variant thereof, which are well known in the art; ligands for biotin, and ways to modify the affinity of biotin for a ligand, are also well known in the art; see, e.g., U.S. Pat.", "Nos.", "6,242,610; 6,150,123; 6,096,508; 6,083,712; 6,022,688; 5,998,155; 5,487,975.The phrase “labeling reagents which .", ".", ".", "do not differ in ionization and detection properties in mass spectrographic analysis” means that the amount and/or mass sequence of the labeling reagents can be detected using the same mass spectrographic conditions and detection devices.", "The term “polypeptide” includes natural and synthetic polypeptides, or mimetics, which can be either entirely composed of synthetic, non-natural analogues of amino acids, or, they can be chimeric molecules of partly natural peptide amino acids and partly non-natural analogs of amino acids.", "The term “polypeptide” as used herein includes proteins and peptides of all sizes.", "The term “sample” as used herein includes any polypeptide-containing sample, including samples from natural sources, or, entirely synthetic samples.", "The term “column” as used herein means any substrate surface, including beads, filaments, arrays, tubes and the like.", "The phrase “do not differ in chromatographic retention properties” as used herein means that two compositions have substantially, but not necessary exactly, the same retention properties in a chromatograph, such as a liquid chromatograph.", "For example, two compositions do not differ in chromatographic retention properties if they elute together, i.e., they elute in what a skilled artisan would consider the same elution fraction.", "Differential Labeling of Peptides and Polypeptides In practicing the methods of the invention, proteins and peptides are subjected to a series of chemical modifications, i.e., differential chemical labeling.", "The chemical modifications can be done before, or after, or before and after fragmentation/digestion of the polypeptide into peptides.", "Differential labeling reagents can differ in their isotope composition (i.e., isotopical reagents), in their structural composition (i.e., homologous reagents), but by a rather small fragment which change does not alter the properties stated above, i.e., the labeling reagent differ in molecular mass but do not differ in chromatographic retention properties and do not differ in ionization and detection properties in mass spectrographic analysis, and the differences in molecular mass are distinguishable by mass spectrographic analysis.", "In one aspect of the invention, mixtures of polypeptides and/or peptides coming from the “standard” protein sample and the “investigated” protein sample(s) are labeled separately with differential reagents, or, one sample is labeled and other sample remains unlabeled.", "As noted above, these differential reagents differ in molecular mass, but do not differ in retention properties regarding the separation method used (e.g., chromatography) and the mass spectrometry methods used will not detect different ionization and detection properties.", "Thus, these differential reagents differ either in their isotope composition (i.e., they are isotopical reagents) or they differ structurally by a rather small fragment which change does not alter the properties stated above (i.e., they are homologous reagents).", "Differential chemical labeling can include esterification of C-termini, amidation of C-termini and/or acylation of N-termini.", "Esterification targets C-termini of peptides and carboxylic acid groups in amino acid side chains.", "Amidation targets C-termini of peptides and carboxylic acid groups in amino acid side chains.", "Amidation may require protection of amine groups first.", "Acylation targets N-termini of peptides and amino and hydroxy groups in amino acid side chains.", "Acylation may require protection of carboxylic groups first.", "The skilled artisan will recognize that the chemical syntheses and differential chemical labeling of peptides and polypeptides (e.g., esterification, amidation, and acylation) used to practice the methods of the invention can be by a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al.", "(Eds), John Wiley & Sons, Inc., NY; Venuti (1989) Pharm.", "Res.", "6: 867-873; the Beilstein Handbook of Organic Chemistry (Beilstein Institut fuer Literatur der Organischen Chemie, Frankfurt, Germany); Beilstein online database and references obtainable therein; “Organic Chemistry,” Morrison & Boyd, 7th edition, 1999, Prentice-Hall, Upper Saddle River, N.J.", "The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature.", "For example, the esterification, amidation, and acylation reactions may be performed on the mixtures of peptides in a fashion similar to other reaction of these types already described in prior art, such as: In alternative aspects, reagents comprise the general formulae: i. ZAOH and ZBOH to esterify peptide C-terminals and/or Glu and Asp side chains; ii.", "ZANH2/ZBNH2 to form amide bond with peptide C-terminals and/or Glu and Asp side chains; or iii.", "ZACO2H/ZBCO2H to form amide bond with peptide N-terminals and/or Lys and Arg side chains; wherein ZA and ZB independently of one another can be R-Z1-A1-Z2-A2-Z3-A3-Z4-A4-, and Z1, Z2, Z3, and Z4 independently of one another can be selected from 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1, OB(OR)(OR1), or, Z1, Z2, Z3, and Z4 independently of one another may be absent, and R is an alkyl group; and, A1, A2, A3, and A4 independently of one another can be selected from (CRR1)n, and R is an alkyl group.", "In alternative aspects, some single C—C bonds from (CRR1)n may be replaced with double or triple bonds, in which case some groups R and R1 will be absent, (CRR1)n can be an o-arylene, an m-arylene, or a p-arylene with up to 6 substituents, carbocyclic, bicyclic, or tricyclic fragments with up to 8 atoms in the cycle with or without heteroatoms (O, N, S) and with or without substituents, or A1, A2, A3, and A4 independently of one another can be absent; R, R1, independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4, can be hydrogen, halogen or an alkyl group, such as an alkenyl, an alkynyl or an aryl group; n in Z1-Z4, independent of n in A1-A4, is an integer that can have value from 0 to about 51; 0 to about 41; 0 to about 31; 0 to about 21, 0 to about 11; 0 to about 6; In alternative aspects, ZA has the same structure as ZB, but they have different isotope compositions.", "Any isotope may be used.", "In alternative aspects, if ZA contains x number of protons, ZB may contain y number of deuterons in the place of protons, and, correspondingly, x−y number of protons remaining; and/or if ZA contains x number of borons-10, ZB may contain y number of borons-11 in the place of borons-10, and, correspondingly, x−y number of borons-10 remaining; and/or if ZA contains x number of carbons-12, ZB may contain y number of carbons-13 in the place of carbons-12, and, correspondingly, x−y number of carbons-12 remaining; and/or if ZA contains x number of nitrogens-14, ZB may contain y number of nitrogens-15 in the place of nitrogens-14, and, correspondingly, x−y number of nitrogens-14 remaining; and/or if ZA contains x number of sulfurs-32, ZB may contain y number of sulfurs-34 in the place of sulfurs-32, and, correspondingly, x−y number of sulfurs-32 remaining; and so on for all elements which may be present and have different stable isotopes; x and y are whole numbers such that x is greater than y.", "In one aspect, x and y are between 1 and about 11, between 1 and about 21, between 1 and about 31, between 1 and about 41, between 1 and about 51.In alternative aspects, reagent pairs/series comprise the general formulae: i. CD3(CD2)nOH/CH3(CH2)nOH to esterify peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; (delta mass=3+2n); ii.", "CD3(CD2)nNH2/CH3(CH2)nNH2 to form amide bond with peptide C-terminals where n=0, 1, 2, .", ".", ".", ", y (delta mass=3+2n); iii.", "D(CD2)nCO2H/H(CH2)nCO2H to form amide bond with peptide N-terminals, where n=0, 1, 2, .", ".", ".", ", y (delta mass=1+2n); wherein y is an integer that can have value of about 51; about 41; about 31; about 21, about 11; about 6, or between about 5 and 51.Other exemplary reagents can be presented by general formulae: i. ZAOH and ZBOH to esterify peptide C-terminals; ii.", "ZANH2/ZBNH2 to form an amide bond with peptide C-terminals; iii.", "ZACO2H/ZBCO2H to form an amide bond with peptide N-terminals; wherein ZA and ZB can be R-Z1-A1-Z2-A2-Z3-A3-Z4-A4- and Z1, Z2, Z3, and Z4, independently of one another, can be selected from 10, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1 or OB(OR)(OR1); or, Z1, Z2, Z3, and Z4, independently of one another, can be absent, and, R is an alkyl group; A1, A2, A3, and A4, independently of one another, can be a moiety comprising the general formulae (CRR1)n. In alternative aspects, single C—C bonds in some (CRR1)n groups may be replaced with double or triple bonds, in which case some groups R and R1 will be absent, or (CRR1)n can be an o-arylene, an m-arylene, or a p-arylene with up to 6 substituents, or a carbocyclic, a bicyclic, or a tricyclic fragments with up to 8 atoms in the cycle, with or without heteroatoms (e.g., O, N or S atoms), or, with or without substituents, or, A1-A4 independently of one another may be absent; In alternative aspects, R, R1, independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4, can be a hydrogen atom, a halogen or an alkyl group, such as an alkenyl, an alkynyl or an aryl group; In alternative aspects, n in Z1-Z4 is independent of n in A1-A4 and is an integer that can have value of about 51; about 41; about 31; about 21, about 11; about 6.In alternative aspects, ZA has a similar structure to that of ZB, but ZA has x extra —CH2— fragment(s) in one or more A1-A4 fragments, and/or ZA has x extra —CF2— fragment(s) in one or more A1-A4 fragments.", "Alternatively, ZA can contain x number of protons and ZB may contain y number of halogens in the place of protons.", "Alternatively, where ZA contains x number of protons and ZB contains y number of halogens, there are x−y number of protons remaining in one or more A1-A4 fragments; and/or ZA has x extra —O— fragment(s) in one or more A1-A4 fragments; and/or ZA has x extra —S— fragment(s) in one or more A1-A4 fragments; and/or if ZA contains x number of —O— fragment(s), ZB may contain y number of-S— fragment(s) in the place of —O— fragment(s), and, correspondingly, x−y number of —O— fragment(s) remaining in one or more A1-A4 fragments; and the like.", "In alternative aspects, x and y are integers that can have value of between 1 about 51; of between 1 about 41; of between 1 about 31; of between 1 about 21, of between 1 about 11; of between 1 about 6, such that x is greater than y. Exemplary homologous reagents pairs/series are i. CH3(CH2)nOH/CH3(CH2)n+mOH to esterify peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y (delta mass=14m) ii.", "CH3(CH2)n NH2/CH3(CH2)n+mNH2 to form amide bond with peptide C-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y (delta mass=14m) iii.", "H(CH2)nCO2H/H(CH2)n+mCO2H to form amide bond with peptide N-terminals, where n=0, 1, 2, .", ".", ".", ", y; m=1, 2, .", ".", ".", ", y (delta mass=14m) wherein y is an integer that can have value of about 51; about 41; about 31; about 21, about 11; about 6, or between about 5 and 51.Methods for Peptide/Protein Separation and Detection The methods of the invention use chromatographic techniques to separate tagged polypeptides and peptides.", "In one aspect, a liquid chromatography is used, e.g., a multidimensional liquid chromatography.", "The chromatogram eluate is coupled to a mass spectrometer, such as a tandem mass spectrometry device (e.g., a “LC-LC-MS/MS” system).", "Any variation and equivalent thereof can be used to separate and detect peptides.", "LC-LC-MS/MS was first developed by Link A. and Yates J. R., as described, e.g., in (Link (1999) Nature Biotechnology 17: 676-682; Link (2000) Electrophoresis 18, 1314-1334.In one aspect, the LC-LC-MS/MS technique is used; it is effective for complexed peptide separation and it is easily automated.", "LC-LC-MS/MS is commonly known by the acronym “MudPIT,” for “Multi-dimensional Protein Identification Technique.” Variations and equivalents of LC-LC-MS/MS used in the methods of the invention include methodologies involving reversed phase columns coupled to either cation exchange columns (as described, e.g., by Opiteck (1997) Anal.", "Chem.", "69: 1518-1524; or, size exclusion columns (as described, e.g., by Opiteck (1997) Anal.", "Biochem.", "258: 349-361).", "In one aspect, an LC-LC-MS/MS technique uses a mixed bed microcapillary column containing strong cation exchange (SCX) and reversed phase (RPC) resins.", "Other exemplary alternatives include protein fractionation combined with one-dimensional LC-ESI MS/MS or peptide fractionation combined MALDI MS/MS.", "Depending on the complexity or the property of the protein samples, any protein fractionation method, including size exclusion chromatography, ion exchange chromatography, reverse phase chromatography, or any of the possible affinity purifications, can be introduced prior to labeling and proteolysis.", "In some circumstances, use of several different methods may be necessary to identify all proteins or specific proteins in a sample.", "Sequence Analysis and Quantification Both quantity and sequence identity of the protein from which the modified peptide originated can be determined by a mass spectrometry device, such as a “multistage mass spectrometry” (MS).", "This can be achieved by the operation of the mass spectrometer in a dual mode in which it alternates in successive scans between measuring the relative quantities of peptides eluting from the capillary column and recording the sequence information of selected peptides.", "Peptides are quantified by measuring in the MS mode the relative signal intensities for pairs or series of peptide ions of identical sequence that are tagged differentially, which therefore differ in mass by the mass differential encoded within the differential labeling reagents.", "Peptide sequence information can be automatically generated by selecting peptide ions of a particular mass-to-charge (m/z) ratio for collision-induced dissociation (CID) in the mass spectrometer operating in the tandem MS mode, as described, e.g., by Link (1997) Electrophoresis 18: 1314-1334; Gygi (1999) Nature Biotechnol.", "17: 994-999; Gygi (1999) Cell Biol.", "19: 1720-1730.The resulting tandem mass spectra can be correlated to sequence databases to identify the protein from which the sequenced peptide originated.", "Exemplary commercial available softwares include TURBO SEQUEST™ by Thermo Finnigan, San Jose, Calif.; MASSSCOT™ by Matrix Science, SONAR MS/MS™ by Proteometrics.", "Routine software modifications may be necessary for automated relative quantification.", "Mass Spectrometry Devices In the methods of the invention use mass spectrometry to identify and quantify differentially labeled peptides and polypeptides.", "Any mass spectrometry system can be used.", "In one aspect of the invention, combined mixtures of peptides are separated by a chromatography method comprising multidimensional liquid chromatography coupled to tandem mass spectrometry, or, “LC-LC-MS/MS,” see, e.g., Link (1999) Biotechnology 17: 676-682; Link (1999) Electrophoresis 18: 1314-1334.Exemplary, mass spectrometry devices include those incorporating matrix-assisted laser desorption-ionization-time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Isola (2001) Anal.", "Chem.", "73: 2126-2131; Van de Water (2000) Methods Mol.", "Biol.", "146: 453459; Griffin (2000) Trends Biotechnol.", "18: 77-84; Ross (2000) Biotechniques 29: 620-626, 628-629).", "The inherent high molecular weight resolution of MALDI-TOF MS conveys high specificity and good signal-to-noise ratio for performing accurate quantitation.", "Use of mass spectrometry, including MALDI-TOF MS, and its use in detecting nucleic acid hybridization and in nucleic acid sequencing, is well known in the art, see, e.g., U.S. Pat.", "Nos.", "6,258,538; 6,238,871; 6,238,869; 6,235,478; 6,232,066; 6,228,654; 6,225,450; 6,051,378; 6,043,031.Fragmentation and Proteolytic Digestion In practicing the methods of the invention, polypeptides are fragmented, e.g., by proteolytic, i.e., enzymatic, digestion and/or other enzymatic reactions or physical fragmenting methodologies.", "The fragmentation can be done before and/or after reacting the peptides/polypeptides with the labeling reagents used in the methods of the invention.", "Methods for proteolytic cleavage of polypeptides are well known in the art, e.g., enzymes include trypsin (see, e.g., U.S. Pat.", "Nos.", "6,177,268; 4,973,554), chymotrypsin (see, e.g., U.S. Pat.", "Nos.", "4,695,458; 5,252,463), elastase (see, e.g., U.S. Pat.", "No.", "4,071,410); subtilisin (see, e.g., U.S. Pat.", "No.", "5,837,516) and the like.", "In one aspect, a chimeric labeling reagent of the invention includes a cleavable linker.", "Exemplary cleavable linker sequences include, e.g., Factor Xa or enterokinase (Invitrogen, San Diego Calif.).", "Other purification facilitating domains can be used, such as metal chelating peptides, e.g., polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.).", "Biological Samples The methods are based on comparison of two or more samples of proteins, one of which can be considered as the standard sample and all others can be considered as samples under investigation.", "For example, in one aspect, the invention provides a method for quantifying changes in protein expression between at least two cellular states, such as, an activated cell versus a resting cell, a normal cell versus a cancerous cell, a stem cell versus a differentiated cell, an injured cell or infected cell versus an uninjured cell or uninfected cell; or, for defining the expressed proteins associated with a given cellular state.", "Sample can be derived from any biological source, including cells from, e.g., bacteria, insects, yeast, mammals and the like.", "Cells can be harvested from any body fluid or tissue source, or, they can be in vitro cell lines or cell cultures.", "Detection Devices and Methods The devices and methods of the invention can also incorporate in whole or in part designs of detection devices as described, e.g., in U.S. Pat.", "Nos.", "6,197,503; 6,197,498; 6,150,147; 6,083,763; 6,066,448; 6,045,996; 6,025,601; 5,599,695; 5,981,956; 5,698,089; 5,578,832; 5,632,957.A number of embodiments of the invention have been described.", "Nevertheless, it will be 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Filed May 22, 1998, Issued Jan. 11, 2000.Wiktorowicz J E, Raysberg Y: Electrophoresis apparatus and method.", "Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K Editors.", "Current Protocols In Molecular Biology, Vol 2.John Wiley & Sons, Inc, C) 2001, 10.21.4-10.21.6, 10.22.5-10.22.10, 10.22.14, 10.22.15-10.22.20.Sambrook J, Russell D W Editors.", "Molecular Cloning A Laboratory Manual 3rd ed.", "Cold Spring Harbor Laboratory Press, New York, © 2001, 18.3, 18.62, 18.66.1.4.8.Additional Methods for Differential Analysis 1.4.8.1.Protein Expression Profiling Using Selective Differential Labeling The use of mass spectrometry to identify proteins whose sequences are present in either DNA or protein databases is well established and integral to the field of Proteomics.", "Protein and peptide mass can be determined at high accuracy by several mass spectrometric techniques.", "Peptide can be further fragmented in a tandem or ion trap mass spectrometer yielding sequence information of the peptide.", "Both types of mass information can be used to identify protein in a sequence database.", "One goal of Proteomics is to define the expressed proteins associated with a given cellular state and another is to quantify changes in protein expression between cellular states.", "One of the new methodologies that have a great impact on proteome research is known as isotope-coded affinity tag (ICAT) peptide labeling (17).", "The method is based on a newly synthesized class of chemical reagents (ICATs) used in combination with tandem mass spectrometry.", "The ICAT reagent contains a biotin affinity tag and a thiol specific reactive group, which are joined by a spacer domain which is available in two forms: regular and isotopically heavy, which includes eight deuterium atoms.", "First, a reduced protein mixture representing one cell state is derivatized with the isotopically light version of the ICAT reagent, while the corresponding reduced protein mixture representing a second cell state is derivatized with the isotopically heavy version of the ICAT reagent.", "Second, the labeled samples are combined and proteolytically digested to produce peptide fragments.", "Third, the tagged cysteine containing peptide fragments are isolated by avidin affinity chromatography.", "Finally, the isolated tagged peptides are separated and analyzed by microcapillary tandem mass spectrometry.", "There are, however, limitations associated with their approach: (i) differential labeling reagents relied on stable isotopes which is expensive and not very flexible to multiplex differential labeling; (ii) The moieties attached to the original peptides are approximately 500 Dalton heavy, which is heavier than some peptides and is likely to affect peptide ionization and fragmentation process; (iii) Some bonds in the labeling reagent are week compared to the amide bond, which might complicate the MS/MS spectrum, (iv) Protein expression profiling is limited to duplex comparison; (v) The affinity interaction between biotin and avidin is too strong to release the immobilized peptide efficiently.", "In one embodiment, this present invention provides a method for simultaneous identification and quantification of expression levels of individual proteins carrying certain functional groups in their side chains.", "The proteins may be analyzed in complex mixtures.", "The method is based on comparison of two or more samples of proteins, one of which can be considered as the standard sample and all others can be considered as samples under investigation.", "The samples of proteins are subjected to a sequence of manipulations including (i) proteolytic digestion into mixtures of peptides, (ii) treatment of the mixtures of peptides with chemical probes, (iii) washing away and discarding the unbound peptides from the mixtures, (iv) cleaving the chemical probes and the consequential release of the peptides still carrying parts of the chemical probes into solution.", "This sequence of manipulations may also include one or more auxiliary chemical and/or enzymatic modifications of functional groups in side chains and/or in the free termini of the proteins and/or peptides in order to achieve selective and the most favorable modification for the next steps in the protocol.", "The auxiliary modifications may be performed between any steps of the main sequence.", "The core structure of the chemical probe consists of (i) a solid support, (ii) a spacer, (iii) a cleavable moiety, (iv) a differential mass labeling unit, and (v) a reactive group.", "The chemical probes perform three functions: (i) they attach peptides carrying specific functional groups in their side chains and/or termini to a solid support by forming covalent chemical bonds to the reactive group of the probe, (ii) they provide means for selective cleavage of the attached peptide from the solid support such that a part of the probe still remains attached to the peptide, and (iii) they serve as differential labeling reagents.", "Differential labeling results from attaching of chemical moieties of different mass but of similar properties to a protein or a peptide such that peptides with the same sequence but with different labels are eluted together in the separation procedure and their ionization and detection properties regarding mass spectrometrical analysis are very similar.", "The differential mass labeling unit remains covalently bound to the peptide after it is cleaved from the solid support part of the probe.", "Signals corresponding to peptides with the same sequence but marked with differential mass labels are assigned to different original protein samples.", "The auxiliary chemical and/or enzymatic modification can be used to introduce additional differential mass labels into the peptides.", "The reactive group on the chemical probe may be activated or modified by a bridging reagent prior to a reaction with mixtures of peptides.", "Such activation or modification provides for a greater flexibility in design of the chemical probe since the same core structure of a chemical probe may be tuned to increase reactivity and/or selectivity towards different functional groups in side chains and/or in termini of the peptides.", "After being cleaved from the solid support part of the chemical probe, the differentially labeled peptide mixtures are combined, subjected to multidimensional chromatographic separation, and analyzed by mass spectrometry methods.", "Mass spectrometry data is processed by special software, which allows for determination and tracing the composition and sequence of peptides in the mixture to identification of the original proteins and their quantification.", "This approach can be used for duplex or potentially multiplex protein expression profiling.", "The complexity of the sample is simplified by targeting peptides containing particular amino acids, which selected by a reaction with chemical probes.", "Novelties of this invention include: (i) design of solid phase-based differential mass labeling reagents for selective peptide modification; (ii) design of various kinds of differential mass unit; (iii) combination of differential mass probes with various bridge reagent to target certain amino acid specifically; (iv) multiplex analysis; (v) combination of proteolytic digestion and chemical and/or enzymatic modifications in side chains and/or in termini of proteins and peptides in order to achieve selective and the most favorable modifications for the next steps in the protocol; (vi) combination of differential chemical labeling with MudPIT, and possible all other protein/peptide separation or purification technologies if necessary.", "One embodiment of this invention provides reagents and procedures for quantification of protein expression using combination of selective differential peptides labeling, and LC MS/MS or LC-LC MS/MS.", "This invention overcomes the limitations inherent in traditional techniques.", "The basic approach described can be employed for quantitative analysis of protein expression in complex samples (such as cells, tissues, and fraction etc.", "), the detection and quantitation of specific proteins in complex samples, and quantitative measurement of specific enzymatic activities in complexed samples.", "1.4.8.2.Technical Description 1.Probe Design: The solid support part of the chemical probe may consist of any of the following materials or any combination of them: gel, glass beads, magnetic beads, polymers, silicon wafer, membrane, or resin.", "The spacer between the solid phase part and the cleavable unit of the chemical probe may be included for convenience and improved yields in synthetic preparation of the chemical probe.", "The spacer may consist of a chain of 2 to 8 atoms, which can be C, O, N, B, Si, S, P, Se .", ".", ".", ", covalently bound to each other.", "In order to satisfy the valence requirements, the atoms may carry hydrogen atoms, halogens, or one of the following groups containing up to 25 atoms: alkyl, hydroxy, alkoxy, amino, alkylamino .", ".", ".", "The spacer may contain cyclic moieties with or without heteroatoms and with or without substituents.", "The cleavable moiety provides means for selective detachment of the solid phase part of the chemical probe-from the differential mass label attached to peptide.", "It is designed such that it can be cleaved by treating the probe with a chemical reagent or any kind of electromagnetic irradiation, photochemically, enzymatically, or thermally.", "Differential mass labeling units differ in molecular mass, but do not differ in retention properties regarding the separation method used and in ionization and detection properties regarding the mass spectrometry methods used.", "These moieties differ either in their isotope composition (isotopic labels) or they differ structurally by a rather small fragment, which change does not alter the properties stated above (homologous labels).", "The isotopic labels can be presented by general formulae: ZA and ZB ZA and ZB=R-Z1-A1-Z2-A2-Z3-A3-Z4-A4- Z1, Z2, Z3, and Z4 independently of one another can be selected from 0, OC(O), OC(S), OC(O) 0, OC(O)NR, OC(S)NR, OSiRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si (RR1)O)n, SnRR1, Sn (RR1) O, BR (OR1), BRR1, B (OR)(OR1), OBR (OR1), OBRR1, OB (OR)(OR1) or Z1-Z4 may be absent; A1, A2, A3, and A4 independently of one another can be selected from (CRR1)n, in which some single C—C bonds may be replaced with double or triple bonds, in which case some groups R and R1 will be absent, o-arylene, m-arylene, p-arylene with up to 6 substituents, carbocyclic, bicyclic, or tricyclic fragments with up to 8 atoms in the cycle with or without heteroatoms (O, N, S) and with or without substituents, or A1-A4 may be absent; R, R1 independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4 is hydrogen, halogen, an alkyl, alkenyl, alkynyl, or aryl group; n in Z1-Z4 is independent of n in A1-A4 and is a whole number that can have value from 0 to 21.ZA has the same structure as ZB, but they have different isotope composition.", "For instance, if ZA contains x number of protons, ZB may contain y number of deuterons in the place of protons, and, correspondingly, x−y number of protons remaining; and/or if ZA contains x number of borons-10, ZB may contain y number of borons-11 in the place of borons-10, and, correspondingly, x−y number of borons-10 remaining; and/or if ZA contains x number of carbons-12, ZB may contain y number of carbons-13 in the place of carbons-12, and, correspondingly, x−y number of carbons-12 remaining; and/or if ZA contains x number of nitrogens-14, ZB may contain y number of nitrogens-15 in the place of nitrogens-14, and, correspondingly, x−y number of nitrogens-14 remaining; and/or if ZA contains x number of sulfurs-32, ZB may contain y number of sulfurs-34 in the place of sulfurs-32, and, correspondingly, x−y number of sulfurs-32 remaining; and so on for all elements which may be present and have different stable isotopes.", "x and y are whole numbers between 1 and 21 such that x is greater than y.", "An example of an isotopical label pairs/series: (CD2)n/(CH2)n, where n=0, 1, 2, .", ".", ".", ", 21; (delta mass=2n).", "The homologous reagents can be presented by general formulae: ZA and ZB where ZA and ZB=R-Z1-A1Z2-A2-Z3-A3-Z4-A4- Z1, Z2, Z3, and Z4 independently of one another can be selected from 0, OC(O), OC(S), OC(O)O, OC(O)NR, OC(S)NR, OSIRR1, S, SC(O), SC(S), SS, S(O), S(O2), NR, NRR1+, C(O), C(O)O, C(S), C(S)O, C(O)S, C(O)NR, C(S)NR, SiRR1, (Si(RR1)O)n, SnRR1, Sn(RR1)O, BR(OR1), BRR1, B(OR)(OR1), OBR(OR1), OBRR1, OB(OR)(OR1) or Z1-Z4 may be absent; A1, A2, A3, and A4 independently of one another can be selected from (CRR1)n, in which some single C—C bonds may be replaced with double or triple bonds, in which case some groups R and R1 will be absent, o-arylene, m-arylene, p-arylene with up to 6 substituents, carbocyclic, bicyclic, or tricyclic fragments with up to 8 atoms in the cycle with or without heteroatoms (O, N, S) and with or without substituents, or A1-A4 may be absent; R, R1 independently from other R and R1 in Z1-Z4 and independently from other R and R1 in A1-A4 is hydrogen, halogen, an alkyl, alkenyl, alkynyl, or aryl group; n in Z1-Z4 is independent of n in A1-A4 and is a whole number that can have value from 0 to 21.ZA has a similar structure to that of ZB, but ZA has x extra —CH2— fragment(s) in one or more A1-A4 fragments, and/or ZA has x extra —CF2— fragment(s) in one or more A1-A4 fragments; and/or if ZA contains x number of protons, ZB may contain y number of halogens in the place of protons, and, correspondingly, x−y number of protons remaining in one or more A1-A4 fragments; and/or ZA has x extra —O— fragment(s) in one or more A1-A4 fragments; and/or ZA has x extra —S— fragment(s) in one or more A1-A4 fragments; and/or if ZA contains x number of —O— fragment(s), ZB may contain y number of —S— fragment(s) in the place of —O— fragment(s), and, correspondingly, x−y number of fragment(s) remaining in one or more A1-A4 fragments; and so on.", "x and y are whole numbers between 1 and 21 such that x is greater than y.", "An examples of homologous label pairs/series: (CH2)n(CH2)n+m, where n=0, 1, 2, .", ".", ".", ", 21; m=1, 2, .", ".", ".", ", 21 (delta mass=14m).", "2.Bridging and Activating Reagents: We may Either Utilize Some Commercial Available Cross Linkers or Synthesized our Own: a. Reactive site 1: probe specific b. Reactive site 2: amino acid specific 3.Methods for Peptide/Protein Separation and Detection: On line 2 dimensional capillary LC ESI MS/MS (MuDPIT) as described in the global differential profiling disclosure, or I D LC ESI MS/MS, MALDI MS. 4.Sequence Analysis and Quantification: Peptides are quantified by measuring in the MS mode the relative signal intensities for pairs or series of peptide ions of identical sequence that are tagged differentially, which therefore differ in mass by the mass differential encoded within the differential labeling reagents.", "Peptide sequence information is automatically generated by selecting peptide ions of a particular mass-to-charge (m/z) ratio for collision-induced dissociation (CID) in the mass spectrometer operating in the tandem MS mode.", "(Link et al, Electrophoresis 18: 1314-34 (1997); Gygi et al Nature Biotechnol 17: 994-9) (1999); Gygi et al., cell Biol 19: 1720-30 (1999)).", "The resulting tandem mass spectra can be correlated to sequence databases to identify the protein from which the sequenced peptide originated.", "Currently commercial available softwares are Turbo SEQUEST by Thermofinigan, MassScot by Matrix Science, and Sonar MS/MS by Proteometrics.", "Special software development will be necessary for automated relative quantification.", "One suggested approach of practicing the invention: 1.Protein sample preparation, which may include protein denaturation, reduction, and proteolytic digestion 2.Treatment of the probe with a desired activating or bridging reagent 3.Treatment of the activated probe with a mixture of peptides 4.Wash off unbound peptides, which don't have the targeted amino acid 5.Combining modified differential labeled peptide mixture 6.Release peptides by cleaving the probe (steps 5 and 6 can be switched) 7.Removing solvent or desalting if necessary 8.Redisoviing peptide in LC loading buffer 9.LC ESI MS and MS/MS analysis MALDI MS and MS/MS analysis 10.Database searching and data analysis 1.5.Metabolomics and Lipidomics Additional holistic monitoring approaches, metabolomics and lipidomics, include profiling metabolite pools, carbohydrates, lipids, glycoproteins, and glycolipids Various chromatographic methods and other qualitative and/or quantitative methods could be utilized to characterize lipid profiles.", "In the area of metabolomics, methods that compare concentrations of metabolites/small molecules, using a variety of chemical analysis tools, e.g.", "mass spec, NMR, other spectroscopic techniques, biosensors could be utilized.", "For some specific method examples, see the following references: J. C. Lindon et al., Prog.", "NMR Spear., 29, 1 (1996) 1-3.C.", "Lindon et al., Drug.", "Met.", "Rev., 29, 705 (1997); B. Vogler et al., J Nat.", "Prod., 61, 175 (1998); and JA.", "Wolfender et al., Curr.", "Org.", "Chem.", "2, 575 (1998); J. K. Nicholson et al., Xenobiotica, 29, 1181(1999).", "1.6.Screening Tools 1.6.1.FACS Fluorescence activated cell sorting (FACS) methods are also a powerful tool for selection/screening.", "In some instances a fluorescent-molecule is made within a cell (e.g., green fluorescent protein).", "The cells producing the protein can simply be sorted by FACS.", "Gel microdrop technology allows screening of cells encapsulated in agarose microdrops (Weaver et al.", "Methods 2: 234-247 (1991)).", "In this technique products secreted by the cell (such as antibodies or antigens) are immobilized with the cell that generated them.", "Sorting and collection of the drops containing the desired product thus also collects the cells that made the product, and provides a ready source for the cloning of the genes encoding the desired functions.", "Desired products can be detected by incubating the encapsulated cells with fluorescent antibodies (Powell et al.", "Bio/Technology 8: 333-337 (1990)).", "FACS sorting can also be used by this technique to assay resistance to toxic compounds and antibiotics by selecting droplets that contain multiple cells (i.e., the product of continued division in the presence of a cytotoxic compound; Goguen et al.", "Nature 363: 189-190 (1995)).", "This method can select for any enzyme that can change the fluorescence of a substrate that can be immobilized in the agarose droplet.", "1.6.2.Reporter Molecule In some embodiments of the invention, screening can be accomplished by assaying reactivity with a reporter molecule reactive with a desired feature of, for example, a gene product.", "Thus, specific functionalities such as antigenic domains can be screened with antibodies specific for those determinants.", "1.6.3.Cell-Cell Indicator In other embodiments of the invention, screening is preferably done with a cell-cell indicator assay.", "In this assay format, separate library cells (Cell A, the cell being assayed) and reporter cells (Cell B, the assay cell) are used.", "Only one component of the system, the library cells, is allowed to evolve.", "The screening is generally carried out in a two-dimensional immobilized format, such as on plates.", "The products of the metabolic pathways encoded by these genes (in this case, usually secondary metabolites such as antibiotics, polyketides, carotenoids, etc.)", "diffuse out of the library cell to the reporter cell.", "The product of the library cell may affect the reporter cell in one of a number of ways.", "The assay system (indicator cell) can have a simple readout (e.g., green fluorescent protein, luciferase, beta-galactosidase) which is induced by the library cell product but which does not affect the library cell.", "In these examples the desired product can be detected by calorimetric changes in the reporter cells adjacent to the library cell.", "1.6.4.Feedback Mechanism In other embodiments, indicator cells can in turn produce something that modifies the growth rate of the library cells via a feedback mechanism.", "Growth rate feedback can detect and accumulate very small differences.", "For example, if the library and reporter cells are competing for nutrients, library cells producing compounds to inhibit the growth of the reporter cells will have more available nutrients, and thus will have more opportunity for growth.", "This is a useful screen for antibiotics or a library of polyketide synthesis gene clusters where each of the library cells is expressing and exporting a different polyketide gene product.", "1.6.5.Screening Secreted Molecules Another variation of this theme is that the reporter cell for an antibiotic selection can itself secrete a toxin or antibiotic that inhibits growth of the library cell.", "Production by the library cell of an antibiotic that is able to suppress growth of the reporter cell will thus allow uninhibited growth of the library cell.", "Conversely, if the library is being screened for production of a compound that stimulates the growth of the reporter cell (for example, in improving chemical syntheses, the library cell may supply nutrients such as amino acids to an auxotrophic reporter, or growth factors to a growth-factor-dependent reporter.", "The reporter cell in turn should produce a compound that stimulates the growth of the library cell.", "Interleukins, growth factors, and nutrients are possibilities.", "Further possibilities include competition based on ability to kill surrounding cells, positive feedback loops in which the desired product made by the evolved cell stimulates the indicator cell to produce a positive growth factor for cell A, thus indirectly selecting for increased product formation.", "In some embodiments of the invention it can be advantageous to use a different organism (or genetic background) for screening than the one that will be used in the final product.", "For example, markers can be added to DNA constructs used for recursive sequence recombination to make the microorganism dependent on the constructs during the improvement process, even though those markers may be undesirable in the final recombinant microorganism.", "Likewise, in some embodiments it is advantageous to use a different substrate for screening an evolved enzyme than the one that will be used in the final product.", "For example, Evnin et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 87: 6659-6663 (1990)) selected trypsin variants with altered substrate specificity by requiring that variant trypsin generate an essential amino acid for an arginine auxotroph by cleaving arginine beta-naphthylamide.", "This is thus a selection for arginine-specific trypsin, with the growth rate of the host being proportional to that of the enzyme activity.", "The pool of cells surviving screening and/or selection is enriched for recombinant genes conferring the desired phenotype (e.g.", "altered substrate specificity, altered biosynthetic ability, etc.).", "Further enrichment can be obtained, if desired, by performing a second round of screening and/or selection without generating additional diversity.", "The recombinant gene or pool of such genes surviving one round of screening/selection forms one or more of the substrates for a second round of recombination.", "Again, recombination can be performed in vivo or in vitro by any of the recursive sequence recombination formats described above.", "If recursive sequence recombination is performed in vitro, the recombinant gene or genes to form the substrate for recombination should be extracted from the cells in which screening/selection was performed.", "Optionally, a subsequence of such gene or genes can be excised for more targeted subsequent recombination.", "If the recombinant gene(s) are contained within episomes, their isolation presents no difficulties.", "If the recombinant genes are chromosomally integrated, they can be isolated by amplification primed from known sequences flanking the regions in which recombination has occurred.", "Alternatively, whole genomic DNA can be isolated, optionally amplified, and used as the substrate for recombination.", "Small samples of genomic DNA can be amplified by whole genome amplification with degenerate primers (Barrett et al.", "Nucleic Acids Research 23: 3488-3492 (1995)).", "These primers result in a large amount of random 3′ ends, which can undergo homologous recombination when reintroduced into cells.", "If the second round of recombination is to be performed in vivo, as is often the case, it can be performed in the cell surviving screening/selection, or the recombinant genes can be transferred to another cell type (e.g., a cell is type having a high frequency of mutation and/or recombination).", "In this situation, recombination can be effected by introducing additional DNA segment(s) into cells bearing the recombinant genes.", "In other methods, the cells can be induced to exchange genetic information with each other by, for example, electroporation.", "In some methods, the second round of recombination is performed by dividing a pool of cells surviving screening/selection in the first round into two subpopulations.", "DNA from one subpopulation is isolated and transfected into the other population, where the recombinant gene(s) from the two subpopulations recombine to form a further library of recombinant genes.", "In these methods, it is not necessary to isolate particular genes from the first subpopulation or to take steps to avoid random shearing of DNA during extraction.", "Rather, the whole genome of DNA sheared or otherwise cleaved into manageable sized fragments is transfected into the second subpopulation.", "This approach is particularly useful when several genes are being evolved simultaneously and/or the location and identity of such genes within chromosome are not known.", "The second round of recombination is sometimes performed exclusively among the recombinant molecules surviving selection.", "However, in other embodiments, additional substrates can be introduced.", "The additional substrates can be of the same form as the substrates used in the first round of recombination, i.e., additional natural or induced mutants of the gene or cluster of genes, forming the substrates for the first round.", "Alternatively, the additional substrate(s) in the second round of recombination can be exactly the same as the substrate(s) in the first round of replication.", "After the second round of recombination, recombinant genes conferring the desired phenotype are again selected.", "The selection process proceeds essentially as before.", "If a suicide vector bearing a selective marker was used in the first round of selection, the same vector can be used again.", "Again, a cell or pool of cells surviving selection is selected.", "If a pool of cells, the cells can be subject to further enrichment.", "1.4.Screening for Various Potential Applications 1.4.1 Novel Drugs: Identifying Targets The invention relates to procedures that can be applied to identifying compounds that bind to and modulate the function of target components of a cell whose function is known or unknown, and cell components that are not amenable to other screening methods.", "The invention relates to generating and/or identifying a compound that binds to and modulates (inhibits or enhances) the function of a component of a cell, thereby producing a phenotypic effect in the cell.", "Such a screen may involve identifying a biomolecule that 1) binds to, in vitro, a component of a cell that has been isolated from other constituents of the cell and that 2) causes, in vivo, as seen in an assay upon intracellular expression of the biomolecule, a phenotypic effect in the cell which is the usual producer and host of the target cell component.", "In an assay demonstrating characteristic 2) above, intracellular production of the biomolecule can be in cells grown in culture or in cells introduced into an animal.", "Further methods within these procedures are those methods comprising an assay for a phenotypic effect in the cell upon intracellular production of the biomolecule, either in cells in culture or in cells that have been introduced into one or more animals, and an assay to identify one or more compounds that behave as competitors of the biomolecule in an assay of binding to the target cell component.", "The target cell component in this embodiment and in other embodiments not limited to pathogens can be one that is found in mammalian cells, especially cells of a type found to cause or contribute to disease or the symptoms of disease (e.g., cells of tumors or cells of other types of hyperproliferative disorders).", "1.7.1.Process for Identifying One or More Compounds that Produce a Phenotypic Effect on a Cell One procedure envisioned in the invention is a process for identifying one or more compounds that produce a phenotypic effect on a cell.", "The process is at the same time a method for target validation.", "The process is characterized by identifying a biomolecule which binds an isolated target cell component, constructing cells comprising the target cell component and further comprising a gene encoding the biomolecular binder which can be expressed to produce the biomolecular binder, testing the constructed cells for their ability to produce, upon expression of the gene encoding the biomolecular binder, a phenotypic effect in the cells (e.g., inhibition of growth), wherein the test of the constructed cells can be a test of the cells in culture or a test of the cells after introducing them into host animals, or both, and further, identifying, for a biomolecular binder that caused the phenotypic effect, one or more compounds that compete with the biomolecular binder for binding to the target cell component.", "A test of the constructed cells after introducing them into host animals is especially well-suited to assessing whether a biomolecular binder can produce a particular phenotype by the expression (regulatable by the researcher) of a gene encoding the biomolecular binder.", "In this method, cells are constructed which have a gene encoding the biomolecular binder, and wherein the biomolecular binder can be produced by regulation of expression of the gene.", "The constructed cells are introduced into a set of animals.", "Expression of the gene encoding the biomolecular binder is regulated in one group of the animals (test animals) such that the biomolecular binder is produced.", "In another group of animals, the gene encoding the biomolecular binder is regulated such that the biomolecular binder is not produced (control animals).", "The cells in the two groups of animals are monitored for a phenotypic change (for example, a change in growth rate).", "If the phenotypic change is observed in cells in the test animals and not in the cells in the control animals, or to a lesser extent in the control animals, then the biomolecular binder has been proven to be effective in binding to its target cell component under in vivo conditions.", "A further embodiment of the invention is a method for determining whether a target cell component of a particular cell type (a “first cell”) is essential to producing a phenotypic effect on the first cell, the method having the steps: isolating the target component of the first cell; identifying a biomolecular binder of the isolated target component of the first cell; constructing a second type of cells (“second cell”) comprising the target component and a regulable, exogenous gene encoding the biomolecular binder; and testing the second cell in culture for an altered phenotypic effect, upon production of the biomolecular binder in the second cell; whereby, if the second cell shows the altered phenotypic effect upon production of the biomolecular binder, then the target component of the first cell is essential to producing the phenotypic effect on the first cell.", "The target cell component in this embodiment and in other embodiments not limited to pathogens can be one that is found in mammalian cells, especially cells of a type found to cause or contribute to disease or the symptoms of disease (e.g., cells of tumors or cells of other types of hyperproliferative disorders).", "1.7.3.Identifying a Biomolecular Inhibitor of Growth of Pathogen Cells One embodiment of the invention is a method for identifying a biomolecular inhibitor of growth of pathogen cells by using cell culture techniques, comprising contacting one or more types of biomolecules with isolated target cell component of the pathogen, applying a means of detecting bound complexes of biomolecules and target cell component, whereby, if the bound complexes are detected, one or more types of biomolecules have been identified as a biomolecular binder of the target cell component, constructing a pathogen strain having a regulatable gene encoding the biomolecular binder, regulating expression of the gene encoding the biomolecular binder to express the gene; and monitoring growth of the pathogen cells in culture relative to suitable control cells, whereby, if growth of the pathogen cells is decreased compared to growth of suitable control cells, then the biomolecule is a biomolecular inhibitor of growth of the pathogen cells.", "1.7.4.Identifying Compounds that Inhibit Infection of a Mammal by a Pathogen A further embodiment of the invention is a method, employing an animal test, for identifying one or more compounds that inhibit infection of a mammal by a pathogen by binding to a target cell component, comprising constructing a pathogen comprising a regulable gene encoding a biomolecule which binds to the target cell component, infecting test animals with the pathogen, regulating expression of the regulable gene to produce the biomolecule, monitoring the test animals and suitable control animals for signs of infection, wherein observing fewer or less severe signs of infection in the test animals than in suitable control animals indicates that the biomolecule is a biomolecular inhibitor of infection, and identifying one or more compounds that compete with the biomolecular inhibitor of growth for binding to the target cell component (as by employing a competitive binding assay), then the compound inhibits infection of a mammal by a pathogen by binding to a target.", "The competitive binding assay to identify binding analogs of biomolecular binders, which have been proven to bind to their targets in an intracellular test of binding, can be applied to any target for which a biomolecular binder has been identified, including targets whose function is unknown or targets for which other types of assays are not easily developed and performed.", "Therefore, the method of the invention offers the advantage of decreasing assay development time when using a gene product of known function as a target cell component and the advantage of bypassing the major hurdle of gene function identification when using a gene product of unknown function as a target cell component.", "Other embodiments of the invention are cells comprising a biomolecule and a target cell component, wherein the biomolecule is produced by expression of a regulable gene, and wherein the biomolecule modulates function of the target cell component, thereby causing a phenotypic change in the cells.", "Yet other embodiments are cells comprising a biomolecule and a target cell component, wherein the biomolecule is a biomolecular binder of the target cell component, and is encoded by a regulatable gene.", "The cells can include mammalian cells or cells of a pathogen, for instance, and the phenotypic change can be a change in growth rate.", "The pathogen can be a species of bacteria, yeast, fungus, or parasite, for example.", "1.7.5.Intracellular Validation of a Biomolecule Described herein are methods that result in the identification of compounds that cause a phenotypic effect on a cell.", "The general steps described herein to find a compound for drug development can be thought of as these: (1) identifying a biomolecule that can bind to an isolated target cell component in vitro, (2) confirming that the biomolecule, when produced in cells with the target cell component, can cause a desired phenotypic effect and (3) identifying, by an in vitro screening method, for example, compounds that compete with the biomolecule for binding to the target cell component.", "Central to these methods is general step (2) above, intracellular validation of a biomolecule comprising one or more steps that determine whether a biomolecule can cause a phenotypic effect on a cell, when the biomolecule is produced by the expression (which can be regulatable) of a gene in the cell.", "As used in general step (2), a biomolecule is a gene product (e.g., polypeptide, RNA, peptide or RNA oligonucleotide) of an exogenous gene—a gene which has been introduced in the course of construction of the cell.", "Biomolecules that bind to and alter the function of a candidate target are identified by various in vitro methods.", "Upon production of the biomolecule within a cell either in vitro or within an animal model system, the biomolecule binds to a specific site on the target, alters its intracellular function, and hence produces a phenotypic change (e.g.", "cessation of growth, cell death).", "When the biomolecule is produced in engineered pathogen cells in an animal model of infection, cessation of growth or death of the engineered pathogen cells leads to the clearing of infection and animal survival, demonstrating the importance of the target in infection and thereby validating the target.", "A further embodiment of this invention provides for identifying a biomolecule that produces a phenotypic effect on a cell (wherein the cell can be, for instance, a pathogen cell or a mammalian cell) and (2) simultaneous intracellular target validation (see reference: patents??).", "1.7.6.Methods for Identifying Compounds that Inhibit the Growth of Cells Having a Target Cell Component The invention includes methods for identifying compounds that inhibit the growth of cells having a target cell component.", "The target cell component can first be identified as essential to the growth of the cells in culture and/or under conditions in which it is desired that the growth of the cells be inhibited.", "These methods can be applied, for example, to various types of cells that undergo abnormal or undesirable proliferation, including cells of neoplasms (tumors or growths, either benign or malignant) which, as known in the art, can originate from a variety of different cell types.", "Such cells can be referred to, for example, as being from adenomas, carcinomas, lymphomas or leukemias.", "The method can also be applied to cells that proliferate abnormally in certain other diseases, such as arthritis, psoriasis or autoimmune diseases.", "If intracellular expression of the biomolecular binder inhibits the function of a target essential for growth (presumably by binding to the target at a biologically relevant site) cells monitored in step (2) will exhibit a slow growth or no growth phenotype.", "Targets found to be essential for growth by these methods are validated starting points for drug discovery, and can be incorporated into assays to identify more stable compounds that bind to the same site on the target as the biomolecule.", "Where the cells are pathogen cells and the desired phenotypic change to be monitored is inhibition of growth, the invention provides a procedure to examine the activity of target (pathogen) cell components in an animal infection model.", "1.7.7.Study as a Target Cell Component a Gene Product of a Particular Cell Type In the course of this method, it may be decided to study as a target cell component a gene product of a particular cell type (e.g., a type of pathogenic bacteria), wherein the target cell component is already known as being encoded by a characterized gene, as a potential target for a modulator to be identified.", "In this case, the target cell component can be isolated directly from the cell type of interest, assuming suitable culture methods are available to grow a sufficient number of cells, using methods appropriate to the type of cell component to be isolated (e.g., protein purification methods such as differential precipitation, ion exchange chromatography, gel chromatography, affinity chromatography, HPLC.", "1.7.8.Target Cell Component can be Produced Recombinantly Alternatively, the target cell component can be produced recombinantly, which requires that the gene encoding the target cell component be isolated from the cell type of interest.", "This can be done by any number of methods, for example known methods such as PCR, using template DNA isolated from the pathogen or a DNA library produced from the pathogen DNA, and using primers based on known sequences or combinations of known and unknown sequences within or external to the chosen gene.", "See, for example, methods described in “The Polymerase Chain Reaction,” Chapter 15 of Current Protocols in Molecular Biology, (Ausubel, F. M. et al., eds), John Wiley & Sons, New York, 1998.Other methods include cloning a gene from a DNA library (e.g., a cDNA library from a eucaryotic pathogen) into a vector (e.g., plasmid, phage, phagemid, virus, etc.)", "and applying a means of selection or screening, to clones resulting from a transformation of vectors (including a population of vectors now having inserted genes) into appropriate host cells.", "The screening method can take advantage of properties given to the host cells by the expression of the inserted chosen gene (e.g., detection of the gene product by antibodies directed against it, detection of an enzymatic activity of the gene product), or can detect the presence of the gene itself (for instance, by methods employing nucleic acid hybridization).", "For methods of cloning genes in E. coli, which also may be applicable to cloning in other bacterial species, see, for example, “Escherichia coli, Plasmids and Bacteriophages,” Chapter I of Current Protocols in Molecular Biology, (Ausubel, F. M. et al., eds), John Wiley & Sons, New York, 1998.For methods applicable to cloning genes of eukaryotic origin, see Chapter 5 (“Construction of Recombinant DNA Libraries”), Chapter 9 (“Introduction of DNA Into Mammalian Cells”) and Chapter 6 (“Screening of Recombinant DNA Libraries”) of Current Protocols in Molecular Biology, (Ausubel, F. M. et al., eds), John Wiley & Sons, New York, 1998.Target proteins can be expressed with E. coli or other prokaryotic gene expression systems, or in eukaryotic gene expression systems.", "Since many eukaryotic proteins carry unique modifications that are required for their activities, e.g.", "glycosylation and methylation, protein expression can in some cases be better carried out in eukaryotic systems, such as yeast, insect, or mammalian cells that can perform these modifications.", "Examples of these expression systems have been reviewed in the following literature: Methods in Enzymology, Volume 185, eds D. V. Goeddel, Academic Press, San Diego, 1990; Geisse et al, Protein Expression and Purification 8: 271-282, 1996; Simonsen and McGrogan, Biologicals 22: 85-94; Jones and Morikawa, Current Opinions in Biotechnologies 7: 512-516, 1996; Possee, Current Opinions in Biotechnologies 8: 569-572.Where a gene encoding a chosen target cell component has not been isolated previously, but is thought to exist because homologs of the gene product are known in other species, the gene can be identified and cloned by a method such as that used in Shiba et al., U.S. Pat.", "No.", "5,759,833, Shiba et al., U.S. Pat.", "No.", "5,629,188, Martinis et al., U.S. Pat.", "No.", "5,656,470 and Sassanfar et al., U.S. Pat.", "No.", "5,756,327.The teachings of these four patents are incorporated herein by reference in their entirety.", "1.7.9.Method Should be Used with Target Cell Components which have not been Previously Isolated or Characterized and whose Functions are Unknown It is an advantage of the target validation method that it can be used with target cell components which have not been previously isolated or characterized and whose functions are unknown.", "In this case, a segment of DNA containing an open reading frame (ORF; a cDNA can also be used, as appropriate to a eukaryotic cell) which has been isolated from a cell of a type that is to be an object of drug action (e.g., tumor cell, pathogen cell) can be cloned into a vector, and the target gene product of the ORF can be produced in host cells harboring the vector.", "The gene product can be purified and further studied in a manner similar to that of a gene product that has been previously isolated and characterized.", "In some cases, the open reading frame (in some cases, cDNA) can be isolated from a source of DNA of the cells of interest (genomic DNA or a library, as appropriate), and inserted into a fusion protein or fusion polypeptide construct.", "This construct can be a vector comprising a nucleic acid sequence which provides a control region (e.g., promoter, ribosome binding site) and a region which encodes a peptide or polypeptide portion of the fusion polypeptide wherein the polypeptide encoded by the fusion vector endows the fusion polypeptide with one or more properties that allow for the purification of the fusion polypeptide.", "For example, the vector can be one from the pGEX series of plasmids (Pharmacia) designed to produce fusions with glutathione S-transferase.", "1.7.10.Host Cells The isolated DNA having an open reading frame, whether encoding a known or an as yet unidentified gene product, when inserted into an expression construct, can be expressed to produce the target cell component in host cells.", "Host cells can be, for example, Gram-negative or Gram-positive bacterial cells such as Escherichia coli or Bacillus subtilis, respectively, or yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Pichia pastoris.", "It is preferable that the target cell component to be used in target validation studies be produced in a host that is genetically related to the pathogen from which the gene encoding it was isolated.", "For example, for a Gram-negative bacterial pathogen, an E. coli host is preferred over a Pichia pastoris host.", "The target cell component so produced can then be isolated from the host cells.", "Many protein purification methods are known that separate proteins on the basis of, for instance, size, charge, or affinity for a binding partner (e.g., for an enzyme, a binding partner can be a substrate or substrate analog), and these methods can be combined in a sequence of steps by persons of skill in the art to produce an effective purification scheme.", "For methods to manipulate RNA, see, for example, Chapter 4 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds), John Wiley & Sons, New York, 1998.An isolated cell component or a fusion protein comprising the cell component can be used in a test to identify one or more biomolecular binders of the isolated product (general step (1)).", "A biomolecular binder of a target cell component can be identified by in vitro assays that test for the formation of complexes of target and biomolecular binder noncovalently, bound to each other.", "For example, the isolated target can be contacted with one or more types of biomolecules under conditions conducive to binding, the unbound biomolecules can be removed from the targets, and a means of detecting bound complexes of biomolecules and targets can be applied.", "The detection of the bound complexes can be facilitated by having either the potential biomolecular binders or the target labeled or tagged with an adduct that allows detection or separation (e.g., radioactive isotope or fluorescent label; streptavidin, avidin or biotin affinity label).", "Alternatively, both the potential biomolecular binders and the target can be differentially labeled.", "For examples of such methods see, e.g., WO 98/19162.1.7.11.Biomolecules to be Tested and Means for Detection The biomolecules to be tested for binding to a target can be from a library of candidate biomolecular binders, (e.g., a peptide or oligonucleotide library).", "For example, a peptide library can be displayed on the coat protein of a phage (see, for examples of the use of genetic packages such as phage display libraries, Koivunen, E. et al., J. Biol.", "Chem.", "268: 20205-20210 (1993)).", "The biomolecules can be detected by means of a chemical tag or label attached to or integrated into the biomolecules before they are screened for binding properties.", "For example, the label can be a radioisotope, a biotin tag, or a fluorescent label.", "Those molecules that are found to bind to the target molecule can be called biomolecular binders.", "1.7.12.Fusion Proteins An isolated target cell component, an antigenically similar portion thereof, or a suitable fusion protein comprising all of or a portion of or the entire target can be used in a method to select and identify biomolecules which bind specifically to the target.", "Where the target cell component comprises a protein, fusion proteins comprising all of, or a portion of, the target linked to a second moiety not occurring in the target as found in nature, can be prepared for use in another embodiment of the method.", "Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope).", "The fusion proteins can be produced by the insertion of a gene encoding a target or a suitable portion of such gene into a suitable expression vector, which encodes an affinity ligand (e.g., pGEX4T-2 and pET-15b, encoding glutathione S— transferase and His-Tag affinity ligands, respectively).", "The expression vector can be introduced into a suitable host cell for expression.", "Host cells are lysed and the lysate, containing fusion protein, can be bound to a suitable affinity matrix by contacting the lysate with an affinity matrix under conditions sufficient for binding of the affinity ligand portion of the fusion protein to the affinity matrix.", "1.7.12.1.Fusion Protein can be Immobilized In one embodiment, the fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more candidate biomolecules (e.g., a mixture of peptides) to be tested as biomolecular binders, under conditions suitable for binding of the biomolecules to the target portion of the bound fusion protein.", "Next, the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound biomolecules and non-specifically bound biomolecules.", "Biomolecules which remain bound can be released by contacting the affinity matrix with fusion protein bound thereto with a suitable elution buffer.", "Wash buffer can be formulated to permit binding of the fusion protein to the affinity matrix, without significantly disrupting binding of specifically bound biomolecules.", "In this aspect, elution buffer can be formulated to permit retention of the fusion protein by the affinity matrix, but can be formulated to interfere with binding of the test biomolecule(s) to the target portion of the fusion protein.", "For example, a change in the ionic strength or pH of the elution buffer can lead to release of biomolecules, or the elution buffer can comprise a release component or components designed to disrupt binding of biomolecules to the target portion of the fusion protein.", "Immobilization can be performed prior to, simultaneous with, or after contacting, the fusion protein with biomolecule, as appropriate.", "Various permutations of the method are possible, depending upon factors such as the biomolecules tested, the affinity matrix-ligand pair selected, and elution buffer formulation.", "For example, after the wash step, fusion protein with biomolecules bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer, such as glutathione for a GST fusion).", "Where the fusion protein comprises a cleavable linker, such as a thrombin cleavage site, cleavage from the affinity ligand can release a portion of the fusion with the biomolecules bound thereto.", "Bound biomolecule can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.", "1.7.12.Various Methods to Identify Biomolecular Binders One or more candidate biomolecular binders can be tested simultaneously.", "Where a mixture of biomolecules is tested, the biomolecules selected by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography).", "Large libraries of biomolecules (e.g., peptides, RNA oligonucleotides) produced by combinatorial chemical synthesis or other methods can be tested (see e. a., Ohimeyer, M. H. J. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90: 10922-10926 (1993) and DeWitt, S. H. et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 90: 6909-6913 (1993), relating to tagged compounds; see also Rutter, W. J. et al.", "U.S. Pat.", "No.", "5,010,175; Huebner, V. D. et al., U.S. Pat.", "No.", "5,182,366; and Geysen, H. M., U.S. Pat.", "No.", "4,833,092).", "Random sequence RNA libraries (see Ellington, A. D. et al., Nature 346: 818-822 (1990); Bock, L. C. et al., Nature 355: 584-566 (1992); and Szostak, J. W., Trends in Biochem.", "Sci.", "17: 89-93 (March, 1992)) can also be screened according to the present method to select RNA molecules which bind to a target.", "Where biomolecules selected from a combinatorial library by the present method carry unique tags, identification of individual biomolecules by chromatographic methods is possible.", "Where biomolecules do not carry tags, chromatographic separation, followed by mass spectrometry to ascertain structure, can be used to identify individual biomolecules selected by the method, for example.", "Other methods to identify biomolecular binders of a target cell component can be used.", "For example, the two-hybrid system or interaction trap is an in vivo system that can be used to identify polypeptides, peptides or proteins (candidate biomolecular binders) that bind to a target protein.", "In this system, both candidate biomolecular binders and target cell component proteins are produced as fusion proteins.", "The two-hybrid system and variations on it have been described (U.S. Pat.", "No.", "5,283,173 and U.S. Pat.", "No.", "5,468,614; Golemis, E. A. et al., pages 20.1.1-20.1.35 In Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., John Wiley and Sons, containing supplements up through Supplement 40, 1997; two-hybrid systems available from Clontech, Palo Alto, Calif.).", "Once one or more biomolecular binders of a cell component have been identified, further steps can be combined with those taken to identify the biomolecular binder, to identify those biomolecular binders that produce a phenotypic effect on a cell (where “a cell” can mean cells of a cell strain or cell line).", "Thus, a method for identifying a biomolecule that produces a phenotypic effect on a first cell can comprise the steps of identifying a biomolecular binder of an isolated target cell component of the first cell, constructing a second cell comprising the target cell component and a regulable exogenous gene encoding the biomolecular binder, and testing the second cell for the phenotypic effect, upon production of the biomolecular binder in the second cell, where the second cell can be maintained in culture or introduced into an experimental animal.", "If the second cell shows the phenotypic effect upon intracellular production of the biomolecular binder, then a biomolecule that produces a phenotypic effect on the first cell has been identified.", "Testing the second cell is general step (2) of the invention, as the three general steps were outlined above.", "1.7.13.Host Cells: Engineered to Control Expression Host cells (also, “second cells” in the terminology used above) of the cell type (e.g., species of pathogenic bacteria) the target was isolated from (or the gene encoding the target was originally isolated from, if the target is produced by recombinant methods), can be engineered to harbor a gene that can regulably express the biomolecular binder (e.g., under an inducible or repressible promoter).", "The ability to regulate the expression of the biomolecular binder is desirable because constitutive expression of the biomolecular binder could be lethal to the cell.", "Therefore, inducible or regulated expression gives the researcher the ability to control if and when the biomolecular binder is expressed.", "The gene expressing the biomolecular binder can be present in one or more copies, either on an extra chromosomal structure, such as on a single or multicopy plasmid, or integrated into the host cell genome.", "Plasmids that provide an inducible gene expression system in pathogenic organisms can be used.", "For example, plasmids allowing tetracycline-inducible expression of a gene in Staphylococcus aureus have been developed.", "1.7.14.Genes for Expression For intracellular expression of a biomolecule to be tested for its phenotypic effect in a eukaryotic cell (e.g., mammalian cell), the genes for expression can be carried on plasmid-based or virus-based vectors, or on a linear piece of DNA or RNA.", "For examples of expression vectors, see Hosfield and Lu, Biotechniques: 306-309, 1998; Stephens and Cockett, Nucleic Acid Research 17: 7110, 1989; Wohigemuth et al, Gene Therapy, 3: 503-512, 1996; Ramirez-Solis et al, Gene 87: 291-294, 1990, Dirks et al, Gene 149: 387-388, 1994; Chenaalvala et al.", "Current Opinion in Biotechnologies 2: 718-722, 1991; Methods in Enzymology, Volume 185, (D. V. Goeddel, ed.)", "Academic Press, San Diego, 1990.The genetic material can be introduced into cells using a variety of techniques, including whole cell or protoplast transformation, electroporation, calcium phosphate-DNA precipitation or DEAE-Dextran transfection, liposome mediated DNA or RNA transfer, or transduction with recombinant viral or retroviral vectors.", "Expression of the gene can be constitutive (e.g., ADHI promoter for expression in S. cerevisiae (Bennetzen, J. L. and Hall, B. D., J. Biol.", "Chem 257: 3026-3031 (1982)), or CMV immediate early promoter and RSV LTR for mammalian expression) or inducible, as the inducible GAL I promoter in yeast (Davis, L. I. and Fink, G. R., Cell 61: 965-978 (1990)).", "A variety of inducible systems can be utilized, for example, E. coli Lac repressor/operator system and Tn 10 Tet repressor/operator systems have been engineered to govern regulated expression in organisms from bacterial to mammalian cells.", "Regulated gene expression can also be achieved by activation.", "For example, gene expression governed by HIV LTR can be activated by HIV or SIV Tat proteins in human cells; GAL4 promoter can be activated by galactose in a nonglucose-containing medium.", "The location of the biomolecule binder genes can be extra chromosomal or chromosomally integrated.", "The chromosome integration can be mediated through homologous or nonhomologous recombinations.", "For proper localization in the cells, it may be desirable to tag the biomolecule binders with certain peptide signal sequences (for example, nuclear localization signal (NLS) sequences, mitochondria localization sequences).", "Secretion sequences have been well documented in the art.", "1.7.15.Fused Biomolecular Binders For presentation of the biomolecular binders in the intracellular system, they can be fused N-terminally, C-terminally, or internally in a carrier protein (if the biomolecular binder is a peptide), and can be fused (5′, 3′ or internally) in a carrier RNA or DNA molecule (if the biomolecular binder is a nucleic acid).", "The biomolecular binder can be presented with a protein or nucleic acid structural scaffold.", "Certain linkages (e.g., a 4-glycine linker for a peptide or a stretch of A's for an RNA can be inserted between the biomolecular binder and the carrier proteins or nucleic acids.", "In such engineered cells, the effect of this biomolecular binder on the phenotype of the cells can be tested, as a manifestation of the binding (implying binding to a functionally relevant site, thus, an activator, or more likely, an inhibitory) effect of the biomolecular binder on the target used in an in vitro binding assay as described above.", "An intracellular test can not only determine which biomolecular binders have a phenotypic effect on the cells, but at the same time can assess whether the target in the cells is essential for maintaining the normal phenotype of the cells.", "For example, a culture of the engineered cells expressing a biomolecular binder can be divided into two aliquots.", "The first aliquot (“test” cells) can be treated in a suitable manner to regulate (e.g., induce or release repression of, as appropriate) the gene encoding the biomolecular binder, such that the biomolecular binder is produced in the cells.", "The second aliquot (“control” cells) can be left untreated so that the biomolecular binder is not produced in the cells.", "In a variation of this method of testing the effect of a biomolecular binder on the phenotype of the cells, a different strain of cells, not having a gene that can express the biomolecular binder, can be used as control cells.", "The phenotype of the cells in each culture (“test” and “control” cells grown under the same conditions, other than the expression of the biomolecular binder), can then be monitored by a suitable means (e.g., enzymatic activity, monitoring, a product of a biosynthetic pathway, antibody to test for presence of cell surface antigen, etc.).", "Where the change in phenotype is a change in growth rate, the growth of the cells in each culture (“test” and “control” cells grown under the same conditions, other than the expression of the biomolecular binder), can be monitored by a suitable means (e.g., turbidity of liquid cultures, cell count, etc).", "If the extent of growth, or rate of growth of the test cells is less than the extent of growth or rate of growth of the control cells, then the biomolecular binder can be concluded to be an inhibitor of the growth of the cells, or a biomolecular inhibitor.", "If the phenotype of the test cells is altered relative to that of the control cells, then the biomolecular binder can be concluded to be one that causes a phenotypic effect.", "In an optional additional test, isolated target cell component having a known function (e.g., an enzyme activity) can be tested for modulation of this known function in the presence of biomolecular binder under conditions conducive to binding of the biomolecular binder to the target cell component.", "Positive results in these tests should encourage the investigator to continue in the drug discovery process with efforts to find a more stable compound (than a peptide, polypeptide or RNA biomolecule) that mimics the binding properties of the biomolecular binder on the tested target cell component.", "1.7.16.Engineering Strain of Cells A further test can, again, employ an engineered strain of cells that comprise both the target cell component and one or more genes encoding a biomolecule tested to be a biomolecular binder of the target cell component.", "The cells of the cell strain can be tested in animals to see if regulable expression of the biomolecular binder in the engineered cells produces an observable or testable change in phenotype of the cells.", "Both the “in culture” test for the effect of intracellular expression of the biomolecular binder and the “in animal” test (described below) for the effect of intracellular expression of the biomolecular binder can be applied not only towards drug discovery in the categories of antimicrobials and anticancer agents, but also towards the discovery of therapeutic agents to treat inflammatory diseases, cardiovascular diseases, diseases associated with metabolic pathways, and diseases associated with the central nervous system, for example.", "Where the engineered strain of cells is a strain of pathogen cells or tumor cells, the object of the test is to see whether production of the biomolecular binder in the engineered strain inhibits growth of these cells after their introduction into an animal by the engineered pathogen.", "Such a test can not only determine which biomolecular binders are inhibitors of growth of the cells, but at the same time can assess whether the target in the cells is essential for maintaining growth of the cells (infection, for a pathogenic organism) in a host mammal.", "Suitable animals for such an experiment are, for example, mammals such as mice, rats, rabbits, guinea pigs, dogs, pigs, and the like.", "Small mammals are preferred for reasons of convenience.", "The engineered cells are introduced into one or more animals (“test” animals) and into one or more animals in a separate group (“control” animals) by a route appropriate to cause symptoms of systemic or local growth of the engineered cells.", "The route of introduction may be, for example, by oral feeding, by inhalation, by subdermal, intramuscular, intravenous, or intraperitoneal injection as appropriate to the desired result.", "After the cell strain has been introduced into the test and control animals, expression of the gene encoding the biomolecular binder is regulated to allow production of the biomolecular binder in the engineered pathogen cells.", "This can be achieved, for instance, by administering to the test animals a treatment appropriate to the regulation system built into the cells, to cause the gene encoding the biomolecular binder to be expressed.", "The same treatment is not administered to the control animals, but the conditions under which they are maintained are otherwise identical to those of the test animals.", "The treatment to express the gene encoding the biomolecular binder can be the administration of an inducer substance (where expression of the biomolecular binder or gene is under the control of an inducible promoter) or the functional removal of a repressor substance (where expression of the biomolecular binder gene is under the control of a repressible promoter).", "After such treatment, the test and control animals can be monitored for a phenotypic effect in the introduced cells.", "Where the introduced cells are constructed pathogen cells, the animals can be monitored for signs of infection (as the simplest endpoint, death of the animal, but also e.g., lethargy, lack of grooming behavior, hunched posture, not eating, diarrhea or other discharges; bacterial titer in samples of blood or other cultured fluids or tissues).", "In the case of testing engineered tumor cells, the test and control animals can be monitored for the development of tumors or for other indicators of the proliferation of the introduced engineered cells.", "If the test animals are observed to exhibit less growth of the introduced cells than the control animals, then the biomolecule can be also called a biomolecular inhibitor of growth, or biomolecular inhibitor of infection, as appropriate, as it can be concluded that the expression in vivo of the biomolecular inhibitor is the cause of the relative reduction in growth of the introduced cells in the test animals.", "1.7.17.In Vitro Assays Further steps of the procedure involve in vitro assays to identify one or more compounds that have binding and activating or inhibitory properties that are similar to those of the biomolecules which have been found to have a phenotypic effect, such as inhibition of growth.", "That is, compounds that compete for binding to a target cell component with the biomolecule would then be structural analogs of the biomolecules.", "Assays to identify such compounds can take advantage of known methods to identify competing molecules in a binding assay.", "These steps comprise general step (3) of the method.", "In one method to identify such compounds, a biomolecular inhibitor (or activator) can be contacted with the isolated target-cell component to allow binding, one or more compounds can be added to the milieu comprising the biomolecular inhibitor and the cell component under conditions that allow interaction and binding between the cell component and the biomolecular inhibitor, and any biomolecular inhibitor that is released from the cell component can be detected.", "1.7.18.Fluorescence One suitable system that allows the detection of released biomolecular inhibitor (or activator) is one in which fluorescence polarization of molecules in the milieu can be measured.", "The biomolecular inhibitor can have bound to it a fluorescent tag or label such as fluorescein or fluorescein attached to a linker.", "Assays for inhibition of the binding of the biomolecular inhibitor to the cell component can be done in microtiter plates to conveniently test a set of compounds at the same time.", "In such assays, a majority of the fluorescently labeled biomolecular inhibitor must bind to the protein in the absence of competitor compound to allow for the detection of small changes in the bound versus free probe population when a compound which is a competitor with a biomolecular inhibitor is added (B.", "A. Lynch, et al., Analytical Biochemistry 247: 77-82 (1997)).", "If a compound competes with the biomolecular inhibitor for a binding site on the target cell component, then fluorescently labeled biomolecular inhibitor is released from the target cell component, lowering the polarization measured in the milieu.", "1.7.19.Radioactive Isotope In a further method for identifying one or more compounds that compete with a biomolecular inhibitor (or activator) for a binding site on a target cell component, the target cell component can be attached to a solid support, contacted with one or more compounds, and contacted with the biomolecular inhibitor.", "One or more washing steps can be employed to remove biomolecular inhibitor and compound not bound to the cell component.", "Either the biomolecular inhibitor bound to the target cell component or the compound bound to the target cell component can be measured.", "Detection of biomolecular inhibitor or compound bound to the cell compound can be facilitated by the use of a label on either molecule type, wherein the label can be, for instance, a radioactive isotope either incorporated into the molecule itself or attached as an adduct, streptavidin or biotin, a fluorescent label or a substrate for an enzyme that can produce from the substrate a colored or fluorescent product.", "An appropriate means of detection of the labeled biomolecular inhibitor or compound moiety of the biomolecular inhibitor-cell component complex or the compound-cell component complex can be applied.", "For example, a scintillation counter can be used to measure radioactivity.", "Radio labeled streptavidin or biotin can be allowed to bind to biotin or streptavidin, respectively, and the resulting complexes detected in a scintillation counter.", "Alkaline phosphatase conjugated to streptavidin can be added to a biotin-labeled biomolecular inhibitor or compound.", "Detection and quantitation of a biotin-labeled complex can then be by addition of pNPP substrate of alkaline phosphatase and detection by spectrophotometry, of a product which absorbs UV light at a wavelength of 405 nm.", "A fluorescent label can also be used, in which case detection of fluorescent complexes can be by a fluorometer.", "Models are available that can read multiple samples, as in a microtiter plate.", "For example, in one type of assay, the method for identifying compounds comprises attaching the target cell component to a solid support, contacting the biomolecular inhibitor with the target cell component under conditions suitable for binding of the biomolecular inhibitor to the cell component, removing unbound biomolecular inhibitor from the solid support, contacting one or more compounds (e.g., a mixture of compounds) with the cell component under conditions suitable for binding of the biomolecular inhibitor to the cell component, and testing for unbound biomolecular inhibitor released from the cell component, whereby if unbound biomolecular inhibitor is detected, one or more compounds that displace or compete with the biomolecular inhibitor for a particular site on the target cell component have been identified.", "Other methods for identifying compounds that are competitive binders with the biomolecule for a target can employ adaptations of fluoresence polarization methods.", "See, for instance, Anal.", "Biochem.", "253(2): 210-218 (1997), Anal.", "Biochem.", "249(1): 29-36 (1997), BioTechniques 17(3): 585-589 (1994) and Nature 373: 254-256 (1995).", "Those compounds that bind competitively to the target cell component can be considered to be drug candidates.", "Further appropriate testing can confirm that those compounds which bind competitively with biomolecular inhibitors (or activators) possess the same activity as seen in an intracellular test of the effect of the biomolecular inhibitor or activator upon the phenotype of cells.", "Derivatives of these compounds having modifications to confer improved solubility, stability, etc., can also be tested for a desired phenotypic effect.", "1.7.20.Combining Steps Combining steps for testing the phenotypic effects of a biomolecule, as can be produced in an intracellular test, with steps for identifying compounds that compete with the biomolecule for sites on a target cell component, yields a method for identifying a compound which is a functional analog of a biomolecule which produces a phenotypic effect on a cell.", "These steps can be to test, for the phenotypic effect, either in culture or in an animal model, or in both, a cell which produces a biomolecule by regulable expression of an exogenous gene in the cell, and to identify, if the biomolecule caused the phenotypic effect, one or more compounds that compete with the biomolecule for binding to a target cell component.", "If a compound is found to compete with the biomolecule for binding to the target cell component, then the compound is a functional analog of a biomolecule which produces a phenotypic effect on the cell.", "Such a functional analog can cause qualitatively a similar effect on the cell, but to a similar degree, lesser degree or greater degree than the biomolecule.", "1.7.21.Method for Determining Whether a Target Component of a Cell is Essential to Producing a Phenotypic Effect on the Cell A further embodiment of the invention combining general steps (1) and (2) is a method for determining whether a target component of a cell is essential to producing a phenotypic effect on the cell, comprising isolating the target component from the cell, identifying a biomolecular binder of the isolated target component of the cell, constructing a second cell comprising the target component and a regulable, exogenous gene encoding the biomolecular binder, and testing the second cell in culture for an altered phenotypic effect, upon production of the biomolecular binder in the second cell, whereby, if the second cell shows the altered phenotypic effect upon production of the bimolecular binder, then the target component of the first cell is essential to producing the phenotypic effect on the first cell.", "1.7.22.Inhibit the Proliferation of the Cells The methods described herein are well suited to the identification of compounds that can inhibit the proliferation of the cells of infectious agents such as bacteria, fungi and the like.", "In addition, a procedure such as the one outlined below can be used in the identification of compounds to inhibit the proliferation of cancer cells.", "The two procedures described below further illustrate the use of the methods described herein and would provide proof of principle of these methods with a known target for anticancer therapy.", "Mammalian dihydrofolate reductase (DHFR) is a proven target for anticancer therapy.", "Methotrexate (MTX) is one of many existing drugs that inhibit DHFR.", "It is widely used for anticancer chemotherapy.", "NIH 3T3 is a mouse fibroblast cell line that is able to develop spontaneous transformed cells when cultured in low concentration (2%) of calf serum in molecular, cellular and developmental biology medium 402 (MCDB) (M. Chow and H. Rubin, Proc., Natl.", "Acad.", "Sci.", "USA 95(8): 4550-4555 (1998)).", "The transformed cells, which can be selectively inhibited by MTX (Chow and Rubin), are isolated.", "Both the normal and transformed NIH3T3 cells are transfected with pTet-On plasmid (Clontech; Palo Alto, Calif.).", "Stable cell lines that express high levels of reverse tetracycline-control led activator (rtTA) are isolated and characterized for their normal or transformed phenotype (Chow and Rubin).", "The DHFR gene (Genbank Accession # L26316) from the NIH 3T3 cell line is amplified by reverse transcription-PCR (RT-PCR) using poly A1RNA isolated from NIH 3T3 cells (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).", "Active DHFR is expressed using the BacPAK Baculovirus Expression System (Clontech) or other appropriate systems.", "The expressed DHFR is purified and biotinylated and subjected to peptide binder identification as exemplified for bacterial proteins.", "The identified peptides are biochemically characterized for in vitro inhibition of DHFR activity.", "Peptides that inhibit DHFR are identified.", "A nucleic acid encoding each peptide can be cloned into a vector such as pGEX4T2 (Pharmacia) to yield a vector which encodes a fusion polypeptide having the peptide fused to the N-terminus of GST.", "This can also be done by PCR amplification as exemplified herein for the peptide Pro-3.The fusion genes are cloned into plasmid pTRE (Clontech) for regulated expression.", "The constructed plasmid or the vector is cotransfected with pTK-Hyg into the stable NIH 3T3 cell line that expresses rtTA.", "The resulting cell lines, termed 3T3N-VITA (normal 3T3 cells that express rtTA and the DHFR inhibitory peptides), 3T3T-VITA (transformed 3T3 cells that express rtTA and the DHFR inhibitory peptides), or 3T3T-VITA control (transformed 3T3 cells that express rtTA and GST), are characterized for their normal or transformed phenotype (loss of contact inhibition, change in morphology, immortalization, etc.).", "102-101 of 3T3T-VITA or 3T3T-VITA control cells are mixed with 105 3T3N-VITA and are grown in MCD 402 medium with 10% calf serum at 37′C for three days.", "Tetracycline is added to the medium to a final concentration of 0 to 1 ug/ml.", "In a control, 200 nM of MTX is added.", "The cultures are incubated for an additional eight days, and the number of foci formed are counted as described by M. Chow and H. Rubin, Proc.", "Natl.", "Acad.", "Sci.", "USA 95(8): 45504555 (1998).", "Peptides that specifically inhibit foci formation of 3T3 transformed cells are identified.", "A murine model of fibroblastoma (Kogerman, P. et al., Oncogene (12): 1407-1416 (1997)) is used for evaluating the DHFR/peptide combination for identification of compounds for cancer therapy.", "Various amounts of 3T3T-VITA or 3T3T-VITA control cells (103, 104, 105, 106 cells) are injected subcutaneously into 5 groups (10 in each group) of athymic nude mice (4-6 weeks old, 18-22 g) to determine the minimal dose needed for development of fibroblastomas in all of the tested animals.", "Upon determination of the minimal tumorigenic dose, 6 groups of athymic nude mice (10 each) are injected subcutaneously (s.c.) with the minimal tumorigenic dose for 3T3T-VITA or 3T3T-VITA control cells to develop fibroblastoma.", "One week after injection, group I mice start receiving MTX s.c. at 2 mg/kg/day as positive control, group 2 to 5 start receiving 1, 2, 5, or 10 mg/kg/day of tetracycline, group 6 start receiving saline (vehicle) as control.", "Five weeks after the introduction of cells, all of the mice are sacrificed and tumors are removed from them.", "Tumor mass is measured and compared among the groups.", "An effective peptide identified by these in vivo experiments can be used for screening libraries of compounds to identify those compounds that competitively bind to DHFR.", "One mechanism of tumorigenesis is overexpression of proto-oncogenes such as Ha-ras (Reviewed by Suarez, H. G., Anticancer Research 9(5): 1331-1343 (1989)).", "Compounds that inhibit the activities of the products of such proto-oncogenes can be used for cancer chemotherapy.", "What follows is a further illustration of the methods described herein, as applied to mammalian cells.", "Transgenic mice that overexpress human Ha-ras have been produced.", "Such transgenic mice develop salivary and/or mammary adenocarcinomas (Nielsen, L. L. et al, In Vivo 8(5): 1331-1343 (1994)).", "Secondary transgenic mice that express rtTA can be generated using the pTet-On plasmid from Clontech.", "Human Ha-ras open reading frame cDNA (Genbank Accession #GO0277) is amplified by RT-PCR using polyA-RNA isolated from human mammary gland or other tissues.", "Active Ha-ras is expressed using the BacPAK Baculovirus Expression System (Clontech) or other appropriate systems.", "The expressed Ha-ras is purified and biotinylated and subjected to peptide binder identification as exemplified herein for bacterial proteins as target cell components.", "The identified peptides are biochemically characterized for in vitro inhibition of Ha-ras GTPase activity.", "Peptides that inhibit Ha-ras are cloned into plasmid pTPE (Clontech) for regulated expression as an N-terminal fusion of GST.", "Such constructs are used to generate tertiary transgenic mice using the secondary transgenic mice.", "Transgenic mice that are able to overexpress peptide genes are identified by Northern and Western analysis.", "Control mice that express GST are also identified.", "Various doses of tetracycline are administered to the tertiary transgenic mice by s.c. or i.p.", "injection before or after tumor onset.", "Prevention or regression of tumors resulting from expression of the peptide genes are analyzed as described above for murine fibroblastoma.", "Peptides found to be effective in in vivo experiments will be used to screen compounds that inhibit human Ha-ras activity for cancer therapy.", "1.7.23.Disease Targets The method of the invention can be applied more generally to mammalian diseases caused by: (1) loss or gain of protein function, (2) over-expression or loss of regulation of protein activity.", "In each case the starting point is the identification of a putative protein target or metabolic pathway involved in the disease.", "The protocol can sometimes vary with the disease indication, depending on the availability of cell culture and animal model systems to study the disease.", "In all cases the process can deliver a validated target and assay combination to support the initiation of drug discovery.", "Appropriate disease indications include, but are not limited to, Alzheimer's, arthritis, cancer, cardiovascular diseases, central nervous system disorders, diabetes, depression, hypertension, inflammation, obesity and pain.", "Appropriate protein targets putatively linked to disease indications include, but are not limited to (1) the leptin protein, putatively linked to obesity and diabetes; (2) a mitogen-activated protein kinase putatively linked to arthritis, osteoporosis and atherosclerosis; (3) the interleukin-1 beta converting protein putatively linked to arthritis, asthma and inflammation; (4) the caspase proteins putatively linked to neurodegenerative diseases such as Alzheimer's, Parkinson's and stroke, and (5) the tumor necrosis factor protein putatively linked to obesity and diabetes.", "Appropriate protein targets include also, but are not limited to, enzymes catalyzing the following types of reactions: (1) oxido-reductases, (2) transferases, (3) hydrolases, (4) lyases, (5) isomerases, and (6) ligases.", "The arachidonic acid pathway constitutes one of the main mechanisms for the production of pain and inflammation.", "The pathway produces different classes of end products, including the prostaglandins, thromboxane and leukotrienes.", "Prostaglandins, an end product of cyclooxygenase metabolism, modulate immune function, mediate vascular phases of inflammation and are potent vasodilators.", "The major therapeutic action of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) is proposed to be inhibition of the enzyme cyclooxygenase (COX).", "Anti-inflammatory potencies of different NSAIDs have been shown to be proportional to their action as COX inhibitors.", "It has also been shown that COX inhibition produces toxic side effects such as erosive gastritis and renal toxicity.", "The knowledge base regarding the toxic side effects of COX inhibitors has been gained through years of monitoring human therapies and human suffering.", "Two kinds of COX enzymes are now known to exist, with inhibition of COX 1related to toxicity, and inhibition of COX2 related to reduction of inflammation.", "Thus, selective COX2 inhibition is a desirable characteristic of new anti-inflammatory drugs.", "The method of the invention can provide a route from identification of potential drug targets to validating these targets (for example, COX1 and COX2) as playing a role in disease (pain and inflammation) to an examination of the phenotype for the inhibition of one or both target isozymes without human suffering.", "Importantly, this information can be collected in vivo.", "As an alternative strategy, the method of the invention can be used to define the phenotype of “genes of unknown function” obtained from various human genome sequencing projects or to assess the phenotype resulting, from inhibition of one isozyme subtype or one member of a family of related protein targets.", "1.5.Definitions Target: (also, “target component of a cell,” or “target cell component”) a constituent of a cell which contributes to and is necessary for the production or maintenance of a phenotype of the cell in which it is found.", "A target can be a single type of molecule or can be a complex of molecules.", "A target can be the product of a single gene, but can also be a complex comprising more than one gene product (for example, an enzyme comprising alpha and beta subunits, mRNA, tRNA, ribosomal RNA or a ribonucleoprotein particle such as a snRNP).", "Targets can be the product of a characterized gene (gene of known function) or the product of an uncharacterized gene (gene of unknown function).", "Target Validation: the process of determining whether a target is essential to the maintenance of a phenotype of the cell type in which the target normally occurs.", "For example, for pathogenic bacteria, researchers developing antimicrobials want to know if a compound which is potentially an antimicrobial agent not only binds to a target in vitro, but also binds to, and modulates the function of, a target in the bacteria in vivo, and especially under the conditions in which the bacteria are producing an infection—those conditions under which the antimicrobial agent must work to inhibit bacterial growth in an infected animal or human.", "If such compounds can be found that bind to a target in vitro and alter the target's function in cells resulting in an altered phenotype, as found by testing cells in culture and/or as found by testing cells in an animal, then the target is validated.", "Phenotypic Effect: a change in an observable characteristic of a cell which can include, e.g., growth rate, level or activity of an enzyme produced by the cell, sensitivity to various agents, antigenic characteristics, and level of various metabolites of the cell.", "A phenotypic effect can be a change away from wild type (normal) phenotype, or can be a change towards wild type phenotype, for example.", "A phenotypic effect can be the causing or curing of a disease state, especially where mammalian cells are referred to herein.", "For cells of a pathogen or tumor cells, especially, a phenotypic effect can be the slowing of growth rate or cessation of growth.", "Biomolecule: a molecule which can be produced as a gene product in cells that have been appropriately constructed to comprise one or more genes encoding the biomolecule.", "Preferably, production of the biomolecule can be turned on, when desired, by an inducible promoter.", "A biomolecule can be a peptide, polypeptide, or an RNA or RNA oligonucleotide, a DNA or DNA oligonucleotide, but is preferably a peptide.", "The same biomolecules can also be made synthetically.", "For peptides, see Merrifield, J., J.", "Am.", "Chem.", "Soc.", "85: 2140-2154 (1963).", "For instance, an Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer) can be used for peptide synthesis.", "Biomolecules produced as gene products intracellularly are tested for their interaction with a target in the intracellular steps described herein (tests performed with cells in culture and tests performed with cells that have been introduced into animals).", "The same biomolecules produced synthetically are tested for their binding to an isolated target in an initial in vitro method described herein.", "Synthetically produced biomolecules can also be used for a final step of the method for finding compounds that are competitive binders of the target.", "Biomolecular Binder (of a target): a biomolecule which has been tested for its ability to bind to an isolated target cell component in vitro and has been found to bind to the target.", "Biomolecular Inhibitor of Growth: a biomolecule which has been tested for its ability to inhibit the growth of cells constructed to produce the biomolecule in an “in culture” test of the effect of the biomolecule on growth of the cells, and has been found, in fact, to inhibit the growth of the cells in this test in culture.", "Biomolecular Inhibitor of Infection: a biomolecule which has been tested for its ability to ameliorate the effects of infection, and has been found to do so.", "In the test, pathogen cells constructed to regulably express the biomolecule are introduced into one or more animals, the gene encoding the biomolecule is regulated so as to allow production of the biomolecule in the cells, and the effects of production of the biomolecule are observed in the infected animals compared to one or more suitable control animals.", "Isolated: term used herein to indicate that the material in question exists in a physical milieu distinct from that in which it occurs in nature.", "For example, an isolated target cell component of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.", "The absolute level of purity is not critical, and those skilled in the art can readily determine appropriate levels of purity according to the use to which the material is to be put.", "In many circumstances the isolated material will form part of a composition (for example, a more or less crude extract containing other substances), buffer system or reagent mix.", "In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC).", "Pathogen or Pathogenic Organism: an organism which is capable of causing disease, detectable by signs of infection or symptoms characteristic of disease.", "Pathogens can include procaryotes (which include, for example, medically significant Gram-positive bacteria such as Streptococcus pneumoniae, Enterococcus faecalis and Staphylococcus aureus, Gram-negative bacteria such as Escherichia coli, Pseudomonas aeroginosa and Klebsiella pneumoniae, and “acid-fast” bacteria such as Mycobacteria, especially M. tuberculosis), eucaryotes such as yeast and fungi (for example, Candida albicans and Aspergillus fumigatus) and parasites.", "It should be recognized that pathogens can include such organisms as soil-dwelling organisms and “normal flora” of the skin, gut and orifices, if such organisms colonize and cause symptoms of infection in a human or other mammal, by abnormal proliferation or by growth at a site from which the organism cannot usually be cultured.", "SECTION 2.WHOLE CELL ENGINEERING USING REAL-TIME METABOLIC FLUX ANALYSIS Technical Field In one embodiment, the present invention provides methods for whole cell engineering, cell biology and molecular biology.", "In particular, the invention is directed to methods for whole cell engineering of new and modified phenotypes by using “on-line” or “real-time” metabolic flux analysis.", "Background In one embodiment of this invention, whole cell metabolic flux analysis is a “horizontal” or “holistic” approach to study the metabolism, or “metabolome,” of an organism.", "A whole cell “horizontal” metabolome approach studies the expression and function of all of the genes of an organism simultaneously.", "By using this whole cell approach to study a cell's metabolism, it is possible to get a complete snapshot of the whole cell's transcriptome (the expressed transcripts, or mRNA messages) and proteome (the expressed polypeptides).", "However, such snapshots are static pictures of one aspect of a cell's physiology and metabolism.", "Development of a means to dynamically monitor many different parameters in a cell culture would be much more effective in detecting new or altered cell phenotypes.", "Summary One embodiment of this invention provides a method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis.", "In one aspect, the genetic composition of the cell is modified by a method comprising addition of a nucleic acid to the cell.", "One or more nucleic acids can be added at the same time, or, in series.", "The genetic composition of the cell can be modified by addition of a nucleic acid heterologous to the cell, or, a nucleic acid homologous to the cell.", "The homologous nucleic acid can comprise a modified homologous nucleic acid, such as a modified homologous gene.", "The coding sequence or transcriptional regulatory sequence of a gene can be modified.", "Alternatively, the genetic composition of the cell can be modified by a method comprising deletion of a sequence or modification of a sequence in the cell.", "The genetic composition of the cell can be modified by a method comprising modifying or knocking out the expression of a gene.", "The method can further comprising selecting a cell comprising a newly engineered phenotype.", "The selected cell can be isolated.", "The method can further comprise culturing the selected or isolated cell, thereby generating a new cell strain or cell line comprising a newly engineered phenotype.", "The methods can further comprise isolating a cell comprising a newly engineered phenotype.", "Any phenotype can be added or modified.", "For example, a phenotype can be specifically targeted for change or addition.", "Thus, specific heterologous genes can be inserted or specific homologous genes can be stochastically or non-stochastically modified.", "For example, the newly engineered phenotype can be, e.g., an increased or decreased expression or amount of a polypeptide, an increased or decreased amount of an mRNA transcript, an increased or decreased expression of a gene, an increased or decreased resistance or sensitivity to a toxin, an increased or decreased resistance use or production of a metabolite, an increased or decreased uptake of a compound by the cell, an increased or decreased rate of metabolism, and an increased or decreased growth rate.", "The newly engineered phenotype can a stable phenotype.", "In another aspect, it can be a transient or an inducible phenotype.", "In one aspect, modifying the genetic composition of a cell comprises insertion of a construct into the cell, wherein construct comprises a nucleic acid operably linked to a constitutively active promoter.", "Alternatively, modifying the genetic composition of a cell can comprise insertion of a construct into the cell, wherein construct comprises a nucleic acid operably linked to an inducible promoter.", "The nucleic acid added to the cell can be stably inserted into the genome of the cell.", "Alternatively, the nucleic acid added to the cell can propagate as an episome in the cell.", "In one aspect, the nucleic acid added to the cell can encode a peptide or a polypeptide.", "The polypeptide can comprise a homologous polypeptide, such as a modified homologous polypeptide.", "Alternatively, the polypeptide can comprise a heterologous polypeptide.", "The nucleic acid added to the cell can encode a transcript comprising a sequence that is antisense to a homologous transcript.", "In one aspect, modifying the genetic composition of the cell can comprise increasing or decreasing the expression of an mRNA transcript.", "Modifying the genetic composition of the cell can comprise increasing or decreasing the expression of a polypeptide, a lipid, a mono- or poly-saccharide or a nucleic acid.", "In one aspect, modifying the homologous gene can comprise knocking out expression of the homologous gene.", "Modifying the homologous gene can comprise increasing the expression of the homologous gene.", "The gene modification can be random, or stochastic, or, non-random, or targeted, i.e., non-stochastic.", "In an exemplary non-stochastic gene modification, a gene to be inserted into a cell to modify a phenotype can be a heterologous gene or a sequence-modified homologous gene, wherein the sequence modification is made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises a homologous gene of the cell (it can also be a heterologous gene that you wish to modify); (b) providing a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; (c) generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide of step (a) with the oligonucleotides of step (b), thereby generating polynucleotides comprising homologous gene sequence variations.", "One variation of this method has been termed “gene site-saturation mutagenesis,” “site-saturation mutagenesis,” “saturation mutagenesis” or simply “GSSM,” and is described in further detail, below.", "It can be used in combination with other mutagenization processes.", "See, e.g., U.S. Pat.", "Nos.", "6,171,820; 6,238,884.Another exemplary non-stochastic gene modification process comprises introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.", "For example, the sequence modification of the gene to be modified (e.g., the heterologous gene or homologous gene) is made by a method comprising the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.", "One variation of this method has been termed “synthetic ligation reassembly,” or simply “SLR,” and is described in further detail, below.", "It can be used in combination with other mutagenization processes.", "See, e.g., U.S. Pat.", "No.", "6,171,820.Any cell can be engineered by the methods the invention, including, e.g., prokaryotic cells and eukaryotic cells.", "Bacteria, Archaebacteria, fungi, yeast, plant cells, insect cells, mammalian cells, including human cells, without limitation, can be engineered by the methods the invention.", "Furthermore, intracellular parasites, bacteria, viruses can be “indirectly” engineered by culturing and monitoring of eukaryotic cells by the methods the invention, including, e.g., immunodeficiency viruses, e.g., HIV, oncoviruses, mycobacteria, protozoan organisms (e.g., trypanosomes, such as Trypanosoma rangeli), plasmodium (e.g., Plasmodium falciparum), toxoplasmosis (e.g., Toxoplasma gondii), Leishmania, and the like.", "In practicing the methods of the invention, any metabolic parameter can be measured.", "In one aspect, several different metabolic parameters are evaluated in the cell culture.", "The metabolic parameters can be measured at the same time or sequentially.", "One exemplary metabolic parameter is rate of cell growth, which can be measured by, e.g., a change in optical density of the cell culture.", "Another exemplary metabolic parameter measured comprises a change in the expression of a polypeptide.", "Changes in the expression of the polypeptide can be measured by any method, e.g., a one-dimensional gel electrophoresis, a two-dimensional gel electrophoresis, a tandem mass spectography, an RIA, an ELISA, an immunoprecipitation and a Western blot.", "In one aspect, the measured metabolic parameter comprises a change in expression of at least one transcript, or, the expression of a transcript of a newly introduced gene.", "The change in expression of the transcript can be measured by a method selected from the group consisting of a hybridization, a quantitative amplification and a Northern blot.", "The transcript expression can be measured by hybridization of a sample comprising transcripts of a cell or nucleic acid representative of or complementary to transcripts of a cell by hybridization to immobilized nucleic acids on an array.", "In one aspect, the measured metabolic parameter comprises a measurement of a metabolite, including primary and secondary metabolites.", "For example, the measured metabolic parameter can comprise an increase or a decrease in a primary or a secondary metabolite.", "The secondary metabolite can be selected from the group consisting of a glycerol and a methanol.", "The measured metabolic parameter can comprise an increase or a decrease in an organic acid, such as an acetate, a butyrate, a succinate and an oxaloacetate.", "In one aspect, the measured metabolic parameter comprises an increase or a decrease in intracellular pH, or, extracellular pH in a culture medium.", "The increase or a decrease in intracellular pH can measured by intracellular application of a dye; the change in fluorescence of the dye can be measured over time.", "In one aspect, the measured metabolic parameter comprises gas exchange rate measurements.", "In one aspect, the measured metabolic parameter comprises an increase or a decrease in synthesis of DNA or RNA over time.", "The increase or a decrease in synthesis, or accumulation, or decay, of DNA or RNA over time can be measured by intracellular application of a dye; the change in fluorescence of the dye can be measured over time.", "In one aspect, the measured metabolic parameter comprises an increase or a decrease in uptake of a composition.", "The composition can be a metabolite, such as a monosaccharide, a disaccharide, a polysaccharide, a lipid, a nucleic acid, an amino acid and a polypeptide.", "The saccharide, disaccharide or polysaccharide can comprise a glucose or a sucrose.", "The composition can also be an antibiotic, a metal, a steroid and an antibody.", "In one aspect, the measured metabolic parameter comprises an increase or a decrease in the secretion of a byproduct or a secreted composition of a cell.", "The byproduct or secreted composition can be a toxin, a lymphokine, a polysaccharide, a lipid, a nucleic acid, an amino acid, a polypeptide and an antibody.", "In one aspect of the methods, the real time monitoring simultaneously measures a plurality of metabolic parameters.", "The real time monitoring of a plurality of metabolic parameters can comprise use of a Cell Growth Monitor device.", "The Cell Growth Monitor device can be a Wedgewood Technology, Inc., Cell Growth Monitor model 652, or similar model or variation thereof.", "In one aspect, the real time simultaneous monitoring measures uptake of substrates, levels of intracellular organic acids and levels of intracellular amino acids.", "The real time simultaneous monitoring can measure: uptake of glucose; levels of acetate, butyrate, succinate or oxaloacetate; and, levels of intracellular natural amino acids.", "In one aspect, the method further comprises use of a computer-implemented program to real time monitor the change in measured metabolic parameters over time.", "The computer-implemented program can comprise a computer-implemented method as set forth in FIG.", "28.The computer-implemented method can comprise metabolic network equations.", "These computer-implemented method can also comprise a pathway analysis, an error analysis, such as a weighted least squares solution, and a flux estimation.", "The computer-implemented method can further comprises a preprocessing unit to filter out the errors for the measurement before the metabolic flux analysis.", "The details of one or more aspects of the invention are set forth in the accompanying drawings and the description below.", "Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.", "All publications, GenBank Accession references (sequences), ATCC Deposits, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.", "Brief Description Of The Drawings FIG.", "28 is a schematic illustrating an exemplary metabolic flux analysis (MFA) procedure of the invention.", "Detailed Description In one embodiment, the invention provides novel methods for whole cell engineering of new and modified phenotypes by using “on-line” or “real-time” metabolic flux analysis.", "In practicing the methods of the invention, as a first step, a cell is modified by changing the genetic composition of the cell.", "The modification can be random, i.e., stochastic, or, by non-stochastic methods, as described herein.", "Specific genes or specific metabolic pathways can be targeted for modification.", "In one aspect, the second step of the methods of the invention comprises culturing the modified cell to generate a plurality of modified cells.", "The cells can be cultured by any means, for example, in cell culture, such as a tissue culture, by fermentation or tissue culture reactors, or in a cell growth monitor device.", "In one aspect, the next step of the methods comprises measuring at least one metabolic parameter of the cell in real time.", "In one aspect, a plurality of metabolome parameters are simultaneously measured.", "Thus, one or several devices can be used to monitor and measure metabolic parameters.", "For example, a cell growth monitor devices can measure a plurality of metabolic parameters of the cells in culture in real time.", "One example is the Wedgewood Technology, Inc. (San Carlos, Calif.), Cell Growth Monitor model 652™, as discussed below.", "Finally, in one embodiment, the methods comprise analyzing these data of to determine if the measured parameters differ from a comparable measurement in an unmodified cell under similar conditions, or, change over time, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis.", "For example, the parameter can be higher, lower or change at a rate that differs from a wild type cell or cell culture.", "It is not necessary to simultaneously monitor an unmodified cell or cell culture in real time to determine if and/or what phenotypic modifications result from the modification of the cell's genetic composition.", "Data and information already known can be used as a reference.", "In one aspect of the invention, the methods further comprise use of a computer-implemented program to real time monitor the change in measured metabolic parameters over time and the analyze and display the resulting processed data.", "One exemplary computer-implemented program comprises a computer-implemented method as set forth in FIG.", "1.In this and other computer-implemented methods that can be used, the paradigm comprises use of metabolic network equations, metabolic pathway analyses, error analysis, such as a weighted least squares solution to give a flux estimation and the like.", "In one aspect of the invention, a nucleic acid (or, the nucleic acid) responsible for the altered phenotype is identified, re-isolated, again modified (e.g., either stochastically or non-stochastically), reinserted into the cell, and the process of real-time metabolic flux analysis is iteratively repeated.", "The process can be iteratively repeated until a desired phenotype is engineered.", "For example, a plant cell and plant cell culture is subjected to iterative repetition of the methods of the invention until a new plant cell is made that comprises a desired new phenotype, e.g., enhanced growth, nutritional value or insect or drought resistance, or all or some of these characteristics.", "A pathogenic microorganism can be subjected to iterative repetition of the methods of the invention until it becomes non-pathogenic.", "A microorganism can be engineered to become lethal to another organism, such as an insect, or, to produce a variety of antibiotics or other compositions.", "Microorganisms can be subjected to iterative repetition of the methods of the invention to engineer, e.g., increased yield of desired products, removal of unwanted co-metabolites, improved utilization of inexpensive carbon and nitrogen sources, and adaptation to fermentor/bioreactor growth conditions, increased production of a primary metabolite, increased production of a secondary metabolite, increased tolerance to acidic conditions, increased tolerance to basic conditions, increased tolerance to organic solvents, increased tolerance to high salt conditions and increased tolerance to high or low temperatures.", "A complete biosynthetic pathway can be inserted into a cell.", "Any cell phenotype can be modified or any phenotype can be added to a cell using the methods of the invention, without limitation.", "The invention can be practiced in combination with other methods for inserting and screening for metabolic pathways, see, e.g., U.S. Pat.", "No.", "6,268,140, which describes producing and screening combinatorial metabolic libraries of multimeric proteins, or, U.S. Pat.", "No.", "5,712,146, which describes vectors encoding polyketide synthases which in turn catalyze the production of a variety of polyketides.", "Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.", "As used herein, the following terms have the meanings ascribed to them unless specified otherwise.", "The terms “array” or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below.", "As used herein, the terms “computer” and “processor” are used in their broadest general contexts and incorporate all such devices, as described in detail, below.", "The term “saturation mutagenesis” or “GSSM” includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail, below.", "The term “optimized directed evolution system” or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.", "The term “synthetic ligation reassembly” or “SLR” includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.", "The term “antibody” includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g.", "Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol.", "Methods 175: 267-73; Yarmush (1992) J. Biochem.", "Biophys.", "Methods 25: 85-97.The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).", "Single chain antibodies are also included by reference in the term “antibody.” Generating and Manipulating Nucleic Acids The methods of the invention include modifying the genetic composition of a cell by addition of a heterologous nucleic acid into the cell or modification of a homologous gene in the cell.", "Nucleic acids can be isolated from a cell, recombinantly generated or made synthetically.", "The sequences can be isolated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like.", "In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein.", "The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.", "General Techniques The nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly.", "Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity.", "Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.", "Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J.", "Am.", "Chem.", "Soc.", "105: 661; Belousov (1997) Nucleic Acids Res.", "25: 3440-3444; Frenkel (1995) Free Radic.", "Biol.", "Med.", "19: 373-380; Blommers (1994) Biochemistry 33: 7886-7896; Narang (1979) Meth.", "Enzymol.", "68: 90; Brown (1979) Meth.", "Enzymol.", "68: 109; Beaucage (1981) Tetra.", "Lett.", "22: 1859; U.S. Pat.", "No.", "4,458,066.Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.", "), Vols.", "1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.", "John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I.", "Theory and Nucleic Acid Preparation, Tijssen, ed.", "Elsevier, N.Y. (1993).", "Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art.", "These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.", "fluid or gel precipitin reactions, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.", "Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones.", "Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat.", "Nos.", "5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat.", "Genet.", "15: 333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50: 306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23: 120-124; cosmids, recombinant viruses, phages or plasmids.", "Amplification of Nucleic Acids In practicing the methods of the invention, nucleic acids encoding heterologous or homologous, or modified nucleic acids, can be reproduced by, e.g., amplification.", "Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample.", "In one aspect of the invention, message isolated from a cell or a cDNA library are amplified.", "The skilled artisan can select and design suitable oligonucleotide amplification primers.", "Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed.", "Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed.", "Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4: 560; Landegren (1988) Science 241: 1077; Barringer (1990) Gene 89: 117); transcription amplification (see, e.g., Kwoh (1989) Proc.", "Natl.", "Acad.", "Sci.", "USA 86: 1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc.", "Natl.", "Acad.", "Sci.", "USA 87: 1874); Q Beta replicase amplification (see, e.g., Smith (1997) J. Clin.", "Microbiol.", "35: 1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) Mol.", "Cell.", "Probes 10: 257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol.", "152: 307-316; Sambrook; Ausubel; U.S. Pat.", "Nos.", "4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13: 563-564.Modification of Nucleic Acids In practicing the methods of the invention, the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell.", "A homologous, heterologous or gene selected by the methods of the invention can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or “directed evolution,” methods.", "Methods for random mutation of genes are well known in the art, see, e.g., U.S. Pat.", "No.", "5,830,696.For example, mutagens can be used to randomly mutate a gene.", "Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination.", "Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylanine, hydrazine or formic acid.", "Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.", "These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence.", "Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.", "Techniques in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 5467-5471; or, combinatorial multiple cassette mutagenesis, see, e.g., Crameri (1995) Biotechniques 18: 194-196.Alternatively, nucleic acids, e.g., genes, can be reassembled after random, or “stochastic,” fragmentation, see, e.g., U.S. Pat.", "Nos.", "6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793.Non-stochastic, or “directed evolution,” methods include, e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof.", "In one aspect of the invention, nucleic acids are selected, using real-time metabolic flux analysis, for conferring a new or modified phenotype on a cell, isolated, modified and reinserted into a cell to reiterate the steps of the methods of the invention.", "Polypeptides encoded by isolated and/or modified nucleic acids can be screened for an activity before their reinsertion into the cell by, e.g., using a capillary array platform.", "See, e.g., U.S. Pat.", "Nos.", "6,280,926; 5,939,250.Saturation mutagenesis, or, GSSM In one aspect of the invention, non-stochastic gene modification, a “directed evolution process,” can be used to modify a gene to be inserted into a cell to add or modify a phenotype.", "Variations of this method have been termed “gene site-saturation mutagenesis,” “site-saturation mutagenesis,” “saturation mutagenesis” or simply “GSSM.” It can be used in combination with other mutagenization processes.", "See, e.g., U.S. Pat.", "Nos.", "6,171,820; 6,238,884.In one aspect, GSSM comprises providing a template polynucleotide and a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide with the oligonucleotides, thereby generating polynucleotides comprising homologous gene sequence variations.", "In one aspect, codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified.", "These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence.", "The downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.", "In one aspect, one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions.", "In another aspect, at least two degenerate cassettes are used—either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.", "For example, more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site.", "This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).", "In another aspect, oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.", "In one aspect, simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e.", "a degenerate (N,N,G/T)n sequence.", "In another aspect, degenerate cassettes having less degeneracy than the N,N,G/T sequence are used.", "For example, it may be desirable in some instances to use (e.g.", "in an oligonucleotide) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet.", "Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet.", "Alternatively, it may be desirable in some instances to use (e.g.", "in an oligo) a degenerate N,N,N triplet sequence.", "In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets) allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position).", "For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e.", "20 possible amino acids per position X 100 amino acid positions) can be generated.", "Through the use of an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet, 32 individual sequences can code for all 20 possible natural amino acids.", "Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides.", "In contrast, the use of a non-degenerate oligonucleotide in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.", "Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide.", "This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.", "In one aspect, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations).", "The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g.", "cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening.", "When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased affinity or avidity to an antigen), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.", "In one aspect, upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position.", "One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions.", "For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions.", "Thus, there are 3×3×3 or 27 total possibilities, including 7 that were previously examined—6 single point mutations (i.e.", "2 at each of three positions) and no change at any position.", "In another aspect, site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents.", "This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.", "Synthetic Ligation Reassembly (SLR) Another non-stochastic gene modification, a “directed evolution process,” that can be can be used in the methods of the invention to modify a gene to be inserted into a cell to add or modify a phenotype has been termed “synthetic ligation reassembly,” or simply “SLR.” SLR is a method of ligating oligonucleotide fragments together non-stochastically.", "This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non-stochastically.", "See, e.g., U.S. patent application Ser.", "No.", "09/332,835 entitled “Synthetic Ligation Reassembly in Directed Evolution” and filed on Jun.", "14, 1999 (“U.S.", "Ser.", "No.", "09/332,835”).", "In one aspect, SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.", "SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged.", "Thus, this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras.", "SLR can be used to generate libraries comprised of over 101000 different progeny chimeras.", "Thus, aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design.", "This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.", "The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be “serviceable” for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.", "Thus the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends.", "If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).", "In one aspect, the annealed building pieces are treated with an enzyme, such as a ligase (e.g.", "T4 DNA ligase), to achieve covalent bonding of the building pieces.", "In one aspect, the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotide molecules.", "These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., chimerized or shuffled.", "In one aspect of this method, the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points.", "The demarcation points can be located at an area of homology, and are comprised of one or more nucleotides.", "These demarcation points are preferably shared by at least two of the progenitor templates.", "The demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides.", "The demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules.", "A demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences.", "Alternatively, a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences.", "Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences.", "In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.", "In one aspect, a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides.", "In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.", "At the same time, in another embodiment, the assembly order (i.e.", "the order of assembly of each building block in the 5′ to 3 sequence of each finalized chimeric nucleic acid) in each combination is by design (or non-stochastic) as described above.", "Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.", "In another aspect, the ligation reassembly method is performed systematically.", "For example, the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g.", "one by one.", "In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels.", "This allows a systematic examination and screening procedure to be performed.", "Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups.", "Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, these methods provide for the generation of a library (or set) comprised of a large number of progeny molecules.", "Because of the non-stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.", "The saturation mutagenesis and optimized directed evolution methods also can be used to generate these amounts of different progeny molecular species.", "It is appreciated that the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings.", "It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention.", "In fact, demarcation points can even be chosen in areas of little or no intermolecular homology.", "For example, because of codon wobble, i.e.", "the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template.", "Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.", "This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecularly homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.", "In another aspect, the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g.", "by mutageneis) or in an in vivo process (e.g.", "by utilizing the gene splicing ability of a host organism).", "It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.", "Thus, according to another aspect, a nucleic acid building block can be used to introduce an intron.", "Thus, functional introns may be introduced into a man-made gene manufactured according to the methods described herein.", "The artificially introduced intron(s) can be functional in a host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.", "Optimized Directed Evolution System In practicing the methods of the invention, nucleic acids can also be modified by a method comprising an optimized directed evolution system.", "Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination.", "Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.", "A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant.", "Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.", "This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events.", "This provides more control over choosing chimeric variants having a predetermined number of crossover events.", "In addition, this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.", "Previously, if one generated, for example, 1013 chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity.", "Moreover, a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity.", "By using these methods, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.", "Thus, although one can still generate 1013 chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events.", "Because the resulting progeny population can be skewed to have a predetermined number of crossover events, the boundaries on the functional variety between the chimeric molecules is reduced.", "This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.", "One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence.", "Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order.", "Additional information can also be found in U.S. Ser.", "No.", "09/332,835.The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.", "For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.", "As one example, a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant.", "Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.", "The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low.", "If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event.", "If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a ⅓ chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.", "Accordingly, a probability density function (PDF) can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction.", "The statistics and mathematics behind determining the PDF is described below.", "By utilizing these methods, one can calculate such a probability density function, and thus enrich the chimeric progeny population for a predetermined number of crossover events resulting from a particular ligation reaction.", "Moreover, a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.", "These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding an polypeptide through recombination.", "This system allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.", "A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant.", "Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence.", "The method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events.", "This provides more control over choosing chimeric variants having a predetermined number of crossover events.", "In addition, these methods provide a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems.", "By using the methods described herein, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events.", "Thus, although one can still generate 1013 chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events.", "Because the resulting progeny population can be skewed to have a predetermined number of crossover events, the boundaries on the functional variety between the chimeric molecules is reduced.", "This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.", "In one aspect, the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides corresponding to fragments or portions of each parental sequence.", "Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order.", "See also U.S. Ser.", "No.", "09/332,835.The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created.", "For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature.", "As one example, a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant.", "Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences.", "The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low.", "If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event.", "If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a ⅓ chance (assuming 3 parents) that a oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.", "Accordingly, a probability density function (PDF) can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction.", "The statistics and mathematics behind determining the PDF is described below.", "One can calculate such a probability density function, and thus enrich the chimeric progeny population for a predetermined number of crossover events resulting from a particular ligation reaction.", "Moreover, a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events.", "Determining Crossover Events Embodiments of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs.", "The output of this program is a “fragment PDF” that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes.", "The processing described herein is preferably performed in MATLAB@ (The Mathworks, Natick, Mass.)", "a programming language and development environment for technical computing.", "Iterative Processes In practicing the methods of the invention, the process can be iteratively repeated.", "For example a nucleic acid (or, the nucleic acid) responsible for an altered phenotype is identified, re-isolated, again modified, reinserted into the cell, and the process of real-time metabolic flux analysis is iteratively repeated.", "The process can be iteratively repeated until a desired phenotype is engineered.", "For example, an entire biochemical pathway can be engineered into a cell.", "Any cell phenotype can be modified or any phenotype can be added to a cell using the methods of the invention, without limitation.", "Nucleic acids can be modified using either stochastic or non-stochastic methods.", "In various aspects, the methods generate sets of chimeric nucleic acid and protein molecules, followed by insertion into a cell, culturing, and then screening by using real-time metabolic flux analysis for a particular activity, such as a changed or added desired phenotype.", "The invention is not limited to only a single round of screening.", "Based on this determination, a second round of reassembly can take place that enriches for progeny having a desired property or incurring a desired phenotype.", "Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait (e.g., a new phenotype), it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed.", "Since incorporating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides.", "This iterative practice of determining which oligonucleotides are most related to the desired trait, and which are unrelated, allows more efficient exploration all of the possible protein variants that might be provide a particular trait or activity.", "Automated Control of Reactions The process of generating any of the reactions of the methods of the invention can be automated with the assistance of automated devices and robotic instruments.", "For example, in one aspect, a cell growth monitor device is used for real-time metabolic flux analysis, such as a Wedgewood Technology, Inc., Cell Growth Monitor model 652.As noted below, this device can be linked to a computer system.", "Another exemplary device is a TECAN GENESIS™ programmable robot made by Tecan Corporation (Hombrechtikon, Switzerland), which can be interfaced with a computer that determines the quantities of each oligonucleotide fragment to yield a resulting PDF.", "By linking a computer system that determines the proper quantities of each oligonucleotide to an automated robot, a complete ligation reassembly system is produced.", "Data links through serial or other interfaces will allow the data files generated from the ligation reassembly calculations to be forwarded in the proper format for the robotic system to automatically begin allocating the proper quantities of each oligonucleotide fragment into a reaction tube.", "The automated system can include a plurality of oligonucleotide fragments derived from a series of nucleic acid sequence variants, wherein said fragments are configured to join one another at unique overhangs.", "The system also has a data input field configured to store a target number of crossover events in for each of the variant sequences.", "Within the system is also a prediction module configured to determine the quantity of each of the fragments to admix together so that mixing the fragments results in a population of progeny molecules that are enriched for crossover events corresponding to the target number.", "The system also provides a robotic arm linked to the prediction module through a communication interface for automatically mixing the fragments in the determined quantities.", "Mutagenized Oligonucleotides While the optimized directed evolution method can use oligonucleotides that have a 100% fidelity to their parent polynucleotide sequence, this level of fidelity is not required.", "For example, if a set of three related parental polynucleotides are chosen to undergo ligation reassembly in order to create, e.g., a new phenotype, a set of oligonucleotides having unique overlapping regions can be synthesized by conventional methods.", "However a set of mutagenized oligonucleotides could also be synthesized.", "These mutagenized oligonucleotides are preferably designed to encode silent, conservative, or non-conservative amino acids.", "The choice to enter a silent mutation might be made to, for example, add a region of nucleotide homology two fragments, but not affect the final translated protein.", "A non-conservative or conservative substitution is made to determine how such a change alters the function of the resultant polypeptide.", "This can be done if, for example, it is determined that mutations in one particular oligonucleotide fragment were responsible for increasing the activity of a peptide.", "By synthesizing mutagenized oligonucleotides (e.g.", ": those having a different nucleotide sequence than their parent), one can explore, in a controlled manner, how resulting modifications to the peptide or protein sequence affect the activity of the peptide or polypeptide.", "Another method for creating variants of a nucleic acid sequence using mutagenized fragments includes first aligning a plurality of nucleic acid sequences to determine demarcation sites within the variants that are conserved in a majority of said variants, but not conserved in all of said variants.", "A set of first sequence fragments of the conserved nucleic acid sequences are then generated, wherein the fragments bind to one another at the demarcation sites.", "A second set of fragments of the not conserved nucleic acid sequences are then generated by, for example, a nucleic acid synthesizer.", "However, the not conserved, sequences are generated to have mutations at their demarcation site so that the second fragments have the same nucleotide sequence at the demarcation sites as said first fragments.", "This allows the not conserved sequences to still hybridize during the ligation reaction to the other parental sequences.", "Once the fragments are generated, a desired number of crossover events can be selected for each of the variants.", "The quantity of each of the first and second fragments is then calculated so that a ligation/incubation reaction between the calculated quantities of the first and second fragments will result in progeny molecules having the desired number of crossover events.", "In Silico, or Computer, Models In silico, or computer program-implemented, paradigms can be used in practicing the methods of the invention to design altered or new nucleic acids to modify cells for the creation of new phenotypes.", "One exemplary in silico method that can be used in practicing the methods of the invention for generating man-made polynucleotide sequences for the creation of new phenotypes detects shared domains between a plurality of template polynucleotides.", "It does so by aligning the template polynucleotides and identifying all sequence strings having a certain percentage of homology, e.g., about 75% to 95% sequence identity, that are shared between all of the template polynucleotides.", "This detects shared domains between the template polynucleotides.", "Next, domain sequences are switched from one template polynucleotide with the sequence of a corresponding domain.", "This is repeated until all domains have been switched with a corresponding domain on another template polynucleotide, thereby generating in silico a library of man-made polynucleotide sequences from a set of template polynucleotides.", "In silico, or computer program-implemented, methods can also be used in practicing the methods of the invention to analyze metabolic flux data; see, e.g., Covert (2001) Trends Biochem.", "Sci.", "26(3): 179-186; Jamshidi (2001) Bioinformatics 17(3): 286-287.For example, the quantitative relationship between a primary carbon source (e.g., for bacteria, acetate or succinate) uptake rate, oxygen uptake rate, and maximal cellular growth rate can be modeled in silico, and used complementary to the “real-time” or “on-line” monitoring of the invention, see, e.g., Edwards (2001) Nat.", "Biotechnol.", "19(2): 125-130.The effects of gene deletions in a central metabolic pathway can also be modeled in silico, and used complementary to the “real-time” or “on-line” monitoring of the invention, see, e.g., Edwards (2000) Proc.", "Natl.", "Acad.", "Sci.", "USA 97(10): 5528-5533.Measuring Metabolic Parameters The methods of the invention involve whole cell evolution, or whole cell engineering, of a cell to develop a new cell strain having a new phenotype.", "To detect the new phenotype, at least one metabolic parameter of a modified cell is monitored in the cell in a “real time” or “on-line” time frame.", "In one aspect, a plurality of cells, such as a cell culture, is monitored in “real time” or “on-line.” In one aspect, a plurality of metabolic parameters is monitored in “real time” or “on-line.” Metabolic flux analysis (MFA) is based on a known biochemistry framework.", "A linearly independent metabolic matrix is constructed based on the law of mass conservation and on the pseudo-steady state hypothesis (PSSH) on the intracellular metabolites.", "In practicing the methods of the invention, metabolic networks are established, including the: identity of all pathway substrates, products and intermediary metabolites identity of all the chemical reactions interconverting the pathway metabolites, the stoichiometry of the pathway reactions, identity of all the enzymes catalysing the reactions, the enzyme reaction kinetics, the regulatory interactions between pathway components, e.g.", "allosteric interactions, enzyme-enzyme interactions etc, intracellular compartmentalisation of enzymes or any other supramolecular organisation of the enzymes, and, the presence of any concentration gradients of metabolites, enzymes or effector molecules or diffusion barriers to their movement.", "Once the metabolic network for a given strain is built, mathematic presentation by matrix notion can be introduced to estimate the intracellular metabolic fluxes if the on-line metabolome data is available.", "Metabolic phenotype relies on the changes of the whole metabolic network within a cell.", "Metabolic phenotype relies on the change of pathway utilization with respect to environmental conditions, genetic regulation, developmental state and the genotype, etc.", "In one aspect of the methods of the invention, after the on-line MFA calculation, the dynamic behavior of the cells, their phenotype and other properties are analyzed by investigating the pathway utilization.", "For example, if the glucose supply is increased and the oxygen decreased during the yeast fermentation, the utilization of respiratory pathways will be reduced and/or stopped, and the utilization of the fermentative pathways will dominate.", "Control of physiological state of cell cultures will become possible after the pathway analysis.", "The methods of the invention can help determine how to manipulate the fermentation by determining how to change the substrate supply, temperature, use of inducers, etc.", "to control the physiological state of cells to move along desirable direction.", "In practicing the methods of the invention, the MFA results can also be compared with transcriptome and proteome data to design experiments and protocols for metabolic engineering or gene shuffling, etc.", "In practicing the methods of the invention, any modified or new phenotype can be conferred and detected, including new or improved characteristics in the cell.", "Any aspect of metabolism or growth can be monitored.", "Monitoring Expression of an mRNA Transcript In one aspect of the invention, the engineered phenotype comprises increasing or decreasing the expression of an mRNA transcript or generating new transcripts in a cell.", "mRNA transcript, or message can be detected and quantified by any method known in the art, including, e.g., Northern blots, quantitative amplification reactions, hybridization to arrays, and the like.", "Quantitative amplification reactions include, e.g., quantitative PCR, including, e.g., quantitative reverse transcription polymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or “real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br.", "J. Haematol.", "114: 313-318; Xia (2001) Transplantation 72: 907-914).", "In one aspect of the invention, the engineered phenotype is generated by knocking out expression of a homologous gene.", "The gene's coding sequence or one or more transcriptional control elements can be knocked out, e.g., promoters enhancers.", "Thus, the expression of a transcript can be completely ablated or only decreased.", "In one aspect of the invention, the engineered phenotype comprises increasing the expression of a homologous gene.", "This can be effected by knocking out of a negative control element, including a transcriptional regulatory element acting in cis- or trans-, or, mutagenizing a positive control element.", "As discussed below in detail, one or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array.", "Monitoring Expression of a Polypeptides, Peptides and Amino Acids In one aspect of the invention, the engineered phenotype comprises increasing or decreasing the expression of a polypeptide or generating new polypeptides in a cell.", "Polypeptides, peptides and amino acids can be detected and quantified by any method known in the art, including, e.g., nuclear magnetic resonance (NMR), spectrophotometry, radiography (protein radiolabeling), electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, various immunological methods, e.g.", "immunoprecipitation, immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE), staining with antibodies, fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry, Raman-spectrometry, GC-MS, and LC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, and the like.", "Novel bioactivities can also be screened using methods, or variations thereof, described in U.S. Pat.", "No.", "6,057,103.Furthermore, as discussed below in detail, one or more, or, all the polypeptides of a cell can be measured using a protein array.", "Biosynthetically directed fractional 13C labeling of proteinogenic amino acids can be monitored by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network.", "Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios of the amino acids; see, e.g., Szyperski (1999) Metab.", "Eng.", "1: 189-197.Monitoring the Expression of a Metabolites and Biosynthetic Pathways In one aspect, primary and secondary metabolites are the measured metabolic parameters.", "Any relevant primary and secondary metabolite can be monitored in real time.", "For example, the measured metabolic parameter can comprise an increase or a decrease in a primary or a secondary metabolite.", "The secondary metabolite can be, e.g., a glycerol or a methanol.", "The measured metabolic parameter can comprise an increase or a decrease in an organic acid, such as an acetate, a butyrate, a succinate and an oxaloacetate.", "In one aspect, the metabolic parameter measured comprises an increase or a decrease in an organic acid, such as an acetate, a butyrate, a succinate and an oxaloacetate.", "The choice of which metabolite or metabolic or biosynthetic pathway to monitor “on-line” or in “real time” depends on which phenotype is desired to be added or modified.", "For example, limonene and other downstream metabolites of geranyl pyrophosphate can be monitored “on-line” or in “real time” as in U.S. Pat.", "No.", "6,291,745, which monitored to generate means for insect control in plants, see, e.g., Metabolites/antibiotics in the supernatant in Bacillus subtilis can be monitored for effective insecticidal, antifungal and antibacterial agents, see, e.g., U.S. Pat.", "No.", "6,291,426.The methods of the invention can also be used to monitor metabolites of the tricarboxylic acid cycle and glycolysis, as in a Bacillus subtilis strain by Sauer (1997) Nat.", "Biotechnol.", "15: 448-452 (who also used fractional 13C-labeling and two-dimensional nuclear magnetic resonance spectroscopy).", "The penicillin biosynthetic pathway can be monitored in real time in, e.g., Penicillium chrysogenum; see, e.g., Nielsen (1995) Biotechnol.", "Prog.", "11(3): 299-305; Jorgensen (1995) Appl.", "Microbiol.", "Biotechnol.", "43(1): 123-130.Asparagine linked (N-linked) glycosylation can be studied in real time; see, e.g., Nyberg (1999) Biotechnol.", "Bioeng.", "62(3): 336-347.The amount of amino acids liberated from peptides in cell cultures grown in a hydrolysate-supplemented medium can be studied in real time; see, e.g., Nyberg (1999) Biotechnol.", "Bioeng.", "62(3): 324-335, who studies pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium.", "The methods of the invention can also be used to monitor flux distributions for maximal ATP production in mitochondria, including ATP yields for glucose, lactate, and palmitate; see, e.g., Ramakrishna (2001) Am.", "J. Physiol.", "Regul.", "Integr.", "Comp.", "Physiol.", "280(3): R695-704.In bacteria, the methods of the invention can also be used to monitor seven essential reactions in the central metabolic pathways, glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, for the growth in a glucose medium, e.g., glucose minimal media.", "For gene modification, the seven genes encoding these enzymes can be grouped into three categories: (1) pentose phosphate pathway genes, (2) three-carbon glycolytic genes, and (3) tricarboxylic acid cycle genes.", "See, e.g., Edwards (2000) Biotechnol.", "Prog.", "16(6): 927-939.Monitoring Intracellular pH In one aspect, the increase or a decrease in intracellular pH is measured “on-line” or in “real time.” The change in intracellular pH can be measured by intracellular application of a dye.", "The change in fluorescence of the dye can be measured over time.", "Any system can be used to determine intracellular pH.", "If a dye if used, in one exemplary method, whole-field time-domain fluorescence lifetime imaging (FLIM) can be used.", "FLIM can be used for the quantitative imaging of concentration ratios of mixed fluorophores and quantitative imaging of perturbations to fluorophore environment; in FLIM, the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image (see, e.g., Cole (2001) J. Microsc.", "203(Pt 3): 246-257).", "Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique that can be used to obtain simultaneous optical and topographic images with spatial resolution of tens of nanometers (see, e.g., Kwak (2001) Anal.", "Chem.", "73(14): 3257-3262).", "A frequency domain fluorescence lifetime imaging microscope (FLIM) enables the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images (see, e.g., Squire (1999) J. Microsc.", "193(Pt 1): 3649).", "Monitoring Expression of Gases In one aspect, the measured metabolic parameter comprises gas exchange rate measurements.", "Any gas can be monitored, e.g., oxygen, carbon monoxide, carbon dioxide, nitrogen and the like.", "See, e.g., Follstad (1999) Biotechnol.", "Bioeng.", "63(6): 675-683.Screening Methodologies and “On-Line” Monitoring Devices In practicing the methods of the invention, “real time” or “on-line” cell monitoring devices are used to identify an engineered phenotype in the cell using real-time metabolic flux analysis.", "Any screening method can be used in conjunction with these “real time” or “on-line” cell monitoring devices.", "Cell Growth Monitor Devices In one aspect, real time monitoring of a plurality of metabolic parameters is done with use of a cell growth monitor device.", "One exemplary such device is a Wedgewood Technology, Inc. (San Carlos, Calif.), Cell Growth Monitor model 652, which can “real time” or “on-line” monitor a variety of metabolic parameters, including: the uptake of substrates, such as glucose; the levels of intracellular intermediates, such as' organic acids, e.g., acetate, butyrate, succinate, oxaloacetate; and, levels of amino acids.", "Any cell growth monitor device can be used, and these devices can be modified to measure any set of parameters, without limitation.", "Cell growth monitor device can be used in conjunction with any other measuring or monitoring devices, such as There are some rapid analysis of metabolites at the whole-cell level, using methods such as pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, and LC-Electrospray and cap-LC-tandem-electrospray mass spectrometries.", "Capillary Arrays In addition to “biochip” arrays (see below), capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, San Diego, Calif., can be used to screen for or monitor a variety of compositions, including polypeptides, nucleic acids, metabolites, by-products, antibiotics, metals, and the like, without limitation.", "Capillary arrays provide another system for holding and screening samples.", "For example, a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample.", "The apparatus can further include interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material.", "A capillary for screening a sample, wherein the capillary is adapted for being bound in an array of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.", "A polypeptide or nucleic acid, e.g., a ligand, can be introduced into a first component into at least a portion of a capillary of a capillary array.", "Each capillary of the capillary array can comprise at least one wall defining a lumen for retaining the first component, and introducing an air bubble into the capillary behind the first component.", "A second component can be introduced into the capillary, wherein the second component is separated from the first component by the air bubble.", "A sample of interest can be introduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall.", "The method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.", "The capillary array can include a plurality of individual capillaries comprising at least one outer wall defining a lumen.", "The outer wall of the capillary can be one or more walls fused together.", "Similarly, the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample.", "The capillaries of the capillary array can be held together in close proximity to form a planar structure.", "The capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by-side.", "The capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries.", "A capillary array can form a microtiter plate having about 100,000 or more individual capillaries bound together.", "Arrays, or “BioChips” In one aspect of the invention, the monitored parameter is transcript expression.", "One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or “biochip.” By using an “array” of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified.", "Arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention.", "“Polypeptide arrays” can also be used to simultaneously quantify a plurality of proteins.", "The present invention can be practiced with any known “array,” also referred to as a “microarray” or “nucleic acid array” or “polypeptide array” or “antibody array” or “biochip,” or variation thereof.", "Arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.", "In practicing the methods of the invention, known arrays and methods of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat.", "Nos.", "6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr.", "Biol.", "8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20: 399-407; Bowtell (1999) Nature Genetics Supp.", "21: 25-32.See also published U.S. patent applications Nos.", "20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.The present invention can use any known array, e.g., GeneChips™, Affymetrix, Santa Clara, Calif.; SpectralChip™ Human BAC Arrays, Spectral Genomics, Houston, Tex.", "; and their accompanying manufacturer's instructions.", "Antibodies and Immunoblots In practicing the methods of the invention, antibodies can be used to isolate, identify or quantify particular polypeptides or polysaccharides.", "The antibodies can be used in immunoprecipitation, staining (e.g., FACS), immunoaffinity columns, and the like.", "If desired, nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.", "Alternatively, the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.", "Furthermore, the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.", "Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.)", "BASIC AND CLINICAL IMMUNOLOGY (7th ed.)", "Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.)", "Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256: 495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York.", "Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals.", "See, e.g., Hoogenboom (1997) Trends Biotechnol.", "15: 62-70; Katz (1997) Annu.", "Rev.", "Biophys.", "Biomol.", "Struct.", "26: 27-45.Sources of Cells and Culturing of Cells The invention provides a method for whole cell engineering of new phenotypes by using real-time metabolic flux analysis.", "Any cell can be engineered, including, e.g., bacterial, Archaebacteria, mammalian, yeast, fungi, insect or plant cell.", "In one aspect of the methods of the invention, a cell is modified by addition of a heterologous nucleic acid into the cell.", "The heterologous nucleic acid can be isolated, cloned or reproduced from a nucleic acid from any source, including any bacterial, mammalian, yeast, insect or plant cell.", "In one aspect, the cell can be from a tissue or fluid taken from an individual, e.g., a patient.", "The cell can be homologous, e.g., a human cell taken from a patient, or, heterologous, e.g., a bacterial or yeast cell taken from the gastrointestinal tract of an individual.", "The cell can be from, e.g., lymphatic or lymph node samples, serum, blood, chord blood, CSF or bone marrow aspirations, fecal samples, saliva, tears, tissue and surgical biopsies, needle or punch biopsies, and the like.", "Any apparatus to grow or maintain cells can be used, e.g., a bioreactor or a fermentor, see, e.g., U.S. Pat.", "Nos.", "6,242,248; 6,228,607; 6,218,182; 6,174,720; 6,168,949; 6,133,022; 6,133,021; 6,048,721; 5,660,977; 5,075,234.Real-time Metabolic Flux Analysis In the methods of the invention, at least one metabolic parameter of the cell is monitored in real time, i.e., by real time, or “on-line,” flux analysis.", "In alternative aspects, many parameters of the cells in culture are monitored simultaneously in real time.", "Because of the real-time distribution of substrates, intermediates and products between alternative metabolic pathways is not accessible by the usual analytical means, the present invention incorporates an MFA method with “on-line” or “real-time” metabolome data.", "Therefore, by calculation, the metabolic flux distributions during the fermentation can be quantified.", "The flux quantification and gene expression analysis, along with sophisticated experimental techniques, can be combined to upgrade the content of information in the physiological and genomic/proteomic data towards the unraveling of cellular function and regulation.", "This allows insight into metabolic pathways, which is highly desirable and necessary in order to understand the behavior of the organism.", "Metabolic Flux Analysis (MFA) is an analysis technique for metabolic engineering.", "It has been used in connection with studies of cell metabolism where the aim is to direct as much carbon as possible from the substrate into the biomass and products.", "Example 1, below, generally describes an exemplary Metabolic Flux Analysis (MFA) that can be used in the methods of the invention.", "“Metabolomics” is a relatively unexplored field and can encompass the analysis of all cellular metabolites.", "Metabolomics provides a powerful new tool for gaining insight into functional biology, and has provided snapshots of the levels of numerous small molecules within a cell, and how those levels change under different conditions.", "These studies are very complementary to gene and polypeptide expression studies (genomics and proteomics), which are actively being applied to studies of infectious diseases, production, and model organisms, as well as human cells and plants.", "The present invention provides an improved methodology to study “metabolomics” by providing a method for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis.", "In practicing the methods of the invention, cellular control can be studied at different hierarchical levels, at the level of the genome, at the level of the transcriptome, at the level of the proteome or at the level of the metabolome.", "Whilst there is much current interest in the genome-wide analysis of cells at the level of transcription (to define the ‘transcriptome’) and translation (to define the ‘proteome’), the third level of analysis, that of the ‘metabolome’, has been curiously unexplored to date.", "The term ‘metabolome’ refers to the entire complement of all the small molecular weight metabolites inside a cell suspension (or other sample) of interest.", "It is likely that measurement of the metabolome in different physiological states, particularly using the methods of the invention, will in fact be much more discriminating for the purposes of functional genomics.", "The genome (the total genetic material in the cell) specifies an organism's total repertoire of responses.", "The genomes of several organisms have now been completely sequenced and several others are near completion or well under way (including a number of parasites).", "Of the genes so far sequenced via the systematic genome sequencing programs, the functions of fewer than half are known with any confidence.", "Technological advances now allow gene expression at any particular stage of development or in any particular physiological state to be analyzed.", "Such analyses can be carried out at the level of transcription using either Northern blots or, more efficiently, using hybridization array technologies to determine which genes are being expressed under different sets of conditions, i.e., the “transcriptome.” Similar analyses can be carried out at the level of translation to define the “proteome,” i.e., the total protein complement of the cell.", "Improvements in 2D electrophoresis and computer software for advanced image analysis allow 1-2×103 proteins to be resolved on a single 20×20 cm plate; and, mass spectrometry coupled with database searching provides a method for rapid protein identification.", "Changes in the transcriptome represent the initial response of a cell to change, while changes in the proteome represent the final response at the level of the macromolecule.", "The third level of analysis, and one analyzed by the methods of the invention, is that of the “metabolome,” which includes the quantitative complement of all the low molecular weight molecules present in cells in a particular physiological or developmental state.", "Metabolite levels, which are monitored in alternative aspects of the invention, are thus the variables of choice to measure in a quantitative analysis of cellular function.", "Metabolites represent the down stream amplification of changes occurring in the transcriptome or the proteome.", "Moreover, metabolites regulate gene expression through a network of feedback pathways such that metabolites drive expression and act as the link between the genome and metabolism.", "The number of metabolites in the metabolome is also lower, by about an order of magnitude than the number of gene products in the transcriptome or the proteome (a typical eukaryotic cell contains around 105 genes and 104 different expressed proteins but only about 103 different known metabolites).", "Therefore, in order to understand intermediary metabolism and to exploit this knowledge changes in the metabolome are much more relevant and will be much easier both to detect and to exploit than changes either in the transcriptome or the proteome.", "The methods of the invention, by identifying sites of specific metabolic lesions via the metabolome, in addition to its inherent scientific interest, will lead to the detection of targets for potentially novel pharmaceuticals or agrochemicals in whole cells.", "The methods of the invention can also be used to design functional assays.", "From these results, they can enable the design of very much simpler assays in which only the targeted metabolites are studied for specific high throughput, mechanistic assays.", "The metabolome analysis of the invention has the advantage of being an online non-invasive technology.", "While static metabolome analysis has some advantages over transcriptome and proteome analysis because, for many organisms, the number of metabolites was far fewer than the number of genes or proteins.", "However, static metabolome analysis had an intrinsic disadvantage as well.", "This was that while biochemistry could generate information about the metabolic pathways, there is no direct link between the metabolites and the genes.", "They were also problems in analysing the concentration or even the very presence of certain metabolites.", "Current identification technologies such as infra-red spectrometry, mass spectrometry, or nuclear magnetic resonance spectroscopy produced some information but their use was limited and could not properly analyze a living cell.", "The methods of the invention, by providing “online” or “real-time” non-invasive technology solved this problem.", "The “online” or “real-time” time dimension of the methods of the invention, lacking in older techniques is one important factor in the methods ability to analyze a living cell.", "Metabolic flux analysis (MFA) is a powerful analysis tool that can couple observed extracellular phenomena, such as uptake/excretion rates, growth rate, product and biomass yields, etc., with the intracellular carbon flux and energy distribution.", "The “on-line” or “real-time” MFA of the invention can be used to investigate the physiology of Escherichia coli, Saccharomyces cerevisiae, and hybridomas (see, e.g., Keasling (1998) Biotechnol.", "Bioeng.", "5; 58(2-3): 231-239; Pramanik (1998) Biotechnol.", "Bioeng.", "60(2): 230-238; Nissen et al., 1997; Schulze et al., 1996; Follstad et al., 1999), lysine production and the effect of mutations in Corynebacterium glutamicum (see, e.g., Vallino (2000) Biotechnol.", "Bioeng.", "67(6): 872-885; Vallino and Stephanopoulos, 1993, 1994; Park et al., 1997; Dominguez (1998) Eur.", "J. Biochem.", "254(1): 96-102), riboflavin production in Bacillus subtilis (see, e.g., Sauer et al., 1996, 1998; Sauer (1997) Nat.", "Biotechnol.", "15: 448-452), penicillin production in Penicillium chrysogenum (Nielsen (1995) Biotechnol.", "Prog.", "11(3): 299-305; Jorgensen (1995) Appl.", "Microbiol.", "Biotechnol.", "43(1): 123-130); and, peptide amino acid metabolism in Chinese hamster ovary (CHO) cells (see, e.g., Nyberg (1999) Biotechnol.", "Bioeng.", "62(3): 324-335; Nyberg (1999) Biotechnol.", "Bioeng.", "62(3): 336-347).", "Moreover, the “on-line” or “real-time” MFA of the invention can be used in combination with NMR, MS, and/or GC-MS to yield hard to get information about futile cycles, the degree of reaction reversibility, as well as active pathways; see, e.g., Szyperski (1999) Metab.", "Eng.", "1: 189-197; Szyperski (1998) Q Rev.", "Biophys.", "31: 41-106; Szyperski (1995) Eur.", "J. Biochem.", "232(2): 433-448; Szyperski et al., 1997; Schmidt et al., 1998; Klapa (1999) Biotechnol.", "Bioeng.", "62(4): 375-391; Mollney et al., 1999; Park et al., 1999; Wiechert et al., 1999; Wittmann and Heinzle, 1999.Schilling, Edwards, and Palsson have even extended the use of MFA to include the analysis of genomic data and the structural properties of cellular networks (Schilling (2000-2001) Biotechnol.", "Bioeng.", "71 (4): 286-306; Edwards and Palsson, 1998; Schilling et al., 1999a,b); to monitor the C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms (see, e.g., Petersen (2000) J. Biol.", "Chem.", "275(46): 35932-35941).", "In MFA, the intracellular fluxes are calculated using a stoichiometric model for all the major intracellular reactions and by applying mass balances around the intracellular metabolites.", "As input to the calculations, a set of measured fluxes, typically the uptake rates of substrates and secretion rates of metabolites is used.", "The novel “real-time” or “on-line” metabolic flux analysis of the invention can provide data regarding a full suite of metabolites synthesized by a biological system under given environmental conditions and/or with genetic regulation.", "The “real-time” or “on-line” MFA methods of the invention can provide metabolomic data sets that are extremely complex.", "The MFA methods of the invention can be an adequate tool to handle, store, normalize, and evaluate the acquired data in order to describe the systemic response of a complex biological system.", "The FIG.", "1 is a schematic illustrating the invention's new application of MFA to determine new phenotypes, pathway utilizations and cell responses to the studied strains during actual cell culture or fermentation periods.", "The results can be either used for post-fermentation analysis, or immediate control of the metabolism.", "The “on-line,” or “real-time” methods of the invention can also incorporate other analytical devices, such as HPLC and GC/MS, to estimate flux distribution in metabolic networks (constructed with our biochemical knowledge and genomic/proteomic information database) from experimental measurements.", "With these devices, “snapshots” of the biological systems under study can be obtained periodically, e.g., about every 1, 5, 10, 15, 20, 25, or 30 minutes, depending on the number of metabolic parameters studied and number of devices used.", "Vector r for Metabolome Data The on-line MFA of the invention uses “rate of change” data, or the difference between current metabolic measurements and last measurements.", "The differences are calculated and stored in the “raw measurement” vector for error analysis before they can be used.", "Thus, in one aspect, a “preprocessing unit” is used to filter out the errors for the measurement before the metabolic flux analysis to make sure that quality data be used.", "See Example 1, below.", "Computer Systems In one aspect, the methods of the invention use computer-implemented methods/programs to real time monitor the change in measured metabolic parameters over time.", "The methods of the invention can be practiced using any program language or computer/processor and in conjunction with any known software or methodology.", "For example, one of the programs called MATHEMATICA™ (Wolfram Research, Inc., Champaign, Ill.), such as MATHEMATICA 4.1™, or variations thereof, can be used, see Example 1, below; and, see also, e.g., Jamshidi (2001) Bioinformatics 17(3): 286-287; Wilson (2001) Biophys.", "Chem.", "91(3): 281-304; Torrecilla (2001) J. Neurochem.", "76(5): 1291-1307.The computer/processor used to practice the methods of the invention can be a conventional general-purpose digital computer, e.g., a personal “workstation” computer, including conventional elements such as microprocessor and data transfer bus.", "The computer/processor can further include any form of memory elements, such as dynamic random access memory, flash memory or the like, or mass storage such as magnetic disc optional storage.", "For example, a conventional personal computer such as those based on an Intel microprocessor and running a Windows operating system can be used.", "Any hardware or software configuration can be used to practice the methods of the invention.", "For example, computers based on other well-known microprocessors and running operating system software such as UNIX, Linux, MacOS and others are contemplated.", "EXAMPLES The following examples are offered to illustrate, but not to limit the claimed invention.", "Example 1 Metabolic Flux Analysis (MFA) The following example describes implementation of an exemplary Metabolic Flux Analysis (MFA), which is applied in the real time analysis of cell cultures in the methods of the invention.", "FIG.", "1.Metabolic Flux Analysis (MFA) is important analysis technique of metabolic engineering.", "A flux balance can be written for each metabolite (yi) within a metabolic system to yield the dynamic mass balance equations that interconnect the various metabolites.", "Generally, for a metabolic network that contains m compounds and n metabolic fluxes, all the transient material balances can be represented by a single matrix equation: dY/dt=AX(t)−r(t) where Y: m dimensional vector of metabolite amounts per cell X: n metabolic fluxes A: Stoichiometric m×n matrix, and r: vector of specific rates from measurements The time constants characterizing metabolic transients are typically very rapid compared to the time constants of cell growth and process dynamics, therefore, the mass balances can be simplified to only consider the steady-state behavior.", "Eliminating the derivative yields: AX(t)=r(t).", "Provided that m>=n and A is full rank, the weighted least squares solution of the above equation is: X=(ATA)−1ATr.", "The sensitivity of the solution can be investigated by the matrix: dX/dr=(ATA)−1AT.", "The elements of the above matrix are useful for the determination of the change of individual fluxes with respect to the error or perturbation in the measurements.", "Inputs Stoichiometric Equations A stoichiometry matrix is derived from the chemical equations to be used in the analysis.", "The matrix consists of coefficients of chemical species involved in the reactions.", "Rows represent the species and columns represent the equations.", "For instance, if we consider the equations of energy production in cells: 2NADH+O2+6ADP→2NAD+2H2O+6ATP 2FADH+O2+4ADP→2FAD+2H2O+4ATP ATP→ADP This system yields a stoichiometry matrix with 3 columns and as many rows as species to be considered in the overall system.", "In this case, 8 species are considered so the NADH −2 0 0 O2 −1 −1 0 NAD 2 0 0 H2O 2 2 0 FADH 0 −2 0 FAD 0 2 0 ATP 6 4 −1 ADP −6 −4 1 matrix is 3×8.Using these templates, the stoichiometric matrix is 35×33, and it is in the EXCEL 97™ file “stoichiex.xls”.", "This is the matrix ‘A’ described above, and it is derived from the 33 chemical equations below.", "1.Central Metabolic Pathways GLC+ATP+NAD→2PYR+ADP+NADH+H2O 1) PYR+NADH→LAC+NAD 2) PYR+NAD→ACCOA+CO2+NADH 3) ACCOA+OAA+NAD+H2O→AKG+CO2+NADH 4) AKG+NAD→SUCCOA+C62+NADH 5) SUCCOA+ADP+H2O+FAD→FUM+ATP+FADH 6) FUM+H2O→MAL 7) MAL+NAD→OAA+NADH 8) GLN+ADP→GLU+NH3+ATP 9) GLU+NAD→AKG+NH3+NADH 10) MAL→PYR+CO2 11) 2.Biomass Synthesis: C50.5% H8.31% 032.93% N8.26% 0.1016 GLC+0.031 GLN+0.008 ARG+0.0003 ASN+0.001 GLU+0.0038 GLY+0.0028 HIS+0.0071 ILE+).008 LEU+).0043 LYS+0.001 MET+0.0152 THR+).0051 VAL→BIOMASS 12) 3.Amino Acid Metabolism PYR+GLU→ALA+AKG 13) SER→PYR+NH3 14) GLY→SER 15) CYS→PYR+NH3 16) ASP+AKG→OAA+GLU 17) ASN→ASP+NH3 18) HIS→GLU+NH3 19) ARG+AKG→2 GLU 20) PRO→GLU 21) ILE+AKG→SUCCOA+ACCOA+GLU 22) VAL+AKG→GLU+CO2+SUCCOA 23) MET→SUCCOA 24) THR→SUCCOA+NH3 25) PHE→TYR 26) TYR+AKG→GLU+FUM+2 ACCOA 27) LYS+2AKG→2GLU+2CO2+2ACCOA 28) LEU+AKG→GLU+3 ACCOA 29) 4.Antibody Formation: 1.05 ARG+1.98 ASN+1.96 ASP+1.42 GLU+1.31 GLY+1.59 ILE+3.79 LEU+1.97 LYS+0.67 MET+0.95 PHE+5.72 SER 1.32 THR 5.05 TYR+2.68 VAL→Ab 30) 5.Energy Production: 2NADH+O2+6ADP→2NAD+2H2O+6ATP 31) 2 FADH+O2+4 ADP→2 FAD+2H20+4 ATP 32) ATP→ADP 33) In order to use this matrix with other mathematics software, it must be converted to a text file.", "Highlight only the cells that contain numbers, select copy from the Edit menu, and paste into a notepad (or simple text editor) document, e.g., the “Notepad” text editor program that comes with Microsoft Windows™ 3.11, 95 and NT.", "The file can be saved in a notepad as a text file “*.txt”.", "Specific Uptake Rates The specific uptake rates are calculated from data from a cell culture reactor.", "This data should also be in a text file as a vector of rates, r, that correspond to the appropriate chemical species, i.e.", "the rows in the stoichiometry matrix above.", "In the provided templates, the specific rates are listed in the EXCEL 97™ file “ratex.xls” as well as a text file (exported from Excel) “rate.txt”.", "Calculations With the inputs in the desired form, it is now time to use a mathematics software package to calculate the estimated internal fluxes.", "This software should be able to handle matrix math and differential equations.", "One template was made in MATHEMATICA™ 3.0 and is named “mfamath.nb”.", "The following section assumes that the calculations are done in MATHEMATICA™ 3.0, but the general procedure can be applied with any suitable package.", "Read in Data First the default directory is set using the SetDirectory command: example: SetDirectory [“a:\\mfa\\”] The data is then read in and saved into the A matrix (for the stoichiometry matrix) and the r vector (for the specific rates).", "example:A=ReadList [“stoichi.txt, Number, RecordLists-->True] r=ReadList[“rate.txt, Number, RecordLists-->True] Sensitivity Analysis Next, the sensitivity matrix (dX/dr) is calculated as (ATA)−1AT.", "example: sens=Inverse [Transpose [A].", "A].", "Transpose [A] Solution and Error Analysis The least squares estimation of the flux distributions, x, and the errors, e, are calculated for the over-determined system of equations.", "example: x=sens.r e=r−A.x Output of Results After calculation of the flux estimations, the results must be written to text files for presentation.", "In the templates provided, 3 results text files are included.", "These files are “flux.txt” that contains the x vector, “error.txt” that holds the error vector, and “sensitivity.txt” that contains the sensitivity matrix.", "An example of creating these text files in MATHEMATICA™ is shown below.", "Example: a1=Openwrite[“flux.txt”.", "FormatType->OutputForm]; Write[a1, TableForm[x, TableSpacing->{0,1}]]; Close[a1] Presentation of Results A critical aspect of this analysis is the efficient and clear presentation of the large number of estimated fluxes.", "The output text files from MATHEMATICA™ can be imported into Excel, and the solution can be plotted as a collection of bar graphs.", "The EXCEL 97™ file “mfaexc.xls” is the template provided that shows the table of data and the bar graphs for each flux.", "It also contains a composite bar graph that plots the fluxes together and grouped by metabolic pathway (see below).", "An additional way to present the data is to show all the internal fluxes overlain on a map of the relevant metabolic pathways.", "The POWERPOINT™ template file “mfa.ppt” shows a metabolic map with bar graphs (linked to the Excel file “mfaexc.xls” which must be opened before the file “mfa.ppt”) to show the magnitude of the fluxes.", "There exists a linking between the Excel file and the POWERPOINT™ presentation.", "When the data in Excel is updated, the linking in the presentation should be updated.", "Devices to Monitor Organic Acids and Amino Acids On-line devices that can monitor organic acids and amino acids can also be used in practicing the methods of the invention.", "For example, in one aspect, the BIO+ON-LINE™ (Lachat Instruments, Milwaukee, Wis.) provides near-real-time monitoring of fermentation and mammalian cell culture processes.", "This device can provide critical information to maximize product yields.", "Mounted on a cart, this device can be rolled up to a fermentation bank and connected via a stream selector valve.", "From there, chemical constituent monitoring occurs automatically for ammonia, glucose, glutamate, glutamine, glycerol, lactate and phosphate individually and organic acids as a profile employing ion exclusion chromatography.", "The BIO+ON-LINE™ is an integrated sampling system that provides a real solution to this challenging problem using a pumping system combined with a FLOWNAMICS® filter probe which exhibits the following benefits: sterilizable in-place; risk-free sampling due to elimination of bypass filters which recirculate material back into the vessel; sterile, cell-free sampling; accommodates all vessel sizes; minimum dead volume to ensure consistent and accurate sampling and to reduce flush time; durable design and construction to withstand temperatures, pressures, viscosities, shear forces and chemical constituents typical of bioprocess environments.", "The BIO+ON-LINE™ can determine up to four analytes simultaneously using flow injection analysis.", "The reaction modules can be removed and substituted with other modules.", "Thus, the user can customize the unit for different fermentation/bioprocess requirements.", "Additionally, the Ion Chromatography channel can be customized to meet other Liquid Chromatography (LC) needs.", "While conductivity detection is the default detector, users can connect UV, RI, or other detectors and their own columns to the unit to meet their customized LC separation needs.", "This system, or variations thereof, is applicable to aerobic and anaerobic bacterial cultures as well as yeast, fungi, algae, insect and mammalian cell cultures.", "Other related devices that can be used to practice the invention include the QUIKCHEM® 8000 (Lachat Instruments, Milwaukee, Wis.) which allows high sample throughput coupled with simple and rapid method changeover to maximize productivity in determining ionic species in a diversity of sample matrices from sub-ppb to percent concentrations.", "One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned as well as those inherent therein.", "The methods described herein are presently representative of exemplary aspects and are not intended as limitations on the scope of the invention.", "Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the claims.", "3.MODIFYING: DIRECTED EVOLUTION METHODS In one aspect the invention described herein is directed to the use of repeated cycles of reductive reassortment, recombination and selection which allow for the directed molecular evolution of highly complex linear sequences, such as DNA, RNA or proteins thorough recombination.", "In vivo shuffling of molecules can be performed utilizing the natural property of cells to recombine multimers.", "While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules.", "The formation of the chiasma requires the recognition of homologous sequences.", "In a preferred embodiment, the invention relates to a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide.", "The present invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.", "The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide.", "The term “hybrid polynucleotide”, as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences.", "Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules.", "In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.", "The invention provides a means for generating hybrid polynucleotides which may encode biologically active hybrid polypeptides.", "In one aspect, the original polynucleotides encode biologically active polypeptides.", "The method of the invention produces new hybrid polypeptides by utilizing cellular processes which integrate the sequence of the original polynucleotides such that the resulting hybrid polynucleotide encodes a polypeptide demonstrating activities derived from the original biologically active polypeptides.", "For example, the original polynucleotides may encode a particular enzyme from different microorganisms.", "An enzyme encoded by a first polynucleotide from one organism may, for example, function effectively under a particular environmental condition, e.g.", "high salinity.", "An enzyme encoded by a second polynucleotide from a different organism may function effectively under a different environmental condition, such as extremely high temperatures.", "A hybrid polynucleotide containing sequences from the first and second original polynucleotides may encode an enzyme which exhibits characteristics of both enzymes encoded by the original polynucleotides.", "Thus, the enzyme encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the enzymes encoded by the first and second polynucleotides, e.g., high salinity and extreme temperatures.", "Enzymes encoded by the original polynucleotides of the invention include, but are not limited to; oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.", "A hybrid polypeptide resulting from the method of the invention may exhibit specialized enzyme activity not displayed in the original enzymes.", "For example, following recombination and/or reductive reassortment of polynucleotides encoding hydrolase activities, the resulting hybrid polypeptide encoded by a hybrid polynucleotide can be screened for specialized hydrolase activities obtained from each of the original enzymes, i.e.", "the type of bond on which the hydrolase acts and the temperature at which the hydrolase functions.", "Thus, for example, the hydrolase may be screened to ascertain those chemical functionalities which distinguish the hybrid hydrolase from the original hydrolyases, such as: (a) amide (peptide bonds), i.e.", "proteases; (b) ester bonds, i.e.", "esterases and lipases; (c) acetals, i.e., glycosidases and, for example, the temperature, pH or salt concentration at which the hybrid polypeptide functions.", "Sources of the original polynucleotides may be isolated from individual organisms (“isolates”), collections of organisms that have been grown in defined media (“enrichment cultures”), or, most preferably, uncultivated organisms (“environmental samples”).", "The use of a culture-independent approach to derive polynucleotides encoding novel bioactivities from environmental samples is most preferable since it allows one to access untapped resources of biodiversity.", "“Environmental libraries” are generated from environmental samples and represent the collective genomes of naturally occurring organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts.", "Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture.", "Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample.", "This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample which may be under-represented by several orders of magnitude compared to the dominant species.", "For example, gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest.", "Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries.", "Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell.", "The host cell is grown under conditions which promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities.", "The microorganisms from which the polynucleotide may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae and protozoa.", "Polynucleotides may be isolated from environmental samples in which case the nucleic acid may be recovered without culturing of an organism or recovered from one or more cultured organisms.", "In one aspect, such microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.", "Polynucleotides encoding enzymes isolated from extremophilic microorganisms are particularly preferred.", "Such enzymes may function at temperatures above 100° C. in terrestrial hot springs and deep sea thermal vents, at temperatures below 0° C. in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge.", "For example, several esterases and lipases cloned and expressed from extremophilic organisms show high activity throughout a wide range of temperatures and pHs.", "Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell.", "A suitable host cell is any cell which is capable of promoting recombination and/or reductive reassortment.", "The selected polynucleotides are preferably already in a vector which includes appropriate control sequences.", "The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bacterial cell.", "Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al, 1986).", "As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera S19; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells.", "The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.", "With particular references to various mammalian cell culture systems that can be employed to express recombinant protein, examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described in “SV40-transformed simian cells support the replication of early SV40 mutants” (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.", "Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.", "DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.", "Host cells containing the polynucleotides of interest can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes.", "The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.", "The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity.", "In another aspect, it is envisioned the method of the present invention can be used to generate novel polynucleotides encoding biochemical pathways from one or more operons or gene clusters or portions thereof.", "For example, bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes.", "The genes are clustered, in structures referred to as “gene clusters,” on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster.", "Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.", "An example of a biochemical pathway encoded by gene clusters are polyketides.", "Polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracyclines and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin).", "Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents.", "Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of an enormous variety of carbon chains differing in length and patterns of functionality and cyclization.", "Polyketide synthase genes fall into gene clusters and at least one type (designated type 1) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins.", "The ability to select and combine desired components from a library of polyketides, or fragments thereof, and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing.", "The method of the present invention makes it possible to facilitate the production of novel polyketide synthases through intermolecular recombination.", "Preferably, gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related array activity from the ligated gene clusters.", "Use of vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E. coli.", "This f-factor of E. coli is a plasmid which affect high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples.", "Once ligated into an appropriate vector, two or more vectors containing different polyketide synthase gene clusters can be introduced into a suitable host cell.", "Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster.", "The novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.", "Therefore, in a preferred embodiment, the present invention relates to a method for producing a biologically active hybrid polypeptide and screening such a polypeptide for enhanced activity by: 1) introducing at least a first polynucleotide in operable linkage and a second polynucleotide in operable linkage, said at least first polynucleotide and second polynucleotide sharing at least one region of partial sequence homology, into a suitable host cell; 2) growing the host cell under conditions which promote sequence reorganization resulting in a hybrid polynucleotide in operable linkage; 3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide; 4) screening the hybrid polypeptide under conditions which promote identification of enhanced biological activity; and 5) isolating the a polynucleotide encoding the hybrid polypeptide.", "Methods for screening for various enzyme activities are known to those of skill in the art and discussed throughout the present specification.", "Such methods may be employed when isolating the polypeptides and polynucleotides of the present invention.", "As representative examples of expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g.", "vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus and yeast).", "Thus, for example, the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide.", "Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences.", "Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.", "The following vectors are provided by way of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).", "However, any other plasmid or other vector may be used as long as they are replicable and viable in the host.", "Low copy number or high copy number vectors may be employed with the present invention.", "A preferred type of vector for use in the present invention contains an f-factor origin replication.", "The f-factor (or fertility factor) in E. coli is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself.", "A particularly preferred embodiment is to use cloning vectors, referred to as “fosmids” or bacterial artificial chromosome (BAC) vectors.", "These are derived from E. coli f-factor which is able to stably integrate large segments of genomic DNA.", "When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.” Another preferred type of vector for use in the present invention is shuttle vector that is optimized for the expression of genes and gene clusters.", "Such systems may include but are not limited to shuttling systems that shuttle between E. coli and another bacteria such as Streptomyces.", "Another preferred type of vector for use in the present invention is a cosmid vector.", "Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA.", "Cloning into cosmid vectors is described in detail in “Molecular Cloning A laboratory Manual” (Sambrook et al, 1989).", "The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct RNA synthesis.", "Particular named bacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, PL and trp.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1.Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.", "The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.", "The vector may also include appropriate sequences for amplifying expression.", "Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.", "In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.", "Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.", "Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), -factor, acid phosphatase, or heat shock proteins, among others.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.", "The cloning strategy permits expression via both vector driven and endogenous promoters; vector promotion may be important with expression of genes whose endogenous promoter will not function in E. coli.", "The DNA isolated or derived from microorganisms can preferably be inserted into a vector or a plasmid prior to probing for selected DNA.", "Such vectors or plasmids are preferably those containing expression regulatory sequences, including promoters, enhancers and the like.", "Such polynucleotides can be part of a vector and/or a composition and still be isolated, in that such vector or composition is not, part of its natural environment.", "Particularly preferred phage or plasmid and methods for introduction and packaging into them are described in detail in the protocol set forth herein.", "The selection of the cloning vector depends upon the approach taken, for example, the vector can be any cloning vector with an adequate capacity to multiply repeated copies of a sequence, or multiple sequences that can be successfully transformed and selected in a host cell.", "One example of such a vector is described in “Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts” (Alting-Mecs and Short, 1993).", "Propagation/maintenance can be by an antibiotic resistance carried by the cloning vector.", "After a period of growth, the naturally abbreviated molecules are recovered and identified by size fractionation on a gel or column, or amplified directly.", "The cloning vector utilized may contain a selectable gene that is disrupted by the insertion of the lengthy construct.", "As reductive reassortment progresses, the number of repeated units is reduced and the interrupted gene is again expressed and hence selection for the processed construct can be applied.", "The vector may be an expression/selection vector which will allow for the selection of an expressed product possessing desirable biologically properties.", "The insert may be positioned downstream of a functional promotor and the desirable property screened by appropriate means.", "In vivo reassortment is focused on “inter-molecular” processes collectively referred to as “recombination” which in bacteria, is generally viewed as a “RecA-dependent” phenomenon.", "The present invention can rely on recombination processes of a host cell to recombine and re-assort sequences, or the cells' ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion.", "This process of “reductive reassortment” occurs by an “intra-molecular”, RecA-independent process.", "Therefore, in another aspect of the present invention, novel polynucleotides can be generated by the process of reductive reassortment.", "The method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropriate host cell.", "The reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units.", "The reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel molecular species.", "Various treatments may be applied to enhance the rate of reassortment.", "These could include treatment with ultra-violet light, or DNA damaging chemicals, and/or the use of host cell lines displaying enhanced levels of “genetic instability”.", "Thus the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution.", "Repeated or “quasi-repeated” sequences play a role in genetic instability.", "In the present invention, “quasi-repeats” are repeats that are not restricted to their original unit structure.", "Quasi-repeated units can be presented as an array of sequences in a construct; consecutive units of similar sequences.", "Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level.", "The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi-repeated sequences.", "The quasi-repeated units provide a practically limitless repertoire of templates upon which slippage events can occur.", "The constructs containing the quasi-repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units.", "When the quasi-repeated sequences are all ligated in the same orientation, for instance head to tail or vice versa, the cell cannot distinguish individual units.", "Consequently, the reductive process can occur throughout the sequences.", "In contrast, when for example, the units are presented head to head, rather than head to tail, the inversion delineates the endpoints of the adjacent unit so that deletion formation will favor the loss of discrete units.", "Thus, it is preferable with the present method that the sequences are in the same orientation.", "Random orientation of quasi-repeated sequences will result in the loss of reassortment efficiency, while consistent orientation of the sequences will offer the highest efficiency.", "However, while having fewer of the contiguous sequences in the same orientation decreases the efficiency, it may still provide sufficient elasticity for the effective recovery of novel molecules.", "Constructs can be made with the quasi-repeated sequences in the same orientation to allow higher efficiency.", "Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following: a) Primers that include a poly-A head and poly-T tail which when made single-stranded would provide orientation can be utilized.", "This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNAseH.", "b) Primers that include unique restriction cleavage sites can be utilized.", "Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required.", "c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules.", "The recovery of the re-assorted sequences relies on the identification of cloning vectors with a reduced RI.", "The re-assorted encoding sequences can then be recovered by amplification.", "The products are re-cloned and expressed.", "The recovery of cloning vectors with reduced RI can be effected by: 1) The use of vectors only stably maintained when the construct is reduced in complexity.", "2) The physical recovery of shortened vectors by physical procedures.", "In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on either an agarose gel, or column with a low molecular weight cut off utilizing standard procedures.", "3) The recovery of vectors containing interrupted genes which can be selected when insert size decreases.", "4) The use of direct selection techniques with an expression vector and the appropriate selection.", "Encoding sequences (for example, genes) from related organisms may demonstrate a high degree of homology and encode quite diverse protein products.", "These types of sequences are particularly useful in the present invention as quasi-repeats.", "However, while the examples illustrated below demonstrate the reassortment of nearly identical original encoding sequences (quasi-repeats), this process is not limited to such nearly identical repeats.", "The following example demonstrates the method of the invention.", "Encoding nucleic acid sequences (quasi-repeats) derived from three (3) unique species are depicted.", "Each sequence encodes a protein with a distinct set of properties.", "Each of the sequences differs by a single or a few base pairs at a unique position in the sequence which are designated “A”, “B” and “C”.", "The quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules.", "The number of quasi-repeat units can be controlled by the assembly conditions.", "The average number of quasi-repeated units in a construct is defined as the repetitive index (RI).", "Once formed, the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell.", "The cells are then propagated and “reductive reassortment” is effected.", "The rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired.", "Whether the reduction in RI is mediated by deletion formation between repeated sequences by an “intra-molecular” mechanism, or mediated by recombination-like events through “inter-molecular” mechanisms is immaterial.", "The end result is a reassortment of the molecules into all possible combinations.", "Optionally, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact (e.g., such as catalytic antibodies) with a predetermined macromolecule, such as for example a proteinaceous receptor, peptide oligosaccharide, viron, or other predetermined compound or structure.", "The displayed polypeptides, antibodies, peptidomimetic antibodies, and variable region sequences that are identified from such libraries can be used for therapeutic, diagnostic, research and related purposes (e.g., catalysts, solutes for increasing osmolarity of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or affinity selection.", "The method can be modified such that the step of selecting for a phenotypic characteristic can be other than of binding affinity for a predetermined molecule (e.g., for catalytic activity, stability oxidation resistance, drug resistance, or detectable phenotype conferred upon a host cell).", "The present invention provides a method for generating libraries of displayed antibodies suitable for affinity interactions screening.", "The method comprises (1) obtaining first a plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotide encoding for said displayed antibody and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and (2) introducing said polynucleotides into a suitable host cell and growing the cells under conditions which promote recombination and reductive reassortment resulting in shuffled polynucleotides.", "CDR combinations comprised by the shuffled pool are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed antibodies comprising CDR permutations and suitable for affinity interaction screening.", "Optionally, the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope (antigen) and thereby selecting a plurality of selected shuffled library members.", "Further, the plurality of selectively shuffled library members can be shuffled and screened iteratively, from 1 to about 1000 cycles or as desired until library members having a desired binding affinity are obtained.", "In another aspect of the invention, it is envisioned that prior to or during recombination or reassortment, polynucleotides generated by the method of the present invention can be subjected to agents or processes which promote the introduction of mutations into the original polynucleotides.", "The introduction of such mutations would increase the diversity of resulting hybrid polynucleotides and polypeptides encoded therefrom.", "The agents or processes which promote mutagenesis can include, but are not limited to: (+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine, see Sun and Hurley, 1992); an N-acelylated or deacetylated 4′-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis (see, for example, van de Poll et al, 1992); or a N-acetylated or deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis (see also, van de Poll et al, 1992, pp.", "751-758); trivalent chromium, a trivalent chromium salt, a polycyclic aromatic hydrocarbon (“PAH”) DNA adduct capable of inhibiting DNA replication, such as 7-bromomethyl-benz[a]anthracene (“BMA”), tris(2,3-dibromopropyl)phosphate (“Tris-BP”), 1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA), benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II) halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline (“N-hydroxy-IQ”), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine (“N-hydroxy-PhIP”).", "Especially preferred “means for slowing or halting PCR amplification consist of UV light (+)-CC-1065 and (+)-CC-1065-(N-3-Adenine).", "Particularly encompassed means are DNA adducts or polynucleotides comprising the DNA adducts from the polynucleotides or polynucleotides pool, which can be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing.", "In another aspect, this invention provides for using UV light to mutagenize polynucleotides.", "One use of such a technique is as follows: one microgram samples of template DNA are obtained and treated with U.V.", "light to cause the formation of dimers, including TT dimers, particularly purine dimers.", "U.V.", "exposure is limited so that only a few photoproducts are generated per gene on the template DNA sample.", "Multiple samples are treated with U.V.", "light for varying periods of time to obtain template DNA samples with varying numbers of dimers from U.V.", "exposure.", "A random priming kit which utilizes a non-proofreading polymease (for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems) is utilized to generate different size polynucleotides by priming at random sites on templates which are prepared by U.V.", "light (as described above) and extending along the templates.", "The priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers.", "The dimers formed by U.V.", "exposure serve as a roadblock for the extension by the non-proofreading polymerase.", "Thus, a pool of random size polynucleotides is present after extension with the random primers is finished.", "In another aspect the present invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions according to the present invention which provide for the production of hybrid or re-assorted polynucleotides.", "The invention also provides the use of polynucleotide shuffling to shuffle a population of viral genes (e.g., capsid proteins, spike glycoproteins, polymerases, and proteases) or viral genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses, reoviruses and rhinoviruses).", "In an embodiment, the invention provides a method for shuffling sequences encoding all or portions of immunogenic viral proteins to generate novel combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes which are likely to arise in the natural environment as a consequence of viral evolution; (e.g., such as recombination of influenza virus strains).", "The invention also provides a method suitable for shuffling polynucleotide sequences for generating gene therapy vectors and replication-defective gene therapy constructs, such as may be used for human gene therapy, including but not limited to vaccination vectors for DNA-based vaccination, as well as anti-neoplastic gene therapy and other general therapy formats.", "In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.", "Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction.", "The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the coding RNA transcript are referred to as “downstream sequences”.", "3.1.Saturation Mutagenesis In one aspect, this invention provides for the use of proprietary codon primers (containing a degenerate N,N,G/T sequence) to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position.", "The oligos used are comprised contiguously of a first homologous sequence, a degenerate N,N,G/T sequence, and preferably but not necessarily a second homologous sequence.", "The downstream progeny translational products from the use of such oligos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.", "In one aspect, one such degenerate oligo (comprised of one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions.", "In another aspect, at least two degenerate N,N,G/T cassettes are used—either in the same oligo or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions.", "Thus, more than one N,N,G/T sequence can be contained in one oligo to introduce amino acid mutations at more than one site.", "This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s).", "In another aspect, oligos serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.", "In a particular exemplification, it is possible to simultaneously mutagenize two or more contiguous amino acid positions using an oligo that contains contiguous N,N,G/T triplets, i.e.", "a degenerate (N,N,G/T)n sequence.", "In another aspect, the present invention provides for the use of degenerate cassettes having less degeneracy than the N,N,G/T sequence.", "For example, it may be desirable in some instances to use (e.g.", "in an oligo) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet.", "Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet.", "Alternatively, it may be desirable in some instances to use (e.g.", "in an oligo) a degenerate N,N,N triplet sequence, or an N,N, G/C triplet sequence.", "It is appreciated, however, that the use of a degenerate triplet (such as N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instant invention is advantageous for several reasons.", "In one aspect, this invention provides a means to systematically and fairly easily generate the substitution of the full range of possible amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide.", "Thus, for a 100 amino acid polypeptide, the instant invention provides a way to systematically and fairly easily generate 2000 distinct species (i.e.", "20 possible amino acids per position X 100 amino acid positions).", "It is appreciated that there is provided, through the use of an oligo containing a degenerate N,N,G/T or an N,N, G/C triplet sequence, 32 individual sequences that code for 20 possible amino acids.", "Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using one such oligo, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides.", "In contrast, the use of a non-degenerate oligo in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.", "This invention also provides for the use of nondegenerate oligos, which can optionally be used in combination with degenerate primers disclosed.", "It is appreciated that in some situations, it is advantageous to use nondegenerate oligos to generate specific point mutations in a working polynucleotide.", "This provides a means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.", "Thus, in a preferred embodiment of this invention, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide.", "The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g.", "cloned into a suitable E. Coli host using an expression vector) and subjected to expression screening.", "When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.", "It is appreciated that upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position.", "One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions.", "For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions.", "Thus, there are 3×3×3 or 27 total possibilities, including 7 that were previously examined—6 single point mutations (i.e.", "2 at each of three positions) and no change at any position.", "In yet another aspect, site-saturation mutagenesis can be used together with shuffling, chimerization, recombination and other mutagenizing processes, along with screening.", "This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.", "In one exemplification, the iterative use of any mutagenizing process(es) is used in combination with screening.", "Thus, in a non-limiting exemplification, this invention provides for the use of saturation mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.", "In addition to performing mutagenesis along the entire sequence of a gene, the instant invention provides that mutagenesis can be use to replace each of any number of bases in a polynucleotide sequence, wherein the number of bases to be mutagenized is preferably every integer from 15 to 100,000.Thus, instead of mutagenizing every position along a molecule, one can subject every a discrete number of bases (preferably a subset totaling from 15 to 100,000) to mutagenesis.", "Preferably, a separate nucleotide is used for mutagenizing each position or group of positions along a polynucleotide sequence.", "A group of 3 positions to be mutagenized may be a codon.", "The mutations are preferably introduced using a mutagenic primer, containing a heterologous cassette, also referred to as a mutagenic cassette.", "Preferred cassettes can have from 1 to 500 bases.", "Each nucleotide position in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/f, C/G, C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E can be referred to as a designer oligo).", "The tables below show exemplary tri-nucleotide cassettes (there are over 3000 possibilities in addition to N,N,G/T and N,N,N and N,N,A/C).", "In a general sense, saturation mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is preferably 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is preferably from 15 to 100,000 bases in length).", "Thusly, a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized.", "A grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of saturation mutagenesis.", "Such groupings are exemplified by deletions, additions, groupings of particular codons, and groupings of particular nucleotide cassettes.", "Defined sequences to be mutagenized (see FIG.", "20) include preferably a whole gene, pathway, cDNA, an entire open reading frame (ORF), and entire promoter, enhancer, repressor/transactivator, origin of replication, intron, operator, or any polynucleotide functional group.", "Generally, a preferred “defined sequences” for this purpose may be any polynucleotide that a 15 base-polynucleotide sequence, and polynucleotide sequences of lengths between 15 bases and 15,000 bases (this invention specifically names every integer in between).", "Considerations in choosing groupings of codons include types of amino acids encoded by a degenerate mutagenic cassette.", "In a particularly preferred exemplification a grouping of mutations that can be introduced into a mutagenic cassette (see Tables 1-85), this invention specifically provides for degenerate codon substitutions (using degenerate oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position, and a library of polypeptides encoded thereby.", "3.2.Chimerizations 3.2.1 “Shuffling” Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce a polynucleotide or polynucleotides.", "Mixtures of related nucleic acid sequences or polynucleotides are subjected to sexual PCR to provide random polynucleotides, and reassembled to yield a library or mixed population of recombinant hybrid nucleic acid molecules or polynucleotides.", "In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other than primers).", "The advantage of the mutagenic shuffling of this invention over error-prone PCR alone for repeated selection can best be explained with an example from antibody engineering.", "Consider DNA shuffling as compared with error-prone PCR (not sexual PCR).", "The initial library of selected pooled sequences can consist of related sequences of diverse origin (i.e.", "antibodies from naive mRNA) or can be derived by any type of mutagenesis (including shuffling) of a single antibody gene.", "A collection of selected complementarity determining regions (“CDRs”) is obtained after the first round of affinity selection.", "In the diagram the thick CDRs confer onto the antibody molecule increased affinity for the antigen.", "Shuffling allows the free combinatorial association of all of the CDR1s with all of the CDR2s with all of the CDR3s, for example.", "This method differs from error-prone PCR, in that it is an inverse chain reaction.", "In error-prone PCR, the number of polymerase start sites and the number of molecules grows exponentially.", "However, the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same.", "In contrast, in nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time.", "For polynucleotides derived from whole plasmids the theoretical endpoint is a single, large concatemeric molecule.", "Since cross-overs occur at regions of homology, recombination will primarily occur between members of the same sequence family.", "This discourages combinations of CDRs that are grossly incompatible (e.g., directed against different epitopes of the same antigen).", "It is contemplated that multiple families of sequences can be shuffled in the same reaction.", "Further, shuffling generally conserves the relative order, such that, for example, CDR 1 will not be found in the position of CDR2.Rare shufflants will contain a large number of the best (eg.", "highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity.", "CDRs from a pool of 100 different selected antibody sequences can be permutated in up to 1006 different ways.", "This large number of permutations cannot be represented in a single library of DNA sequences.", "Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired.", "Error-prone PCR, in contrast, keeps all the selected CDRs in the same relative sequence, generating a much smaller mutant cloud.", "The template polynucleotide which may be used in the methods of this invention may be DNA or RNA.", "It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.", "Preferably, the template polynucleotide is from 50 bp to 50 kb.", "It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention, and in fact have been successfully used.", "The template polynucleotide may be obtained by amplification using the PCR reaction (U.S. Pat.", "No.", "4,683,202 and U.S. Pat.", "No.", "4,683,195) or other amplification or cloning methods.", "However, the removal of free primers from the PCR products before subjecting them to pooling of the PCR products and sexual PCR may provide more efficient results.", "Failure to adequately remove the primers from the original pool before sexual PCR can lead to a low frequency of crossover clones.", "The template polynucleotide often should be double-stranded.", "A double-stranded nucleic acid molecule is recommended to ensure that regions of the resulting single-stranded polynucleotides are complementary to each other and thus can hybridize to form a double-stranded molecule.", "It is contemplated that single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, at this step.", "It is also contemplated that two different but related polynucleotide templates can be mixed at this step.", "The double-stranded polynucleotide template and any added double- or single-stranded polynucleotides are subjected to sexual PCR which includes slowing or halting to provide a mixture of from about 5 bp to 5 kb or more.", "Preferably the size of the random polynucleotides is from about 10 bp to 1000 bp, more preferably the size of the polynucleotides is from about 20 bp to 500 bp.", "Alternatively, it is also contemplated that double-stranded nucleic acid having multiple nicks may be used in the methods of this invention.", "A nick is a break in one strand of the double-stranded nucleic acid.", "The distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.", "This can provide areas of self-priming to produce shorter or smaller polynucleotides to be included with the polynucleotides resulting from random primers, for example.", "The concentration of any one specific polynucleotide will not be greater than 1% by weight of the total polynucleotides, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.", "The number of different specific polynucleotides in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.At this step single-stranded or double-stranded polynucleotides, either synthetic or natural, may be added to the random double-stranded shorter or smaller polynucleotides in order to increase the heterogeneity of the mixture of polynucleotides.", "It is also contemplated that populations of double-stranded randomly broken polynucleotides may be mixed or combined at this step with the polynucleotides from the sexual PCR process and optionally subjected to one or more additional sexual PCR cycles.", "Where insertion of mutations into the template polynucleotide is desired, single-stranded or double-stranded polynucleotides having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded polynucleotides may be added in a 10 fold excess by weight as compared to the total nucleic acid.", "Where a mixture of different but related template polynucleotides is desired, populations of polynucleotides from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1:40.For example, a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for).", "In such an example, the ratio of randomly provided wild-type polynucleotides which may be added to the randomly provided sexual PCR cycle hybrid polynucleotides is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.The mixed population of random polynucleotides are denatured to form single-stranded polynucleotides and then re-annealed.", "Only those single-stranded polynucleotides having regions of homology with other single-stranded polynucleotides will re-anneal.", "The random polynucleotides may be denatured by heating.", "One skilled in the art could determine the conditions necessary to completely denature the double-stranded nucleic acid.", "Preferably the temperature is from 80° C. to 100° C., more preferably the temperature is from 90° C. to 96° C. other methods which may be used to denature the polynucleotides include pressure (36) and pH.", "The polynucleotides may be re-annealed by cooling.", "Preferably the temperature is from 20° C. to 75° C., more preferably the temperature is from 40° C. to 65° C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult.", "The degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded polynucleotides.", "Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt.", "The salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mm.", "The salt may be KCl or NaCl.", "The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.", "The annealed polynucleotides are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e.", "dATP, dCTP, DGTP and dTTP).", "The nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.", "The approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers.", "If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65° C. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30° C. One skilled in the art could vary the temperature of annealing to increase the number of crossovers achieved.", "The polymerase may be added to the random polynucleotides prior to annealing, simultaneously with annealing or after annealing.", "The cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid.", "This cycle is repeated for a desired number of times.", "Preferably the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.", "The resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.", "This larger polynucleotide may contain a number of copies of a polynucleotide having the same size as the template polynucleotide in tandem.", "This concatemeric polynucleotide is then denatured into single copies of the template polynucleotide.", "The result will be a population of polynucleotides of approximately the same size as the template polynucleotide.", "The population will be a mixed population where single or double-stranded polynucleotides having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.", "These polynucleotides are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.", "It is contemplated that the single polynucleotides may be obtained from the larger concatemeric polynucleotide by amplification of the single polynucleotide prior to cloning by a variety of methods including PCR (U.S. Pat.", "No.", "4,683,195 and U.S. Pat.", "No.", "4,683,202), rather than by digestion of the concatemer.", "The vector used for cloning is not critical provided that it will accept a polynucleotide of the desired size.", "If expression of the particular polynucleotide is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the polynucleotide to allow expression of the polynucleotide in the host cell.", "Preferred vectors include the pUC series and the pBR series of plasmids.", "The resulting bacterial population will include a number of recombinant polynucleotides having random mutations.", "This mixed population may be tested to identify the desired recombinant polynucleotides.", "The method of selection will depend on the polynucleotide desired.", "For example, if a polynucleotide which encodes a protein with increased binding efficiency to a ligand is desired, the proteins expressed by each of the portions of the polynucleotides in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e.", "panning, affinity chromatography).", "If a polynucleotide which encodes for a protein with increased drug resistance is desired, the proteins expressed by each of the polynucleotides in the population or library may be tested for their ability to confer drug resistance to the host organism.", "One skilled in the art, given knowledge of the desired protein, could readily test the population to identify polynucleotides which confer the desired properties onto the protein.", "It is contemplated that one skilled in the art could use a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee Wis.).", "The recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule.", "The phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell.", "The leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle.", "Thus the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.", "It is further contemplated that a number of cycles of nucleic acid shuffling may be conducted with polynucleotides from a subpopulation of the first population, which sub-population contains DNA encoding the desired recombinant protein.", "In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.", "It is also contemplated that a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type polynucleotides and a sub-population of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the sub-population.", "Any source of nucleic acid, in purified form can be utilized as the starting nucleic acid.", "Thus the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded.", "In addition, a DNA-RNA hybrid which contains one strand of each may be utilized.", "The nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated.", "Preferably the specific nucleic acid sequence is from 50 to 50000 base pairs.", "It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of this invention.", "The nucleic acid may be obtained from any source, for example, from plasmids such a pBR322, from cloned DNA or RNA or from natural DNA or RNA from any source including bacteria, yeast, viruses and higher organisms such as plants or animals.", "DNA or RNA may be extracted from blood or tissue material.", "The template polynucleotide may be obtained by amplification using the polynucleotide chain reaction (PCR, see U.S. Pat.", "No.", "4,683,202 and U.S. Pat.", "No.", "4,683,195).", "Alternatively, the polynucleotide may be present in a vector present in a cell and sufficient nucleic acid may be obtained by culturing the cell and extracting the nucleic acid from the cell by methods known in the art.", "Any specific nucleic acid sequence can be used to produce the population of hybrids by the present process.", "It is only necessary that a small population of hybrid sequences of the specific nucleic acid sequence exist or be created prior to the present process.", "The initial small population of the specific nucleic acid sequences having mutations may be created by a number of different methods.", "Mutations may be created by error-prone PCR.", "Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence.", "Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis.", "In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence.", "The polynucleotide sequence can also be altered by chemical mutagenesis.", "Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.", "Other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.", "Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence.", "Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.", "Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.", "Generally, plasmid polynucleotides so mutagenized are introduced into E. coli and propagated as a pool or library of hybrid plasmids.", "Alternatively the small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes).", "Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.", "Once the mixed population of the specific nucleic acid sequences is generated, the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.", "The choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.", "The templates of this invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).", "For example, cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large polynucleotides.", "If the mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector.", "This is referred to as clonal amplification because while the absolute number of nucleic acid sequences increases, the number of hybrids does not increase.", "Utility can be readily determined by screening expressed polypeptides.", "The DNA shuffling method of this invention can be performed blindly on a pool of unknown sequences.", "By adding to the reassembly mixture oligonucleotides (with ends that are homologous to the sequences being reassembled) any sequence mixture can be incorporated at any specific position into another sequence mixture.", "Thus, it is contemplated that mixtures of synthetic oligonucleotides, PCR polynucleotides or even whole genes can be mixed into another sequence library at defined positions.", "The insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template.", "Thus, the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.", "This approach of mixing two genes may be useful for the humanization of antibodies from murine hybridomas.", "The approach of mixing two genes or inserting alternative sequences into genes may be useful for any therapeutically used protein, for example, interleukin 1, antibodies, tPA and growth hormone.", "The approach may also be useful in any nucleic acid for example, promoters or introns or 3′ untranslated region or 5′ untranslated regions of genes to increase expression or alter specificity of expression of proteins.", "The approach may also be used to mutate ribozymes or aptamers.", "Shuffling requires the presence of homologous regions separating regions of diversity.", "Scaffold-like protein structures may be particularly suitable for shuffling.", "The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding.", "Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle which are well-known in the art.", "This shuffling can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.", "3.2.1.1.In Vitro Shuffling The equivalents of some standard genetic matings may also be performed by shuffling in vitro.", "For example, a “molecular backcross” can be performed by repeatedly mixing the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.", "As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice.", "It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e.", "immunogenicity).", "Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not, an advantage which cannot be achieved by error-prone mutagenesis or cassette mutagenesis methods.", "Large, functional genes can be assembled correctly from a mixture of small random polynucleotides.", "This reaction may be of use for the reassembly of genes from the highly fragmented DNA of fossils.", "In addition random nucleic acid fragments from fossils may be combined with polynucleotides from similar genes from related species.", "It is also contemplated that the method of this invention can be used for the in vitro amplification of a whole genome from a single cell as is needed for a variety of research and diagnostic applications.", "DNA amplification by PCR is in practice limited to a length of about 40 kb.", "Amplification of a whole genome such as that of E. coli (5,000 kb) by PCR would require about 250 primers yielding 125 forty kb polynucleotides.", "This approach is not practical due to the unavailability of sufficient sequence data.", "On the other hand, random production of polynucleotides of the genome with sexual PCR cycles, followed by gel purification of small polynucleotides will provide a multitude of possible primers.", "Use of this mix of random small polynucleotides as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatamer containing many copies of the genome.", "100 fold amplification in the copy number and an average polynucleotide size of greater than 50 kb may be obtained when only random polynucleotides are used.", "It is thought that the larger concatamer is generated by overlap of many smaller polynucleotides.", "The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA.", "It is expected that this approach will be useful for the mapping of genomes.", "The polynucleotide to be shuffled can be produced as random or non-random polynucleotides, at the discretion of the practitioner.", "Moreover, this invention provides a method of shuffling that is applicable to a wide range of polynucleotide sizes and types, including the step of generating polynucleotide monomers to be used as building blocks in the reassembly of a larger polynucleotide.", "For example, the building blocks can be fragments of genes or they can be comprised of entire genes or gene pathways, or any combination thereof.", "3.2.1.2.In Vivo Shuffling In an embodiment of in vivo shuffling, the mixed population of the specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell.", "The polynucleotides can be introduced into the host cells by a variety of different methods.", "The host cells can be transformed with the smaller polynucleotides using methods known in the art, for example treatment with calcium chloride.", "If the polynucleotides are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences.", "Alternatively, the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.", "In general, in this embodiment, the specific nucleic acids sequences will be present in vectors which are capable of stably replicating the sequence in the host cell.", "In addition, it is contemplated that the vectors will encode a marker gene such that host cells having the vector can be selected.", "This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.", "However, it is contemplated that the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence.", "Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the polynucleotides into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein.", "It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.", "It has been found that when two polynucleotides which have regions of identity are inserted into the host cells homologous recombination occurs between the two polynucleotides.", "Such recombination between the two mutated specific nucleic acid sequences will result in the production of double or triple hybrids in some situations.", "It has also been found that the frequency of recombination is increased if some of the mutated specific nucleic acid sequences are present on linear nucleic acid molecules.", "Therefore, in a preferred embodiment, some of the specific nucleic acid sequences are present on linear polynucleotides.", "After transformation, the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired.", "For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected.", "If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand.", "Alternatively, the protein can be expressed in another system to ensure proper processing.", "Once a subset of the first recombined specific nucleic acid sequences (daughter sequences) having the desired characteristics are identified, they are then subject to a second round of recombination.", "In the second cycle of recombination, the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above.", "In this way a set of second recombined specific nucleic acids sequences can be identified which have enhanced characteristics or encode for proteins having enhanced properties.", "This cycle can be repeated a number of times as desired.", "It is also contemplated that in the second or subsequent recombination cycle, a backcross can be performed.", "A molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type sequence, such that at least one wild-type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation.", "Recombination with the wild-type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.", "In another embodiment of this invention, it is contemplated that during the first round a subset of the specific nucleic acid sequences can be generated as smaller polynucleotides by slowing or halting their PCR amplification prior to introduction into the host cell.", "The size of the polynucleotides must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences.", "The size of the polynucleotides will range from 0.03 kb to 100 kb more preferably from 0.2 kb to 10 kb.", "It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be utilized to generate PCR polynucleotides prior to introduction into the host cells.", "The shorter polynucleotide sequences can be single-stranded or double-stranded.", "If the sequences were originally single-stranded and have become double-stranded they can be denatured with heat, chemicals or enzymes prior to insertion into the host cell.", "The reaction conditions suitable for separating the strands of nucleic acid are well known in the art.", "The steps of this process can be repeated indefinitely, being limited only by the number of possible hybrids which can be achieved.", "After a certain number of cycles, all possible hybrids will have been achieved and further cycles are redundant.", "In an embodiment the same mutated template nucleic acid is repeatedly recombined and the resulting recombinants selected for the desired characteristic.", "Therefore, the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. coli.", "The particular vector is not essential, so long as it is capable of autonomous replication in E. coli.", "In a preferred embodiment, the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector.", "It is also preferred that the vector contain a gene encoding for a selectable marker.", "The population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells.", "The vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage.", "The concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell.", "Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors.", "This results in the generation of hybrids (daughters) having a combination of mutations which differ from the original parent mutated sequences.", "The host cells are then clonally replicated and selected for the marker gene present on the vector.", "Only those cells having a plasmid will grow under the selection.", "The host cells which contain a vector are then tested for the presence of favorable mutations.", "Such testing may consist of placing the cells under selective pressure, for example, if the gene to be selected is an improved drug resistance gene.", "If the vector allows expression of the protein encoded by the mutated nucleic acid sequence, then such selection may include allowing expression of the protein so encoded, isolation of the protein and testing of the protein to determine whether, for example, it binds with increased efficiency to the ligand of interest.", "Once a particular daughter mutated nucleic acid sequence has been identified which confers the desired characteristics, the nucleic acid is isolated either already linked to the vector or separated from the vector.", "This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.", "It has been shown that by this method nucleic acid sequences having enhanced desired properties can be selected.", "In an alternate embodiment, the first generation of hybrids are retained in the cells and the parental mutated sequences are added again to the cells.", "Accordingly, the first cycle of Embodiment 1 is conducted as described above.", "However, after the daughter nucleic acid sequences are identified, the host cells containing these sequences are retained.", "The parent mutated specific nucleic acid population, either as polynucleotides or cloned into the same vector is introduced into the host cells already containing the daughter nucleic acids.", "Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above.", "This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained.", "It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the preferred hybrids may come from the parental hybrids or any subsequent generation.", "In an alternative embodiment, the invention provides a method of conducting a “molecular” backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations.", "Neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties.", "Such mutations may however confer on the nucleic acid or peptide undesirable characteristics.", "Accordingly, it is desirable to eliminate such neutral mutations.", "The method of this invention provide a means of doing so.", "In this embodiment, after the hybrid nucleic acid, having the desired characteristics, is obtained by the methods of the embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated.", "The nucleic acid or vector is then introduced into the host cell with a large excess of the wild-type nucleic acid.", "The nucleic acid of the hybrid and the nucleic acid of the wild-type sequence are allowed to recombine.", "The resulting recombinants are placed under the same selection as the hybrid nucleic acid.", "Only those recombinants which retained the desired characteristics will be selected.", "Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA.", "This cycle can be repeated a number of times until all of the silent mutations are eliminated.", "Thus the methods of this invention can be used in a molecular backcross to eliminate unnecessary or silent mutations.", "3.2.2.Exonuclease-Mediated Reassembly In a particular embodiment, this invention provides for a method for shuffling, assembling, reassembling, recombining, &/or concatenating at least two polynucleotides to form a progeny polynucleotide (e.g.", "a chimeric progeny polynucleotide that can be expressed to produce a polypeptide or a gene pathway).", "In a particular embodiment, a double stranded polynucleotide end (e.g.", "two single stranded sequences hybridized to each other as hybridization partners) is treated with an exonuclease to liberate nucleotides from one of the two strands, leaving the remaining strand free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner.", "In a particular aspect, a double stranded polynucleotide end (that may be part of—or connected to—a polynucleotide or a nonpolynucleotide sequence) is subjected to a source of exonuclease activity.", "Serviceable sources of exonuclease activity may be an enzyme with 3′ exonuclease activity, an enzyme with 5′ exonuclease activity, an enzyme with both 3′ exonuclease activity and 5′ exonuclease activity, and any combination thereof.", "An exonuclease can be used to liberate nucleotides from one or both ends of a linear double stranded polynucleotide, and from one to all ends of a branched polynucleotide having more than two ends.", "The mechanism of action of this liberation is believed to be comprised of an enzymatically-catalyzed hydrolysis of terminal nucleotides, and can be allowed to proceed in a time-dependent fashion, allowing experimental control of the progression of the enzymatic process.", "By contrast, a non-enzymatic step may be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks that is comprised of subjecting a working sample to denaturing (or “melting”) conditions (for example, by changing temperature, pH, and/or salinity conditions) so as to melt a working set of double stranded polynucleotides into single polynucleotide strands.", "For shuffling, it is desirable that the single polynucleotide strands participate to some extent in annealment with different hybridization partners (i.e.", "and not merely revert to exclusive reannealment between what were former partners before the denaturation step).", "The presence of the former hybridization partners in the reaction vessel, however, does not preclude, and may sometimes even favor, reannealment of a single stranded polynucleotide with its former partner, to recreate an original double stranded polynucleotide.", "In contrast to this non-enzymatic shuffling step comprised of subjecting double stranded polynucleotide building blocks to denaturation, followed by annealment, the instant invention further provides an exonuclease-based approach requiring no denaturation—rather, the avoidance of denaturing conditions and the maintenance of double stranded polynucleotide substrates in annealed (i.e.", "non-denatured) state are necessary conditions for the action of exonucleases (e.g., exonuclease III and red alpha gene product).", "Additionally in contrast, the generation of single stranded polynucleotide sequences capable of hybridizing to other single stranded polynucleotide sequences is the result of covalent cleavage—and hence sequence destruction—in one of the hybridization partners.", "For example, an exonuclease III enzyme may be used to enzymatically liberate 3′ terminal nucleotides in one hybridization strand (to achieve covalent hydrolysis in that polynucleotide strand); and this favors hybridization of the remaining single strand to a new partner (since its former partner was subjected to covalent cleavage).", "By way of further illustration, a specific exonuclease, namely exonuclease III is provided herein as an example of a 3′ exonuclease; however, other exonucleases may also be used, including enzymes with 5′ exonuclease activity and enzymes with 3′ exonuclease activity, and including enzymes not yet discovered and enzymes not yet developed.", "It is particularly appreciated that enzymes can be discovered, optimized (e.g.", "engineered by directed evolution), or both discovered and optimized specifically for the instantly disclosed approach that have more optimal rates &/or more highly specific activities &/or greater lack of unwanted activities.", "In fact it is expected that the instant invention may encourage the discovery &/or development of such designer enzymes.", "In sum, this invention may be practiced with a variety of currently available exonuclease enzymes, as well as enzymes not yet discovered and enzymes not yet developed.", "The exonuclease action of exonuclease III requires a working double stranded polynucleotide end that is either blunt or has a 5′ overhang, and the exonuclease action is comprised of enzymatically liberating 3′ terminal nucleotides, leaving a single stranded 5′ end that becomes longer and longer as the exonuclease action proceeds (see FIG.", "1).", "Any 5′ overhangs produced by this approach may be used to hybridize to another single stranded polynucleotide sequence (which may also be a single stranded polynucleotide or a terminal overhang of a partially double stranded polynucleotide) that shares enough homology to allow hybridization.", "The ability of these exonuclease III-generated single stranded sequences (e.g.", "in 5′ overhangs) to hybridize to other single stranded sequences allows two or more polynucleotides to be shuffled, assembled, reassembled, &/or concatenated.", "Furthermore, it is appreciated that one can protect the end of a double stranded polynucleotide or render it susceptible to a desired enzymatic action of a serviceable exonuclease as necessary.", "For example, a double stranded polynucleotide end having a 3′ overhang is not susceptible to the exonuclease action of exonuclease III.", "However, it may be rendered susceptible to the exonuclease action of exonuclease III by a variety of means; for example, it may be blunted by treatment with a polymerase, cleaved to provide a blunt end or a 5′ overhang, joined (ligated or hybridized) to another double stranded polynucleotide to provide a blunt end or a 5′ overhang, hybridized to a single stranded polynucleotide to provide a blunt end or a 5′ overhang, or modified by any of a variety of means).", "According to one aspect, an exonuclease may be allowed to act on one or on both ends of a linear double stranded polynucleotide and proceed to completion, to near completion, or to partial completion.", "When the exonuclease action is allowed to go to completion, the result will be that the length of each 5′ overhang will extend far towards the middle region of the polynucleotide in the direction of what might be considered a “rendezvous point” (which may be somewhere near the polynucleotide midpoint).", "Ultimately, this results in the production of single stranded polynucleotides (that can become dissociated) that are each about half the length of the original double stranded polynucleotide (see FIG.", "1).", "Alternatively, an exonuclease-mediated reaction can be terminated before proceeding to completion.", "Thus this exonuclease-mediated approach is serviceable for shuffling, assembling &/or reassembling, recombining, and concatenating polynucleotide building blocks, which polynucleotide building blocks can be up to ten bases long or tens of bases long or hundreds of bases long or thousands of bases long or tens of thousands of bases long or hundreds of thousands of bases long or millions of bases long or even longer.", "This exonuclease-mediated approach is based on the action of double stranded DNA specific exodeoxyribonuclease activity of E. coli exonuclease 111.Substrates for exonuclease III may be generated by subjecting a double stranded polynucleotide to fragmentation.", "Fragmentation may be achieved by mechanical means (e.g., shearing, sonication, etc.", "), by enzymatic means (e.g.", "using restriction enzymes), and by any combination thereof.", "Fragments of a larger polynucleotide may also be generated by polymerase-mediated synthesis.", "Exonuclease III is a 28K monomeric enzyme, product of the xthA gene of E. coli with four known activities: exodeoxyribonuclease (alternatively referred to as exonuclease herein), RNaseH, DNA-3′-phosphatase, and AP endonuclease.", "The exodeoxyribonuclease activity is specific for double stranded DNA.", "The mechanism of action is thought to involve enzymatic hydrolysis of DNA from a 3′ end progressively towards a 5′ direction, with formation of nucleoside 5′-phosphates and a residual single strand.", "The enzyme does not display efficient hydrolysis of single stranded DNA, single-stranded RNA, or double-stranded RNA; however it degrades RNA in an DNA-RNA hybrid releasing nucleoside 5′-phosphates.", "The enzyme also releases inorganic phosphate specifically from 3′phosphomonoester groups on DNA, but not from RNA or short oligonucleotides.", "Removal of these groups converts the terminus into a primer for DNA polymerase action.", "Additional examples of enzymes with exonuclease activity include red-alpha and venom phosphodiesterases.", "Red alpha (red gene product (also referred to as lambda exonuclease) is of bacteriophage origin.", "The red gene is transcribed from the leftward promoter and its product is involved (24 kD) in recombination.", "Red alpha gene product acts processively from 5′-phosphorylated termini to liberate mononucleotides from duplex DNA (Takahashi & Kobayashi, 1990).", "Venom phosphodiesterases (Laskowski, 1980) is capable of rapidly opening supercoiled DNA.", "3.2.3.Non-Stochastic Ligation Reassembly In one aspect, the present invention provides a non-stochastic method termed synthetic ligation reassembly (SLR), that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or chimerized randomly, but rather are assembled non-stochastically.", "A particularly glaring difference is that the instant SLR method does not depend on the presence of a high level of homology between polynucleotides to be shuffled.", "In contrast, prior methods, particularly prior stochastic shuffling methods require that presence of a high level of homology, particularly at coupling sites, between polynucleotides to be shuffled.", "Accordingly these prior methods favor the regeneration of the original progenitor molecules, and are suboptimal for generating large numbers of novel progeny chimeras, particularly full-length progenies.", "The instant invention, on the other hand, can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras.", "Conceivably, SLR can even be used to generate libraries comprised of over 101000 different progeny chimeras with (no upper limit in sight).", "Thus, in one aspect, the present invention provides a method, which method is non-stochastic, of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.", "The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be “serviceable” for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders.", "Thus, in one aspect, the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and, if more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s).", "FIG.", "4, Panel C illustrates an exemplary assembly process comprised of 2 sequential steps to achieve a designed (non-stochastic) overall assembly order for five nucleic acid building blocks.", "In a preferred embodiment of this invention, the annealed building pieces are treated with an enzyme, such as a ligase (e.g.", "T4 DNA ligase), achieve covalent bonding of the building pieces.", "In a preferred embodiment, the design of nucleic acid building blocks is obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules.", "These progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i.e.", "chimerized or shuffled.", "In one exemplification, this invention provides for the chimerization of a family of related genes and their encoded family of related products.", "In a particular exemplification, the encoded products are enzymes.", "As a representative list of families of enzymes which may be mutagenized in accordance with the aspects of the present invention, there may be mentioned, the following enzymes and their functions: 1 Lipase/Esterase a. Enantioselective hydrolysis of esters (lipids)/thioesters 1) Resolution of racemic mixtures 2) Synthesis of optically active acids or alcohols from meso-diesters b.", "Selective syntheses 1) Regiospecific hydrolysis of carbohydrate esters 2) Selective hydrolysis of cyclic secondary alcohols c. Synthesis of optically active esters, lactones, acids, alcohols 1) Transesterification of activated/nonactivated esters 2) Interesterification 3) Optically active lactones from hydroxyesters 4) Regio- and enantioselective ring opening of anhydrides d. Detergents e. Fat/Oil conversion f. Cheese ripening 2 Protease a. Ester/amide synthesis b. Peptide synthesis c. Resolution of racemic mixtures of amino acid esters d. Synthesis of non-natural amino acids e. Detergents/protein hydrolysis 3 Glycosidase/Glycosyl transferase a. Sugar/polymer synthesis b. Cleavage of glycosidic linkages to form mono, di- and oligosaccharides c. Synthesis of complex oligosaccharides d. Glycoside synthesis using UDP-galactosyl transferase e. Transglycosylation of disaccharides, glycosyl fluorides, aryl galactosides f. Glycosyl transfer in oligosaccharide synthesis g. Diastereoselective cleavage of -glucosylsulfoxides h. Asymmetric glycosylations i.", "Food processing j.", "Paper processing 4.Phosphatase/Kinase a. Synthesis/hydrolysis of phosphate esters 1) Regio-, enantioselective phosphorylation 2) Introduction of phosphate esters 3) Synthesize phospholipid precursors 4) Controlled polynucleotide synthesis b. Activate biological molecule c. Selective phosphate bond formation without protecting groups 5.Mono/Dioxygenase a.", "Direct oxyfunctionalization of unactivated organic substrates b. Hydroxylation of alkane, aromatics, steroids c. Epoxidation of alkenes d. Enantioselective sulphoxidation e. Regio- and stereoselective Bayer-Villiger oxidations 6.Haloperoxidase a. Oxidative addition of halide ion to nucleophilic sites b.", "Addition of hypohalous acids to olefinic bonds c. Ring cleavage of cyclopropanes d. Activated aromatic substrates converted to ortho and para derivatives e. 1.3 diketones converted to 2-halo-derivatives f. Heteroatom oxidation of sulfur and nitrogen containing substrates g. Oxidation of enol acetates, alkynes and activated aromatic rings 7.Lignin peroxidase/Diarylpropane peroxidase a. Oxidative cleavage of C—C bonds b. Oxidation of benzylic alcohols to aldehydes c. Hydroxylation of benzylic carbons d. Phenol dimerization e. Hydroxylation of double bonds to form diols f. Cleavage of lignin aldehydes 8.Epoxide hydrolase a. Synthesis of enantiomerically pure bioactive compounds b. Regio- and enantioselective hydrolysis of epoxide c. Aromatic and olefinic epoxidation by monooxygenases to form epoxides d. Resolution of racemic epoxides e. Hydrolysis of steroid epoxides 9.Nitrile hydmtase/nitrilase a. Hydrolysis of aliphatic nitriles to carboxamides b. Hydrolysis of aromatic, heterocyclic, unsaturated aliphatic nitriles to corresponding acids c. Hydrolysis of acrylonitrile d. Production of aromatic and carboxamides, carboxylic acids (nicotinamide, picolinamide, isonicotinamide) e. Regioselective hydrolysis of acrylic dinitrile f. -amino acids from -hydroxynitriles 10.Transaminase a.", "Transfer of amino groups into oxo-acids 11.Amidase/Acylase a. Hydrolysis of amides, amidines, and other C—N bonds b. Non-natural amino acid resolution and synthesis These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.", "Thus according to one aspect of this invention, the sequences of a plurality of progenitor nucleic acid templates are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology, and are comprised of one or more nucleotides, and which demarcation points are shared by at least two of the progenitor templates.", "The demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be generated.", "Thus, the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the progeny molecules.", "Preferably a serviceable demarcation point is an area of homology (comprised of at least one homologous nucleotide base) shared by at least two progenitor templates.", "More preferably a serviceable demarcation point is an area of homology that is shared by at least half of the progenitor templates.", "More preferably still a serviceable demarcation point is an area of homology that is shared by at least two thirds of the progenitor templates.", "Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the progenitor templates.", "Even more preferably still a serviceable demarcation points is an area of homology that is shared by at almost all of the progenitor templates.", "Even more preferably still a serviceable demarcation point is an area of homology that is shared by all of the progenitor templates.", "The process of designing nucleic acid building blocks and of designing the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled is illustrated in FIGS.", "6 and 7.As shown, the alignment of a set of progenitor templates reveals several naturally occurring demarcation points, and the identification of demarcation points shared by these templates helps to non-stochastically determine the building blocks to be generated and used for the generation of the progeny chimeric molecules.", "In a preferred embodiment, this invention provides that the ligation reassembly process is performed exhaustively in order to generate an exhaustive library.", "In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules.", "At the same time, in a particularly preferred embodiment, the assembly order (i.e.", "the order of assembly of each building block in the 5′ to 3′ sequence of each finalized chimeric nucleic acid) in each combination is by design (or non-stochastic).", "Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.", "In another preferred embodiment, this invention provides that the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened systematically, e.g.", "one by one.", "In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, an experimental design can be achieved where specific sets of progeny products are made in each of several reaction vessels.", "This allows a systematic examination and screening procedure to be performed.", "Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups.", "Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules.", "Because of the non-stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design.", "In a particularly preferred embodiment of this invention, such a generated library is comprised of preferably greater than 103 different progeny molecular species, more preferably greater than 105 different progeny molecular species, more preferably still greater than 1010 different progeny molecular species, more preferably still greater than 1015 different progeny molecular species, more preferably still greater than 1020 different progeny molecular species, more preferably still greater than 1030 different progeny molecular species, more preferably still greater than 1040 different progeny molecular species, more preferably still greater than 1050 different progeny molecular species, more preferably still greater than 1060 different progeny molecular species, more preferably still greater than 1070 different progeny molecular species, more preferably still greater than 1080 different progeny molecular species, more preferably still greater than 10100 different progeny molecular species, more preferably still greater than 10110 different progeny molecular species, more preferably still greater than 10120 different progeny molecular species, more preferably still greater than 10130 different progeny molecular species, more preferably still greater than 10140 different progeny molecular species, more preferably still greater than 10150 different progeny molecular species, more preferably still greater than 10175 different progeny molecular species, more preferably still greater than 10200 different progeny molecular species, more preferably still greater than 10300 different progeny molecular species, more preferably still greater than 10400 different progeny molecular species, more preferably still greater than 10500 different progeny molecular species, and even more preferably still greater than 101000 different progeny molecular species.", "In one aspect, a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide.", "According to one preferred embodiment, this polynucleotide is a gene, which may be a man-made gene.", "According to another preferred embodiment, this polynucleotide is a gene pathway, which may be a man-made gene pathway.", "This invention provides that one or more man-made genes generated by this invention may be incorporated into a man-made gene pathway, such as a pathway operable in a eukaryotic organism (including a plant).", "It is appreciated that the power of this invention is exceptional, as there is much freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings.", "It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention.", "In fact, demarcation points can even be chosen in areas of little or no intermolecular homology.", "For example, because of codon wobble, i.e.", "the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template.", "Alternatively, a codon can be altered such that the coding for an originally amino acid is altered.", "This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecularly homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.", "In another exemplifaction, the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g.", "one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g.", "by mutageneis) or in an in vivo process (e.g.", "by utilizing the gene splicing ability of a host organism).", "It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.", "Thus, according to another embodiment, this invention provides that a nucleic acid building block can be used to introduce an intron.", "Thus, this invention provides that functional introns may be introduced into a man-made gene of this invention.", "This invention also provides that functional introns may be introduced into a man-made gene pathway of this invention.", "Accordingly, this invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced intron(s).", "Accordingly, this invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced intron(s).", "Preferably, the artificially introduced intron(s) are functional in one or more host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.", "This invention provides a process of producing man-made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing.", "The ability to achieve chimerizations, using couplings as described herein, in areas of little or no homology among the progenitor molecules, is particularly useful, and in fact critical, for the assembly of novel gene pathways.", "This invention thus provides for the generation of novel man-made gene pathways using synthetic ligation reassembly.", "In a particular aspect, this is achieved by the introduction of regulatory sequences, such as promoters, that are operable in an intended host, to confer operability to a novel gene pathway when it is introduced into the intended host.", "In a particular exemplification, this invention provides for the generation of novel man-made gene pathways that is operable in a plurality of intended hosts (e.g.", "in a microbial organism as well as in a plant cell).", "This can be achieved, for example, by the introduction of a plurality of regulatory sequences, comprised of a regulatory sequence that is operable in a first intended host and a regulatory sequence that is operable in a second intended host.", "A similar process can be performed to achieve operability of a gene pathway in a third intended host species, etc.", "The number of intended host species can be each integer from 1 to 10 or alternatively over 10.Alternatively, for example, operability of a gene pathway in a plurality of intended hosts can be achieved by the introduction of a regulatory sequence having intrinsic operability in a plurality of intended hosts.", "Thus, according to a particular embodiment, this invention provides that a nucleic acid building block can be used to introduce a regulatory sequence, particularly a regulatory sequence for gene expression.", "Preferred regulatory sequences include, but are not limited to, those that are man-made, and those found in archeal, bacterial, eukaryotic (including mitochondrial), viral, and prionic or prion-like organisms.", "Preferred regulatory sequences include but are not limited to, promoters, operators, and activator binding sites.", "Thus, this invention provides that functional regulatory sequences may be introduced into a man-made gene of this invention.", "This invention also provides that functional regulatory sequences may be introduced into a man-made gene pathway of this invention.", "Accordingly, this invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced regulatory sequence(s).", "Accordingly, this invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced regulatory sequence(s).", "Preferably, an artificially introduced regulatory sequence(s) is operatively linked to one or more genes in the man-made polynucleotide, and are functional in one or more host cells.", "Preferred bacterial promoters that are serviceable for this invention include lac, lacZ, T3, T7, gpt, lambda PR, PL and trp.", "Serviceable eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1.Particular plant regulatory sequences include promoters active in directing transcription in plants, either constitutively or stage and/or tissue specific, depending on the use of the plant or parts thereof.", "These promoters include, but are not limited to promoters showing constitutive expression, such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley et al., 1982), those for leaf-specific expression, such as the promoter of the ribulose bisphosphate carboxylase small subunit gene (Coruzzi et al., 1984), those for root-specific expression, such as the promoter from the glutamin synthase gene (Tingey et al., 1987), those for seed-specific expression, such as the cruciferin A promoter from Brassica napus (Ryan et al., 1989), those for tuber-specific expression, such as the class-I patatin promoter from potato (Rocha-Sasa et al., 1989; Wenzler et al., 1989) or those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato (Bird et al., 1988).", "Other regulatory sequences that are preferred for this invention include terminator sequences and polyadenylation signals and any such sequence functioning as such in plants, the choice of which is within the level of the skilled artisan.", "An example of such sequences is the 3′ flanking region of the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan, 1984).", "The regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV, and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Cirus (AIMV) RNA4 (Brederode et al., 1980) or any other sequences functioning in a like manner.", "Man-made genes produced using this invention can also serve as a substrate for recombination with another nucleic acid.", "Likewise, a man-made gene pathway produced using this invention can also serve as a substrate for recombination with another nucleic acid.", "In a preferred instance, the recombination is facilitated by, or occurs at, areas of homology between the man-made intron-containing gene and a nucleic acid with serves as a recombination partner.", "In a particularly preferred instance, the recombination partner may also be a nucleic acid generated by this invention, including a man-made gene or a man-made gene pathway.", "Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced intron(s) in the man-made gene.", "The synthetic ligation reassembly method of this invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends.", "The two ligatable ends on each nucleic acid building block may be two blunt ends (i.e.", "each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.", "A serviceable overhang for this purpose may be a 3′ overhang or a 5′ overhang.", "Thus, a nucleic acid building block may have a 3′ overhang or alternatively a 5′ overhang or alternatively two 3′ overhangs or alternatively two 5′ overhangs.", "The overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by purposeful experimental design and is not random.", "According to one preferred embodiment, a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single-stranded oligos) and contacting them so as to allow them to anneal to form a double-stranded nucleic acid building block.", "A double-stranded nucleic acid building block can be of variable size.", "The sizes of these building blocks can be small or large depending on the choice of the experimenter.", "Preferred sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs).", "Other preferred size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100,000 bp (including every integer value in between).", "It is appreciated that current methods of polymerase-based amplification can be used to generate double-stranded nucleic acids of up to thousands of base pairs, if not tens of thousands of base pairs, in length with high fidelity.", "Chemical synthesis (e.g.", "phosphoramidite-based) can be used to generate nucleic acids of up to hundreds of nucleotides in length with high fidelity; however, these can be assembled, e.g.", "using overhangs or sticky ends, to form double-stranded nucleic acids of up to thousands of base pairs, if not tens of thousands of base pairs, in length if so desired.", "A combination of methods (e.g.", "phosphoramidite-based chemical synthesis and PCR) can also be used according to this invention.", "Thus, nucleic acid building block made by different methods can also be used in combination to generate a progeny molecule of this invention.", "The use of chemical synthesis to generate nucleic acid building blocks is particularly preferred in this invention & is advantageous for other reasons as well, including procedural safety and ease.", "No cloning or harvesting or actual handling of any biological samples is required.", "The design of the nucleic acid building blocks can be accomplished on paper.", "Accordingly, this invention teaches an advance in procedural safety in recombinant technologies.", "Nonetheless, according to one preferred embodiment, a double-stranded nucleic acid building block according to this invention may also be generated by polymerase-based amplification of a polynucleotide template.", "In a non-limiting exemplification, as illustrated in FIG.", "2, a first polymerase-based amplification reaction using a first set of primers, F2 and R1, is used to generate a blunt-ended product (labeled Reaction 1, Product 1), which is essentially identical to Product A.", "A second polymerase-based amplification reaction using a second set of primers, F1 and R2, is used to generate a blunt-ended product (labeled Reaction 2, Product 2), which is essentially identical to Product B.", "These two products are mixed and allowed to melt and anneal, generating potentially useful double-stranded nucleic acid building blocks with two overhangs.", "In the example of FIG.", "2, the product with the 3′ overhangs (Product C) is selected by nuclease-based degradation of the other 3 products using a 3′ acting exonuclease, such as exonuclease III.", "It is appreciated that a 5′ acting exonuclease (e.g.", "red alpha) may be also be used, for example to select Product D instead.", "It is also appreciated that other selection means can also be used, including hybridization-based means, and that these means can incorporate a further means, such as a magnetic bead-based means, to facilitate separation of the desired product.", "Many other methods exist by which a double-stranded nucleic acid building block can be generated that is serviceable for this invention; and these are known in the art and can be readily performed by the skilled artisan.", "According to particularly preferred embodiment, a double-stranded nucleic acid building block that is serviceable for this invention is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block.", "The two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang; thus containing no mismatches, apart from any overhang(s).", "According to another embodiment, the two strands of a double-stranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang.", "Thus, according to this embodiment, a double-stranded nucleic acid building block can be used to introduce codon degeneracy.", "Preferably the codon degeneracy is introduced using the site-saturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes.", "Contained within an exemplary experimental design for achieving an ordered assembly according to this invention are: 1) The design of specific nucleic acid building blocks.", "2) The design of specific ligatable ends on each nucleic acid building block.", "3) The design of a particular order of assembly of the nucleic acid building blocks.", "An overhang may be a 3′ overhang or a 5′ overhang.", "An overhang may also have a terminal phosphate group or alternatively may be devoid of a terminal phosphate group (having, e.g., a hydroxyl group instead).", "An overhang may be comprised of any number of nucleotides.", "Preferably an overhang is comprised of 0 nucleotides (as in a blunt end) to 10,000 nucleotides.", "Thus, a wide range of overhang sizes may be serviceable.", "Accordingly, the lower limit may be each integer from 1-200 and the upper limit may be each integer from 2-10,000.According to a particular exemplification, an overhang may consist of anywhere from 1 nucleotide to 200 nucleotides (including every integer value in between).", "The final chimeric nucleic acid molecule may be generated by sequentially assembling 2 or more building blocks at a time until all the designated building blocks have been assembled.", "A working sample may optionally be subjected to a process for size selection or purification or other selection or enrichment process between the performance of two assembly steps.", "Alternatively, the final chimeric nucleic acid molecule may be generated by assembling all the designated building blocks at once in one step.", "Utility The in vivo recombination method of this invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence.", "However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide.", "The approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin 1, antibodies, tPA and growth hormone.", "This approach may be used to generate proteins having altered specificity or activity.", "The approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 3′ untranslated regions or 5′ untranslated regions of genes.", "Thus this approach may be used to generate genes having increased rates of expression.", "This approach may also be useful in the study of repetitive DNA sequences.", "Finally, this approach may be useful to mutate ribozymes or aptamers.", "Scaffold-like regions separating regions of diversity in proteins may be particularly suitable for the methods of this invention.", "The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding.", "Examples of such scaffolds are the immunoglobulin beta barrel, and the four-helix bundle.", "The methods of this invention can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.", "The equivalents of some standard genetic matings may also be performed by the methods of this invention.", "For example, a “molecular” backcross can be performed by repeated mixing of the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.", "As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice.", "It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e.", "immunogenicity).", "Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not.", "3.2.4.End-Selection This invention provides a method for selecting a subset of polynucleotides from a starting set of polynucleotides, which method is based on the ability to discriminate one or more selectable features (or selection markers) present anywhere in a working polynucleotide, so as to allow one to perform selection for (positive selection) &/or against (negative selection) each selectable polynucleotide.", "In a preferred aspect, a method is provided termed end-selection, which method is based on the use of a selection marker located in part or entirely in a terminal region of a selectable polynucleotide, and such a selection marker may be termed an “end-selection marker”.", "End-selection may be based on detection of naturally occurring sequences or on detection of sequences introduced experimentally (including by any mutagenesis procedure mentioned herein and not mentioned herein) or on both, even within the same polynucleotide.", "An end-selection marker can be a structural selection marker or a functional selection marker or both a structural and a functional selection marker.", "An end-selection marker may be comprised of a polynucleotide sequence or of a polypeptide sequence or of any chemical structure or of any biological or biochemical tag, including markers that can be selected using methods based on the detection of radioactivity, of enzymatic activity, of fluorescence, of any optical feature, of a magnetic property (e.g.", "using magnetic beads), of immunoreactivity, and of hybridization.", "End-selection may be applied in combination with any method serviceable for performing mutagenesis.", "Such mutagenesis methods include, but are not limited to, methods described herein (supra and infra).", "Such methods include, by way of non-limiting exemplification, any method that may be referred herein or by others in the art by any of the following terms: “saturation mutagenesis”, “shuffling”, “recombination”, “re-assembly”, “error-prone PCR”, “assembly PCR”, “sexual PCR”, “crossover PCR”, “oligonucleotide primer-directed mutagenesis”, “recursive (&/or exponential) ensemble mutagenesis (see Arkin and Youvan, 1992)”, “cassette mutagenesis”, “in vivo mutagenesis”, and “in vitro mutagenesis”.", "Moreover, end-selection may be performed on molecules produced by any mutagenesis &/or amplification method (see, e.g., Arnold, 1993; Caldwell and Joyce, 1992; Stemmer, 1994; following which method it is desirable to select for (including to screen for the presence of) desirable progeny molecules.", "In addition, end-selection may be applied to a polynucleotide apart from any mutagenesis method.", "In a preferred embodiment, end-selection, as provided herein, can be used in order to facilitate a cloning step, such as a step of ligation to another polynucleotide (including ligation to a vector).", "This invention thus provides for end-selection as a serviceable means to facilitate library construction, selection &/or enrichment for desirable polynucleotides, and cloning in general.", "In a particularly preferred embodiment, end-selection can be based on (positive) selection for a polynucleotide; alternatively end-selection can be based on (negative) selection against a polynucleotide; and alternatively still, end-selection can be based on both (positive) selection for, and on (negative) selection against, a polynucleotide.", "End-selection, along with other methods of selection &/or screening, can be performed in an iterative fashion, with any combination of like or unlike selection &/or screening methods and serviceable mutagenesis methods, all of which can be performed in an iterative fashion and in any order, combination, and permutation.", "It is also appreciated that, according to one embodiment of this invention, end-selection may also be used to select a polynucleotide that is at least in part: circular (e.g.", "a plasmid or any other circular vector or any other polynucleotide that is partly circular), &/or branched, &/or modified or substituted with any chemical group or moiety.", "In accord with this embodiment, a polynucleotide may be a circular molecule comprised of an intermediate or central region, which region is flanked on a 5′ side by a 5′ flanking region (which, for the purpose of end-selection, serves in like manner to a 5′ terminal region of a non-circular polynucleotide) and on a 3′ side by a 3′ terminal region (which, for the purpose of end-selection, serves in like manner to a 3′ terminal region of a non-circular polynucleotide).", "As used in this non-limiting exemplification, there may be sequence overlap between any two regions or even among all three regions.", "In one non-limiting aspect of this invention, end-selection of a linear polynucleotide is performed using a general approach based on the presence of at least one end-selection marker located at or near a polynucleotide end or terminus (that can be either a 5′ end or a 3′ end).", "In one particular non-limiting exemplification, end-selection is based on selection for a specific sequence at or near a terminus such as, but not limited to, a sequence recognized by an enzyme that recognizes a polynucleotide sequence.", "An enzyme that recognizes and catalyzes a chemical modification of a polynucleotide is referred to herein as a polynucleotide-acting enzyme.", "In a preferred embodiment, serviceable polynucleotide-acting enzymes are exemplified non-exclusively by enzymes with polynucleotide-cleaving activity, enzymes with polynucleotide-methylating activity, enzymes with polynucleotide-ligating activity, and enzymes with a plurality of distinguishable enzymatic activities (including non-exclusively, e.g., both polynucleotide-cleaving activity and polynucleotide-ligating activity).", "Relevant polynucleotide-acting enzymes thus also include any commercially available or non-commercially available polynucleotide endonucleases and their companion methylases including those catalogued at the website http://www.neb.com/rebase, and those mentioned in the following cited reference (Roberts and Macelis, 1996).", "Preferred polynucleotide endonucleases include—but are not limited to—type II restriction enzymes (including type IIS), and include enzymes that cleave both strands of a double stranded polynucleotide (e.g.", "Not 1, which cleaves both strands at 5′ .", ".", ".", "GC/GGCCGC .", ".", ".", "3′) and enzymes that cleave only one strand of a double stranded polynucleotide, i.e.", "enzymes that have polynucleotide-nicking activity, (e.g.", "N. BstNB 1, which cleaves only one strand at 5′ .", ".", ".", "GAGTCNNNN/N .", ".", ".", "3′).", "Relevant polynucleotide-acting enzymes also include type III restriction enzymes.", "It is appreciated that relevant polynucleotide-acting enzymes also include any enzymes that may be developed in the future, though currently unavailable, that are serviceable for generating a ligation compatible end, preferably a sticky end, in a polynucleotide.", "In one preferred exemplification, a serviceable selection marker is a restriction site in a polynucleotide that allows a corresponding type II (or type IIS) restriction enzyme to cleave an end of the polynucleotide so as to provide a ligatable end (including a blunt end or alternatively a sticky end with at least a one base overhang) that is serviceable for a desirable ligation reaction without cleaving the polynucleotide internally in a manner that destroys a desired internal sequence in the polynucleotide.", "Thus it is provided that, among relevant restriction sites, those sites that do not occur internally (i.e.", "that do not occur apart from the termini) in a specific working polynucleotide are preferred when the use of a corresponding restriction enzyme(s) is not intended to cut the working polynucleotide internally.", "This allows one to perform restriction digestion reactions to completion or to near completion without incurring unwanted internal cleavage in a working polynucleotide.", "According to a preferred aspect, it is thus preferable to use restriction sites that are not contained, or alternatively that are not expected to be contained, or alternatively that are unlikely to be contained (e.g.", "when sequence information regarding a working polynucleotide is incomplete) internally in a polynucleotide to be subjected to end-selection.", "In accordance with this aspect, it is appreciated that restriction sites that occur relatively infrequently are usually preferred over those that occur more frequently.", "On the other hand it is also appreciated that there are occasions where internal cleavage of a polypeptide is desired, e.g.", "to achieve recombination or other mutagenic procedures along with end-selection.", "In accord with this invention, it is also appreciated that methods (e.g.", "mutagenesis methods) can be used to remove unwanted internal restriction sites.", "It is also appreciated that a partial digestion reaction (i.e.", "a digestion reaction that proceeds to partial completion) can be used to achieve digestion at a recognition site in a terminal region while sparing a susceptible restriction site that occurs internally in a polynucleotide and that is recognized by the same enzyme.", "In one aspect, partial digest are useful because it is appreciated that certain enzymes show preferential cleavage of the same recognition sequence depending on the location and environment in which the recognition sequence occurs.", "For example, it is appreciated that, while lambda DNA has 5 EcoR I sites, cleavage of the site nearest to the right terminus has been reported to occur 10 times faster than the sites in the middle of the molecule.", "Also, for example, it has been reported that, while Sac II has four sites on lambda DNA, the three clustered centrally in lambda are cleaved 50 times faster than the remaining site near the terminus (at nucleotide 40,386).", "Summarily, site preferences have been reported for various enzymes by many investigators (e.g., Thomas and Davis, 1975; Forsblum et al, 1976; Nath and Azzolina, 1981; Brown and Smith, 1977; Gingeras and Brooks, 1983; Krüger et al, 1988; Conrad and Topal, 1989; Oller et al, 1991; Topal, 1991; and Pein, 1991; to name but a few).", "It is appreciated that any empirical observations as well as any mechanistic understandings of site preferences by any serviceable polynucleotide-acting enzymes, whether currently available or to be procured in the future, may be serviceable in end-selection according to this invention.", "It is also appreciated that protection methods can be used to selectively protect specified restriction sites (e.g.", "internal sites) against unwanted digestion by enzymes that would otherwise cut a working polypeptide in response to the presence of those sites; and that such protection methods include modifications such as methylations and base substitutions (e.g.", "U instead of T) that inhibit an unwanted enzyme activity.", "It is appreciated that there are limited numbers of available restriction enzymes that are rare enough (e.g.", "having very long recognition sequences) to create large (e.g.", "megabase-long) restriction fragments, and that protection approaches (e.g.", "by methylation) are serviceable for increasing the rarity of enzyme cleavage sites.", "The use of M.Fnu II (mCGCG) to increase the apparent rarity of Not I approximately twofold is but one example among many (Qiang et al, 1990; Nelson et al, 1984; Maxam and Gilbert, 1980; Raleigh and Wilson, 1986).", "According to a preferred aspect of this invention, it is provided that, in general, the use of rare restriction sites is preferred.", "It is appreciated that, in general, the frequency of occurrence of a restriction site is determined by the number of nucleotides contained therein, as well as by the ambiguity of the base requirements contained therein.", "Thus, in a non-limiting exemplification, it is appreciated that, in general, a restriction site composed of, for example, 8 specific nucleotides (e.g.", "the Not I site or GC/GGCCGC, with an estimated relative occurrence of 1 in 48, i.e.", "1 in 65,536, random 8-mers) is relatively more infrequent than one composed of, for example, 6 nucleotides (e.g.", "the Sma I site or CCC/GGG, having an estimated relative occurrence of 1 in 46, i.e.", "1 in 4,096, random 6-mers), which in turn is relatively more infrequent than one composed of, for example, 4 nucleotides (e.g.", "the Msp I site or C/CGG, having an estimated relative occurrence of 1 in 44, i.e.", "1 in 256, random 4-mers).", "Moreover, in another non-limiting exemplification, it is appreciated that, in general, a restriction site having no ambiguous (but only specific) base requirements (e.g.", "the Fin I site or GTCCC, having an estimated relative occurrence of 1 in 45, i.e.", "1 in 1024, random 5-mers) is relatively more infrequent than one having an ambiguous W (where W=A or T) base requirement (e.g.", "the Ava II site or G/GWCC, having an estimated relative occurrence of 1 in 4×4×2×4×4—i.e.", "1 in 512-random 5-mers), which in turn is relatively more infrequent than one having an ambiguous N (where N=A or C or G or T) base requirement (e.g.", "the Asu I site or G/GNCC, having an estimated relative occurrence of 1 in 4×4×1×4×4, i.e.", "1 in 256-random 5-mers).", "These relative occurrences are considered general estimates for actual polynucleotides, because it is appreciated that specific nucleotide bases (not to mention specific nucleotide sequences) occur with dissimilar frequencies in specific polynucleotides, in specific species of organisms, and in specific groupings of organisms.", "For example, it is appreciated that the % G+C contents of different species of organisms are often very different and wide ranging.", "The use of relatively more infrequent restriction sites as a selection marker include—in a non-limiting fashion—preferably those sites composed at least a 4 nucleotide sequence, more preferably those composed of at least a 5 nucleotide sequence, more preferably still those composed at least a 6 nucleotide sequence (e.g.", "the BamH I site or G/GATCC, the Bgl II site or A/GATCT, the Pst I site or CTGCA/G, and the Xba I site or T/CTAGA), more preferably still those composed at least a 7 nucleotide sequence, more preferably still those composed of an 8 nucleotide sequence nucleotide sequence (e.g.", "the Asc I site or GG/CGCGCC, the Not I site or GC/GGCCGC, the Pac I site or TTAAT/TAA, the Pme I site or GTTT/AAAC, the Srf I site or GCCC/GGGC, the Sse838 I site or CCTGCA/GG, and the Swa I site or ATTT/AAAT), more preferably still those composed of a 9 nucleotide sequence, and even more preferably still those composed of at least a 10 nucleotide sequence (e.g.", "the BspG I site or CG/CGCTGGAC).", "It is further appreciated that some restriction sites (e.g.", "for class IIS enzymes) are comprised of a portion of relatively high specificity (i.e.", "a portion containing a principal determinant of the frequency of occurrence of the restriction site) and a portion of relatively low specificity; and that a site of cleavage may or may not be contained within a portion of relatively low specificity.", "For example, in the Eco57 I site or CTGAAG(16/14), there is a portion of relatively high specificity (i.e.", "the CTGAAG portion) and a portion of relatively low specificity (i.e.", "the N 16 sequence) that contains a site of cleavage.", "In another preferred embodiment of this invention, a serviceable end-selection marker is a terminal sequence that is recognized by a polynucleotide-acting enzyme that recognizes a specific polynucleotide sequence.", "In a preferred aspect of this invention, serviceable polynucleotide-acting enzymes also include other enzymes in addition to classic type II restriction enzymes.", "According to this preferred aspect of this invention, serviceable polynucleotide-acting enzymes also include gyrases, helicases, recombinases, relaxases, and any enzymes related thereto.", "Among preferred examples are topoisomerases (which have been categorized by some as a subset of the gyrases) and any other enzymes that have polynucleotide-cleaving activity (including preferably polynucleotide-nicking activity) &/or polynucleotide-ligating activity.", "Among preferred topoisomerase enzymes are topoisomerase I enzymes, which is available from many commercial sources (Epicentre Technologies, Madison, Wis.; Invitrogen, Carlsbad, Calif.; Life Technologies, Gathesburg, Md.)", "and conceivably even more private sources.", "It is appreciated that similar enzymes may be developed in the future that are serviceable for end-selection as provided herein.", "A particularly preferred topoisomerase I enzyme is a topoisomerase I enzyme of vaccinia virus origin, that has a specific recognition sequence (e.g.", "5′ .", ".", ".", "AAGGG .", ".", ".", "3′) and has both polynucleotide-nicking activity and polynucleotide-ligating activity.", "Due to the specific nicking-activity of this enzyme (cleavage of one strand), internal recognition sites are not prone to polynucleotide destruction resulting from the nicking activity (but rather remain annealed) at a temperature that causes denaturation of a terminal site that has been nicked.", "Thus for use in end-selection, it is preferable that a nicking site for topoisomerase-based end-selection be no more than 100 nucleotides from a terminus, more preferably no more than 50 nucleotides from a terminus, more preferably still no more than 25 nucloetides from a terminus, even more preferably still no more than 20 nucleotides from a terminus, even more preferably still no more than 15 nucleotides from a terminus, even more preferably still no more than 10 nucleotides from a terminus, even more preferably still no more than 8 nucleotides from a terminus, even more preferably still no more than 6 nucleotides from a terminus, and even more preferably still no more than 4 nucleotides from a terminus.", "In a particularly preferred exemplification that is non-limiting yet clearly illustrative, it is appreciated that when a nicking site for topoisomerase-based end-selection is 4 nucleotides from a terminus, nicking produces a single stranded oligo of 4 bases (in a terminal region) that can be denatured from its complementary strand in an end-selectable polynucleotide; this provides a sticky end (comprised of 4 bases) in a polynucleotide that is serviceable for an ensuing ligation reaction.", "To accomplish ligation to a cloning vector (preferably an expression vector), compatible sticky ends can be generated in a cloning vector by any means including by restriction enzyme-based means.", "The terminal nucleotides (comprised of 4 terminal bases in this specific example) in an end-selectable polynucleotide terminus are thus wisely chosen to provide compatibility with a sticky end generated in a cloning vector to which the polynucleotide is to be ligated.", "On the other hand, internal nicking of an end-selectable polynucleotide, e.g.", "500 bases from a terminus, produces a single stranded oligo of 500 bases that is not easily denatured from its complementary strand, but rather is serviceable for repair (e.g.", "by the same topoisomerase enzyme that produced the nick).", "This invention thus provides a method—e.g.", "that is vaccinia topoisomerase-based &/or type II (or IIS) restriction endonuclease-based &/or type III restriction endonuclease-based &/or nicking enzyme-based (e.g.", "using N. BstNB I)— for producing a sticky end in a working polynucleotide, which end is ligation compatible, and which end can be comprised of at least a 1 base overhang.", "Preferably such a sticky end is comprised of at least a 2-base overhang, more preferably such a sticky end is comprised of at least a 3-base overhang, more preferably still such a sticky end is comprised of at least a 4-base overhang, even more preferably still such a sticky end is comprised of at least a 5-base overhang, even more preferably still such a sticky end is comprised of at least a 6-base overhang.", "Such a sticky end may also be comprised of at least a 7-base overhang, or at least an 8-base overhang, or at least a 9-base overhang, or at least a 10-base overhang, or at least 15-base overhang, or at least a 20-base overhang, or at least a 25-base overhang, or at least a 30-base overhang.", "These overhangs can be comprised of any bases, including A, C, G, or T. It is appreciated that sticky end overhangs introduced using topoisomerase or a nicking enzyme (e.g.", "using N. BstNB I) can be designed to be unique in a ligation environment, so as to prevent unwanted fragment reassemblies, such as self-dimerizations and other unwanted concatamerizations.", "According to one aspect of this invention, a plurality of sequences (which may but do not necessarily overlap) can be introduced into a terminal region of an end-selectable polynucleotide by the use of an oligo in a polymerase-based reaction.", "In a relevant, but by no means limiting example, such an oligo can be used to provide a preferred 5′ terminal region that is serviceable for topoisomerase I-based end-selection, which oligo is comprised of: a 1-10 base sequence that is convertible into a sticky end (preferably by a vaccinia topoisomerase I), a ribosome binding site (i.e.", "and “RB S”, that is preferably serviceable for expression cloning), and optional linker sequence followed by an ATG start site and a template-specific sequence of 0-100 bases (to facilitate annealment to the template in the polymerase-based reaction).", "Thus, according to this example, a serviceable oligo (which may be termed a forward primer) can have the sequence: 5′[terminal sequence=(N)1-10][topoisomerase I site & RBS=AAGGGAGGAG][linker=(N)1-100][start codon and template-specific sequence=ATG(N)0-100]3′.", "Analogously, in a relevant, but by no means limiting example, an oligo can be used to provide a preferred 3′ terminal region that is serviceable for topoisomerase I-based end-selection, which oligo is comprised of: a 1-10 base sequence that is convertible into a sticky end (preferably by a vaccinia topoisomerase 1), and optional linker sequence followed by a template-specific sequence of 0-100 bases (to facilitate annealment to the template in the polymerase-based reaction).", "Thus, according to this example, a serviceable oligo (which may be termed a reverse primer) can have the sequence: 5′[terminal sequence=(N)1-10][topoisomerase I site=AAGGG][linker=(N)1-100][template-specific sequence=(N)0-100]3′.", "It is appreciated that, end-selection can be used to distinguish and separate parental template molecules (e.g.", "to be subjected to mutagenesis) from progeny molecules (e.g.", "generated by mutagenesis).", "For example, a first set of primers, lacking in a topoisomerase I recognition site, can be used to modify the terminal regions of the parental molecules (e.g.", "in polymerase-based amplification).", "A different second set of primers (e.g.", "having a topoisomerase I recognition site) can then be used to generate mutated progeny molecules (e.g.", "using any polynucleotide chimerization method, such as interrupted synthesis, template-switching polymerase-based amplification, or interrupted synthesis; or using saturation mutagenesis; or using any other method for introducing a topoisomerase I recognition site into a mutagenized progeny molecule as disclosed herein) from the amplified template molecules.", "The use of topoisomerase I-based end-selection can then facilitate, not only discernment, but selective topoisomerase 1-based ligation of the desired progeny molecules.", "Annealment of a second set of primers to thusly amplified parental molecules can be facilitated by including sequences in a first set of primers (i.e.", "primers used for amplifying a set parental molecules) that are similar to a toposiomerase I recognition site, yet different enough to prevent functional toposiomerase I enzyme recognition.", "For example, sequences that diverge from the AAGGG site by anywhere from 1 base to all 5 bases can be incorporated into a first set of primers (to be used for amplifying the parental templates prior to subjection to mutagenesis).", "In a specific, but non-limiting aspect, it is thus provided that a parental molecule can be amplified using the following exemplary—but by no means limiting—set of forward and reverse primers: Forward Primer: 5′ CTAGAAGAGAGGAGAAAACCATG(N)10-100 3′, and Reverse Primer: 5′ GATCAAAGGCGCGCCTGCAGG(N)10-100 3′ According to this specific example of a first set of primers, (N)10-100 represents preferably a 10 to 100 nucleotide-long template-specific sequence, more preferably a 10 to 50 nucleotide-long template-specific sequence, more preferably still a 10 to 30 nucleotide-long template-specific sequence, and even more preferably still a 15 to 25 nucleotide-long template-specific sequence.", "According to a specific, but non-limiting aspect, it is thus provided that, after this amplification (using a disclosed first set of primers lacking in a true topoisomerase I recognition site), amplified parental molecules can then be subjected to mutagenesis using one or more sets of forward and reverse primers that do have a true topoisomerase I recognition site.", "In a specific, but non-limiting aspect, it is thus provided that a parental molecule can be used as templates for the generation of a mutagenized progeny molecule using the following exemplary—but by no means limiting—second set of forward and reverse primers: Forward Primer: 5′ CTAGAAGGGAGGAGAAAACCATG 3′ Reverse Primer: 5′ GATCAAAGGCGCGCCTGCAGG 3′ (contains Asc I recognition sequence) It is appreciated that any number of different primers sets not specifically mentioned can be used as first, second, or subsequent sets of primers for end-selection consistent with this invention.", "Notice that type II restriction enzyme sites can be incorporated (e.g.", "an Asc I site in the above example).", "It is provided that, in addition to the other sequences mentioned, the experimentalist can incorporate one or more N,N,G/T triplets into a serviceable primer in order to subject a working polynucleotide to saturation mutagenesis.", "Summarily, use of a second and/or subsequent set of primers can achieve dual goals of introducing a topoisomerase I site and of generating mutations in a progeny polynucleotide.", "Thus, according to one use provided, a serviceable end-selection marker is an enzyme recognition site that allows an enzyme to cleave (including nick) a polynucleotide at a specified site, to produce a ligation-compatible end upon denaturation of a generated single stranded oligo.", "Ligation of the produced polynucleotide end can then be accomplished by the same enzyme (e.g.", "in the case of vaccinia virus topoisomerase 1), or alternatively with the use of a different enzyme.", "According to one aspect of this invention, any serviceable end-selection markers, whether like (e.g.", "two vaccinia virus topoisomerase I recognition sites) or unlike (e.g.", "a class II restriction enzyme recognition site and a vaccinia virus topoisomerase I recognition site) can be used in combination to select a polynucleotide.", "Each selectable polynucleotide can thus have one or more end-selection markers, and they can be like or unlike end-selection markers.", "In a particular aspect, a plurality of end-selection markers can be located on one end of a polynucleotide and can have overlapping sequences with each other.", "It is important to emphasize that any number of enzymes, whether currently in existence or to be developed, can be serviceable in end-selection according to this invention.", "For example, in a particular aspect of this invention, a nicking enzyme (e.g.", "N. BstNB I, which cleaves only one strand at 5′ .", ".", ".", "GAGTCNNNN/N .", ".", ".", "3′) can be used in conjunction with a source of polynucleotide-ligating activity in order to achieve end-selection.", "According to this embodiment, a recognition site for N. BstNB 1—instead of a recognition site for topoisomerase 1—should be incorporated into an end-selectable polynucleotide (whether end-selection is used for selection of a mutagenized progeny molecule or whether end-selection is used apart from any mutagenesis procedure).", "It is appreciated that the instantly disclosed end-selection approach using topoisomerase-based nicking and ligation has several advantages over previously available selection methods.", "In sum, this approach allows one to achieve direction cloning (including expression cloning).", "Specifically, this approach can be used for the achievement of: direct ligation (i.e.", "without subjection to a classic restriction-purification-ligation reaction, that is susceptible to a multitude of potential problems from an initial restriction reaction to a ligation reaction dependent on the use of T4 DNA ligase); separation of progeny molecules from original template molecules (e.g.", "original template molecules lack topoisomerase I sites that not introduced until after mutagenesis), obviation of the need for size separation steps (e.g.", "by gel chromatography or by other electrophoretic means or by the use of size-exclusion membranes), preservation of internal sequences (even when topoisomerase I sites are present), obviation of concerns about unsuccessful ligation reactions (e.g.", "dependent on the use of T4 DNA ligase, particularly in the presence of unwanted residual restriction enzyme activity), and facilitated expression cloning (including obviation of frame shift concerns).", "Concerns about unwanted restriction enzyme-based cleavages especially at internal restriction sites (or even at often unpredictable sites of unwanted star activity) in a working polynucleotide—that are potential sites of destruction of a working polynucleotide can also be obviated by the instantly disclosed end-selection approach using topoisomerase-based nicking and ligation.", "3.3 Tunable 3.4 Transposons 3.4.1.General Applications In one aspect, the present invention relates generally to the field of transposable nucleic acid and for introducing genetic changes to nucleic acid.", "In one embodiment this invention relates to transposable elements isolated from maize and a process for using the same to identify and isolate genes and to insert desired gene sequences into plants in a heritable manner.", "In another embodiment, this invention provides for using transposons as a high molecular weight cloning system.", "3.4.2.Specific Methodologies 3.4.2.1.Description of Transposable Elements Transposable genetic elements are DNA sequences, found in a wide variety of prokaryotic and eukaryotic organisms, that can move or transpose from one position to another position in a genome.", "In vivo, intra-chromosomal transpositions as well as transpositions between chromosomal and non-chromosomal genetic material are known.", "In several systems, transposition is known to be under the control of a transposase enzyme that is typically encoded by the transposable element.", "The genetic structures and transposition mechanisms of various transposable elements are summarized, for example, in “Transposable Genetic Elements” in “The Encyclopedia of Molecular Biology,” Kendrew and Lawrence, Eds., Blackwell Science, Ltd., Oxford (1994), incorporated herein by reference.", "Scientists have taken advantage of transposons to transport reporter genes for use in studying gene expression.", "These include transcriptional (Type I) fusions and translational (Type II) fusions.", "Transcriptional fusions, unlike translational fusions, place a reporter gene under the control of another promoter, but do not translationally fuse two protein domains.", "Translational fusions have generally been made to link a reporter gene carried inside the transposon to the translational frame of the target gene so that the reporter gene is expressed under direct control of the transcription and translation signals of the target gene of interest to study gene regulation.", "This requires that an open reading frame extend through the end of the transposable element to join an internal reporter protein to external translational sequences.", "This usually results in complete inactivation of the target gene.", "3.4.2.2.In Vitro Transposition Systems In vitro transposition systems that utilize the particular transposable elements of bacteriophage Mu and bacterial transposon Tn10 have been described, by the research groups of Kiyoshi Mizuuchi and Nancy Kleckner, respectively.", "The bacteriophage Mu system was first described by Mizuuchi, K., “In Vitro Transposition of Bacteria Phage Mu: A Biochemical Approach to a Novel Replication Reaction,” Cell:785-794 (1983) and Craigie, R. et al., “A Defined System for the DNA Strand-Transfer Reaction at the Initiation of Bacteriophage Mu Transposition: Protein and DNA Substrate Requirements,” P.N.A.S.", "U.S.A. 82: 7570-7574 (1985).", "The DNA donor substrate (mini-Mu) for Mu in vitro reaction normally requires six Mu transposase binding sites (three of about 30 bp at each end) and an enhancer sequence located about 1 kb from the left end.", "The donor plasmid must be supercoiled.", "Proteins required are Mu-encoded A and B proteins and host-encoded HU and IHF proteins.", "Lavoie, B.", "D, and G. Chaconas, “Transposition of phage Mu DNA,” Curr.", "Topics Microbiol.", "Immunol.", "204: 83-99 (1995).", "The Mu-based system is disfavored for in vitro transposition system applications because the Mu termini are complex and sophisticated and because transposition requires additional proteins above and beyond the transposase.", "The Tn10 system was described by Morisato, D. and N. Kleckner, “Tn10 Transposition and Circle Formation in vitro,” Cell 51: 101-111 (1987) and by Benjamin, H. W. and N. Kleckner, “Excision Of Tn10 from the Donor Site During Transposition Occurs By Flush Double-Strand Cleavages at the Transposon Termini,” P.N.A.S.", "U.S.A. 89: 4648-4652 (1992).", "The Tn10 system involves a supercoiled circular DNA molecule carrying the transposable element (or a linear DNA molecule plus E. coli IHF protein).", "The transposable element is defined by complex 42 bp terminal sequences with IHF binding site adjacent to the inverted repeat.", "In fact, even longer (81 bp) ends of Tn10 were used in reported experiments.", "Sakai, J. et al., “Identification and Characterization of Pre-leavage Synaptic Complex that is an Early Intermediate in Tn10 transposition,” E.M.B.O.", "J.", "14: 4374-4383 (1995).", "In the Tn10 system, chemical treatment of the transposase protein is essential to support active transposition.", "In addition, the termini of the Tn10 element limit its utility in a generalized in vitro transposition system.", "Both the Mu- and Tn10-based in vitro transposition systems are further limited in that they are active only on covalently closed circular, supercoiled DNA targets.", "What is desired is a more broadly applicable in vitro transposition system that utilizes shorter, more well defined termini and which is active on target DNA of any structure (linear, relaxed circular, and supercoiled circular DNA).", "According to alternative embodiments of this invention, the steps of introducing a plurality of traits and/or generating a set of mutagenized organisms may include the step of cloning.", "In a preferred embodiment, this invention provides that the step of cloning may comprise using a Tn7 transposon-based system, such as but not limited to GPS-1.GPS-1 is an in vitro system (New England BioLabs Inc., Catalog #E7100S) that uses TnsABC Transposase to insert a transposon (Transprimer™) randomly into the DNA target (See references Craig, N. L. (1996) Curr Top Microbiol Immunol 204, 2748; Stellwagen, A. E. and Craig, N. L. (1997) Genetics 145, 573-85; Biery, M. C., Stewart, F. J., Stellwagen, A. E., Raleigh, E. A. and Craig, N. L., (2000) Nucleic Acids Res 28, 1067-1077).", "Such a system or modifications thereof that take advantage of transposon insertion sequences can be utilized to aid in the cloning of high molecular weight DNA.", "Such cloning approaches may also be provided for by this invention as a step in cell screening.", "3.4.23.Importance of Transposons in Agriculture Currently, there is a great deal of interest in the development of gene transfer vectors for use with agriculturally important plants (See Outlook for Science and Technology, The Next Five Years, Vol.", "III (National Science Foundation (1982); and O.T.A.", "Report, Impact of Applied Genetics (1981)).", "Although the United States presently has an excess productivity in the agricultural sector, this is recognized as a local and short term condition.", "Thus, agricultural research and planning must be based on long term considerations.", "The variety of problems surrounding increases in population, degradation of prime farm land and decreasing availability of good farm land necessitates the increased use of marginal land, as well as exogenous fertilizers and chemical pest control compositions.", "Classical plant breeding programs have thus far been successful in increasing agricultural productivity.", "However, a substantial fraction of the increase in farm productivity experienced in the United States in the past 40 years is attributable to the use of fertilizers and modern energy intensive cultivation practices, both of which are increasingly costly.", "The ability of plant breeding alone to sustain productivity is a matter of some question.", "Plant breeders are divided in their views on whether genetic improvements will continue at the rate that has occurred over the past few decades or will begin to level out.", "Since such questions cannot be resolved a priori, it is prudent to explore a variety of additional means by which agronomically useful traits can be accumulated and improved in major crop plants.", "The unconventional areas that are presently receiving the most attention in the academic research establishment, as well as in both small and large firms with plant-oriented research programs are wide genetic crosses, tissue culture and the development of gene transfer systems that circumvent fertility barriers.", "In the past, many attempts have been made to transform plant cells with DNA from a variety of sources.", "The first unequivocal demonstration that DNA transfer can and does occur in plants emerged from the work described above on Agrobacterium tumefaciens Ti plasmid.", "However, Ti-plasmid mediated gene transfer is presently accomplished only in dicotyledonous plants that interact with the plasmid's natural host bacterium.", "Since most major crop species are monocotyledonous, ti-plasmid mediated gene transfer has limited applications.", "3.4.2.3.1.Use of Transposons on the Ti Plasmid of Agrobacterium In higher organisms, transposons have been, or are being, used in several ways.", "For example, transposons are used as mutagens on the Ti plasmid of Agrobacterium tumefaciens.", "That is, a method for using bacterial transposons to cause insertion mutations in the Agrobacterium tumefaciens Ti plasmid, the causative agent of crown gall disease in dicotyledonous plants, has been developed.", "(See Zambriski, P., Goodman, H., Van Montagu, M. and Schell, J., Mobile Genetic Elements, J. Shapiro, Ed., (Academic Press) New York, pp.", "506-535 (1983)).", "By this technique, it has become possible to identify the plasmid-borne genes that are responsible for virulence, as well as those that are responsible for the tumorous transformation of plant cells caused by the Ti plasmid.", "Further, it has become possible to show by using transposable elements, that a portion of the Ti plasmid can be integrated into plant genomes and can act as a vehicle for transferring genes from virtually any organism to any dicotyledonous plant that is susceptible to Agrobacterium tumefaciens.", "3.4.2.3.2.Use of Transposons in Maize In maize, a monocotyledon, transposable elements were first genetically identified in the mid-1940s.", "These elements have been studied extensively and their genetic behavior has been extensively reviewed (See McClintock, B., Cold Spring Harbor Symp.", "Quant.", "Biol.", "16: 1347 (1951); McClintock, B., Cold Spring Harbor Symp.", "Quant.", "Biol.", "21: 197-216 (1956); McClintock, B., Brookhaven Symp.", "Biol.", "18: 162-184 (1965); Fincham, J. R. S., and Sastry, G. R. K., Ann.", "Rev.", "Genet.", "8: 15-50 (1974); and Fedoroff, N., Mobile Genetic Elements, J. Shapiro, Ed., (Academic Press) New York, pp.", "1-63 (1983)).", "It has been demonstrated that transposons are normal, although cryptic, residents of the maize genome and that upon activation, they are responsible for various types of genetic rearrangements, including chromosome breakage, deletions, duplications, inversions and translocations.", "In addition, it has been shown that certain common types of unstable mutations, which have been studied for decades in both maize and in other organisms, are attributable to the insertion of transposons into genes or genetic loci.", "3.4.2.4.1.Type I Fusions Type I transcriptional fusions have been used to study gene expression and regulation by co-opting the native transcriptional signal to express the exogenous reporter gene.", "For example for gene expression in E. coli, yeast, and Drosophila development.", "3.4.2.4.2.Type II Fusions Type II fusions have also been used to study gene expression and regulation, but in this case not only co-opt the transcriptional signals, but any translational signals as well to express the reporter gene.", "In this type of system the protein product usually only expresses the activity of the reporter exogenous gene.", "MudII elements are mini-Mu deletion elements which are type II Mu transposable elements.", "Examples of these include beta-galactosidase fusion elements, where a beta-galactosidase (lacZ) reporter gene is inserted via transposable elements to detect transcription and translation of regulated gene systems.", "This usually results in the inactivation of the targeted gene.", "Two types of Mu protein fusions have been developed, lacZ fusion elements and nptI fusion elements (Symonds, Toussaint et al.", "(1987).", "Phage Mu) The lacZ elements have been used to study translation regulation, determination of the translation phase of target genes, infer the location of a protein fusion by hybrid protein size, determine amino terminal sequence, and raise antibodies to regions of the protein of interest.", "By far the major goal of these studies has been to determine mechanisms of gene expression in the studied organisms.", "The nptI system was designed to perform transposon-tagging since nptI is known to function as an aminoglycoside resistance gene in a variety of organisms.", "Transposon tagging is a method of creating an mutant by inserting a transposon with a selectable marker into the gene of interest so that mutants which inactivate the gene can be identified and maintained.", "This element is useful since it allows the nptI to be directly linked to the transcription/translation system of the organism being studied.", "In these studies there has been no emphasis on creating novel proteins with new activities using these transposable elements.", "More importantly, these Mu elements are restricted to making amino-terminal fusions to the reporter protein.", "In these cases the inserted reporter gene is fused to the carboxy-end of the truncated targeted protein, terminating inside the Mu.", "If the transposable element were to insert before the amino terminal of a targeted gene, functional translation could only occur on the marker gene by itself, and no translation of the target gene would occur.", "3.4.2.43.Problems with Mu Unfortunately, available Mu elements had several problems.", "First, it has not been demonstrated that Mu elements can be readily used as a general method for the development of fusion proteins with two active domains.", "Second, the Mu elements used thus far for creation of protein fusions can not be used for construction of “carboxy-terminal” fusions since they did not have an open reading frame extending into the element.", "Third, the Mu elements previously used have long linker regions which incorporate a 40 amino acid linker between the fused domains.", "This could create protein folding problems or unwanted domain interactions.", "Fourth the currently existing Mu elements had only a single restriction site for the insertion of protein domains.", "Finally, although Mu elements which had deleted ends existed, it was not known whether they would transpose well with additional sequences added in such close proximity to the right end and whether the intervening linker region which would join the two protein domains would interfere with the construction of active chimeric proteins.", "3.4.2.5.Other Transposons Other transposons have been used in a similar manner as Mu to create lac fusions to study gene expression.", "These include Tn10 and Tn917 (Berg and Howe.", "(1989).", "Mobile DNA).", "The Tn5 element has also been used to construct phoA fusions in vivo.", "Fusions with alkaline phosphatase (phoA) have also been used to probe the structure of membrane bound proteins (Lloyd and Kadner.", "(1990).", "J. Bacteriol.", "172: 1688-93.).", "In general, these transposons have been used to study the membrane topology structure of a particular gene and protein secretion.", "The resultant fusion proteins are also limited to amino-terminal fusion of the reporter PhoA reporter protein resulting in fusion at the carboxy end of the targeted gene.", "In general, these types of fusions have been applied to the study of gene expression.", "These elements were constructed with truncated marker proteins that extend through the end of the transposon.", "Transposition of the element can create an in-frame fusion with a target gene, thereby activating expression.", "Mini-Mu elements are used because they transpose at high frequencies, insert randomly, and can be packaged along with a target plasmid and transduced to a new cell (Symonds, Toussaint et al.", "(1987).", "Phage Mu).", "Some of the more pertinent work that has been done in the area of transposable elements are detailed in the following.", "Namgoong et al., (1994), teach that the Mu transposition reaction attachment sites attL and attR can promote the assembly of higher order complexes held together by non-covalent protein-DNA and protein-protein interactions.", "(Namgoong, Jayaram et al.", "(1994).", "J Mol.", "Biol.", "238: 514-527.)", "Harel et al., (1990), teach that in Mu helper-mediated transposition packaging the left end contains an essential domain defined by nucleotides 1 to 54 of the left end (attL).", "At the right end (attR), they teach that the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increase transposition frequency about 15-fold.", "(Harel, Dupliessis et al.", "(1990).", "Arch Microbiol.", "154: 67-72.)", "Groenen and van de Putte (1986), teach that the Mu A protein binds weakly to sequences between nucleotides 1 to 30 on the right end (RI) and between nucleotides 110 and 135 on the left end (L2).", "Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base pairs in the stronger A binding sites, located adjacent to these weak A binding sites.", "(Groenen and van de Putte.", "(1986).", "J Mol.", "Biol.", "189: 597-602.)", "Groenen and et al.", "(1985) teach the DNA sequences at the end of the genome of bacteriophage Mu that are essential for transposition.", "(Groenen, Timmers et al.", "(1985).", "Proc Natl Acad Sci, USA.", "82: 2087-2091.)", "Lloyd and Kadner teach the how to probe the topology of the uhpT sugar phosphate transporter using a Tn5phoA element.", "(Lloyd and Kadner.", "(1990).", "J. Bacteriol.", "172: 1688-93.)", "Phage Mu (1987), Cold Spring Harbor Laboratory Press (Symonds, et al eds.)", "teaches general methods for handling and working with bacteriophage Mu as a transposon, and describes the various uses of mini-Mu elements including the construction of Mu transcriptional and translational fusions.", "Silhavy and Beckwith (1985) teaches the various uses of lac fusions for the study of biological problems.", "(Silhavy and Beckwith.", "(1985).", "Microbiol Rev.", "49: 398-418.)", "Mobile DNA, (1989), American Society for Microbiology, Publishers.", "(Berg, Howe, eds) describes transposons.", "Casadaban, et al.", "(1983) Methods in Enzymol, provides a good general review of beta-galactosidase gene fusions for the study of gene expression.", "(Casadaban, Martinez-Arias et al.", "(1983).", "Recombinant DNA.", "Methods in Enzymology.", "100: 293-308.)", "3.4.3.In Vitro Transposition System The present invention is summarized in that an in vitro transposition system comprises a preparation of a suitably modified transposase of bacterial transposon Tn5, a donor DNA molecule that includes a transposable element, a target DNA molecule into which the transposable element can transpose, all provided in a suitable reaction buffer.", "3.4.3.1.Donor DNA Molecule: Transposable DNA Sequence of Interest The transposable element of the donor DNA molecule is characterized as a transposable DNA sequence of interest, the DNA sequence of interest being flanked at its 5′- and 3′-ends by short repeat sequences that are acted upon in trans by Tn5 transposase.", "3.4.3.1.1.Modified Transposase Enzyme Comprises Two Classes of Differences From Wild Type Tn5 Transposase The invention is further summarized in that the suitably modified transposase enzyme comprises two classes of differences from wild type Tn5 transposase, where each class has a separate measurable effect upon the overall transposition activity of the enzyme and where a greater effect is observed when both modifications are present.", "The suitably modified enzyme both (1) binds to the repeat sequences of the donor DNA with greater avidity than wild type Tn5 transposase (“class (1) mutation”) and (2) is less likely than the wild type protein to assume an inactive multimeric form (“class (2) mutation”).", "A suitably modified Tn5 transposase of the present invention that contains both class (1) and class (2) modifications induces at least about 100-fold (.+/−.", "10%) more transposition than the wild type enzyme, when tested in combination in an in vivo conjugation assay as described by Weinreich, M. D., “Evidence that the cis Preference of the Tn5 Transposase is Caused by Nonproductive Multimerization,” Genes and Development 8: 2363-2374 (1994), incorporated herein by reference.", "Under optimal conditions, transposition using the modified transposase may be higher.", "A modified transposase containing only a class (1) mutation binds to the repeat sequences with sufficiently greater avidity than the wild type Tn5 transposase that such a Tn5 transposase induces about 5- to 50-fold more transposition than the wild type enzyme, when measured in vivo.", "A modified transposase containing only a class (2) mutation is sufficiently less likely than the wild type Tn5 transposase to assume the multimeric form that such a Tn5 transposase also induces about 5- to 50-fold more transposition than the wild type enzyme, when measured in vivo.", "3.4.4.Transposons—Specialized Applications 3.4.4.1.In Vitro System for Introducing any Transposable Element from a Donor DNA into a Target DNA It will be appreciated that this technique provides a simple, in vitro system for introducing any transposable element from a donor DNA into a target DNA.", "It is generally accepted and understood that Tn5 transposition requires only a pair of OE termini, located to either side of the transposable element.", "These OE termini are generally thought to be 18 or 19 bases in length and are inverted repeats relative to one another.", "Johnson, R. C., and W. S. Reznikoff, Nature 304: 280 (1983), incorporated herein by reference.", "The Tn5 inverted repeat sequences, which are referred to as “termini” even though they need not be at the termini of the donor DNA molecule, are well known and understood.", "Apart from the need to flank the desired transposable element with standard Tn5 outside end (“OE”) termin, few other requirements on either the donor DNA or the target DNA are envisioned.", "It is thought that Tn5 has few, if any, preferences for insertion sites, so it is possible to use the system to introduce desired sequences at random into target DNA.", "Therefore, it is believed that this method, employing the modified transposase described herein and a simple donor DNA, is broadly applicable to introduce changes into any target DNA, without regard to its nucleotide sequence.", "3.4.4.2.Generation of Functional Fusion Protein Products The instant invention provides constructs and methods for the rapid and efficient generation of functional fusion protein products with either carboxy-terminal or amino-terminal fusions.", "Functional fusion proteins are those which retain some of the activity of the original domains, and/or those which have a newly created activity.", "Throughout this specification, reference is made to two types of fusions: carboxy terminal fusions and amino terminal fusions.", "In this text we use amino and carboxy terminal fusions to refer to the end of the domain inside of the Mu elements which is fused to the target molecule.", "Thus, carboxy terminal fusion elements are those with a protein domain inside of the Mu which extends out of the Mu element such that the exogenous protein is fused to the amino end of the endogenous protein.", "The amino terminal fusion elements are those that create fusions with a target gene extending into the element such that the exogenous protein is fused to the carboxy terminal of the endogenous protein (as referenced within U.S. Pat.", "Nos.", "5,965,443 and 4,732,856).", "3.4.5.Additional Applications It is envisioned that in addition to the uses specifically noted herein, other applications will be apparent to the skilled molecular biologist.", "In particular, methods for introducing desired mutations into prokaryotic or eukaryotic DNA are very desirable.", "For example, at present it is difficult to knock out a functional eukaryotic gene by homologous recombination with an inactive version of the gene that resides on a plasmid.", "The difficulty arises from the need to flank the gene on the plasmid with extensive upstream and downstream sequences.", "Using this system, however, an inactivating transposable element containing a selectable marker gene (e.g., neo) can be introduced in vitro into a plasmid that contains the gene that one desires to inactivate.", "After transposition, the products can be introduced into suitable host cells.", "Using standard selection means, one can recover only cell colonies that contain a plasmid having the transposable element.", "Such plasmids can be screened, for example by restriction analysis, to recover those that contain a disrupted gene.", "Such clones can then be introduced directly into eukaryotic cells for homologous recombination and selection using the same marker gene.", "Also, one can use the system to readily insert a PCR-amplified DNA fragment into a vector, thus avoiding traditional cloning steps entirely.", "This can be accomplished by (1) providing suitable a pair of PCR primers containing OE termini adjacent to the sequence-specific parts of the primers, (2) performing standard PCR amplification of a desired nucleic acid fragment, (3) performing the in vitro transposition reaction of the present invention using the double-stranded products of PCR amplification as the donor DNA.", "The invention is not intended to be limited to the foregoing examples, but to encompass all such modifications and variations as come within the scope of the appended claims.", "3.5.Homologous Recombination 3.5.1.Homologous Recombination for the Generation of Deletions and Insertions The invention relates to compositions and methods of rapidly evolving specific protein domains using a library of nucleic acid filaments and a recombinase polypeptide or peptide.", "The invention relates to compositions and methods for targeting sequence modifications in one or more genes of a related family of genes using enhanced homologous recombination techniques.", "The invention also relates to compositions and methods for isolating and identifying novel members of homologous sequences families.", "These techniques may be used to create animal or plant models of disease as well as to identify new targets for drug or pathogen screening.", "3.5.1.1.Evolution of Genes In nature, the evolution of genes and their encoded proteins occurs through an equilibrium between recombination or mutation and selection.", "While evolution in nature takes millions of years, in vitro methods and compositions have been developed to evolve proteins, with improved and novel functions, in a matter of hours to days.", "3.5.1.1.1.Through Mutagenesis Current in vitro gene evolution methods utilize repeated cycles of random mutagenesis or random nicking and mixing of related genes containing mutations in PCR-based random recombination.", "These methods couple multiple rounds of in vitro mutagenesis with screening systems to produce and identify the desired mutants or recombinants (Stemmer 1994.Nature 370: 389-391; Arnold 1996.Chemical Engineering Science 51: 5091-5102).", "Research has shown, however, that the mutations of interest tend to occur in those regions or domains that are directly related to function (Chen and Arnold.", "1993.PNAS USA 90: 5618-5622).", "However, these mutagenesis methods produce random mutations throughout the gene of interest which requires the need to screen large numbers of uninteresting or deleterious mutants.", "The labor-intensive and time consuming aspects of these methods are further complicated by the necessity of multiple rounds of subcloning and can be extremely challenging if the screening system is complex and does not utilize a selection system.", "3.5.1.1.2.Homologous Recombination (HR) Homologous recombination (HR) is defined as the exchange of homologous or similar DNA sequences between two DNA molecules.", "As essential feature of HR is that the enzymes responsible for the recombination event can pair any homologous sequences as substrates.", "The ability of HR to transfer genetic information between DNA molecules makes targeted homologous recombination a very powerful method in genetic engineering and gene manipulation.", "Both genetic and cytological studies have indicated that such a crossing-over process occurs between pairs of homologous chromosomes during meiosis in higher organisms.", "3.5.1.1.3.Site-Specific Recombination Alternatively, in site-specific recombination, exchange occurs at a specific site, as in the integration of phage A into the E coli chromosome and the excision of lambda DNA from it.", "Site-specific recombination involves specific inverted repeat sequences; e.g.", "the Cre-loxP and FLP—FRT systems.", "Within these sequences there is only a short stretch of homology necessary for the recombination event, but not sufficient for it.", "The enzymes involved in this event generally cannot recombine other pairs of homologous (or nonhomologous) sequences, but act specifically.", "3.5.1.1.4.Advantage of Homologous Recombination over Site-Specific Recombination Although both site-specific recombination and homologous recombination are useful mechanisms for genetic engineering of DNA sequences, targeted homologous recombination provides a basis for targeting and altering essentially any desired sequence in a duplex DNA molecule, such as targeting a DNA sequence in a chromosome for replacement by another sequence.", "Site-specific recombination has been proposed as one method to integrate transfected DNA at chromosomal locations having specific recognition sites (O'Gorman et al.", "(1991) Science 251: 1351; Onouchi et al.", "(1991) Nucleic Acids Res.", "19: 6373).", "Unfortunately, since this approach requires the presence of specific target sequences and recombinases, its utility for targeting recombination events at any particular chromosomal location is severely limited in comparison to targeted general recombination.", "3.5.1.1.5.HR to Create Transgenic Plants, Animals, and Organisms Homologous recombination has also been used to create transgenic plants and animals.", "Transgenic organisms contain stably integrated copies of genes or gene constructs derived from another species in the chromosome of the transgenic organism.", "In addition, gene targeted animals can be generated by introducing cloned DNA constructs of the foreign genes into totipotent cells by a variety of methods, including homologous recombination.", "For example, animals that develop from genetically altered totipotent cells can contain the foreign gene in all somatic cells and also in germ-line cells.", "3.5.1.1.5.1.Current Methods Using Embryonic Stem Cells Currently methods for producing transgenic and targeted animals have been performed on totipotent embryonic stem cells (ES) and with fertilized zygotes.", "ES cells have an advantage in that large numbers of cells can be manipulated easily by homologous recombination in vitro before they are used to generate targeted animals.", "Currently, however, only embryonic stem cells from mice have been shown to contribute to the germ line.", "Alternatively, DNA can also be introduced into fertilized oocytes by micro-injection into pronuclei which are then transferred into the uterus of a pseudo-pregnant recipient animal to develop to term.", "The ability of mammalian and human cells to incorporate exogenous genetic material into genes residing on chromosomes has demonstrated that these cells have the general enzymatic machinery for carrying out homologous recombination required between resident and introduced sequences.", "These targeted recombination events can be used to correct mutations at known sites, replace genes or gene segments with defective ones, or introduce foreign genes into cells.", "3.5.1.1.5.2.Frequency and Efficiency of HR HR can be used to add subtle mutations at known sites, replace wild type genes or gene segments or introduce completely foreign genes into cells.", "However, HR efficiency is very low in living cells and is dependent on several parameters, including the method of DNA delivery, how it is packaged, its size and conformation, DNA length and position of sequences homologous to the target, and the efficiency of hybridization and recombination at chromosomal sites.", "These variables severely limit the use of conventional HR approaches for gene evolution in cell based systems.", "(Kucherlapati et al., 1984.PNAS; USA 81: 3153-3157; Smithies et al.", "1985.Nature 317: 230-234; Song et al.", "1987.PNAS USA 84: 6820-6824; Doetschman et al.", "1987.Nature 330: 576-578; Kim and Smithies.", "1988.Nuc.", "Acids.", "Res.", "16: 8887-8903; Koller and Smithies.", "1989.PNAS USA 86: 8932-8935; Shesely et al.", "1991.PNAS USA 88: 4294-4298; Kim et al.", "1991.Gene 103: 227-233).", "3.5.1.1.5.2.1.Enhancement by the Presence of Recombinase Activities The frequency of HR is significantly enhanced by the presence of recombinase activities in cellular and cell free systems.", "Several proteins or purified extracts that promote HR (i.e., recombinase activity) have been identified in prokaryotes and eukaryotes (Cox and Lehman., 1987.Annu.", "Rev.", "Biochem.", "56: 229-262; Radding.", "1982.Annual Review of Genetics 16: 405-547; McCarthy et al.", "1988.PNAS; USA 85: 5854-5858).", "These recombinases promote one or more steps in the formation of homologously-paired intermediates, strand-exchange, and/or other steps.", "Recent advances have resulted in techniques allowing enhanced homologous recombination (EHR) using recombinases such as recA and Rad51 and single-stranded nucleic acids that have sequence heterologies.", "This allows sequence modifications to be specifically targeted to virtually any genomic position.", "See for example, PCT US93/03868 and PCT US98/05223, both of which are expressly incorporated herein by reference.", "3.5.1.1.5.2.1.1.Recombinase Rec A: A Bacterial Protein that Catalyses Homologous Pairing and Strand Exchange Between Two Homologous DNA Molecules The most studied recombinase to date is the RecA recombinase of E coli, which is involved in homology search and strand exchange reactions (Cox and Lehman, 1987, supra).", "The bacterial RecA protein (Mr 37,842) catalyses homologous pairing and strand exchange between two homologous DNA molecules (Kowalczykowski et al.", "1994.Microbiol.", "Rev.", "58: 401-465; West.", "1992.Annu.", "Rev.", "Biochem.", "61: 603-640); Roca and Cox.", "1990.CRC Cit.", "Rev.", "Biochem.", "Mol.", "Biol.", ": 415-455; Radding.", "1989.Biochim.", "Biophys.", "Acta.", "1008: 131-145; Smith.", "1989.Cell 58: 807-809).", "RecA protein binds cooperatively to any given sequence of single-stranded DNA with a stochiometry of one RecA protein monomer for every three to four nucleotides in DNA (Cox and Lehman, 1987, supra).", "This forms unique right handed helical nucleoprotein filaments in which the DNA is extended by 1.5 times its usual length (Yu and Egelman 1992.J.", "Mol.", "Biol.", "227: 334-346).", "These nucleoprotein filaments, which are referred to as DNA probes, are crucial “homology search engines” which catalyze DNA pairing.", "Once the filament finds its homologous target gene sequence, the DNA probe strand invades the target and forms a hybrid DNA structure, referred to as a joint molecule or D-loop (DNA displacement loop) (McEntee et al.", "1979.PNAS USA 76: 2615-2619; Shibata et al.", "1979.PNAS USA 76: 1638-1642).", "The phosphate backbone of DNA inside the RecA nucleoprotein filaments is protected against digestion by phosphodiesterases and nucleases.", "RecA protein is the prototype of a universal class of recombinase enzymes which promote probe-target pairing reactions.", "Recently, genes homologous to E. coli RecA (the Rad51 family of proteins) were isolated from all groups of eukaryotes, including yeast and humans.", "Rad5]protein promotes homologous pairing and strand invasion and exchange between homologous DNA molecules in a similar manner to RecA protein (Sung.", "1994.Science 265: 1241-1243; Sung and Robberson.", "1995.Cell 82: 453-461; Gupta et al.", "1997.PNAS USA 94: 463-468; Baumann et al.", "1996.Cell 87: 757-766).", "3.5.1.1.5.3.Functional Genomics: the Correlation of Genotype and Phenotype One area of pressing interest in biology is within the area of “functional genomics”, i.e.", "the correlation of genotype and phenotype.", "This requires animal systems, since phenotypic changes must be evaluated in vivo.", "Similarly, and related to this idea, is the elucidation and characterization of gene families, i.e.", "genes or proteins that are structurally related, i.e.", "they have sequence homologies between the members of the family.", "Since presumably many, if not most, disease states are caused by multiple gene interactions, the ability to evaluate interactions among genes, and particularly within or between gene families, at the phenotype level, would be extremely valuable.", "The functional genomics tools that allow facile identification and engineering of gene family members in animals and cells, however, are not yet available.", "While the amino acid sequence motifs shared between gene family members may be identical, due to degeneracy in the DNA code, the DNA sequence identity may be significantly less.", "Hence, one criterion necessary for genetic modifications of gene family members is development of homologous recombination technologies that can be used to clone and modify similar DNA sequences that share little sequence identity.", "This is particularly important since homologous recombination in cells normally requires significant sequence identity to work efficiently.", "Relaxing the amount of sequence identity needed for homologous recombination allows greater flexibility to target related genes for creating transgenic animals and cells containing modifications in gene family consensus sequences, and also will allow the rapid cloning, generation of gene family specific libraries, and evolution of gene family members.", "Accordingly, it is an object of the invention to provide an efficient method of domain specific gene evolution that generates maximal diversity but increases the probability of identifying a gene of interest.", "3.5.2.Domain Specific Gene Evolution 3.5.2.1.Domain Specific Gene Evolution—Comprising Forming a Plurality of Recombination Intermediates Comprising a Target Nucleic Acid Encoding an Amino Acid Sequence of Interest, a Recombinase and a Plurality of Targeting Polynucleotides The present invention provides methods of domain specific gene evolution comprising forming a plurality of recombination intermediates comprising a target nucleic acid encoding an amino acid sequence of interest, a recombinase and a plurality of targeting polynucleotides.", "The targeting polynucleotides are substantially complementary to each other and each comprises a homology clamp that substantially correspond to or is substantially complementary to a predetermined sequence of the target nucleic acid and comprise random or degenerate sequences.", "The predetermined sequence encodes a domain of the amino acid sequence.", "The method further comprises contacting the intermediate with a recombination proficient cell, whereby a library of altered target nucleic acids are produced.", "The altered target nucleic acids are expressed in the cell to generate a pool of variant amino acid sequences.", "The method further comprises selecting and isolating a cell comprising an altered target nucleic acid that expressed a variant amino acid having a desired activity.", "3.5.2.2.Comprising Forming a Recombination Intermediate Comprising a Target Nucleic Acid Encoding an Amino Acid Sequence of Interest, a Recombinase and a Pair of Targeting Poly Nucleotides In another aspect of the invention, a method of domain specific gene evolution comprises forming a recombination intermediate comprising a target nucleic acid encoding an amino acid sequence of interest, a recombinase and a pair of targeting polynucleotides.", "The targeting polynucleotides are substantially complementary to each other and each comprises a homology clamp that substantially corresponds to or is substantially complementary to a predetermined sequence of the target nucleic acid.", "The predetermined sequence encodes a domain of the amino acid sequence.", "The method further comprises contacting the intermediate with a single-strand specific nuclease or junction-specific nuclease to form a nicked or open-ended target nucleic acid.", "The regions adjacent to the hybridized region or junctions are susceptible to nucleases.", "The target nucleic acid is reassembled and recombined to produce a library of altered target nucleic acids.", "The target nucleic acids are expressed to generate a pool of variant amino acid sequences.", "The variant amino acid sequences are selected and characterized to identify an altered target nucleic acid encoding a variant amino acid sequence of interest.", "In a further aspect, each method is repeated one or more times to further evolve a variant amino acid sequence having a desired activity.", "In yet another aspect, more than one domain or a protein is evolved simultaneously.", "3.5.2.3.Compositions It is an object of the present invention to provide compositions comprising at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each having a consensus homology clamp for a gene family.", "In an additional aspect, the invention provides compositions comprising at least one recombinase and a plurality of pairs of single stranded targeting polynucleotides, where the plurality of pairs comprises a set of degenerate probes encoding the consensus sequence.", "In a further aspect, the invention provides kits comprising the compositions of the invention and at least one reagent.", "3.5.2.4.Methods for Targeting a Sequence Modification in at Least One Member of a Consensus Family of Genes in a Cell by Homologous Recombination.", "In an additional aspect, the invention provides methods for targeting a sequence modification in at least one member of a consensus family of genes in a cell by homologous recombination.", "The method comprises introducing into at least one cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each having a consensus homology clamp for the family.", "The method can additionally comprise identifying a target cell having a targeted sequence modification.", "3.5.2.4.1.Methods of Making a Non-Human Organism with a Targeted Sequence Modification in at Least One Member of a Gene Family In a further aspect, the invention provides methods of making a non-human organism with a targeted sequence modification in at least one member of a gene family.", "The method comprises introducing into a cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each having a consensus homology clamp for said family.", "The cell is then subjected to conditions that result in the formation of an animal, and the animal has at least one modification in at least one member of a consensus family of genes.", "In a further aspect, the invention provides non-human organisms containing a sequence modification in an endogenous consensus functional domain of a gene member of a gene family.", "3.5.2.5.Methods of Isolating a Member of a Gene Family Comprising a Protein Consensus Sequence In an additional aspect, the invention provides methods of isolating a member of a gene family comprising a protein consensus sequence.", "The method comprises adding to a complex mixture of nucleic acids at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each having a consensus homology clamp for said family.", "At least one of the targeting polynucleotides comprises a purification tag.", "The method is done under conditions whereby the targeting polynucleotides form a complex with the member, and the family member is isolated using said purification tag.", "The complex nucleic acid mixture may be a cDNA library, a cell, RNA or a restriction endonucleases genomic digest.", "3.5.3.Targeting a Predetermined Nucleic Acid Sequence that Encodes a Specific Protein Domain, to Make a Plurality of Targeted Sequence Modifications The present invention provides methods and compositions for domain specific gene evolution.", "In one aspect of the invention, the method comprises targeting a predetermined nucleic acid sequence that encodes a specific protein domain, to make a plurality of targeted sequence modifications.", "That is, by targeting the recombinogenic probes of the invention to particular protein domains, gene evolution and selection are targeted to specific domains known or believed to harbor specific activities or functions.", "These methods create maximal diversity in specific domains of interest, thereby, decreasing the size of the library of mutations that are to be screened and increasing the probability of finding a gene with improved or desired attributes.", "Therefore, the libraries of the present invention are enriched for advantageous or interesting mutations or recombinant sequence(s).", "3.5.3.1.Combining A Plurality Of Pairs Of Single-Stranded Targeting Poly Nucleotides, A Predetermined Target Nucleic Acid, And A Recombinase To Form A Polynucleotide: Target Nucleic Acid Complex.", "Accordingly, the methods comprise combining a plurality of pairs of single-stranded targeting poly nucleotides, a predetermined target nucleic acid, and a recombinase to form a polynucleotide: target nucleic acid complex.", "The targeting polynucleotides comprise at least one homology clamp for targeting a predetermined domain of a target nucleic acid and randomized or degenerate sequences.", "The complex is optionally introduced into a plurality of recombination proficient cells which catalyze strand exchange and homologous recombination intracellularly to produce a library of modified nucleic acids.", "Cells are selected and isolated that comprise a modified nucleic acid that encodes a polypeptide having a desired property.", "The process is preferably repeated iteratively to further evolve the target domain of interest.", "3.5.3.2.Domain Specific DNA Nicking In another aspect of the invention, methods of domain specific DNA nicking are provided for domain specific gene evolution.", "This method comprises combining a pair of single-stranded targeting polynucleotides, a predetermined target nucleic acid, and a recombinase to form a polynucleotide: target nucleic acid complex.", "The targeting polynucleotides are substantially complementary and comprise at least one homology clamp for targeting a predetermined domain of a target nucleic acid.", "The polynucleotide: target nucleic acid complex is treated with a single-strand specific nuclease, which preferentially nicks the regions flanking the polynucleotide: target nucleic acid complex region (Ferrin and Camerini-Otero.", "1991.Science.", "254 1494-1497).", "That is, the domain is protected from recombination by the initial presence of the recombinase in the complex.", "The nuclease is inactivated and the complex dissociated.", "The nicked target nucleic acid is reassembled and recombined by PCR to produce a library of nucleic acids with preferential modifications in the nicked regions.", "The library of modified nucleic acids can be introduced into a host cell and expressed.", "Cells are selected and isolated that comprise a modified nucleic acid that encodes a polypeptide having a desired property.", "This process is repeated iteratively to further evolve the predetermined targeted domain of interest.", "In each of the methods described above, single domains and optionally multiple domains are targeted.", "The methods and compositions described above are optionally used in combination for domain specific gene evolution.", "For example, individual or multiple rounds of domain specific DNA nicking are followed or interspersed with one or more rounds of domain specific evolution employing a plurality of targeting polynucleotides described above.", "The methods of the present invention also avoid multiple subcloning steps.", "This is particularly relevant when large complex vectors such as lambda, BACS, PACS, YACS, MACS and other genomic DNAs are used and where multiple subcloning steps make mutagenesis and shuffling of unique sites in large vectors particularly tedious and time consuming.", "3.5.3.3.Generating Homologous Recombination Intermediates In Vitro, Panels or Libraries of Mutagenized and Shuffled Genes to Generate In Vitro Evolution Accordingly, the present invention provides methods to introduce recombinogenic probe or hybrid complexes into recombination proficient cells to link in vitro and in vivo recombination and evolution processes.", "By generating homologous recombination intermediates in vitro, panels or libraries of mutagenized and shuffled genes are generated for in vitro evolution.", "The link to in vivo systems allows in vivo selection of evolved genes encoding proteins of a desired characteristic.", "The present invention can thus be used in a variety of important ways.", "3.5.3.3.1.Methods Can Be Used in the Creation of Transgenic Organisms, Animal, and Plant Models of Disease First, these methods can be used in the creation of transgenic organisms, animal, and plant models of disease.", "Thus, for example, domain-specific targeting polynucleotides used in homologous recombination methods can generate animals that have a wide variety of mutations in a wide variety of functionally related genes, potentially resulting in a wide variety of phenotypes, including phenotypes related to disease states.", "This may also be done on a cellular level, to identify genes involved in cellular phenotypes, i.e.", "target identification.", "3.5.3.3.2.Identity “Reversion” Genes, Genes That Can Modulate Disease States Secondly, domain targeting can be used in cells or animals that are diseased or altered; in essence, domain targeting can be done to identify “reversion” genes, genes that can modulate disease states caused by different genes, either genes within the same gene family or a completely different gene family.", "Thus, for example the loss of one type of enzymatic activity, resulting in a disease phenotype, may be compensated by alterations in a different but homologous enzymatic activity.", "3.5.3.3.3.Creation of Libraries of Altered Nucleic Acids In addition, the methods may be used in the creation of libraries of altered nucleic acids, including extrachromosomal sequences, and can be expressed in cells to produce libraries of altered proteins, which then can be screened for any number of useful or interesting properties, including, but not limited to, increased or altered stability (thermal, pH, oxidants, to proteases, etc.", "); altered specificity (for example, in the case of enzymes); altered binding; modified activity and other desirable properties, such as, altered immunogenicity.", "3.5.3.4.Use of Homology Motif Tags (HMTs) in Targeted Homologous Recombination to Elucidate Disease Mechanisms and to Identify Disease Targets Contained within Gene Families The present invention is directed to the use of homology motif tags (HMTs) in targeted homologous recombination to elucidate disease mechanisms and to identify disease targets contained within gene families related by the presence of one or more common domains.", "That is, there are a large number of gene families that contain genes related by the presence of similar functional domains, i.e.", "binding domains for substrates or other proteins, enzymatic domains such as kinase or protease domains, signaling and regulator domains, receptor binding domains, ATP binding domains, leucine zipper domains, zinc finger domains, etc.", "These functional domains frequently result in primary sequence homology; that is, related functional domains have related sequences.", "Many of these functional domains have been studied and so-called “consensus sequences” identified; that is, an average sequence derived from a number of related sequences.", "Each residue (or set of residues) of the consensus sequence is the most frequent at that position in the set under consideration.", "Consensus sequences can be either amino acid or nucleic acid consensus sequences, with amino acid sequences being used to generate nucleic acid consensus sequences.", "Interestingly, while a wide variety of gene families are known, the majority of drug targets come from only four of these gene families.", "These are the G-protein coupled or seven-transmembrane domain receptors, nuclear (hormone) receptors, ion channels, esterases.", "Other important gene families are enzymes, including recombinases.", "Of the top 100 pharmaceutical drugs, 18 bind to seven-transmembrane receptors, 10 to nuclear receptors and 16 to ion channels.", "By using HMTs directed to the consensus sequences of gene families for homologous recombination and particularly enhanced homologous recombination methods, sequence modifications may be made to any number of targeted genes in a related family.", "3.53.4.1.Methods and Compositions Utilizing Homology Motif Tags (HMTs) or Consensus Sequences Accordingly, the present invention provides methods and compositions utilizing homology motif tags (HMTs) or consensus sequences.", "By “homology motif tag” or “protein consensus sequence” herein is meant an amino acid consensus sequence of a gene family.", "By “consensus nucleic acid sequence” herein is meant a nucleic acid that encodes a consensus protein sequence of a functional domain of a gene family.", "In addition, “consensus nucleic acid sequence” can also refer to cis sequences that are non-coding but can serve a regulatory or other role.", "As outlined below, generally a library of consensus nucleic acid sequences are used, that comprises a set of degenerate nucleic acids encoding the protein consensus sequence.", "A wide variety of protein consensus sequences for a number of gene families are known.", "A “gene family” therefore is a set of genes that encode proteins that contain a functional is domain for which a consensus sequence can be identified.", "However, in some instances, a gene family includes non-coding sequences; for example, consensus regulatory regions can be identified.", "For example, gene family/consensus sequences pairs are known for the G-protein coupled receptor family, the AAA-protein family, the bZIP transcription factor family, the mutS family, the recA family, the Rad51 family, the dmel family, the recF family, the SH2 domain family, the Bcl-2 family, the single-stranded binding protein family, the TFIID transcription family, the TGF-beta family, the TNF family, the XPA family, the XPG family, actin binding proteins, bromodomain GDP exchange factors, MCM family, ser/thr phosphatase family, etc.", "As will be appreciated by those in the art, the proteins of the gene families generally do not contain the exact consensus sequences; generally consensus sequences are artificial sequences that represent the best comparison of a variety of sequences.", "The actual sequence that corresponds to the functional sequence within a particular protein is termed a “consensus functional domain” herein; that is, a consensus functional domain is the actual sequence within a protein that corresponds to the consensus sequence.", "A consensus functional domain may also be a “predetermined endogenous DNA sequence” (also referred to herein as a “predetermined target sequence”) that is a polynucleotide sequence contained in a target cell.", "Such sequences can include, for example, chromosomal sequences (e.g., structural genes, regulatory sequences including promoters and enhancers, recombinatorial hotspots, repeat sequences, integrated proviral sequences, hairpins, palindromes), episomal or extrachromosomal sequences (e.g., replicable plasmids or viral replication intermediates) including chloroplast and mitochondrial DNA sequences.", "By “predetermined” or “pre-selected” it is meant that the consensus functional domain target sequence may be selected at the discretion of the practitioner on the basis of known or predicted sequence information, and is not constrained to specific sites recognized by certain site-specific recombinases (e.g., FLP recombinase or CRE recombinase).", "In some embodiments, the predetermined endogenous DNA target sequence will be other than a naturally occurring germline DNA sequence (e.g., a transgene, parasitic, mycoplasmal or viral sequence).", "3.5.3.4.1.1.Gene Family is the G-Protein Coupled Receptor Family 3.5.3.4.1.1.1.Subfamily 1 Also Called R7G Proteins In a preferred embodiment, the gene family is the G-protein coupled receptor family, which has over 900 identified members, including several subfamilies.", "In a preferred embodiment, the G-protein coupled receptors are from subfamily I and are also called R7G proteins.", "They are an extensive group of receptors which recognize hormones, neurotransmitters, odorants and light and transduce extracellular signals by interaction with guanine (G) nucleotide-binding proteins.", "The structure of all these receptors is thought to be virtually identical, and they contain seven hydrophobic regions, each of which putatively spans the membrane.", "The N-terminus is extracellular and is frequently glycosylated, and the C-terminus is cytoplasmic and generally phosphorylated.", "Three extracellular loops alternate with three cytoplasmic loops to link the seven transmembrane regions.", "G-protein coupled receptors include, but are not limited to: the class A rhodopsin first subfamily, including amine (acetylcholine (muscarinic), adrenoceptors, domamine, histamine, serotonin, octopamine), peptides (angiotensin, bombesin, bradykinin, C5a anaphylatoxin, Finet-leu-phe, interleukin-8, chemokine, CCK, endothelin, mealnocortin, neuropeptide Y, neurotensin, opioid, somatostatin, tachykinin, thrombin, vasopressin-like, galanin, proteinase activated), hormone proteins (follicle stimulating hormone, lutropin-choriogonadotropic hormone, thyrotropin), rhodopsin (vertebrate), olfactory (olfactory type I-II, gustatory), prostanoid (prostaglandin, prostacyclin, thromboxane), nucleotide (adenosine, purinoceptors), cannabis, platelet activating factor, gonadotropin-releasing hormone (gonadotropin releasing hormone, thyrotropin-releasing hormone, growth hormone secretagogue), melatonin, viral proteins, MHC receptor, Mas proto-oncogene, EBV-induced and glucocorticoid induced; the class B secretin second subfamily, including calcitonin, corticotropin releasing factor, gastric inhibitory peptide, glucagon, growth hormone releasing hormone, parathyroid hormone, secretin, vasoactive intestinal polypeptide, and diuretic hormone; the class C metabotropic glutamate third subfamily, including metabrotropic glutamate and extracellular calcium-sensing agents; and the class D pheromone fourth subfamily.", "Because of the large number of family members, these large classes of GPCRs can be further subdivided into subfamilies.", "Examples of these subfamilies are calcitonin, glucagon, vasoactive and parathyroid are from class B; and acetylcholine, histamine angiotensin, alpha2- and beta-adrenergic are from class A.", "From each subfamily small protein consensus sequences can be derived from sequence alignments.", "For example, there are 6 motifs for the metabotripic glutamate like GPRCs derived from the indicated number of family members.", "Using the protein consensus sequence, degenerate nucleic acid probes are made to encode the protein consensus sequence, as is well known in the art.", "The protein sequence is encoded by DNA triplets which are deduced using standard tables.", "In some cases additional degeneracy is used to enable production in one oligonucleotide synthesis.", "In many cases motifs were chosen to minimize degeneracy.", "Amplification of neighboring sequences can utilize two motifs as indicated by faithful or error prone amplification.", "Alternatively outside sequences can be used as is indicated using vector sequence.", "In addition degenerate oligos can be synthesized and used directly in the procedure without amplification.", "Double stranded (ds) DNA probes are denatured and coated with RecA or another recombinase such as Rad51.This material can be used to bind to and allow capture of specific clones from cDNA or genomic libraries.", "Alternatively this material can be introduced into cells producing transgenic cells or animals with alterations in related family members.", "3.5.3.4.1.1.2.Second Subfamily Encoding Receptors that Bind Peptide Hormones that do not Show Sequence Similarity to the First R7G Subfamily In addition to the first subfamily of G-protein coupled receptors, there is a second subfamily encoding receptors that bind peptide hormones that do not show sequence similarity to the first R7G subfamily.", "All the characterized receptors in this subfamily are coupled to G-proteins that activate both adenylyl cyclase and the phosphatidylinositol-calcium pathway.", "However, they are structurally similar; like classical R7G proteins they putatively contain seven transmembrane regions, a glycosylated extracellular N-terminus and a cytoplasmic C-terminus.", "Known receptors in this subfamily are encoded on multiple exons, and several of these genes are alternatively spliced to yield functionally distinct products.", "The N-terminus contains five conserved cysteine residues putatively important in disulfide bonds.", "Known G-protein coupled receptors in this subfamily are listed above.", "3.5.3.4.1.1.3.Third Subfamily Encoding Receptors that Bind Glutamate and Calcium but do not Show Sequence Similarity to Either of the Other Subfamilies In addition to the first and second subfamilies of G-protein coupled receptors, there is a third subfamily encoding receptors that bind glutamate and calcium but do not show sequence similarity to either of the other subfamilies.", "Structurally, this subfamily has signal sequences, very large hydrophobic extracellular regions of about 540 to 600 amino acids that contain 17 conserved cysteines (putatively involved in disulfides), a region of about 250 residues that appear to contain seven transmembrane domains, and a C-terminal cytoplasmic domain of variable length (50 to 350 residues).", "Known G-protein coupled receptors of this subfamily are listed above.", "3.5.3.4.1.2.Gene Family is the bZIP Transcription Factor Family In a preferred embodiment, the gene family is the bZIP transcription factor family.", "This eukaryotic gene family encodes DNA binding transcription factors that contain a basic region that mediates sequence specific DNA binding, and a leucine zipper, required for dimerization.", "The bZIP family includes, but is not limited to, AP-1, ATF, CREB, CREM, FOS, FRA, GBF, GCN4, HBP, JUN, MET4, OCS1, OP, TAFI, XBP1, and YBBO.", "In a preferred embodiment, the gene family is involved in DNA mismatch repair, such as mutL, hexB and PMS1.Members of this family include, but are not limited to, MLH1, PMS1, PMS2, HexB and MulL.", "The protein consensus sequence is G-F—R-G-E-A-L.", "In a preferred embodiment, the gene family is the mutS family, also involved in mismatch repair of DNA, directed to the correction of mismatched base pairs that have been missed by the proofreading element of the DNA polymerase complex.", "MutS gene family members include, but are not limited to, MSH2, MSH3, MSH6 and MutS.", "In a preferred embodiment, the gene family is the recA family.", "The bacterial recA is essential for homologous recombination and recombinatorial repair of DNA damage.", "RecA has many activities, including the formation of nucleoprotein filaments, binding to single stranded and double stranded DNA, binding and hydrolyzing ATP, recombinase activity and interaction with lexA causing lexA activation and autocatalytic cleavage.", "RecA family members include those from E. coli, drosophila, human, lily, etc.", "specifically including but not limited to, E. coli recA, Rec1, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51 E, XRCC2 and DMCI.", "3.5.3.4.1.3.Gene Family is the RecF Family In a preferred embodiment, the gene family is the recF family.", "The prokaryotic recF protein is a single-stranded DNA binding protein which also putatively binds ATP.", "RecF is involved in DNA metabolism; it is required for recombinatorial DNA repair and for induction of the SOS response.", "RecF is a protein of about 350 to 370 amino acid residues; there is a conserved ATP-binding site motif “A” in the N-terminal section of the protein as well as two other conserved regions, one located in the central section and the other in the C-terminal section.", "3.5.3.4.1.4.Gene Family is the Bcl-2 Family In a preferred embodiment, the gene family is the Bcl-2 family.", "Programmed cell death (PCD), or apoptosis, is induced by events such as growth factor withdrawal and toxins.", "It is generally controlled by regulators, which have either an inhibitory effect (i.e.", "anti-apoptotic) or block the protective effect of inhibitors (pro-apoptotic).", "Many viruses have found a way of countering defensive apoptosis by encoding their own anti-apoptotic genes thereby preventing their target cells from dying too soon.", "All proteins belonging to the Bcl-2 family contain at least one of a BH1, BH2, BH3 or BH4 domain.", "All anti-apoptotic proteins contain BH1 and BH2 domains, some of them contain an additional N-terminal BH4 domain (such as Bcl-2, Bcl-x(L), Bcl-W, etc.", "), which is generally not found in pro-apoptotic proteins (with the exception of Bcl-x(S).", "Generally all pro-apoptotic proteins contain a BH3 domain (except for Bad), thought to be crucial for the dimerization of the proteins with other Bcl-2 family members and crucial for their killing activity.", "In addition, some of the pro-apoptotic proteins contain BH1 and BH2 domains (such as Bax and Bak).", "The BH3 domain is also present in some anti-apoptosis proteins, such as Bcl-2 and Bcl-x(L).", "Known Bcl-2 proteins include, but are not limited to, Bcl-2, Bcl-x(L), Bcl-W, Bcl-x(S), Bad, Bax, and Bak.", "3.5.3.4.1.5.Gene Family is the Site-Specific Recombinase Family In a preferred embodiment, the gene family is the site-specific recombinase family.", "Site-specific recombination plays an important role in DNA rearrangement in a) recombination between inverted repeats resulting in the reversal of a DNA segment; and b) recombination between repeat sequences on two DNA molecules resulting in their cointegration, or between repeats on one DNA molecule resulting the excision of a DNA fragment.", "Site-specific recombination is characterized by a strand exchange mechanism that requires no DNA synthesis or high energy cofactor; the phosphodiester bond energy is conserved in a phospho-protein linkage during strand cleavage and re-ligation.", "Two unrelated families of recombinases are currently known.", "The first, called the “phage integrase” family, groups a number of bacterial, phage and yeast plasmid enzymes.", "The second, called the “resolvase” family, groups enzymes which share the following structural characteristics: an N-terminal catalytic and dimerization domain that contains a conserved serine residue involved in the transient covalent attachment to DNA, and a C-terminal helix-turn-helix DNA-binding domain.", "3.5.3.4.1.6.Gene Family is the Single-Stranded Binding Protein Family In a preferred embodiment, the gene family is the single-stranded binding protein family.", "The E coli single-stranded binding protein (ssb), also known as the helix-destabilizing protein, is a protein of 177 amino acids.", "It binds tightly as a homotetramer to a single-stranded DNA ss-DNA) and plays an important role in DNA replication, recombination and repair.", "Members of the ssb family include, but are not limited to, E. coli ssb and eukaryotic RPA proteins.", "3.5.3.4.1.7.Gene Family Is The TFIID Transcription Family In a preferred embodiment, the gene family is the TFIID transcription family.", "Transcription factor TRID (or TATA-binding protein, TBP), is a general factor that plays a major role in the activation of eukaryotic genes transcribed by RNA polymerase II.", "TRID binds specifically to the TATA box promoter element which lies close to the position of transcription initiation.", "There is a remarkable degree of sequence conservation of a C-terminal domain of about 180 residues in TFIID from various eukaryotic sources.", "This region is necessary and sufficient for TATA box binding.", "The most significant structural feature of this domain is the presence of two conserved repeats of a 77 amino-acid region.", "3.5.3.4.1.8.Gene Family is the TGF-beta Family In a preferred embodiment, the gene family is the TGF-beta family.", "Transforming growth factor-beta (TGF-beta) is a multifunctional protein that controls proliferation, differentiation and other functions in many cell types.", "TGF-beta-1 is a protein of 112 amino acid residues derived by proteolytic cleavage from the C-terminal portion of the precursor protein.", "Members of the TGF-beta family include, but are not limited to, the TGF-1-3 subfamily (including TGF1, TGF2, and TGF3); the BMP3 subfamily (BM3B, BMP3); the BMP5-8 subfamily (BM8A, BMP5, BMP6, BMP7, and BMP8); and the BMP 2 & 4 subfamily (BMP2, BMP4, DECA).", "In a preferred embodiment, the gene family is the TNF family.", "A number of cytokines can be grouped into a family on the basis of amino acid sequence, as well as structural and functional similarities.", "These include (1) tumor necrosis factor (TNF), also known as cachectin or TNF-alpha, which is a cytokine with a wide variety of functions.", "TNF-alpha can cause cytolysis of certain tumor cell lines; it is involved in the induction of cachexia; it is a potent pyrogen, causing fever by direct action or by stimulation of interleukin-1 secretion; and it can stimulate cell proliferation and induce cell differentiation under certain conditions; (2) lymphotoxin-alpha (LT-alpha) and lymphotoxin-beta (LT-beta), two related cytokines produced by lymphocytes and which are cytotoxic for a wide range of tumor cells in vitro and in vivo; (3) T cell antigen gp39 (CD40L), a cytokine that seems to be important in B-cell development and activation; (4) CD27L, a cytokine that plays a role in T-cell activation; it induces the proliferation of costimulated T cells and enhances the generation of cytolytic T cells; (5) CD30L, a cytokine that induces proliferation of T-cells; (6) FASL, a cytokine involved in cell death; (8) 4-1 BBL, an inducible T cell surface molecule that contributes to T-cell stimulation; (9) OX40L, a cytokine that co-stimulates T cell proliferation and cytokine production; and (10), TNF-related apoptosis inducing ligand (TRAIL), a cytokine that induces apoptosis.", "3.5.3.4.1.9.Gene Family is the XPA Family In a preferred embodiment, the gene family is the XPA family.", "Xeroderma pigmentosa (XP) is a human autosomal recessive disease, characterized by a high incidence of sunlight-induced skin cancer.", "Skin cells associated with this condition are hypersensitive to ultaviolet light, due to defects in the incision step of DNA excision repair.", "There are a minimum of 7 genetic complementation groups involved in this disorder: XPA to XPG.", "XPA is the most common form of the disease and is due to defects in a 30 kD nuclear protein called XPA or (XPAC).", "The sequence of XPA is conserved from higher eukaryotes to yeast (gene RAD14).", "XPA is a hydrophilic protein of 247 to 296 amino acid residues that has a C4-type zinc finger motif in its central section.", "3.5.3.4.1.10.Gene Family is the XPG Family In a preferred embodiment, the gene family is the XPG family.", "The defect in XPG can be corrected by a 133 kD nuclear protein called XPG (or XPGC).", "Members of the XPG family include, but are not limited to, FENI, XPG, RAD2, EXO1, and DIN7.Once having identified a gene family and a consensus sequence, the compositions of the invention can be made.", "The compositions of the invention comprise at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each have a consensus homology clamp for a gene family.", "3.53.5.Homologous Recombination Accordingly, the present invention provides methods of homologous recombination.", "By “homologous recombination” (HR) herein is meant an exchange of homologous or similar DNA sequence between two DNA molecules.", "An essential feature of HR is that the enzyme responsible for the recombination event can pair any homologous sequences as substrates.", "The ability of HR to transfer genetic information between DNA molecules makes targeted homologous recombination a very powerful method in genetic engineering and gene manipulation.", "HR can be used to insert, delete, and/or substitute any one or more nucleotides in a gene or gene segment or to introduce or delete genes in a targeted nucleic acid.", "Once having identified a protein domain, the compositions of the invention can be made.", "The compositions of the invention comprise at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other and each have a domain homology clamp.", "3.5.3.6.Recombinase By “recombinase” herein is meant a protein or peptide (e.g.", "L2 peptide) that, when included with an exogenous targeting polynucleotide, provide a measurable increase in the recombination frequency and/or localization frequency between the targeting polynucleotide and an endogenous predetermined DNA sequence.", "Thus, in a preferred embodiment, increases in recombination frequency from the normal range of 10−8 to 10−4, to 10−4 to 101, preferably 10−3 to 101, and most preferably 10−2 to 100, may be achieved.", "In the present invention, recombinase refers to a family of RecA-like and Rad51-like recombination proteins all having essentially all or most of the same functions, particularly: (i) the recombinase protein's ability to properly bind to and position targeting polynucleotides on their homologous targets and (ii) the ability of recombinase protein/targeting polynucleotide complexes to efficiently find and bind to complementary endogenous sequences.", "The best characterized RecA protein is from E coli, in addition to the wild-type protein a number of mutant RecA proteins have been identified (e.g., RecA803; see Madiraju et al., PNAS USA 85(18): 6592 (1988); Madiraju et al, Biochem.", "31: 10529 (1992); Layery et al., J. Biol.", "Chem.", "267: 20648 (1992)).", "Further, many organisms have RecA-like recombinases with strand-transfer activities (e.g., Fugisawa et al., (1985) Nucl.", "Acids Res.", "13: 7473; Hsieh et al., (1986) Cell 44: 885; Hsieh et al., (1989) J. Biol.", "Chem.", "264: 5089; Fishel et al., (1988) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 85: 3683; Cassuto et al., (1987) Mol.", "Gen. Genet.", "208: 10; Ganea et al., (1987) Mol.", "Cell Biol.", "7: 3124; Moore et al., (1990) J. Biol.", "Chem.", "19: 11108; Keene et al., (1984) Nucl.", "Acids Res.", "12: 3057; Kimeic, (1984) Cold Spring Harbor Symp.", "48: 675; Kmeic, (1986) Cell 44: 545; Kolodner et al., (1987) Proc.", "Natl.", "Acad.", "Sci.", "USA 84: 5560; Sugino et al., (1985) Proc.", "Natl.", "Acad.", "Sci.", "USA 85: 3683; Halbrook et al., (1989) J. Biol.", "Chem.", "264: 21403; Eisen et al., (1988) Proc.", "Natl.", "Acad.", "Sci.", "USA 85: 7481; McCarthy et al., (1988) Proc.", "Natl.", "Acad.", "Sci.", "US 85: 5854; Lowenhaupt et al., (1989) J. Biol.", "Chem 264: 20568, which are incorporated herein by reference.", "Examples of such recombinase proteins include, for example but not limited to: RecA, RecA803, uvsX, and other RecA mutants and RecA-like recombinases (Roca, A.", "1.", "(1990) Crit.", "Rev.", "Biochem.", "Molec.", "Biol.", "25: 415), sep1 (Kolodner et al.", "(1987) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.6.1 B4: 5560; Tishkoff et al.", "Molec.", "Cell.", "Biol.", "11: 2593), RuvC (Dunderdale et al.", "(1991) Nature 354: 506), DST2, KEMI, XRN I (Dykstra et al.", "(1991) Molec.", "Cell.", "Biol.", "11: 2583), STPalpha/DSTI (Clark et al.", "(1991) Molec.", "Cell.", "Biol.", "11: 2576), HPP—I (Moore et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.", "I B8: 9067), other target recombinases (Bishop et al.", "(1992) Cell 69 439; Shinohara et al.", "(1992) Cell 69 457); incorporated herein by reference.", "RecA may be purified from E coli strains, such as E coli strains JC 12772 and JC 1 5369 (available from A. J. Clark and M. Madiraju, University of California-Berkeley, or purchased commercially).", "These strains contain the RecA coding sequences on a “runaway” replicating plasmid vector present at a high copy numbers per cell.", "The RecA803 protein is a high-activity mutant of wild-type RecA.", "The art teaches several examples of recombinase proteins, for example, from Drosophila, yeast, plant, human, and non-human mammalian cells, including proteins with biological properties similar to RecA (i.e., RecA-like recombinases), such as Rad51, Rad55, Rad57, dmcl from mammals and yeast.", "In addition, the recombinase may actually be a complex of proteins, i.e.", "a “recombinosome”.", "In addition, included within the definition of a recombinase are portions or fragments of recombinases which retain recombinase biological activity, as well as variants or mutants of wild-type recombinases which retain biological activity, such as the E. coli RecA803 mutant with enhanced recombinase activity.", "3.5.3.6.1.RecA or rad51 In a preferred embodiment, RecA or rad51 is used.", "For example, RecA protein is typically obtained from bacterial strains that overproduce the protein: wild-type E coli RecA protein and mutant RecA803 protein may be purified from such strains.", "Alternatively, RecA protein can also be purchased from, for example, Pharmacia (Piscataway, N.J.) or Boehringer Mannheim (Indianapolis, Ind.).", "RecA proteins, and its homologs, form a nucleoprotein filament when it coats a single-stranded DNA.", "In this nucleoprotein filament, one monomer of RecA protein is bound to about 3 nucleotides.", "This property of RecA to coat single-stranded DNA is essentially sequence independent, although particular sequences favor initial loading of RecA onto a polynucleotide (e.g., nucleation sequences).", "The nucleoprotein filament(s) can be formed on essentially any DNA molecule and can be formed in cells (e.g., mammalian cells), forming complexes with both single-stranded and double-stranded DNA, although the loading conditions for dsDNA are somewhat different than for ssDNA.", "3.5.3.6.1.1.The Recombinase is Combined with Targeting Polynucleotides The recombinase is combined with targeting polynucleotides as is more fully outlined below.", "By “nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.", "A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org.", "Chem.", "35: 3800 (1970); Sprinzl et al., Eur.", "J. Biochem.", "81: 579 (1977); Letsinger et al., Nucl.", "Acids Res.", "14: 3487 (1986); Sawai et al, Chem.", "Lett.", "805 (1984), Letsinger et al., J.", "Am.", "Chem.", "Soc.", "110: 4470 (1988); and Pauwels et al., Chemica Scripta 26: 141 91986)), phosphorothioate, phosphorodithioate, O— methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J.", "Am.", "Chem.", "Soc.", "114: 1895 (1992); Meier et al., Chem.", "Int.", "Ed.", "Engl.", "31: 1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207 (1996), all of which are incorporated by reference).", "These modifications of the ribose-phosphate backbone or bases may be done to facilitate the addition of other moieties such as chemical constituents, including 2′ O-methyl and 5′ modified substituents, as discussed below, or to increase the stability and half-life of such molecules in physiological environments.", "The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.", "The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine and hypoxathanine, etc.", "Thus, for example, chimeric DNA-RNA molecules may be used such as described in Cole-Strauss et al., Science 273: 1386 (1996) and Yoon et al., PNAS USA 93: 2071 (1996), both of which are hereby incorporated by reference.", "In general, the targeting polynucleotides may comprise any number of structures, as long as the changes do not substantially effect the functional ability of the targeting polynucleotide to result in homologous recombination.", "For example, recombinase coating of alternate structures should still be able to occur.", "By “targeting polynucleotides” herein is meant the polynucleotides used to make alterations in the protein domains as described herein.", "Targeting polynucleotides are generally ssDNA or dsDNA, most preferably two complementary single stranded DNAs.", "Targeting polynucleotides are generally at least about 5 to 2000 nucleotides long, preferably about 12 to 200 nucleotides long, at least about 200 to 500 nucleotides long, more preferably at least about 500 to 2000 nucleotides long, or longer; however, as the length of a targeting polynucleotide increases beyond about 20,000 to 50,000 to 400,000 nucleotides, the efficiency or transferring an intact targeting polynucleotide into the cell decreases.", "The length of homology may be selected at the discretion of the practitioner on the basis of the sequence composition and complexity of the predetermined endogenous target DNA sequence(s) and guidance provided in the art, which generally indicates that 1.3 to 6.8 kilobase segments of homology are preferred when non-recombinase mediated methods are utilized (Hasty et al.", "(1991) Molec.", "Cell.", "Biol.", "11: 5586; Shulman et al.", "(1990) Molec.", "Cell.", "Biol.", "10: 4466, which are incorporated herein by reference).", "Targeting polynucleotides have at least one sequence that substantially corresponds to, or is substantially complementary to, a predetermined endogenous DNA sequence.", "As used herein, the terms “predetermined target nucleic acid” and “predetermined target sequence” and “predetermined domain of a target nucleic acid” refer to polynucleotide sequences contained in a target nucleic acid.", "Such sequences include, for example, chromosomal sequences (e.g., structural genes, regulatory sequences including promoters and enhancers, recombinatorial hotspots, repeat sequences, integrated proviral sequences, hairpins, palindromes), episomal or extrachromosomal sequences (e.g., replicable plasmids or viral replication intermediates) including chloroplast and mitochondrial DNAsequences.", "By “predetermined” or “pre-selected” it is meant that the target sequence may be selected at the discretion of the practitioner on the basis of known or predicted sequence information, and is not constrained to specific sites recognized by certain site-specific recombinases (e.g., FLIP recombinase or CRE recombinase).", "In some embodiments, the predetermined endogenous DNA target sequence will be other than a naturally occurring germline DNA sequence (e.g., a transgene, parasitic, mycoplasmal or viral sequence).", "An exogenous polynucleotide is a polynucleotide which is transferred into a target cell but which has not been replicated in that host cell; for example, a virus genome polynucleotide that enters a cell by fusion of a virion to the cell is an exogenous polynucleotide, however, replicated copies of the viral polynucleotide subsequently made in the infected cell are endogenous sequences (and may, for example, become integrated into a cell chromosome).", "Similarly, transgenes which are microinjected or transfected into a cell are exogenous polynucleotides, however integrated and replicated copies of the transgene(s) are endogenous sequences.", "3.5.3.6.1.1.1.Target Nucleic Acid Comprises a Nucleotide Sequence Encoding a Protein or Polypeptide or can be Made to Comprise Non-Coding Regions as Well In a preferred embodiment, the target nucleic acid comprises a nucleotide sequence encoding a protein or polypeptide, although as outlined herein, target nucleic acids may be made to non-coding regions as well.", "By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.", "Thus “amino acid” or “peptide residue”, as used herein means naturally occurring and naturally modified amino acids.", "For example, “amino acid” also includes imino acid residues such as proline and hydroxyproline.", "A “naturally modified amino acid” includes for examples, amino acids that are modified to contain carbohydrate structures, such as high-mannose or complex carbohydrates, phosphate, or lipids.", "In the preferred embodiment, the amino acids are in the (S) or L-configuration.", "The nucleotide sequence encoding the polypeptide is preferably operably linked to transcription and translation control elements operable in a host cell of interest, such that, introduction of the target nucleic acid results in expression of the encoded protein.", "The transcription control elements include a promoter, such as, a constitutive or inducible promoter.", "When the host cell of interest is a eukaryotic cell, enhancer elements are optionally employed.", "In a preferred embodiment the target nucleic acid is an extrachromosomal vector such as a plasmid.", "In other embodiments, the target nucleic acid is a viral vector, such as, a retrovirus, a phage, a BAC, PAC, YAC, MAC or other types of genomic and chromosomal DNA.", "The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature.", "For example, a polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.", "3.5.3.6.1.1.2.The Target Nucleic Acid Comprises a Nucleic Acid Encoding a Protein Domain The methods of the invention are used for alteration and evolution of protein domains; that is, in a preferred embodiment, the target nucleic acid comprises a nucleic acid encoding a protein domain.", "By “protein domain” and grammatical equivalents as used herein are meant a region of a protein that provides a specific structural and/or functional characteristic.", "Accordingly, a protein domain is an enzymatic active site, a ligand binding site, an allosteric effector region, an epitope, a region of a protein that is modified, such as, by addition of a carbohydrate, phosphate or lipid.", "A domain also relates to the hydrophobicity or hydrophilicity of a region and, therefore, also includes extracellular, intracellular, and transmembrane domains.", "Cell targeting sequences, such as, a signal peptide, nuclear localization sequence, mitochondrial localization sequences, etc.", "that direct proteins to either an extracellular or subcellular locale are domains.", "Additional domains include regions of proteins that interact with other proteins or nucleic acids, for example, include multimerization sequences, zinc-finger motifs, and the like.", "In another aspect, a protein domain is a region encoded by an exon.", "Targeting polynucleotides have at least one sequence that substantially corresponds to, or is substantially complementary to, a target nucleic acid; in a preferred embodiment, it corresponds or complements a nucleic acid encoding a protein domain.", "By “corresponds to” herein is meant that a polynucleotide sequence is homologous (i.e., may be similar or identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.", "In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence can hybridize to all or a portion of a reference polynucleotide sequence.", "Thus, one of the complementary single stranded targeting polynucleotides is complementary to one strand of the endogenous target domain sequence (i.e.", "Watson) and corresponds to the other strand of the endogenous target domain sequence (i.e.", "Crick).", "Thus, the complementarity between two single-stranded targeting polynucleotides need not be perfect.", "For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is perfectly complementary to a reference sequence “GTATA.", "The terms “substantially corresponds to” or “substantial identity” or “homologous” as used herein denotes a characteristic of a nucleic acid sequence, wherein a nucleic acid sequence has at least about 50 percent sequence identity as compared to a reference sequence, typically at least about 70 percent sequence identity, and preferably at least about 85 percent sequence identity as compared to a reference sequence.", "The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.", "The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.", "However, the reference sequence is at least 18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long.", "“Substantially complementary” as used herein refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence.", "In general, targeting efficiency increases with the length of the targeting polynucleotide portion that is substantially complementary to a reference sequence present in the target DNA.", "By “sequence homology” herein is meant sequence similarity or sequence identity.", "Nucleic acid similarity can be determined using, for example, BLASTN (Altschul et al.", "1990.J.", "Mol.", "Biol.", "147: 195-197).", "BLASTN uses a simple scoring system in which matches count +5 and mismatches −4.To achieve computational efficiency, the default parameters have been incorporated directly into the source code.", "3.5.3.7.Percent Nucleic Acid Sequence Identity is Determined In an alternative embodiment, percent nucleic acid sequence identity is determined.", "In percent identity calculations relative weight is not assigned to the various types of sequence variation, such as, insertions, deletions, substitutions, etc.", "Only identities are scored positively (+1) and all forms of sequence variation given a value of “0”, which obviates the need for a weighted scale or parameters as described above for sequence similarity calculations.", "Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the “shorter” sequence in the aligned region and multiplying by 100.The “longer” sequence is the one having the most actual residues in the aligned region.", "3.5.3.8.Domain Homology Clamps: a Portion of the Targeting Polynucleotide that can Specifically Hybridize to a Nucleic Acid Encoding a Domain within a Gene of Interest These corresponding/complementary sequences are sometimes referred to herein as “domain homology clamps”, as they serve as templates for homologous pairing with the predetermined endogenous sequence(s).", "Thus, a “domain homology clamp” is a portion of the targeting polynucleotide that can specifically hybridize to a nucleic acid encoding a domain within a gene of interest.", "“Specific hybridization” is defined herein as the formation of hybrids between a targeting polynucleotide (e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions as compared to the predetermined target nucleic acid sequence) and a predetermined target nucleic acid, wherein the targeting polynucleotide preferentially hybridizes to the predetermined target nucleic acid such that, for example, at least one discrete band can be identified on a Southern blot of nucleic acid prepared from target cells that contain the target nucleic acid sequence, and/or a targeting polynucleotide in an intact nucleus localizes to a discrete chromosomal location characteristic of a unique or repetitive sequence.", "As will be appreciated by those in the art, a target domain sequence may be present in more than one target polynucleotide species (e.g., a particular target sequence may occur in multiple members of a gene family).", "It is evident that optimal hybridization conditions will vary depending upon the sequence composition and length(s) of the targeting polynucleotide(s) and target(s), and the experimental method selected by the practitioner.", "Various guidelines may be used to select appropriate hybridization conditions (see, Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology.", "Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.), which are incorporated herein by reference.", "Methods for hybridizing a targeting polynucleotide to a discrete chromosomal location in intact nuclei are known in the art, see for example WO 93/05177 and Kowalczykowski and Zarling (1994) in Gene Targeting, Ed.", "Manuel Vega.", "In targeting polynucleotides, domain homology clamps are typically located at or near the 5′ or 3′ end, preferably domain homology clamps are internal or located at each end of the polynucleotide (Berinstein et al.", "(1992) Molec, Cell.", "Biol.", "12: 360, which is incorporated herein by reference).", "Without wishing to be bound by any particular theory, it is believed that the addition of recombinases permits efficient gene targeting with targeting polynucleotides having short (i.e., about 10 to 1000 basepair long) segments of homology, as well as with targeting polynucleotides having longer segments of homology.", "3.5.3.9.Targeting Polynucleotides 3.5.3.9.1.Targeting Polynucleotides that have Domain Homology Clamps that are Highly Homologous to the Predetermined Target Endogenous Domain Functional Domain Nucleic Acid Sequence Therefore, it is preferred that targeting polynucleotides of the invention have domain homology clamps that are highly homologous to the predetermined target endogenous domain functional domain nucleic acid sequence(s).", "Typically, targeting polynucleotides of the invention have at least one domain homology clamp that is at least about 18 to 35 nucleotides long, and it is preferable that domain homology clamps are at least about 20 to 100 nucleotides long, and more preferably at least about 100-500 nucleotides long, although the degree of sequence homology between the domain homology clamp and the targeted sequence and the base composition of the targeted sequence will determine the optimal and minimal clamp lengths (e.g., G-C rich sequences are typically more thermodynamically stable and will generally require shorter clamp length).", "Therefore, both domain homology clamp length and the degree of sequence homology can only be determined with reference to a particular predetermined sequence, but domain homology clamps generally must be at least about 10 nucleotides long and must also substantially correspond or be substantially complementary to a predetermined target sequence.", "Preferably, a homology clamp is at least about 10, and preferably at least about 50 nucleotides long and is substantially identical to or complementary to a predetermined target sequence.", "Without wishing to be bound by a particular theory, it is believed that the addition of recombinases to a targeting polynucleotide enhances the efficiency of homologous recombination between homologous, nonisogenic sequences (e.g., between an exon 2 sequence of an albumin gene of a Balb/c mouse and a homologous albumin gene exon 2 sequence of a C57/BL6 mouse), as well as between isogenic sequences.", "3.5.3.9.2.Targeting Polynucleotides Comprising a Plurality of Targeting Polynucleotides Comprising at Least one Shared Homology Clamp and a Degenerate Sequence In one aspect of the invention, the targeting polynucleotides comprise a plurality of targeting polynucleotides comprising at least one shared homology clamp and a degenerate sequence.", "By “plurality” herein is meant more than one.", "The targeting polynucleotides find use in the mutagenesis and evolution of a target nucleic acid sequence that encodes specific protein domain by insertion, deletion and/or substitution of the nucleic acid sequence encoding the domain.", "In one embodiment the degenerate sequence is completely randomized, representing all possible combinations of nucleotides.", "In another embodiment, the degenerate sequence is biased, for example, to eliminate sequences encoding for transcriptional or translational stop signals.", "In another embodiment, the degenerate sequence is biased, to represent the codon bias of a host cell or class of organisms.", "The degenerate sequence is optionally biased to randomize specific sequence while maintaining other sequences constant.", "The length of the degenerate sequence is determined by the practitioner and is based on the desired number of nucleotides within the predetermined sequence to be modified.", "35.3.9.3.Targeting Polynucleotides Substantially Identical to the Predetermined Target Sequence In an alternative embodiment, the targeting polynucleotides are substantially identical to the predetermined target sequence.", "In the presence of a recombinase, the targeting polynucleotides form complexes with a predetermined target sequence of a target nucleic acid.", "As a part of the complex, the predetermined target sequence is resistant to nuclease digestion.", "The regions flanking the polynucleotide: target complex are susceptible to single-strand specific exonucleases.", "Accordingly, to effect domain specific evolution, these regions are nicked and the resultant fragments are reassembled and recombined by PCR as described below and by Stemmer et al.", "Nature.", "370: 389-391 and Stemmer et al.", "PNAS USA 91: 10747-10751, hereby incorporated by reference.", "The formation of heteroduplex joints is not a stringent process; genetic evidence supports the view that the classical phenomena of meiotic gene conversion and aberrant meiotic segregation results in part from the inclusion of mismatched base pairs in heteroduplex joints, and the subsequent correction of some of these mismatched base pairs before replication.", "Observations on RecA protein have provided information on parameters that affect the discrimination of relatedness from perfect or near-perfect homology and that affect the inclusion of mismatched base pairs in heteroduplex joints.", "The ability of RecA protein to drive strand exchange past all single base-pair mismatches and to form extensively mismatched joints in superhelical DNA reflect its role in recombination and gene conversion.", "This error-prone process may also be related to its role in mutagenesis.", "RecA-mediated pairing reactions involving DNA of X174 and G4, which are about 70 percent homologous, have yielded homologous recombinants (Cunningham et al.", "(1981) Cell 24: 213), although RecA preferentially forms homologous joints between highly homologous sequences, and is implicated as mediating a homology search process between an invading DNA strand and a recipient DNA strand, producing relatively stable heteroduplexes at regions of high homology.", "Accordingly, it is the fact that recombinases can drive the homologous recombination reaction between strands which are significantly, but not perfectly, homologous, which allows gene conversion and the modification of target sequences.", "Thus, targeting polynucleotides may be used to introduce nucleotide substitutions, insertions and deletions into an endogenous functional domain nucleic acid sequence, and thus the corresponding amino acid substitutions, insertions and deletions in proteins expressed from the endogenous domain functional domain nucleic acid sequence.", "By “endogenous” in this context herein is meant the naturally occurring sequence, i.e.", "sequences or substances originating from within a cell or organism.", "Similarly, “exogenous” refers to sequences or substances originating outside the cell or organism.", "3.5.3.9.4.Method Where Two Substantially Complementary Targeting Polynucleotides are Used In a preferred embodiment, two substantially complementary targeting polynucleotides are used.", "3.53.9.5.Method where the Targeting Polynucleotides form a Double Stranded Hybrid, which may be Coated with Recombinase In one embodiment, the targeting polynucleotides form a double stranded hybrid, which may be coated with recombinase, although when the recombinase is RecA, the loading conditions may be somewhat different from those used for single stranded nucleic acids.", "3.5.3.9.6.Method where Two Substantially Complementary Single-Stranded Targeting Polynucleotides are Used In a preferred embodiment, two substantially complementary single-stranded targeting polynucleotides are used.", "The two complementary single-stranded targeting polynucleotides are usually of equal length, although this is not required.", "However, as noted below, the stability of the four strand hybrids of the invention is putatively related, in part, to the lack of significant unhybridized single-stranded nucleic acid, and thus significant unpaired sequences are not preferred.", "Furthermore, as noted above, the complementarity between the two targeting polynucleotides need not be perfect.", "The two complementary single-stranded targeting polynucleotides are simultaneously or contemporaneously introduced into a target cell harboring a predetermined endogenous target sequence, generally with at lease one recombinase protein (e.g., RecA).", "Under most circumstances, it is preferred that the targeting polynucleotides are incubated with RecA or other recombinase prior to introduction into a target cell, so that the recombinase protein(s) may be “loaded” onto the targeting polynucleotide(s), to coat the nucleic acid, as is described below.", "Incubation conditions for such recombinase loading are described infra.", "A targeting polynucleotide may contain a sequence that enhances the loading process of a recombinase, for example a RecA loading sequence is the recombinogenic nucleation sequence poly[d(A-C)], and its complement, poly[d(G-T)].", "The duplex sequence poly[d(A-C)*d(G-T)n, where n is from 5 to 25, is a middle repetitive element in target DNA.", "There appears to be a fundamental difference in the stability of RecA-protein-mediated D-loops formed between one single-stranded DNA (ssDNA) probe hybridized to negatively supercoiled DNA targets in comparison to relaxed or linear duplex DNA targets.", "Internally located dsDNA target sequences on relaxed linear DNA targets hybridized by ssDNA probes produce single D-loops, which are unstable after removal of RecA protein (Adzuma, Genes Devel.", "6: 1679 (1992); Hsieh et al, PNAS USA 89: 6492 (1992); Chiu et al., Biochemistry 32: 13146 (1993)).", "This probe DNA instability of hybrids formed with linear duplex DNA targets is most probably due to the incoming ssDNA probe W—C base pairing with the complementary DNA strand of the duplex target and disrupting the base pairing in the other DNA strand.", "The required high free-energy of maintaining a disrupted DNA strand in an unpaired ssDNA conformation in a protein-free single-D-loop apparently can only be compensated for either by the stored free energy inherent in negatively supercoiled DNA targets or by base pairing initiated at the distal ends of the joint DNA molecule, allowing the exchanged strands to freely intertwine.", "However, the addition of a second complementary ssDNA to the three-strand-containing single-D-loop stabilizes the deproteinized hybrid joint molecules by allowing W-C base pairing of the probe with the displaced target DNA strand.", "The addition of a second RecA-coated complementary ssDNA (cssDNA) strand to the three-strand containing single D-loop stabilizes deproteinized hybrid joints located away from the free ends of the duplex target DNA (Sena & Zarling, Nature Genetics 3: 365 (1993); Revet et al.", "J. Mol.", "Biol.", "232: 779 (1993); Jayasena and Johnston, J. Mol.", "Bio.", "230: 1015 (1993)).", "The resulting four-stranded structure, named a double D-loop by analogy with the three-stranded single D-loop hybrid has been shown to be stable in the absence of RecA protein.", "This stability likely occurs because the restoration of W-C basepairing in the parental duplex would require disruption of two W-C basepairs in the double-D-loop (one W-C pair in each heteroduplex D-loop).", "Since each base-pairing in the reverse transition (double-D-loop to duplex) is less favorable by the energy of one W-C basepair, the pair of cssDNA probes are thus kinetically trapped in duplex DNA targets in stable hybrid structures.", "The stability of the double-D loop joint molecule within internally located probe: target hybrids is an intermediate stage prior to the progression of the homologous recombination reaction to the strand exchange phase.", "The double D-loop permits isolation of stable multistranded DNA recombination intermediates.", "In addition, when the targeting polynucleotides are used to generate insertions or deletions in an endogenous nucleic acid sequence, as is described herein, the use of two complementary single-stranded targeting polynucleotides allows the use of internal homology clamps.", "The use of internal homology clamps allows the formation of stable deproteinized cssDNA: probe target hybrids with homologous DNA sequences containing either relatively small or large insertions and deletions within a homologous DNA target.", "Without being bound by theory, it appears that these probe: target hybrids, with heterologous inserts in the cssDNA probe, are stabilized by the re-annealing of cssDNA probes to each other within the double-D-loop hybrid, forming a novel DNA structure with an internal homology clamp.", "Similarly stable double-D-loop hybrids formed at internal sites with heterologous inserts in the linear DNA targets (with respect to the cssDNA probe) are equally stable.", "Because cssDNA probes are kinetically trapped within the duplex target, the multi-stranded DNA intermediates of homologous DNA pairing are stabilized and strand exchange is facilitated.", "3.5.3.10.Length of the Internal Homology Clamp (i.e.", "the Length of the Insertion or Deletion) In a preferred embodiment, the length of the internal homology clamp (i.e.", "the length of the insertion or deletion) is from about 1 to 50% of the total length of the targeting polynucleotide, with from about 1 to about 20% being preferred and from about 1 to about 10% being especially preferred, although in some cases the length of the deletion or insertion may be significantly larger.", "As for the domain homology clamps, the complementarity within the internal homology clamp need not be perfect.", "A targeting polynucleotide used in a method of the invention typically is a single-stranded nucleic acid, usually a DNA strand, or derived by denaturation of a duplex DNA, which is complementary to one (or both) strand(s) of the target duplex nucleic acid.", "Thus, one of the complementary single stranded targeting polynucleotides is complementary to one strand of the endogenous target sequence (i.e.Watson) and the other complementary single stranded targeting polynucleotide is complementary to the other strand of the endogenous target sequence (i.e.", "Crick).", "The domain homology clamp sequence preferably contains at least 90-95% sequence homology with the target sequence (although as outlined above, less sequence homology can be tolerated), to insure sequence-specific targeting of the targeting polynucleotide to the endogenous DNA domain target.", "Each single-stranded targeting polynucleotide is typically about 50-600 bases long, although a shorter or longer polynucleotide may also be employed.", "3.53.11.Method for Making the Targeting Polynucleotides Once the gene family and domain sequence is selected, the targeting polynucleotides are made, as will be appreciated by those in the art.", "For example, for large targeting polynucleotides, plasmids are engineered to contain an appropriately sized gene sequence with a deletion or insertion in the gene of interest and at least one flanking homology clamp which substantially corresponds or is substantially complementary to an endogenous target DNA sequence.", "Vectors containing a targeting polynucleotide sequence are typically grown in E coli and then isolated using standard molecular biology methods.", "Alternatively, targeting polynucleotides may be prepared in single-stranded form by oligonucleotide synthesis methods, which may first require, especially with larger targeting polynucleotides, formation of subfragments of the targeting polynucleotide, typically followed by splicing of the subfragments together, typically by enzymatic ligation.", "In general, as will be appreciated by those in the art, targeting polynucleotides may be produced by chemical synthesis of oligonucleotides, nick-translation of a double-stranded DNA template, polymerase chain-reaction amplification of a sequence (or ligase chain reaction amplification), purification of prokaryotic or target cloning vectors harboring a sequence of interest (e.g., a cloned cDNA or genomic clone, or portion thereof) such as plasmids, phagemids, YACs, cosmids, bacteriophage DNA, other viral DNA or replication intermediates, or purified restriction fragments thereof, as well as other sources of single and double-stranded polynucleotides having a desired nucleotide sequence.", "When using microinjection procedures it may be preferable to use a transfection technique with linearized sequences containing only modified target gene sequence and without vector or selectable sequences.", "The modified gene site is such that a homologous recombinant between the exogenous targeting polynucleotide and the endogenous DNA target sequence can be identified by using carefully chosen primers and PCR, followed by analysis to detect if PCR products specific to the desired targeted event are present (Erlich et al., (1991) Science 252: 1643, which is incorporated herein by reference).", "Several studies have already used PCR to successfully identify and then clone the desired transfected cell lines (Zimmer and Gruss, (1989) Nature 338: 150; Mouellic et al., (1990) Proc.", "Natl.", "Acad.", "Sci.", "USA 87: 4712; Shesely et al., (1991) Proc.", "Natl.", "Acad.", "Sci.", "USA 88: 4294, which are incorporated herein by reference).", "This approach is very effective when the number of cells receiving exogenous targeting polynucleotide(s) is high (i.e., with microinjection, or with liposomes) and the treated cell populations are allowed to expand to cell groups of approximately 1×104 cells (Capecchi, (11989) Science 244: 1288).", "When the target gene is not on a sex chromosome, or the cells are derived from a female, both alleles of a gene can be targeted by sequential inactivation (Mortensen et al., (1991) Proc.", "Natl.", "Acad.", "Sci.", "US 88: 7036).", "Alternatively, animals heterologous for the target gene can be bred to homologously as is known in the art.", "The invention may also be practiced with individual targeting polynucleotides which do not comprise part of a complementary pair.", "In each case, a targeting polynucleotide is introduced into a target cell simultaneously or contemporaneously with a recombinase protein, typically in the form of a recombinase coated targeting polynucleotide as outlined herein (i.e., a polynucleotide pre-incubated with recombinase wherein the recombinase is noncovalently bound to the polynucleotide; generally referred to in the art as a nucleoprotein filament).", "3.5.3.12.Alterations in the Target Nucleic Acid Comprising a Domain or Domains of Interest The present invention allows for the introduction of alterations in the target nucleic acid comprising a domain or domains of interest.", "That is, the fact that heterologies are tolerated in targeting polynucleotides allows for two things: first, the use of a heterologous domain homology clamps that may target genes encoding functional domains of a protein or multiple proteins, resulting in a variety of genotypes and phenotypes, and secondly, the introduction of alterations to the target sequence.", "Thus typically, a targeting polynucleotide (or complementary polynucleotide pair) has a portion or region having a sequence that is not present in the preselected endogenous targeted sequence(s) (i.e., a nonhomologous portion or mismatch) which may be as small as a single mismatched nucleotide, several mismatches, or may span up to about several kilobases or more of nonhomologous sequence.", "3.5.3.12.1.Methods and Compositions for Inactivation of a Domain of a Gene Accordingly, in a preferred embodiment, the methods and compositions of the invention are used for inactivation of a domain of a gene.", "That is, exogenous targeting polynucleotides can be used to inactivate, decrease or alter the biological activity of one or more domains in a gene of a cell (or transgenic nonhuman animal or plant).", "This finds particular use in the generation of animal models of disease states, or in the elucidation of gene function and activity, similar to “knock out” experiments.", "Alternatively, the biological activity of the wild-type gene may be either decreased, or the wild-type activity altered to mimic disease states.", "This includes genetic manipulation of non-coding gene sequences that affect the transcription of genes, including, promoters, repressors, enhancers and transcriptional activating sequences.", "3.5.3.12.1.1.Amino Acid Substitutions, Insertions or Deletions in the Endogenous Target Sequences Thus in a preferred embodiment, homologous recombination of the targeting polynucleotide and endogenous target sequence will result in amino acid substitutions, insertions or deletions in the endogenous target sequences, potentially both within the functional domain region and outside of it, for example as a result of the incorporation of PCR tags.", "This will generally result in modulated or altered gene function of the endogenous gene, including both a decrease or elimination of function as well as an enhancement of function.", "Nonhomologous portions are used to make insertions, deletions, and/or replacements in a predetermined endogenous targeted DNA sequence, and/or to make single or multiple nucleotide substitutions in a predetermined endogenous target DNA sequence so that the resultant recombined sequence (i.e., a targeted recombinant endogenous sequence) incorporates some or all of the sequence information of the nonhomologous portion of the targeting polynucleotide(s).", "Thus, the nonhomologous regions are used to make variant sequences, i.e.", "targeted sequence modifications.", "In this way, site directed modifications may be done in a variety of systems for a variety of purposes.", "3.5.3.12.1.1.1.Disruption by Either the Substitution, Insertion, Deletion or Frame Shifting of Nucleotides The endogenous target sequence, generally nucleic acid encoding a domain, may be disrupted in a variety of ways.", "The term “disrupt” as used herein comprises a change in the coding or non-coding sequence of an endogenous nucleic acid.", "In one preferred embodiment, a disrupted gene will no longer produce a functional gene product.", "In another preferred embodiment, a disrupted gene produces a variant gene product.", "Generally, disruption may occur by either the substitution, insertion, deletion or frame shifting of nucleotides.", "3.5.3.12.1.1.2.Disruption by Amino Acid Substitutions In one embodiment, amino acid substitutions are made.", "This can be the result of either the incorporation of a non-naturally occurring domain sequence into a target, or of more specific changes to a particular sequence outside of the domain sequence.", "3.5.3.12.1.1.3.Disruption by an Insertion Sequence In one embodiment, the endogenous sequence is disrupted by an insertion sequence.", "The term “insertion sequence” as used herein means one or more nucleotides which are inserted into an endogenous gene to disrupt it.", "In general, insertion sequences can be as short as 1 nucleotide or as long as a gene, as outlined herein.", "For non-gene insertion sequences, the sequences are at least 1 nucleotide, with from about 1 to about 50 nucleotides being preferred, and from about 10 to 25 nucleotides being particularly preferred.", "An insertion sequence may comprise a polylinker sequence, with from about 1 to about 50 nucleotides being preferred, and from about 10 to 25 nucleotides being particularly preferred.", "Insertion sequence may be a PCR tag used for identification of the first gene.", "In a preferred embodiment, an insertion sequence comprises a gene which not only disrupts the endogenous gene, thus preventing its expression, but also can result in the expression of a new gene product.", "Thus, in a preferred embodiment, the disruption of an endogenous gene by an insertion sequence gene is done in such a manner to allow the transcription and translation of the insertion gene.", "An insertion sequence that encodes a gene may range from about 50 bp to 5000 bp of cDNA or about 5000 bp to 50000 bp of genomic DNA.", "As will be appreciated by those in the art, this can be done in a variety of ways.", "In a preferred embodiment, the insertion gene is targeted to the endogenous gene in such a manner as to utilize endogenous regulatory sequences, including promoters, enhancers or a regulatory sequence.", "In an alternate embodiment, the insertion sequence gene includes its own regulatory sequences, such as a promoter, enhancer or other regulatory sequence etc.", "Particularly preferred insertion sequence genes include, but are not limited to, genes which encode selection or reporter proteins.", "In addition, the insertion sequence genes may be modified or variant genes.", "3.5.3.12.1.1.4.Disruption by Deletions The term “deletion” as used herein comprises removal of a portion of the nucleic acid sequence of an endogenous gene.", "Deletions range from about 1 to about 100 nucleotides, with from about 1 to 50 nucleotides being preferred and from about 1 to about 25 nucleotides being particularly preferred, although in some cases deletions may be much larger, and may effectively comprise the removal of the entire functional domain, the entire endogenous gene and/or its regulatory sequences.", "Deletions may occur in combination with substitutions or modifications to arrive at a final modified endogenous gene.", "3.5.3.12.1.1.5.Disruption Simultaneously by an Insertion and a Deletion In a preferred embodiment, endogenous genes may be disrupted simultaneously by an insertion and a deletion.", "For example, a domain of an endogenous gene, with or without its regulatory sequences, may be removed and replaced with an insertion sequence gene.", "Thus, for example, all but the regulatory sequences of an endogenous gene may be removed, and replaced with an insertion sequence gene, which is now under the control of the endogenous gene's regulatory elements.", "The term “regulatory element” is used herein to describe a non-coding sequence which affects the transcription or translation of a gene including, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, enhancer or activator sequences, dimerizing sequences, etc.", "In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequence.", "Promoter sequences encode either constitutive or inducible promoters.", "The promoters may be either naturally occurring promoters or hybrid promoters.", "Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.", "In addition to domain homology clamps and optional internal homology clamps, the targeting polynucleotides of the invention may comprise additional components, such as cell-uptake components, chemical substituents, purification tags, etc.", "3.53.12.2.Targeting Polynucleotide Comprising A Cell-Uptake Component In a preferred embodiment, at least one of the targeting polynucleotides comprises at least one cell-uptake component.", "As used herein, the term “cell-uptake component” refers to an agent which, when bound, either directly or indirectly, to a targeting polynucleotide, enhances the intracellular uptake of the targeting polynucleotide into at least one cell type (e.g., hepatocytes).", "A targeting polynucleotide of the invention may optionally be conjugated, typically by covalently or preferably noncovalent, binding, to a cell-uptake component.", "Various methods have been described in the art for targeting DNA to specific cell types.", "A targeting polynucleotide of the invention can be conjugated to essentially any of several cell-uptake components known in the art.", "For targeting to hepatocytes, a targeting polynucleotide can be conjugated to an asialoorosomucoid (ASOR)-poly-L-lysine conjugate by methods described in the art and incorporated herein by reference (Wu G Y and Wu C H (1987) J. Biol.", "Chem.", "262: 4429; Wu G Y and Wu C H (1988) Biochemistry 27: 887; Wu G Y and Wu C H (1988) J. Biol.", "Chem.", "263: 1462 1; Wu G Y and Wu C H (1992) J. Biol.", "Chem.", "267: 12436; Wu et al.", "(1991) J. Biol.", "Chem.", "266: 14338; and Wilson et al.", "0 992) J. Biol.", "Chem.", "267: 963, WO92/06180; WO92/05250; and WO91/17761, which are incorporated herein by reference).", "Alternatively, a cell-uptake component may be formed by incubating the targeting polynucleotide with at least one lipid species and at least one protein species to form protein-lipid-polynucleotide complexes consisting essentially of the targeting polynucleotide and the lipid-protein cell-uptake component.", "Lipid vesicles made according to Feigner (WO91/17424, incorporated herein by reference) and/or cationic lipidization (WO91/16024, incorporated herein by reference) or other forms for polynucleotide administration (EP 465,529, incorporated herein by reference) may also be employed as cell-uptake components.", "Nucleases, DNA damaging chemicals, UV radiation or gamma-radiation may also be used.", "In addition to cell-uptake components, targeting components such as nuclear localization signals may be used, as is known in the art.", "See for example Kido et al., Exper.", "Cell Res.", "198: 107-114 (1992), hereby expressly incorporated by reference.", "Typically, a targeting polynucleotide of the invention is coated with at least one recombinase and is conjugated to a cell-uptake component, and the resulting cell targeting complex is contacted with a target cell under uptake conditions (e.g., physiological conditions) so that the targeting polynucleotide and the recombinase(s) are internalized in the target cell.", "A targeting polynucleotide may be contacted simultaneously or sequentially with a cell-uptake component and also with a recombinase; preferably the targeting polynucleotide is contacted first with a recombinase, or with a mixture comprising both a cell-uptake component and a recombinase under conditions whereby, on average, at least about one molecule of recombinase is noncovalently attached per targeting polynucleotide molecule and at least about one cell-uptake component also is noncovalently attached.", "Most preferably, coating of both recombinase and cell-uptake component saturates essentially all of the available binding sites on the targeting polynucleotide.", "A targeting polynucleotide may be preferentially coated with a cell-uptake component so that the resultant targeting complex comprises, on a molar basis, more cell-uptake component than recombinase(s).", "Alternatively, a targeting polynucleotide may be preferentially coated with recombinase(s) so that the resultant targeting complex comprises, on a molar basis, more recombinase(s) than cell-uptake component.", "Cell-uptake components are included with recombinase-coated targeting polynucleotides of the invention to enhance the uptake of the recombinase-coated targeting polynucleotide(s) into cells, particularly for in vivo gene targeting applications, such as gene therapy to treat genetic diseases, including neoplasia, and targeted homologous recombination to treat viral infections wherein a viral sequence (e.g., an integrated hepatitis B virus (HBV) genome or genome fragment) may be targeted by homologous sequence targeting and inactivated.", "Alternatively, a targeting polynucleotide may be coated with the cell-uptake component and targeted to cells with a contemporaneous or simultaneous administration of a recombinase (e.g., liposomes or immunoliposomes containing a recombinase, a viral-based vector encoding and expressing a recombinase).", "In addition to recombinase and cellular uptake components, at least one of the targeting polynucleotides may include chemical substituents.", "Exogenous targeting polynucleotides that have been modified with appended chemical substituents may be introduced along with recombinase (e.g., RecA) into a metabolically active target cell to homologously pair with a predetermined endogenous DNA target sequence in the cell.", "In a preferred embodiment, the exogenous targeting polynucleotides are derivatized, and additional chemical substituents are attached, either during or after polynucleotide synthesis, respectively, and are thus localized to a specific endogenous target sequence where they produce an alteration or chemical modification to a local DNA sequence.", "Preferred attached chemical substituents include, but are not limited to: cross-linking agents (see Podyminogin et al., Biochem.", "34: 13098 (1995) and 35: 7267 (1996), both of which are hereby incorporated by reference), nucleic acid cleavage agents, metal chelates (e.g., iron/EDTA chelate for iron catalyzed cleavage), topoisomerases, endonucleases, exonucleases, ligases, phosphodiesterases, photodynamic porphyrins, chemotherapeutic drugs (e.g., adriamycin, doxirubicin), intercalating agents, labels, base-modification agents, agents which normally bind to nucleic acids such as labels, etc.", "(see for example Afonina et al., PNAS USA 93: 3199 (1996), incorporated herein by reference) immunoglobulin chains, and oligonucleotides.", "Iron/EDTA chelates are particularly preferred chemical substituents where local cleavage of a DNA sequence is desired (Hertzberg et al.", "(1982) J.", "Am.", "Chem.", "Soc.", "104: 313; Hertzberg and Dervan (1984) Biochemistry 23: 3934; Taylor et al.", "(1984) Tetrahedron 40: 457; Dervan, P B (1986) Science 232: 464, which are incorporated herein by reference).", "Further preferred are groups that prevent hybridization of the complementary single stranded nucleic acids to each other but not to unmodified nucleic acids; see for example Kutryavin et al., Biochem.", "35: 11170 (1996) and Woo et al., Nucleic Acid.", "Res.", "24(13): 2470 (1996), both of which are incorporated by reference.", "2′-0 methyl groups are also preferred; see Cole-Strauss et al., Science 273: 1386 (1996); Yoon et al., PNAS 93: 2071 (1996)).", "Additional preferred chemical substituents include labeling moieties, including fluorescent labels.", "Preferred attachment chemistries include: direct linkage, e.g., via an appended reactive amino group (Corey and Schultz (1988) Science 238: 1401, which is incorporated herein by reference) and other direct linkage chemistries, although streptavidin/biotin and digoxigenin/antidigoxigenin antibody linkage methods may also be used.", "Methods for linking chemical substituents are provided in U.S. Pat.", "Nos.", "5,135,720, 5,093,245, and 5,055,556, which are incorporated herein by reference.", "Other linkage chemistries may be used at the discretion of the practitioner.", "3.5.3.12.3.Targeting Polynucleotides Comprises at Least One Purification Tag or Capture Moiety In a preferred embodiment, at least one of the targeting polynucleotides comprises at least one purification tag or capture moiety, some of which are discussed above as chemical substituents, for example biotin, digoxigenin, psoralen, etc.", "Alternatively, the domain oligonucleotide could be directly attached to beads with the targeting reaction performed on a solid phase support.", "3.5.3.12.4.Targeting Polynucleotides are Coated with Recombinase Prior to Introduction to the Domain Target In a preferred embodiment, the targeting polynucleotides are coated with recombinase prior to introduction to the domain target.", "The procedures below are directed to the use of E. coli RecA, although as will be appreciated by those in the art, other recombinases may be used as well.", "Targeting polynucleotides can be coated using GTPgammaS, mixes of ATPgammaS with rATP, rGTP and/or dATP, or dATP or rATP alone in the presence of an rATP generating system (Boehringer Mannheim).", "Various mixtures of GTPgammaS, ATPgammaS, ATP, AIDP, dATP and/or rATP or other nucleosides may be used, particularly preferred are mixes of ATPgammaS and ATP or ATPgammaS and ADP.", "The targeting polynucleotide, whether double-stranded or single-stranded, is denatured by heating in an aqueous solution at 95-100′C for five minutes, then placed in an ice bath for 20 seconds to about one minute followed by centrifugation at 0′C for approximately 20 sec, before use.", "When denatured targeting polynucleotides are not placed in a freezer at −20′C they are usually immediately added to standard RecA coating reaction buffer containing ATPgammaS, at room temperature, and to this is added the RecA protein.", "Alternatively, RecA protein may be included with the buffer components and ATPgammaS before the polynucleotides are added.", "RecA coating of targeting polynucleotide(s) is initiated by incubating polynucleotide-RecA mixtures at 37′C for 10-15 min.", "RecA protein concentration tested during reaction with polynucleotide varies depending upon polynucleotide size and the amount of added polynucleotide, and the ratio of RecA molecule: nucleotide preferably ranges between about 3:1 and 1:3.When single-stranded polynucleotides are RecA coated independently of their homologous polynucleotide strands, the mM and microM concentrations of ATPgammaS and RecA, respectively, can be reduced to one-half those used with double-stranded targeting polynucleotides (i.e., RecA and ATPgammaS concentration ratios are usually kept constant at a specific concentration of individual polynucleotide strand, depending on whether a single- or double-stranded polynucleotide is used).", "RecA protein coating of targeting polynucleotides is normally carried out in a standard 10× RecA coating reaction buffer.", "10× RecA reaction buffer (i.e., 10×AC buffer) consists of.", "100 mM Tris acetate (pH 7.5 at 37° C.), 20 mM magnesium acetate, 500 mM sodium acetate, 10 mM DTT, and 50% glycerol).", "All of the targeting polynucleotides, whether double-stranded or single-stranded, typically are denatured before use by heating to 95-100° C. for five minutes, placed on ice for one minute, and subjected to centrifugation (10,000 rpm) at 0° C. for approximately 20 seconds (e.g., in a Tomy centrifuge).", "Denatured targeting polynucleotides usually are added immediately to room temperature RecA coating reaction buffer mixed with ATPgammaS and diluted with double-distilled H2O as necessary.", "A reaction mixture typically contains the following components: (i) 0.2-4.8 mM ATPgammaS; and (ii) between 1-100 ng/ul of targeting polynucleotide.", "To this mixture is added about 1-20 μl of RecA protein per 10-100 ul of reaction mixture, usually at about 2-10 mg/ml (purchased from Pharmacia or purified), and is rapidly added and mixed.", "The final reaction volume-for RecA coating of targeting polynucleotide is usually in the range of about 10-500 ul.", "RecA coating of targeting polynucleotide is usually initiated by incubating targeting polynucleotide-RecA mixtures at 37° C. for about 10-15 min.", "RecA protein concentrations in coating reactions varies depending upon targeting polynucleotide size and the amount of added targeting polynucleotide: RecA protein concentrations are typically in the range of 5 to 50 uM.", "When single-stranded targeting polynucleotides are coated with RecA, independently of their complementary strands, the concentrations of ATPgammaS and RecA protein may optionally be reduced to about one-half of the concentrations used with double-stranded targeting polynucleotides of the same length: that is, the RecA protein and ATPgammaS concentration ratios are generally kept constant for a given concentration of individual polynucleotide strands.", "3.5.3.12.4.1.Evaluation of Coating of Targeting Polynucleotides with RecA Protein The coating of targeting polynucleotides with RecA protein can be evaluated in a number of ways.", "First, protein binding to DNA can be examined using band-shift gel assays (McEntee et al., (1981) 1.Biol.", "Chem.", "256: 8835).", "Labeled polynucleotides can be coated with RecA protein in the presence of ATPgammaS and the products of the coating reactions may be separated by agarose gel electrophoresis.", "Following incubation of RecA protein with denatured duplex DNAs the RecA protein effectively coats single-stranded targeting polynucleotides derived from denaturing a duplex DNA.", "As the ratio of RecA protein monomers to nucleotides in the targeting polynucleotide increases from 0, 1:27, 1:2.7 to 3.7:1 for 121-mer and 0, 1:22, 1:2.2 to 4.5:1 for 159-mer, targeting polynucleotide's electrophoretic mobility decreases, i.e., is retarded, due to RecA-binding to the targeting polynucleotide.", "Retardation of the coated polynucleotide's mobility reflects the saturation of targeting polynucleotide with RecA protein.", "An excess of RecA monomers to DNA nucleotides is required for efficient RecA coating of short targeting polynucleotides (Leahy et al., (1986) J. Biol.", "Chem.", "261: 954).", "A second method for evaluating protein binding to DNA is in the use of nitrocellulose fiber binding assays (Leahy et al., (1986) J. Biol.", "Chem.", "261: 6954; Woodbury, et al., (1983) Biochemistry 22(20): 4730-4737.The nitrocellulose filter binding method is particularly useful in determining the dissociation-rates for protein: DNA complexes using labeled DNA.", "In the filter binding assay, DNA: protein complexes are retained on a filter while free DNA passes through the filter.", "This assay method is more quantitative for dissociation-rate determinations because the separation of DNA: protein complexes from free targeting polynucleotide is very rapid.", "Alternatively, recombinase protein(s) (prokaryotic, eukaryotic or endogeneous to the target cell) may be exogenously induced or administered to a target cell simultaneously or contemporaneously (i.e., within about a few hours) with the targeting polynucleotide(s).", "Such administration is typically done by micro-injection, although electroporation, lipofection, and other transfection methods known in the art may also be used.", "Alternatively, recombinase-proteins may be produced in vivo.", "For example, they may be produced from a homologous or heterologous expression cassette in a transfected cell or targeted cell, such as a transgenic totipotent cell (e.g.", "a fertilized zygote) or an embryonal stem cell (e.g., a murine ES cell such as AB-1) used to generate a transgenic non-human animal line or a somatic cell or a pluripotent hematopoietic stem cell for reconstituting all or part of a particular stem cell population (e.g.", "hematopoietic) of an individual.", "Conveniently, a heterologous expression cassette includes a modulatable promoter, such as an ecdysone-inducible promoter-enhancer combination, an estrogen-induced promoter-enhancer combination, a CMV promoter-enhancer, an insulin gene promoter, or other cell-type specific, developmental stage-specific, hormone-inducible drug inducible, or other modulatable promoter construct so that expression of at least one species of recombinase protein from the cassette can by modulated for transiently producing recombinase(s) in vivo simultaneous or contemporaneous with introduction of a targeting polynucleotide into the cell.", "When a hormone-inducible promoter-enhancer combination is used, the cell must have the required hormone receptor present, either naturally or as a consequence of expression a co-transfected expression vector encoding such receptor.", "Alternatively, the recombinase may be endogeneous and produced in high levels.", "In this embodiment, preferably in eukaryotic target cells such as tumor cells, the target cells produce an elevated level of recombinase.", "In other embodiments the level of recombinase may be induced by DNA damaging agents, such as mitomycin C, UV or gamma-irradiation.", "Alternatively, recombinase, levels may be elevated by transfection of a plasmid encoding the recombinase gene into the cell.", "3.53.13.Specialized Applications 3.5.3.13.1.Identification of New Members of Gene Families Which may be Useful in Functional Genomic Studies as well as in the Identification of New Drug Targets Once made, the compositions of the invention find use in a number of applications upon administration to target cells.", "In general, the compositions and methods of the invention are useful to identify new members of gene families which may be useful in functional genomic studies as well as in the identification of new drug targets; both of these may be accomplished through the generation of “knock out” animal models.", "In addition, the present invention allows the modification of functional domain targets, the creation of transgenic plants and animals, the cloning of genes containing domain functional domains, etc.", "3.5.3.13.2.Domain Specific Gene Evolution Once made and administered to a target host cell, the compositions of the invention find use in a number of applications, including domain specific gene evolution.", "The polypeptide or protein encoded by the targeted nucleic undergoes homologous recombination with the plurality of polynucleotides to produce a plurality of modified target nucleic acids that are expressed to produce a plurality of modified proteins.", "Selection systems are employed to identify and isolate host cells expressing proteins having a desired property or phenotype.", "For example, if the expressed protein is an enzyme, cells having a modified enzyme activity are identified.", "The desired activity can be an increased or decreased or altered activity.", "Proteins having the desired phenotype are selected and isolated, the modified nucleic acid is sequenced to identify sequences effecting the desired activity, and the process is repeated iteratively as needed to produce a protein having a desired activity or property.", "In this and other embodiments, suitable target sequences include nucleic acid sequences encoding therapeutically or commercially relevant proteins, including, but not limited to, enzymes (proteases, recombinases, lipases, kinases, carbohydrases, isomerases, tautomerases, nucleases etc.", "), hormones, receptors, transcription factors, growth factors, cytokines, globin genes, immunosupppressive genes, tumor suppressors, oncogenes, complement-activating genes, milk proteins (casein, alpha-lactalbumin, beta-lactoglobulin, bovine and human serum albumin), immunoglobulins, milk proteins, and pharmaceutical proteins and vaccines.", "In a preferred embodiment, the methods of the invention are used to generate pools or libraries of variant nucleic acid sequences, and cellular libraries containing the variant sequences.", "This idea is somewhat similar to the “gene shuffling” techniques of the literature (see Stemmer et al., 1994, Natuere 370: 389 which attempt to rapidly “evolve” genes by making multiple random changes simultaneously.", "In the present invention, this end is accomplished by using at least one cycle, and preferably reiterative cycles, of enhanced homologous recombination with targeting polynucleotides containing random mismatches, substitutions, insertions, or deletions.", "By using a library of targeting polynucleotides comprising a plurality of random mutations, and repeating the homologous recombination steps as many times as needed, a rapid “gene evolution” can occur, wherein the new genes may contain large numbers of mutations.", "Thus, in this embodiment, a plurality of targeting polynucleotides are used.", "The targeting polynucleotides each have at least one homology clamp that substantially corresponds to or is substantially complementary to the target sequence.", "Generally, the targeting polynucleotides are generated in pairs; that is, pairs of two single stranded targeting polynucleotides that are substantially complementary to each other are made (i.e.", "a Watson strand and a Crick strand).", "However, as will be appreciated by those in the art, less than a one to one ratio of Watson to Crick strands may be used; for example, an excess of one of the single stranded target polynucleotides (i.e.", "Watson) may be used.", "Preferably, sufficient numbers of each of Watson and Crick strands are used to allow the majority of the targeting polynucleotides to form double D-loops, which are preferred over single D-loops as outlined above.", "In addition, the pairs need not have perfect complementarity; for example, an excess of one of the single stranded target polynucleotides (i.e.", "Watson), which may or may not contain mismatches, may be paired to a large number of variant Crick strands, etc.", "Due to the random nature of the pairing, one or both of any particular pair of single-stranded targeting polynucleotides may not contain any mismatches.", "However, generally, at least one of the strands will contain at least one mismatch.", "The plurality of pairs preferably comprise a pool or library of mismatches.", "The size of the library will depend on a number of factors, including the number of residues to be mutagenized, the succeptibility of the protein to mutation, etc., as will be appreciated by those in the art.", "Generally, a library in this instance preferably comprises at least 10% different mismatches over the length of the targeting polynucleotides, with at least 30% mismatches being preferred and at least 40% being particularly preferred, although as will be appreciated by those in the art, lower (1, 2, 5%, etc.)", "or higher amounts of mismatches being both possible and desirable in some instances.", "That is, the plurality of pairs comprise a pool of random and preferably degenerate mismatches over some regions or all of the entire targeting sequence.", "As outlined herein, “mismatches” include substitutions, insertions and deletions, with the former being preferred.", "Thus, for example, a pool of degenerate variant targeting polynucleotides covering some, or preferably all, possible mismatches over some region are generated, as outlined above, using techniques well known in the art.", "Preferably, but not required, the variant targeting polynucleotides each comprise only one or a few mismatches (less than 10), to allow complete multiple randomization.", "That is, by repeating the homologous recombination steps any number of times, as is more fully outlined below, the mismatches from a plurality of probes can be incorporated into a single target sequence.", "The mismatches can be either non-random (i.e.", "targeted) or random, including biased randomness.", "That is, in some instances specific changes are desirable, and thus the sequence of the targeting polynucleotides are specifically chosen.", "In a preferred embodiment, the mismatches are random.", "The targeting polynucleotides can be chemically synthesized, and thus may incorporate any nucleotide at any position.", "The synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized targeting polynucleotides.", "Preferred methods maximize library size and diversity.", "It is important to understand that in any library system encoded by oligonucleotide synthesis one cannot have complete control over the codons that will eventually be incorporated into the peptide structure.", "This is especially true in the case of codons encoding stop signals (TAA, TGA, TAG).", "In a synthesis with NNN as the random region, there is a {fraction (3/64)}, or 4.69%, chance that the codon will be a stop codon.", "To alleviate this, random residues are encoded as NNK, where K=T or G. This allows for encoding of all potential amino acids (changing their relative representation slightly), but importantly preventing the encoding of two stop residues TAA and TGA.", "3.5.3.13.2.1.Mismatches are Fully Randomized, with No Sequence Preferences or Constants at any Position In one embodiment, the mismatches are fully randomized, with no sequence preferences or constants at any position.", "3.5.3.13.2.2.Biased Library In a preferred embodiment, the library is biased.", "That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.", "For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.", "As will be appreciated by those in the art, the introduction of a pool of variant targeting polynucleotides (in combination with recombinase) to a target sequence, in vitro to an extrachromosomal sequence, can result in a large number of homologous recombination reactions occuring over time.", "That is, any number of homologous recombination reactions can occur on a single target sequence, to generate a wide variety of single and multiple mismatches within a single target sequence, and a library of such variant target sequences, most of which will contain mismatches and be different from other members of the library.", "This thus works to generate a library of mismatches.", "3.5.3.13.2.2.1.Generating a Large Number of Different Variants within a Particular Region of a Sequence, Similar to Cassette Mutagenesis but not Limited by Sequence Length In a preferred embodiment, the variant targeting polynucleotides are made to a particular region or domain of a sequence (i.e.", "a nucleotide sequence that encodes a particular protein domain).", "For example, it may be desirable to generate a library of all possible variants of a binding domain of a protein, without affecting a different biologically functional domain, etc.", "Thus, the methods of the present invention find particular use in generating a large number of different variants within a particular region of a sequence, similar to cassette mutagenesis but not limited by sequence length.", "This idea is sometimes referred to herein as “domain specific gene evolution”.", "In addition, two or more regions may also be altered simultaneously using these techniques; thus “single domain” and “multi-domain” shuffling can be done.", "Suitable domains include, but are not limited to, kinase domains, nucleotide-binding sites, DNA binding sites, signaling domains, receptor binding domains, transcriptional activating regions, promoters, origins, leader sequences, terminators, localization signal domains, and, in immunoglobulin genes, the complementarity determining regions (CDR), Fc, VH, and VL.", "In a preferred embodiment, the variant targeting polynucleotides are made to the entire target sequence.", "In this way, a large number of single and multiple mismatches may be made in an entire sequence.", "Thus, this embodiment proceeds as follows.", "A pool of, targeting polynucleotides are made each containing one or more mismatches.", "The probes are coated with recombinase as generally described herein, and introduced to the target sequence.", "Upon binding of the probes to form D-loops, the recombinase is preferably removed.", "These polynucleotide: target sequences can then introduced into recombinant proficient cells, to produce target protein which can then be tested for biological activity, based on the identification of the target sequence.", "Depending on the results, the altered target sequence can be used as the starting target sequence in reiterative rounds of homologous recombination, generally using the same library.", "Preferred embodiments utilize at least two rounds of homologous recombination, with at least 5 rounds being preferred and at least 10 rounds being particularly preferred.", "Again, the number of reiterative rounds that are performed will depend on the desired end-point, the resistance or succeptibility of the protein to mutation, the number of mismatches in each probe, etc.", "3.5.3.14.Target Sequence 3.5.3.14.1.Target Sequences—an Immunoglobulin In a preferred embodiment, the target sequence is an immunoglobulin.", "The amino terminal region of the light and heavy chains of an antibody that come together to form the antigen binding site and the variability of their amino acid sequences provides the structural basis for the diversity of antigen binding sites.", "The variability of the variable regions of both the heavy and light chains is for the most part restricted to three small hypervariable regions in each chain.", "The remianing part of the variable regions, known as framework regions, is relatively constant.", "Each of the hypervariable regions consists of only about 5 to 10 amino acids; the corresponding regions in the DNA encoding these regions are known as the complementarity determining regions, or CDRs.", "Thus to engineer an antibody library, for example an antibody phage library, one can change the sequences in the CDR regions of both the heavy and light chains.", "Different permutations and combinations of CDRs can be changed and evolved to engineer antibody-phage libraries.", "3.5.3.14.2.Target Sequence—a Single-Chain Fv Framework for any Number of Specific Antigens In a preferred embodiment, the target sequence is a single-chain Fv framework for any number of specific antigens.", "Single chain Fv (scFv) consists Of VL and VH domains of an immunoglobulin linked by a peptide spacer and thus contains the minimal antigen-binding domains of an antibody.", "3.5.3.14.3.Target Sequence—an Antibody-Phage Fusion In a preferred embodiment, antibody-phage fusions are used as the target sequence.", "As is known in the art, single-chain Fv fusions with the pill minor coat protein allows expression of the antibody on the surface of a phage, wherein it is available to bind antigen.", "Five copies of pill are expressed on the surface of the phage.", "It is therefor possible to express five scFv on the phage.", "This antibody-phage display system has been used previously to isolate novel antibodies.", "By starting with antibodies to any antigen, higher affinity antibodies may be made, as well as novel antibodies.", "3.5.3.14.4.Target Sequence—the Coding Sequence for beta-Lactamase In a preferred embodiment, the target sequence is the coding sequence for beta-lactamase.", "Thus, the methods of the invention may be used to create superior recombinant reporter genes such as lacZ and green fluorescent protein (GFP); superior antibiotic and drug resistance genes; superior recombinase genes; superior recombinant vectors; and other superior recombinant genes and proteins, including immunoglobulins, vaccines or other proteins with therapeutic value.", "For example, targeting polynucleotides containing any number of alterations may be made to one or more functional or structural domains of a protein, and then the products of homologous recombination evaluated.", "Once made and administered to target cells, the target cells may be screened to identify a cell that contains the targeted sequence modification.", "This will be done in any number of ways, and will depend on the target gene and targeting polynucleotides as will be appreciated by those in the art.", "The screen may be based on phenotypic, biochemical, genotypic, or other functional changes, depending on the target sequence.", "In an additional embodiment, as will be appreciated by those in the art, selectable markers or marker sequences may be included in the targeting polynucleotides to facilitate later identification.", "3.5.3.15.Kits Containing the Compositions of the Invention are Provided In a preferred embodiment, kits containing the compositions of the invention are provided.", "The kits include the compositions, particularly those of libraries or pools of degenerate cssDNA probes, along with any number of reagents or buffers, including recombinases, buffers, ATP, etc.", "3.5.3.16.Targeting Polynucleotide: Target Nucleic Acid Complexes Serve as Substrates for Single-Stranded Endonucleases In an alternate embodiment, the targeting polynucleotide: target nucleic acid complexes serve as substrates for single-stranded endonucleases, such as, S1 and mung bean nuclease.", "Preferably the targeting polynucleotides are substantially complementary and form double D-loops with the target nucleic acid.", "The junctions of the complexes are single-stranded in nature, and thus are suceptible to single-strand specific nucleases and junction-specific nucleases.", "Accordingly, treatment of the complex with a single-strand nuclease results in defined nicks in the selected region encoding a predetermined domain of a protein encoded by the target nucleic acid.", "The nicked target nucleic acid is disassociated from the targeting polynucleotides and are reassembled and “shuffled” in vitro by PCR (Stemmer.", "1994.Nature 370: 389-391) to produce a plurality modified nucleic acids.", "The modified nucleic acids are introduced into an appropriate host cell, as described above, for expression of the plurality of modified proteins.", "Selection techniques are used as described herein to identify and isolate a cell expressing a modified protein.", "The process is repeated iteratively as needed to further evolve the targeted nucleic acid.", "3.5.3.17.Isolation of New Members of Gene Families that Comprise Particular Domains In a preferred embodiment, the present invention finds use in the isolation of new members of gene families that comprise particular domains.", "The use of domain filaments (i.e.", "domain homology clamps preferably containing a purification tag such as biotin, disoxisenin, or one purification method such as the use of a RecA antibody), allows the identification of genes containing the domain.", "Once identified, the new genes can be cloned, sequenced and the protein gene products purified.", "As will be appreciated by those in the art, the functional importance of the new genes can be assessed in a number of ways, including functional studies on the protein level, as well as the generation of “knock out” animal models.", "By choosing domain sequences for therapeutically relevant protein domains, novel targets can be identified that can be used in screening of drug candidates.", "3.5.3.18.Utilizing the Purification Tag to Isolate the Gene(s) Thus, in a preferred embodiment, the present invention provides methods for isolating new members of gene families containing protein domains comprising introducing targeting polynucleotides comprising domain homology clamps and at least one purification tag, preferably biotin, to a mix of nucleic acid, such as a plasmid cDNA library or a cell, and then utilizing the purification tag to isolate the gene(s).", "The exact methods will depend on the purification tag; a preferred method utilizes the attachment of the binding ligand for the tag to a bead, which is then used to pull out the sequence.", "Alternatively anti-RecA antibodies could be used to capture RecA-coated probes.", "The genes are then cloned, sequenced, and reassembled if necessary, as is well known in the art.", "3.5.3.19.Use in Functional Genomic Studies, by Providing the Creation of Transgenic Animal Models of Disease In an alternate preferred embodiment, the present invention finds use in functional genomic studies, by providing the creation of transgenic animal models of disease.", "Thus, for example, domain sequences used in homologous recombination methods can generate animals that have a wide variety of mutations in a wide variety of related domains of genes, potentially resulting in a wide variety of phenotypes, including phenotypes related to disease states.", "That is, by targeting a domain family, one, two or multiple genes in the family may be altered in any given experiment, thus creating a wide variety of genotypes and phenotypes to evaluate.", "Thus, in a preferred embodiment, the compositions and methods of the invention are used to generate pools or libraries of variant nucleic acid sequences, wherein the mutations are within the functional domain coding region, cellular libraries containing the variant libraries, and libraries of animals containing the variant libraries.", "Furthermore, domain targeting can be used in cells or animals that are diseased or altered; in essence, domain targeting can be done to identify “reversion” genes, genes that can modulate disease states caused by domains of different genes.", "Thus for example the loss of one type of enzymatic activity, resulting in a disease phenotype, may be compensated by alterations in a different but homologous enzymatic activity.", "Accordingly, once the recombinase-targeting polynucleotide compositions are formulated, they are introduced or administered into target cells.", "The administration is typically done as is known for the administration of nucleic acids into cells, and, as those skilled in the art will appreciate, the methods may depend on the choice of the target cell.", "Suitable methods include, but are not limited to, microinjection, electroporation, lipofection, etc.", "By “target cells” herein is meant prokaryotic or eukaryotic cells.", "Suitable prokaryotic cells include, but are not limited to, bacteria such as E coli, Bacillus species, and the extremophile bacteria such as thermophiles, halophiles, etc.", "Preferably, the procaryotic target cells are recombination competent.", "Suitable eukaryotic cells include, but are not limited to, fungi such as yeast and filamentous fungi, including species of Aspergillus, Trichoderma, and Neurospora; plant cells including those of corn, sorghum, tobacco, canola, soybean, cotton, tomato, potato, alfalfa, sunflower, etc.", "; and animal cells, including fish, reptiles, amphibia, birds and mammals.", "Suitable fish cells include, but are not limited to, those from species of salmon, trout, tilapia, tuna, carp, flounder, halibut, swordfish, cod and zebrafish.", "Suitable bird cells include, but are not limited to, those of chickens, ducks, quail, pheasants, ostrich, and turkeys, and other jungle foul or game birds.", "Suitable mammalian cells include, but are not limited to, cells from horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, marine mammals including dolphins and whales, as well as cell lines, such as human cell lines of any tissue or stem cell type, and stem cells, including pluripotent and non-pluripotent, and non-human zygotes.", "Particular human cells including, but are not limited to, tumor cells of all types (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells, osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.", "Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, mouse La, HT1080, Cl 27, Rat2, CV-1, NIH3T3 cells, CHO, COS, 293 cells, etc.", "See the ATCC cell line catalog, hereby expressly incorporated by reference.", "3.5.3.20.Procaryotic Cells are Used to Identify, Clone, or Alter Target Sequences In a preferred embodiment, procaryotic cells are used to identify, clone, or alter target sequences, preferably protein domains.", "In this embodiment, a pre-selected target DNA sequence is chosen for alteration.", "Preferably, the pre-selected target DNA sequence is contained within an extrachromosomal sequence.", "By “extrachromosomal sequence” herein is meant a sequence separate from the chromosomal or genomic sequences.", "Preferred extrachromosomal sequences include plasmids (particularly procaryotic plasmids such as bacterial plasmids), p1 vectors, viral genomes, yeast, bacterial and mammalian artificial chromosomes (YAC, BAC and MAC, respectively), and other autonomously self-replicating sequences, although this is not required.", "As described herein, a recombinase and at least two single stranded targeting polynucleotides which are substantially complementary to each other, each of which contain a homology clamp to the target sequence contained on the extrachromosomal sequence, are added to the extrachromosomal sequence, preferably in vitro.", "The two single stranded targeting polynucleotides are preferably coated with recombinase, and at least one of the targeting polynucleotides contain at least one nucleotide substitution, insertion or deletion.", "The targeting polynucleotides then bind to the target sequence in the extrachromosomal sequence to effect homologous recombination and form an altered extrachromosomal sequence which contains the substitution, insertion or deletion.", "The altered extrachromosomal sequence is then introduced into the procaryotic cell using techniques known in the art.", "Preferably, the recombinase is removed prior to introduction into the target cell, using techniques known in the art.", "For example, the reaction may be treated with proteases such as proteinase K, detergents such as SDS, and phenol extraction (including phenol:chloroform:isoamyl alcohol extraction).", "These methods may also be used for eukaryotic cells.", "The cells are then grown under conditions which allow the expression of the variant nucleic acids to form variant proteins, particularly with alterations in domains.", "3.53.20.1.Proteins Having the Desired Phenotype are Selected and Isolated In a preferred embodiment, proteins having the desired phenotype are selected and isolated, the modified nucleic acid is sequenced to identify sequences effecting the desired activity, and the process is repeated iteratively as needed to produce a protein having a desired activity or property.", "Thus, in a preferred embodiment, the methods of the invention are repeated until the desired protein or phenotype is seen.", "Alternatively, the pre-selected target DNA sequence is a chromosomal sequence.", "In this embodiment, the recombinase with the targeting polynucleotides are introduced into the target cell, preferably eukaryotic target cells.", "In this embodiment, it may be desirable to bind (generally non-covalently) a nuclear localization signal to the targeting polynucleotides to facilitate localization of the complexes in the nucleus.", "See for example Kido et al., Exper.", "Cell Res.", "198: 107-114 (1992), hereby expressly incorporated by reference.", "The targeting polynucleotides and the recombinase function to effect homologous recombination, resulting in altered chromosomal or genomic sequences.", "3.5.3.21.Eukaryotic Cells are Used In a preferred embodiment, eukaryotic cells are used.", "Basically, any mammalian cells may be used, with mouse, rat, primate and human cells being particularly preferred.", "Accordingly, suitable cell types include, but are not limited to, tumor cells of all types, i.e., fibroblasts, epithelial cells (particularly melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.", "Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH 3T3 cells, CHO, Cos, etc.", "See the ATCC cell line catalog, hereby expressly incorporated by reference.", "For making transgenic non-human animals (which include homologously targeted non-human animals) embryonal stem cells (ES cells), donor cells for nuclear transfer and fertilized zygotes are preferred.", "In a preferred embodiment, embryonal stem cells are used.", "Murine ES cells, such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62: 1073-1085 (1990)) essentially as described (Robertson, E. J.", "(1987) in Teratocarcinomas and Embcyonic Stem Cells: A Practical Approach.", "E. J. Robertson, ed.", "(oxford: IRL Press), p. 71-112; Zjilstra et al., Nature 342: 435-438 (1989); and Schwartzberg et al., Science 246: 799-803 (1989), each of which is incorporated herein by reference) may be used for homologous gene targeting.", "Other suitable ES lines include, but are not limited to, the E14 line (Hooper et al.", "(1987) Nature 326: 292-295), the D3 line (Doetschman et al.", "(1985) J. Embryol.", "Exp.", "Morph.", "87: 21-45), and the CCE line (Robertson et al.", "(1986) Nature 323: 445448).", "The success of generating a mouse line from ES cells bearing a specific targeted mutation depends on the pluripotence of the ES cells (i.e., their ability, once injected into a host blastocyst, to participate in embryogenesis and contribute to the germ cells of the resulting animal).", "The pluripotence of any given ES cell line can vary with time in culture and the care with which it has been handled.", "The only definitive assay for pluripotence is to determine whether the specific population of ES cells to be used for targeting can give rise to chimeras capable of germline transmission of the ES genome.", "For this reason, prior to gene targeting, a portion of the parental population of AB-1 cells is injected into C57B 1/6J blastocysts to ascertain whether the cells are capable of generating chimeric mice with extensive ES cell contribution and whether the majority of these chimeras can transmit the ES genome to progeny.", "3.5.3.22.Non-Human Zygotes are Used In a preferred embodiment, non-human zygotes are used, for example to make transgenic animals, using techniques known in the art (see U.S. Pat.", "No.", "4,873,191; Brinster et al., PNAS 86: 7007 (1989); Susulic et al., J. Biol.", "Chem.", "49: 29483 (1995), and Cavard et al., Nucleic Acids Res.", "16: 2099 (1988), hereby incorporated by reference).", "Preferred zygotes include, but are not limited to, animal zygotes, including fish, avian, reptilian, amphibian and mammalian zygotes.", "Suitable fish zygotes include, but are not limited to, those from species of salmon, trout, tuna, carp, flounder, halibut, swordfish, cod, tilapia and zebrafish.", "Suitable bird zygotes include, but are not limited to, those of chickens, ducks, quail, pheasant, turkeys, and other jungle fowl and game birds.", "Suitable mammalian zygotes include, but are not limited to, cells from horses, cows, buffalo, deer, sheep, rabbits, rodents such as mice, rats, hamsters and guinea pigs, goats, pigs, primates, and marine mammals including dolphins and whales.", "See Hogan et al., Manipulating the Mouse Embryo (A Laboratory Manual), 2nd Ed.", "Cold Spring Harbor Press, 1994, incorporated by reference.", "The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.", "For example, micro-injection is commonly utilized for target cells, although calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection also may be used.", "Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, and others (see, generally, Sambrook et al.", "Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference).", "Direct injection of DNA and/or recombinase-coated targeting polynucleotides into target cells, such as skeletal or muscle cells also may be used (Wolff et al.", "(1990) Science 247: 1465, which is incorporated herein by reference).", "3.5.3.23.Precursor Animals or Cells Already Contain a Disease Allele In a preferred embodiment, the precursor animals or cells already contain a disease allele.", "As used herein, the term “disease allele” refers to an allele of a gene which is capable of producing a recognizable disease.", "A disease allele may be dominant or recessive and may produce disease directly or when present in combination with a specific genetic background or preexisting pathological condition.", "A disease allele may be present in the gene pool or may be generated de novo in an individual by somatic mutation.", "For example and not limitation, disease alleles include: activated oncogenes, a sickle cell anemia allele, a Tay-Sachs allele, a cystic fibrosis allele, a Lesch-Nyhan allele, a retinoblastoma-susceptibility allele, a Fabry's disease allele, a Huntington's chorea allele, and a xenoderma pigmentosa allele.", "As used herein, a disease allele encompasses both alleles associated with human diseases and alleles associated with recognized veterinary diseases.", "For example, the deltaF508 CFTR allele in a human disease allele which is associated with cystic fibrosis in North Americans.", "Once made and administered to target cells, new domains of genes may be isolated as outlined herein.", "Alternatively, the target cells may be screened to identify a cell that contains the targeted functional domain sequence modification.", "This will be done in any number of ways, and will depend on the target domain and targeting polynucleotides as will be appreciated by those in the art.", "The screen may be based on phenotypic, biochemical, genotypic, or other functional changes, depending on the target sequence.", "For example, IgE levels may be evaluated for inflammation or asthma; vascular tone or blood pressure can be evaluated for hypertension, behavior screens can be done for neurologic effects, lipoprotein profiles can be screened for cardiovascular effects; secreted molecules can be evaluated for endocrine processes; CBCs can be done for hematology studies, etc.", "In an additional embodiment, as will be appreciated by those in the art, selectable markers or marker sequences may be included in the targeting polynucleotides to facilitate later identification.", "The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the invention in any manner.", "All patents, patent applications, and publications cited herein are expressly incorporated by reference in their entirety.", "3.5.4.Gene Deletion in Bacteria This invention relates to a method and means for deleting a gene from a bacterial chromosome in a single step.", "3.5.4.1 Applications 3.5.4.1.1 The Construction of Special Bacterial Strains which have a Particular Genetic Background Many applications require the construction of special bacterial strains which have a particular genetic background.", "These genetic backgrounds are the framework in which specific recombinant DNA plasmid constructions are tested to determine whether they can provide functions which are missing from the background of the bacteria.", "If such functions are provided by the recombinant plasmid, then there is positive evidence that a particular genetic locus or loci is encoded by the plasmid.", "The construction of genetic backgrounds is therefore a vital step in the subsequent cloning and investigation of specific genes.", "3.5.4.1.2.The Gene in Question has been Deleted, so there is No Production whatsoever of a Mutant Protein Making Analysis Much Less Ambiguous The most common backgrounds are those in which a single mutation is present in a specific gene on the bacterial chromosome.", "This may result in synthesis of a defective version of the protein encoded by that gene, resulting in a specific cellular dysfunction.", "Correction of the cellular dysfunction, by introduction of a specific recombinant plasmid, is evidence that the relevant gene has been cloned onto the specific plasmid.", "However, a mutant protein may not be silent and may undergo interactions with other components, thereby creating the appearance that the plasmid gene encodes the entire active protein when it does not.", "This can seriously confuse the analysis.", "A much less ambiguous and therefore more desirable approach is one in which the gene in question has been deleted.", "In these circumstances, there is no production whatsoever of a mutant protein.", "3.5.4.13.If Bacteria is Being Used to Genetically Engineer a Protein, Deletion of the Gene that Manufactures a Contaminating Protein Made by the Same Bacteria can Reduce Purification Steps Another situation where it is desirable to delete a complete gene from the chromosome of a bacteria is when the bacteria is being used in the production of a genetically engineered protein.", "Examples of these situations include the expression of insulin, growth hormone, protein A, and various vaccines from recombinant genes inserted into E. coli.", "Many times the E. coli produces proteins which contaminate the purified product produced by the genetic engineering.", "Although it is possible to add additional purification steps to remove this contaminant, it would be preferable to avoid the problem entirely by deleting the gene encoding the contaminating protein.", "Methods that are presently used to alter a gene include random mutation or inactivation of the gene sequence by mutation, insertion, or deletion of some portion of the gene.", "However, this can still lead to the production of inactive protein fragments or deletion of more of the chromosome than is necessary or desirable.", "It is therefore an object of the present invention to provide a method and means for deleting a specific gene from a bacteria.", "It is another object of the present invention to provide a method and means for inserting and/or inactivating or deleting the recA gene in a variety of bacteria.", "3.5.4.2.Miscellaneous Applications 3.5.4.2.1 Creating a Deletion in a Defined Target within the Bacterial Chromosome There are two obstacles which have to be overcome.", "One is to develope a method which will create a deletion in a defined target within the bacterial chromosome.", "3.5.4.2.2 Generalizing the Approach so that Even Essential Genes can be Deleted The second is to generalize the approach so that even essential genes can be deleted.", "This is because many of the genes of interest are essential ones.", "The deletion of an essential gene normally would result in cell death.", "Essential proteins includes enzymes of glycolysis, enzymes associated with amino acid or sugar biosynthesis, enzymes and factors associated with protein and nucleic acid biosynthesis (including both RNA and DNA), enzymes required for the synthesis of cofactors for oxidation, reduction, methylation and transamination processes, and enzymes necessary for synthesis of essential lipids and polysaccharides or of any other essential molecule, including various nucleic acids, such as transfer or ribosomal RNAs, and segments of nucleic acids, such as gene regulatory elements.", "It is therefore an object of the present invention to provide a method wherein an organism is produced which does not contain genetic material coding for the molecule which is to be cloned and expressed in the organism.", "A further object of the present invention is to provide a method whereby a deficient organism which is to be used for cloning an essential gene remains viable even under restrictive conditions.", "3.5.43 Specialized Applications 3.5.43.1.Method for the Deletion of a Gene from a Bacteria Using a Single Step Procedure that is Applicable to any Gene that has been Cloned Disclosed is a method for the deletion of a gene from a bacteria using a single step procedure that is applicable to any gene that has been cloned.", "The procedure depends upon site-directed recombination of linear DNA fragments with sequences on the chromosome as a function of recA in combination with the subsequent inactivation or deletion of the recA gene.", "The method is analogous to a procedure used to give insertions by homologous recombination into specific plasmid genes.", "3.5.43.1.1.Strategy for Construction of Chromosomal Deletions The basic strategy for construction of chromosomal deletions is to transform the bacteria with linear DNA fragments which contain an antibiotic resistant or other phenotypically detectable gene segment (a “marker”) flanked by sequences homologous to a closely spaced region on the cell chromosome containing the gene to be deleted A double-crossover event within the homologous sequences, effectively deleting the entire gene, is selected for by screening for the antibiotic resistant phenotype.", "The linear fragment is not integrated into the chromosome in the absence of enzymes expressed by the recA gene.", "Accordingly, if the gene is absent or inactive, a recA gene must be inserted into the cell prior to the linear recombination event, and then inactivated or removed to prevent subsequent incorporation of other non-chromosomal sequences into the chromosome.", "This is particularly important if the bacteria is used as a host for the expression of genetically engineered proteins from sequences carried on plasmids or other extrachromosomal elements.", "The recA gene can be provided either in the form of an extrachromosomal element such as a plasmid or through incorporation of the gene into the chromosome.", "The recA gene is preferably inactivated or deleted by means of a double reciprocal recombination event utilizing linear sequences containing sequences homologous to the flanking sequences on either side of the recA gene in the chromosome.", "This is essentially the same method used to delete or insert a gene into the chromosome by homologous recombination as described above.", "The present invention includes isolated linear DNA fragments constructed for use in the method for deleting a gene from the chromosome and for inserting or deleting the recA gene.", "The method and sequences are applicable to a variety of bacteria including strains of Escherichia, Pseudomonas, Agrobacterium, Proteus, Erwinia, Shigella, Bacillus, Rhizobium, Vibrio, Salmonella, Streptococcus, and Haemophilus.", "A plasmid which has a temperature-sensitive replicon and a wild-type allele of the desired gene is used to restore or maintain the phenotype produced by the deleted gene.", "This plasmid maintains production of the desired protein, and therefore cell viability if the encoded protein is essential to cell growth, when the chromosomal copy of the desired gene has been deleted.", "However, since the resulting cells have a temperature-sensitive phototype, the expression of the plasmid gene may be easily prevented by culturing the host strain at an elevated temperature.", "The resulting deficient host strain may then be used to screen other mutated and cloned genes for their ability to produce the desired protein.", "3.5.5.Additional Considerations 3.5.5.1.Maintaining the Viability of the Bacteria The present invention is a method for deleting any gene from a bacterial strain, while maintaining the viability of the bacteria if the gene encodes an essential molecule and deletion of the essential gene results in a lethal phenotype.", "The gene to be deleted is provided on a plasmid with a temperature sensitive replicon.", "The cells now have a temperature sensitive phenotype.", "When the cells are grown at an elevated temperature under conditions which allow rapid detection of the absence of the desired molecule, complementation of this phenotype by introduction of a DNA fragment fused onto a stable plasmid is strong evidence for cloning of the gene which has been deleted from the chromosome.", "3.5.5.2.Homologous Recombination The present invention is a method for deleting any gene from a bacterial strain employing linear DNA fragments incorporating sequences homologous to the sequences flanking the gene to be deleted in the chromosome and sequences allowing insertion or removal/inactivation of recA in a variety of bacteria.", "3.5.5.2.1.Roles for Several Enzymes Needed in Recombination Homologous recombination has been detected in a wide variety of organisms, from simple bacteriophages to complex eukaryotic cells.", "Genetic and biochemical investigations have defined roles for several enzymes needed in recombination.", "3.5.5.2.2.RecA 3.5.5.2.2.1.Alignment of DNA Molecules before Exchange The RecA protein participates in the early steps of synapse, allowing alignment of DNA molecules before exchange, in strand transfer, where there is transfer of a single-stranded segment to a recipient duplex to form a limited heteroduplex region between the interacting DNAs and in the extension of this heteroduplex region by a reaction involving the concerted winding and unwinding of incoming and outgoing DNA chains, respectively.", "The hydrolysis of ATP by RecA protein is required for these events in vitro.", "3.5.5.2.2.2.Controlling Expression of a Group of Unlinked Genes that Aid in Recovery of Cells after Exposure to DNA Damaging Agents The recA gene performs another equally important role in cell metabolism by controlling expression of a group of unlinked genes that aid in recovery of cells after exposure to DNA-damaging agents.", "This response, termed the SOS response, involves genes that participate in repair of DNA damage, mutagenesis, and coordination of cell division events.", "3.5.5.2.2.3.Characterization The purified RecA protein is a single polypeptide ranging in weight from about 37,000 to about 42,000.Although there is some variation in the sequences between bacterial strains, the recA proteins from a variety of bacteria in general have been isolated and characterized by interspecies complementation and assays utilizing comparisons with isolated, characterized proteins.", "The present method and the linear fragments for use in the present method are not limited to organisms such as E. coli.", "The recA gene is found in a variety of bacteria including both gram negative and gram positive organisms.", "The recA gene has been isolated or identified and characterized in several organisms.", "It is possible to prepare a genomic library from any bacterial species and to isolate a clone containing a sequence homologous to a characterized recA gene by interspecific complementation using one of the available DNA clones containing a recA sequence or cross-reactivity with antisera to RecA proteins from a well-characterized organism such as E. coli.", "RecA+ and RecA-strains are available from a variety of sources.", "For example, cloning and characterization of recA genes and recA proteins from Proteus vulgaris, Erwinia carotovoria, Shigella flexneria and Escherichia coli are described by S. L. Keener, et al., in 1.Bacter.", "160(1), 153-160 (1984).", "The RecA proteins produced by these organisms were demonstrated to be highly conserved among the species.", "In fact, the protein produced by one species could be introduced into another species where it complemented repair and regulatory defects of recA mutations.", "Other bacterial recA genes and gene products have been described by C. A.", "Miles, et al, in Mol.", "Gen. Genet.", "204,161-165 (1986) (Agrobacterium tumefaciens C58), 1.Goldberg et al, J. Bacteriol.", "165(3), 715-722(1986), (Vibrio cholera), M. Better et al, J. Bacteriol.", "155(1), 311-316 (1983), (Rhizobium meliloti), T. A. Kokjohn et al, J. Bacteriol.", "163(2), 568-572 (1985), (Pseudomonas aeruginosa), and C. M. Lovett, Jr. et al, J. Bio.", "Chem.", "260(6), 3305-3313 (1985) (Bacillus subtilis).", "These articles detail the isolation and characterization of gene libraries and the proteins encoded by the recA genes using techniques known to those skilled in the art including construction of gene libraries, identification of homologous genes using hybridization to probes from other more well characterized species such as E. coli, isolation and characterization of RecA proteins using antisera to RecA proteins from E. coli, and interspecies complementation of deficient strains of E. coli using gene segments from the libraries.", "The isolated proteins were useful for in vitro complementation studies.", "RecA deficient strains and RecA clones are available from many of the laboratories cited in the above articles and from the E. coli Genetic Stock Center at Yale University run by Dr. Barbara Bachman.", "3.5.5.2.3.Construction of Linear DNA Fragments which have Sequences Homologous to Closely Spaced Regions on the Chromosome in the Bacteria which Flank Each Side of the Gene of Interest The method whereby the DNA fragment is introduced into the chromosome to delete a gene or to regulate recA involves the construction of linear DNA fragments which have sequences homologous to closely spaced regions on the chromosome in the bacteria which flank each side of the gene of interest.", "In general, they must contain at least 80 to 100 nucleotides homologous to sequences flanking the gene to be deleted or the recA gene.", "An antibiotic resistant locus such as Kanr, which encodes a protein making the bacterial resistant to the antibiotic, or other marker, is placed between these flanking sequences.", "The linear DNA segment is introduced into a specific cell strain and selection is done for a double, reciprocal recombination which will delete the target gene and insert the marker into its place.", "As a result of the insertion of the antibiotic resistant gene, the cells are now resistant to the antibiotic.", "Cells which do not contain the insertion are eliminated by growing the bacteria in a medium containing the antibiotic.", "Any other gene which allows for rapid screening of the cells containing a double, reciprocal recombination may be used in place of a gene for antibiotic resistance.", "For example, any gene for an essential protein, including enzymes, cofactors, proteins which are necessary for the synthesis of essential lipids, polysaccharides, nucleic acids, and other protein molecules such as receptors, as well as nucleic acids which have functional activity such as ribozymes, may be used.", "Other genes which confer a detectable phenotype on the cell strain such as sensitivity to temperature or ultraviolet radiation, auxotrophism for a sugar, amino acid, protein or nucleotide, or any other phenotype which can be detected by chemical indicators either in vitro or in vivo assay, or an immunoassay for a specific cellular component may also be used.", "Such chemical, radioactive, or immunological screening assays are well known to those skilled in the art.", "3.5.53.Eliminating Contamination from the Host Producing its Own Protein by Deletion and Replacement In one application, in which the goal is to produce and purify a foreign protein, and the microorganism encodes its own version of the protein, the gene for the microorganism's own protein is eliminated.", "The gene for the foreign protein is then inserted and the protein produced.", "The purification process is thereby simplified since there is no contamination by the host protein, whether analogous to the protein being produced or unrelated which copurifies with, or interferes with the purification of, the protein being produced.", "For example, a protein may interfere with binding of the protein to be purified to a column.", "3.5.5.4.A Plasmid is Used to Introduce a Mutated Gene into an Organism In a second application, a plasmid is used to introduce a gene into an organism which typically contains a mutation in the gene to be investigated resulting in a negative phenotype for the product of the gene to be investigated.", "Failure of the organism to produce a biologically active form of the protein encoded by the mutated gene may confer a lethal phenotype under certain defined conditions.", "For example, this can be at a temperature, designated as the restrictive temperature, at which the mutant protein denatures or otherwise undergoes inactivation.", "The cloned gene which is introduced is selected for by virtue of its ability to confer cell viability or any other detectable phenotype for the desired protein at the restrictive temperature.", "The acquisition of viability or other detectable phenotype at the normally restrictive temperature is evidence that the gene of interest has been cloned.", "3.5.5.4.1.Problems: The Defective Host Protein May Interact With a Protein Produced from the Introduced Plasmid The major problem with this second system is that, unless the inactive gene is deleted in entirety, the defective host protein may interact with a protein produced from the introduced plasmid.", "This interaction may stabilize the defective host Protein enough so that its activity is restored even at the restrictive temperature.", "In this case, the restoration of growth at the restrictive temperature would be a false positive, that is, the growth would not be due to activity encoded by the cloned DNA segment.", "This problem holds true for all selections based on complementation of a phenotype which is due to a defect in a specific protein.", "Such phenotypes include temperature sensitivity, amino acid auxotrophies or any other auxotrophies which result from a lack of synthesis of a key ingredient such as a sugar, nucleotide, critical protein or nucleic acid, or cofactor used for oxidation, reduction, or transamination reactions.", "3.5.5.5.False Positives The problem of “false positives” also exists for cloned DNA pieces which are created to encode enzyme fragments as a means to define the catalytic core or to define any segment which achieves a specific purpose, such as a piece which undergoes self-association, binds to a specific ligand or receptor, or forms a specific complex or array with one or more additional components.", "In these cases, the engineering of protein fragments, which are tested in a host cell that encodes a defective version of the protein of interest, is seriously hampered if the defective host protein interacts in any way with the engineered pieces.", "3.5.5.6.Use of Temperature Sensitive Replicons In the present invention, specifically designed linear DNA fragments are used to create a deletion of a gene by site-specific recombination.", "These fragments are transformed into the host cell.", "Cell viability or the detectable phenotype can be maintained during the procedure by provision of the gene encoding the desired protein on a recombinant plasmid that has a temperature-sensitive replicon, so that the cells which contain the deletion have a temperature sensitive phenotype.", "To achieve the deletion by recombination with the linear DNA fragments, it is necessary for the cells to have a RecA+ phenotype which is derived from recA, or its equivalent.", "Once recombination has occurred, the cell must immediately be changed to RecA or else the temperature sensitive plasmid will recombine with homologous sequences on the chromosome.", "The same would apply to any other extrachromosomal element where integration into the host chromosome would be undesirable.", "The RecA− phenotype may be achieved by simultaneous inactivation of recA during the transformation with linear fragments or, after the transformation, by immediately introducing RecA− by mating with an appropriate RecA− strain or by transduction with a phage which carries a RecA− gene segment.", "Although mutagenesis may also be an effective means of making the cell RecA−, this is a “hit or miss” approach.", "The preferred method is to use homologous recombination of linear DNA sequences bounded by sequences hybridizing to the sequences flanking the recA gene.", "The recA gene is necessary in order for the gene encoding the desired protein to be incorporated into the organism.", "However, any plasmids or other extrachromosomal elements in the cell will be incorporated unless the recA gene is immediately removed.", "This is a particular concern where the bacteria serves as a host for the expression of a genetically engineered protein from multicopy plasmids.", "3.6.Artificial Chromosomes 3.6.1 Artificial Chromosomes, Uses Thereof and Methods for Preparing Artificial Chromosomes Methods for preparing cell lines that contain artificial chromosomes, methods for preparation of artificial chromosomes, methods for isolation of the artificial chromosomes, methods for purification of artificial chromosomes, methods for targeted insertion of heterologous DNA into artificial chromosomes, methods for delivery of the chromosomes to selected cells and tissues, and methods for isolation and large-scale production of the chromosomes are provided.", "Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods.", "In particular, satellite artificial chromosomes that, except for inserted heterologous DNA, are substantially composed of heterochromatin are provided.", "Cell-based methods for use of the artificial chromosomes, including for gene therapy, production of gene products and production of transgenic plants and animals are also provided.", "3.6.2 Limitations of Existing Gene Delivery Technologies Several viral vectors, non-viral, and physical delivery systems for gene therapy and recombinant expression of heterologous nucleic acids have been developed [see, e.g., Mitani et al.", "(1993) Trends Biotech.", "11: 162-166].", "The presently available systems, however, have numerous limitations, particularly where persistent, stable, or controlled gene expression is required.", "These limitations include: (1) size limitations because there is a limit, generally on order of about ten kilobases [kB], at most, to the size of the DNA insert [gene] that can be accepted by viral vectors, whereas a number of mammalian genes of possible therapeutic importance are well above this limit, especially if all control elements are included; (2) the inability to specifically target integration so that random integration occurs which carries a risk of disrupting vital genes or cancer suppressor genes; (3) the expression of randomly integrated therapeutic genes may be affected by the functional compartmentalization n the nucleus and are affected by chromatin-based position effects; (4) the copy number and consequently the expression of a given gene to be integrated into the genome cannot be controlled.", "Thus, improvements in gene delivery and stable expression systems are needed [see, e.g., Mulligan (1993) Science 260: 926-932].", "In addition, safe and effective vectors and gene therapy methods should have numerous features that are not assured by the presently available systems.", "For example, a safe vector should not contain DNA elements that can promote unwanted changes by recombination or mutation in the host genetic material, should not have the potential to initiate deleterious effects in cells, tissues, or organisms carrying the vector, and should not interfere with genomic functions.", "In addition, it would be advantageous for the vector to be non-integrative, or designed for site-specific integration.", "Also, the copy number of therapeutic gene(s) carried by the vector should be controlled and stable, the vector should secure the independent and controlled function of the introduced gene(s); and the vector should accept large (up to Mb size) inserts and ensure the functional stability of the insert.", "The limitations of existing gene delivery technologies, however, argue for the development of alternative vector systems suitable for transferring large [up to Mb size or larger] genes and gene complexes together with regulatory elements that will provide a safe, controlled, and persistent expression of the therapeutic genetic material.", "At the present time, none of the available vectors fulfill all these requirements.", "Most of these characteristics, however, are possessed by chromosomes.", "Thus, an artificial chromosome would be an ideal vector for gene therapy, as well as for stable, high-level, controlled production of gene products that require coordination of expression of numerous genes or that are encoded by large genes, and other uses.", "Artificial chromosomes for expression of heterologous genes in yeast are available, but construction of defined mammalian artificial chromosomes has not been achieved.", "Such construction has been hindered by the lack of an isolated, functional, mammalian centromere and uncertainty regarding the requisites for its production and stable replication.", "Unlike in yeast, there are no selectable genes in close proximity to a mammalian centromere, and the presence of long runs of highly repetitive pericentric heterochromatic DNA makes the isolation of a mammalian centromere using presently available methods, such as chromosome walking, virtually impossible.", "Other strategies are required for production of mammalian artificial chromosomes, and some have been developed.", "For example, U.S. Pat.", "No.", "5,288,625 provides a cell line that contains an artificial chromosome, a minichromosome, that is about 20 to 30 megabases.", "Methods provided for isolation of these chromosomes, however, provide preparations of only about 10-20% purity.", "Thus, development of alternative artificial chromosomes and perfection of isolation and purification methods as well as development of more versatile chromosomes and further characterization of the minichromosomes is required to realize the potential of this technology.", "Therefore, it is an object herein to provide mammalian artificial chromosomes and methods for introduction of foreign DNA into such chromosomes.", "It is also an object herein to provide methods of isolation and purification of the chromosomes.", "It is also an object herein to provide methods for introduction of the mammalian artificial chromosome into selected cells, and to provide the resulting cells, as well as transgenic animals, birds, fish and plants that contain the artificial chromosomes.", "It is also an object herein to provide methods for gene therapy and expression of gene products using artificial chromosomes.", "It is a further object herein to provide methods for constructing species-specific artificial chromosomes de novo.", "Another object herein is to provide methods to generate de novo mammalian artificial chromosomes.", "3.6.3Methods for Preparing Artificial Chromosomes Mammalian artificial chromosomes [MACs] are provided.", "Also provided are artificial chromosomes for other higher eukaryotic species, such as insects, birds, fowl and fish, produced using the MACS and methods provided herein.", "Methods for generating and isolating such chromosomes are provided.", "Methods using the MACs to construct artificial chromosomes from other species, such as insect, bird, fowl and fish species are also provided.", "The artificial chromosomes are fully functional stable chromosomes.", "Two types of artificial chromosomes are provided.", "One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the other type are minichromosomes based on-amplification of euchromatin.", "Artificial chromosomes provide an extra-genomic locus for targeted integration of megabase pair size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters.", "Thus, methods using the MACs to introduce the genes into cells, tissues, and animals, as well as species such as birds, fowl, fish and plants, are also provided.", "The artificial chromosomes with integrated heterologous DNA may be used in methods of gene therapy, in methods of production of gene products, particularly products that require expression of multigenic biosynthetic pathways, and also are intended for delivery into the nuclei of germline cells, such as embryo-derived stem cells [ES cells], for production of transgenic animals, birds, fowl and fish.", "Transgenic plants, including monocots and dicots, are also contemplated herein.", "Mammalian artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway or a very large gene, such as the cystic fibrosis [CF; about 250 kb] genomic DNA gene [cystic fibrosis [CF; about 600 kb] gene], several genes, such as multiple genes encoding a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell.", "Vectors for targeted introduction of such genes, including the tumor suppressor genes, such as p53, the cystic fibrosis transmembrane regulator cDNA [CFTR], and the genes for anti-HIV ribozymes, such as an anti-HIV gag ribozyme gene, into the artificial chromosomes are also provided.", "The chromosomes provided herein are generated by introducing heterologous DNA that includes DNA encoding one or multiple selectable marker(s) into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting clones those that include chromosomes with more than one centromere and/or fragments thereof.", "The amplification that produces the additional centromere occurs in cells that contain chromosomes in which the heterologous DNA has integrated near the centromere in the pericentric region of the chromosome.", "The selected clonal cells are then used to generate artificial chromosomes.", "In preferred embodiments, the DNA with the selectable marker that is introduced into cells to generate artificial chromosomes includes sequences that target it to the pericentric region of the chromosome.", "For example, vectors, such as pTEMPUD, which includes such DNA specific for mouse satellite DNA, are provided.", "Also provided are derivatives of pTEMPUD containing human satellite DNA sequences that specifically target human chromosomes or human satellite sequences.", "Upon integration, these vectors can induce the amplification that results in generation of additional centromeres.", "Artificial chromosomes are generated by culturing the cells with the multi-centric, typically dicentric, chromosomes under conditions whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome.", "Among the MACs provided herein are the SATACs, which are primarily made up of repeating units of short satellite DNA and are fully heterochromatic, so that without insertion of heterologous or foreign DNA, the chromosomes preferably contain no genetic information.", "They can thus be used as “safe” vectors for delivery of DNA to mammalian hosts because they do not contain any potentially harmful genes.", "The SATACs are generated, not from the minichromosome fragment as, for example, in U.S. Pat.", "No.", "5,288,625, but from the fragment of the formerly dicentric chromosome.", "In addition, methods for generating euchromatic minichromosomes and the use thereof are also provided herein.", "Methods for generating one type of MAC, the minichromosome, previously described in U.S. Pat.", "No.", "5,288,625, and the use thereof for expression of heterologous DNA are provided.", "Cell lines containing the minichrombsome and the use thereof for cell fusion are also provided.", "In one embodiment, a cell line containing the mammalian minichromosome is used as recipient cells for donor DNA encoding a selected gene or multiple genes.", "To facilitate integration of the donor DNA into the minichromosome, the recipient cell line preferably contains the minichromosome but does not also contain the formerly dicentric chromosome.", "This may be accomplished by methods disclosed herein such as cell fusion and selection of cells that contain a minichromosome and no formerly dicentric chromosome.", "The donor DNA is linked to a second selectable marker and is targeted to and integrated into the minichromosome.", "The resulting chromosome is transferred by cell fusion into an appropriate recipient cell line, such as a Chinese hamster cell line [CHO].", "After large-scale production of the cells carrying the engineered chromosome, the chromosome is isolated.", "In particular, metaphase chromosomes are obtained, such as by addition of colchicine, and they are purified from the cell lysate.", "These chromosomes are used for cloning, sequencing and for delivery of heterologous DNA into cells.", "Also provided are SATACs of various sizes that are formed by repeated culturing under selective conditions and subcloning of cells that contain chromosomes produced from the formerly dicentric chromosomes.", "The exemplified SATACs are based on repeating NA units that are about 15 Mb [two about 7.5 Mb blocks].", "The repeating DNA unit of SATACs formed from other species and other chromosomes may vary, but typically would be on the order of about 7 to about 20 Mb.", "The repeating DNA units are referred to herein as megareplicons, which in the exemplified SATACs contain tandem blocks of satellite DNA flanked by non-satellite DNA, including heterologous DNA and non-satellite DNA.", "Amplification produces an array of chromosome segments [each called an amplicon] that contain two inverted megareplicons bordered by heterologous [“foreign”] DNA.", "Repeated cell fusion, growth on selective medium and/or BrdU [5-bromodeoxyuridine] treatment or other treatment with other genome destabilizing reagent or agent, such as ionizing radiation, including X-rays, and subcloning results in cell lines that carry stable heterochromatic or partially heterochromatic chromosomes, including a 150-200 Mb “sausage” chromosome, a 500-1000 Mb gigachromosome, a stable 250400 Mb megachromosome and various smaller stable chromosomes derived therefrom.", "These chromosomes are based on these repeating units and can include heterologous DNA that is expressed.", "Thus, methods for producing MACs of both types (i.e., SATACS and minichromosomes) are provided.", "These methods are applicable to the production of artificial chromosomes containing centromeres derived from any higher eukaryotic cell, including mammals, irds, fowl, fish, insects and plants.", "The resulting chromosomes can be purified by methods provided herein to provide vectors for introduction of heterologous DNA into selected cells for production of the gene product(s) encoded by the heterologous DNA, for production of transgenic animals, birds, fowl, fish and plants or for gene therapy.", "In addition, methods and vectors for fragmenting the minichromosomes and SATACs are provided.", "Such methods and vectors can be used for in vivo generation of smaller stable artificial chromosomes.", "Vectors for chromosome fragmentation are used to produce an artificial chromosome that contains a megareplicon, a centromere and two telomeres and will be between about 7.5 Mb and about 60 Mb, preferably between about 10 Mb-15 Mb and 30-50 Mb.", "As exemplified herein, the preferred range is between about 7.5 Mb and 50 Mb.", "Such artificial chromosomes may also be produced by other methods.", "Isolation of the 15 Mb [or 30 Mb amplicon containing two 15 Mb inverted repeats] or a 30 Mb or higher multimer, such as 60 Mb, thereof should provide a stable chromosomal vector that can be manipulated in vitro.", "Methods for reducing the size of the MACs to generate smaller stable self-replicating artificial chromosomes are also provided.", "Methods and vectors for targeting heterologous DNA into the artificial chromosomes are also provided as are methods and vectors for fragmenting the chromosomes to produce smaller but stable and self-replicating artificial chromosomes.", "The chromosomes are introduced into cells to produce stable transformed cell lines or cells, depending upon the source of the cells.", "Introduction is effected by any suitable method including, but not limited to electroporation, direct uptake, such as by calcium phosphate precipitation, uptake of isolated chromosomes by lipofection, by microcell fusion, by lipid-mediated carrier systems or other suitable method.", "The resulting cells can be used for production of proteins in the cells.", "The chromosomes can be isolated and used for gene delivery.", "Methods for isolation of the chromosomes based on the DNA content of the chromosomes, which differs in MACs versus the authentic chromosomes, are provided.", "These artificial chromosomes can be used in gene therapy, gene product production systems, production of humanized genetically transformed animal organs, production of transgenic plants and animals, including mammals, birds, fowl, fish, invertebrates, vertebrate, reptiles and insects, any organism or device that would employ chromosomal elements as information storage vehicles, and also for analysis and study of centromere function, for the production of artificial chromosome vectors that can be constructed in vitro, and for the preparation of species-specific artificial chromosomes.", "The artificial chromosomes can be introduced into cells using microinjection, cell fusion, microcell fusion, electroporation, electrofusion, projectile bombardment, calcium phosphate precipitation, site-specific targeting, lipid-mediated transfer systems and other such methods.", "Cells particularly suited for use with the artificial chromosomes include, but are not limited to plant cells, particularly tomato, arabidopsis, and others, insect cells, including silk worm cells, insect larvae, fish, reptiles, amphibians, arachnids, mammalian cells, avian cells, embryonic stem cells, haematopoietic stem cells, embryos and cells for use in methods of genetic therapy, such as lymphocytes that are used in methods of adoptive immunotherapy and nerve or neural cells.", "Thus methods of producing gene products and transgenic animals and plants are provided.", "Also provided are the resulting transgenic animals and plants.", "Exemplary cell lines that contain these chromosomes are also provided.", "Methods for preparing artificial chromosomes for particular species and for cloning centromeres are also provided.", "For example, two methods for generating artificial chromosomes for use in different species are provided.", "First, the methods herein may applied to different species.", "Second, means for generating species-specific artificial chromosomes and for cloning centromeres are provided.", "In particular, a method for cloning a centromere from an animal or plant by preparing a library of DNA fragments that contain the genome of the plant or animal, introducing each of the fragments into a mammalian satellite artificial chromosome [SATAC] that contains a centromere from a different species, generally a mammal, from the selected plant or animal, generally a non-mammal, and a selectable marker.", "The selected plant or animal is one in which the mammalian species centromere does not function.", "Each of the SATACs is introduced into the cells, which are grown under selective conditions, and cells with SATACs are identified.", "Such SATACS should contain a centromere encoded by the DNA from the library or should contain the necessary elements for stable replication in the selected species.", "Also provided are libraries in which the relatively large fragments of DNA are contained on artificial chromosomes.", "Transgenic animals, invertebrates and vertebrates, plants and insects, fish, reptiles, amphibians, arachnids, birds, fowl, and mammals are also provided.", "Of particular interest are transgenic animals that express genes that confer resistance or reduce susceptibility to disease.", "Since multiple genes can be introduced on a MAC, a series of genes encoding an antigen can be introduced, which upon expression will serve to immunize [in a manner similar to a multivalent vaccine) the host animal against the diseases for which exposure to the antigens provide immunity or some protection.", "Also of interest are transgenic animals that serve as models of certain diseases and disorders for use in studying the disease and developing therapeutic treatments and cures thereof.", "Such animal models of disease express genes [typically carrying a disease-associated mutation], which are introduced into the animal on a MAC and which induce the disease or disorder in the animal.", "Similarly, MACs carrying genes encoding antisense RNA may be introduced into animal cells to generate conditional “knock-out” transgenic animals.", "In such animals, expression of the antisense RNA results in decreased or complete elimination of the products of genes corresponding to the antisense RNA.", "Of further interest are transgenic mammals that harbor MAC-carried genes encoding therapeutic proteins that are expressed in the animal's milk.", "Transgenic animals for use in xenotransplantation, which express MAC— carried genes that serve to humanize the animal's organs, are also of interest.", "Genes that might be used in humanizing animal organs include those encoding human surface antigens.", "Methods for cloning centromeres, such as mammalian centromeres, are also provided.", "In particular, in one embodiment, a library composed of fragments of SATACs are cloned into YACs [yeast artificial chromosomes] that include a detectable marker, such as DNA encoding tyrosinase, and then introduced into mammalian cells, such as albino mouse embryos.", "Mice produced from embryos containing such YACs that include a centromere that functions in mammals will express the detectable marker.", "Thus, if mice are produced from albino mouse embryos into which a functional mammalian centromere was introduced, the mice will be pigmented or have regions of pigmentation.", "3.6.4 Particularly Relevant Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.", "All patents and publications referred to herein are incorporated by reference.", "As used herein, a mammalian artificial chromosome [MAC] is a piece of DNA that can stably replicate and segregate alongside endogenous chromosomes.", "It has the capacity to accommodate and express heterologous genes inserted therein.", "It is referred to as a mammalian artificial chromosome because it includes an active mammalian centromere(s).", "Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include plant and insect centromeres, respectively.", "A human artificial chromosome [HAC] refers to chromosomes that include human centromeres, BUGACs refer to insect artificial chromosomes, and AVACs refer to avian artificial chromosomes.", "As used herein, stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95%, of the cells retain the chromosome.", "Stability is measured in the presence of a selective agent.", "Preferably these chromosomes are also maintained in the absence of a selective agent.", "Stable chromosomes also retain their structure during cell culturing, suffering neither intrachromosomal nor interchromosomal rearrangements.", "As used herein, growth under selective conditions means growth of a cell under conditions that require expression of a selectable marker for survival.", "As used herein, euchromatin and heterochromatin have their recognized meanings, euchromatin refers to DNA that contains genes, and heterochromatin refers to chromatin that has been thought to be inactive.", "Highly repetitive DNA sequentes [satellite DNA], at least with respect to mammalian cells, are usually located in regions of centromeric heterochromatin [pericentric heterochromatin].", "Constitutive heterochromatin refers to heterochromatin that contains the highly repetitive DNA which is constitutively condensed and genetically inactive.", "As used herein, BrdU refers to 5-bromodeoxyuridine, which during replication is inserted in place of thymidine.", "BrdU is used as a mutagen; it also inhibits condensation of metaphase chromosomes during cell division.", "As used herein, a dicentric chromosome is a chromosome that contains two centromeres.", "A multicentric chromosome contains more than two centromeres.", "As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced.", "Each of the fragments are replicable chromosomes.", "If one of the chromosomes undergoes amplification of euchromatic DNA to produce a full functionally chromosome that contains the newly introduced heterologous DNA and primarily [at least more than 50%] euchromatin, it is a minichromosome.", "The remaining chromosome is a formerly dicentric chromosome.", "If one of the chromosomes undergoes amplification, whereby heterochromatin (satellite DNA] is amplified and a euchromatic portion [or arm] remains, it is referred to as a sausage hromosome.", "A chromosome that is substantially all heterochroinatin, except for portions of heterologous DNA, is called a SATAC.", "Such chromosomes [SATACs] can be produced from sausage chromosomes by culturing the cell containing the sausage chromosome under conditions, such as BrdU treatment and/or growth under selective conditions, that destabilize the chromosome so that a satellite artificial chromosomes [SATAC] is produced.", "For purposes herein, it is understood that SATACs may not necessarily be produced in multiple steps, but may appear after the initial introduction of the heterologous DNA and growth under selective conditions, or they may appear after several cycles of growth under selective conditions and BrdU treatment.", "As used herein an amplicon is a repeated DNA amplification unit that contains a set of inverted repeats of the megareplicon.", "A megareplicon represents a higher order replication unit.", "For example, with reference to the SATACs, the megareplicon contains a set of tandem DNA blocks each containing satellite DNA flanked by non-satellite DNA.", "Contained within the megareplicon is a primary replication site, referred to as the megareplicator, which may be involved in organizing and facilitating replication of the pericentric heterochromatin and possibly the centromeres.", "Within the megareplicon there may be smaller [e.g., 50-300 kb in some mammalian cells] secondary replicons.", "In the exemplified SATACS, the megareplicon is defined by two tandem about 7.5 Mb DNA blocks [see, e.g., FIG.", "3].", "Within each artificial chromosome [AC] or among a population thereof, each amplicon has the same gross structure but may contain sequence variations.", "Such variations will arise as a result of movement of mobile genetic elements, deletions or insertions or mutations that arise, particularly in culture.", "Such variation does not affect the use of the ACs or their overall structure as described herein.", "As used herein, the minichromosome refers to a chromosome derived from a multicentric, typically dicentric, chromosome [see, e.g., FIG.", "1] that contains more euchromatic than heterochromatic DNA.", "As used herein, a megachromosome refers to a chromosome that, except for introduced heterologous DNA, is substantially composed of heterochromatin.", "Megachromosomes are made of an array of repeated amplicons that contain two inverted megareplicons bordered by introduced heterologous DNA [see, e.g., FIG.", "3 for a schematic drawing of a megachromosome].", "For purposes herein, a megachromosome is about 50 to 400 Mb, generally about 250400 Mb.", "Shorter variants are also referred to as truncated megachromosomes [about 90 to 120 or 150 Mb], dwarf megachromosomes [about 150-200 Mb] and cell lines, and a micro-megachromosome [about 60-90 Mb].", "For purposes herein, the term megachromosome refers to the overall repeated structure based on an array of repeated chromosomal segments [amplicons] that contain two inverted megareplicons bordered by any inserted heterologous DNA.", "The size will be specified.", "As used herein, genetic therapy involves the transfer or insertion of heterologous DNA into certain cells, target cells, to produce specific gene products that are involved in correcting or modulating disease.", "The DNA is introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a product encoded thereby is produced.", "Alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product.", "It may encode a product, uch as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product.", "Genetic therapy may also be used to introduce therapeutic compounds, such as TNF, that are not normally produced in the host or that are not produced in therapeutically effective amounts or at a therapeutically useful time.", "Expression of the heterologous DNA by the target cells within an organism afflicted with the disease thereby enables modulation of the disease.", "The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.", "As used herein, heterologous or foreign DNA and RNA are used interchangeably and refer to DNA or RNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differ from hat in which it occurs in nature.", "It is DNA or RNA that is not endogenous to the cell and has been exogenously introduced into the cell.", "Examples of heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest, introduced for purposes of gene therapy or for production of an encoded protein.", "Other examples of heterologous DNA include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.", "Antibodies that are encoded by heterologous DNA may be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.", "As used herein, a therapeutically effective product is a product that is encoded by heterologous DNA that, upon introduction of the DNA into a host, a product is expressed that effectively ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease or that cures said disease.", "As used herein, transgenic plants refer to plants in which heterologous or foreign DNA is expressed or in which the expression of a gene naturally present in the plant has been altered.", "As used herein, operative linkage of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences refers to the relationship between such DNA and such sequences of nucleotides.", "For example, operative linkage of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame.", "As used herein, isolated, substantially pure DNA refers to DNA fragments purified according to standard techniques employed by those skilled in the art, such as that found in Maniatis et al.", "[(1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.].", "As used herein, expression refers to the process by which nucleic acid is transcribed into mRNA and translated into peptides, polypeptides, or proteins.", "If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host ell or organism is selected, include splicing of the mRNA.", "As used herein, vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression of the heterologous DNA or for replication of the cloned heterologous DNA.", "Selection and use of such vectors and lasmids are well within the level of skill of the art.", "As used herein, transformation/transfection refers to the process by which DNA or RNA is introduced into cells.", "Transfection refers to the taking up of exogenous nucleic acid, e.g., an expression vector, by a host cell whether or not any coding sequences are in fact expressed.", "Numerous methods of transfection are known to the ordinarily skilled artisan, for example, by direct uptake using calcium phosphate [CaPO4; see, e.g., Wigler et al.", "(1979) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 76: 1373-1376], polyethylene glycol [PEG]-mediated DNA uptake, electroporation, lipofection [see, e.g., Strauss (1996) Meth.", "Mol.", "Biol.", "54: 307-327], microcell fusion [see, EXAMPLES, see, also Lambert (1991) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 88: 5907-5911; U.S. Pat.", "No.", "5,396,767, Sawford et al.", "(1987) Somatic Cell Mol.", "Genet.", "13: 279-284; Dhar et al.", "(1984) Somatic Cell Mol.", "Genet.", "10: 547-559; and McNeill-Killary et al.", "(1995) Meth.", "Enzymol.", "254: 133-152], lipid-mediated carrier systems [see, e.g., Teifel et al.", "(1995) Biotechnigues 19: 79-80; Albrecht et al.", "(1996) Ann.", "Hematol.", "72: 73-79; Holmen et al.", "(1995) In Vitro Cell Dev.", "Biol.", "Anim.", "31: 347-351; REmy et al.", "(1994) Bioconjug.", "Chem.", "5: 647-654; Le Bolch et al.", "(1995) Tetrahedron Lett.", "36: 6681-6684; Loeffler et al.", "(1993) Meth.", "Enzymol.", "217: 599-618] or other suitable method.", "Successful transfection is generally recognized by detection of the presence of the heterologous nucleic acid within the transfected cell, such as any indication of the operation of a vector within the host cell.", "Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.", "As used herein, injected refers to the microinjection [use of a small syringe] of DNA into a cell.", "As used herein, substantially homologous DNA refers to DNA that includes a sequence of nucleotides that is sufficiently similar to another such sequence to form stable hybrids under specified conditions.", "It is well known to those of skill in this art that nucleic acid fragments with different sequences may, under the same conditions, hybridize detectably to the same “target” nucleic acid.", "Two nucleic acid fragments hybridize detectably, under stringent conditions over a sufficiently long hybridization period, because one fragment contains a segment of at least about 14 nucleotides in a sequence which is complementary [or nearly complementary] to the sequence of at least one segment in the other nucleic acid fragment.", "If the time during which hybridization is allowed to occur is held constant, at a value during which, under preselected stringency conditions, two nucleic acid fragments with exactly complementary base-pairing segments hybridize detectably to each other, departures from exact complementarity can be introduced into the base-pairing segments, and base-pairing will nonetheless occur to an extent sufficient to make hybridization detectable.", "As the departure from complementarity between the base-pairing segments of two nucleic acids becomes larger, and as conditions of the hybridization become more stringent, the probability decreases that the two segments will hybridize detectably to each other.", "Two single-stranded nucleic acid segments have “substantially the same sequence,” within the meaning of the present specification, if (a) both form a base-paired duplex with the same segment, and (b) the melting temperatures of said two duplexes in a solution of 0.5.times.", "SSPE differ by less than 10.degree.", "C. If the segments being compared have the same number of bases, then to have “substantially the same sequence”, they will typically differ in their sequences at fewer than 1 base in 10.Methods for determining melting temperatures of nucleic acid duplexes are well known [see, e.g., Meinkoth and Wahl (1984) Anal.", "Biochem.", "138: 267-284 and references cited therein].", "As used herein, a nucleic acid probe is a DNA or RNA fragment that includes a sufficient number of nucleotides to specifically hybridize to DNA or RNA that includes identical or closely related sequences of nucleotides.", "A probe may contain any number of nucleotides, from as few as about 10 and as many as hundreds of thousands of nucleotides.", "The conditions and protocols for such hybridization reactions are well known to those of skill in the art as are the effects of probe size, temperature, degree of ismatch, salt concentration and other parameters on the hybridization reaction.", "For example, the lower the temperature and higher the salt concentration at which the hybridization reaction is carried out, the greater the degree of mismatch that may be present in the hybrid molecules.", "To be used as a hybridization probe, the nucleic acid is generally rendered detectable by labelling it with a detectable moiety or label, such as .sup.32 p, .sup.3H and .sup.", "14 C, or by other means, including chemical labelling, such as by nick-translation in the presence of deoxyuridylate biotinylated at the 5′-position of the uracil moiety.", "The resulting probe includes the biotinylated uridylate in place of thymidylate residues and can be detected [via the biotin moieties] by any of a number of commercially available detection systems based on binding of streptavidin to the biotin.", "Such commercially available detection systems can be obtained, for example, from Enzo Biochemicals, Inc. [New York, N.Y.].", "Any other label known to those of kill in the art, including non-radioactive labels, may be used as long as it renders the probes sufficiently detectable, which is a function of the sensitivity of the assay, the time available [for culturing cells, extracting DNA, and hybridization assays], the quantity of DNA or RNA available as a source of the probe, the particular label and the means used to detect the label.", "Once sequences with a sufficiently high degree of homology to the probe are identified, they can readily be isolated by standard techniques, which are described, for example, by Maniatis et al.", "((1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).", "As used herein, conditions under which DNA molecules form stable hybrids and are considered substantially homologous are such that DNA molecules with at least about 60% complementarity form stable hybrids.", "Such DNA fragments are herein considered to be “substantially homologous”.", "For example, DNA that encodes a particular protein is substantially homologous to another DNA fragment if the DNA forms stable hybrids such that the sequences of the fragments are at least about 60% complementary and if a protein encoded by the DNA retains its activity.", "For purposes herein, the following stringency conditions are defined: 1) high stringency: 0.1.times.SSPE, 0.1% SDS, 65.degree.", "C. 2) medium stringency: 0.2.times.SSPE, 0.1% SDS, 50.degree.", "C. 3) low stringency: 1.0×SSPE, 0.1% SDS, 50.degree.", "C. or any combination of salt and temperature and other reagents that result in selection of the same degree of mismatch or matching.", "As used herein, immunoprotective refers to the ability of a vaccine or exposure to an antigen or immunity-inducing agent, to confer upon a host to whom the vaccine or antigen is administered or introduced, the ability to resist infection by a disease-causing pathogen or to have reduced symptoms.", "The selected antigen is typically an antigen that is presented by the pathogen.", "As used herein, all assays and procedures, such as hybridization reactions and antibody-antigen reactions, unless otherwise specified, are conducted under conditions recognized by those of skill in the art as standard conditions.", "3.6.5 Preparation of Cell Lines Containing MACs The methods, cells and MACs provided herein are produced by virtue of the discovery of the existence of a higher-order replication unit [megareplicon] of the centromeric region.", "This megareplicon is delimited by a primary replication initiation site [megareplicator], and appears to facilitate replication of the centromeric heterochromatin, and most likely, centromeres.", "Integration of heterologous DNA into the megareplicator region or in close proximity thereto, initiates a large-scale amplification of megabase-size chromosomal segments, which leads to de novo chromosome formation in living cells.", "Cell lines containing MACs can be prepared by transforming cells, preferably a stable cell line, with a heterologous DNA fragment that encodes a selectable marker, culturing under selective conditions, and identifying cells that have a multicentric, typically dicentric, chromosome.", "These cells can then be manipulated as described herein to produce the minichromosomes and other MACs, particularly the heterochromatic SATACs as described herein.", "Development of a multicentric, particularly dicentric, chromosome typically is effected through integration of the heterologous DNA in the pericentric heterochromatin.", "Thus, the probability of incorporation can be increased by including DNA, such as satellite DNA, in the heterologous fragment that encodes the selectable marker.", "The resulting cell lines can then be treated as the exemplified cells herein to produce cells in which the dicentric chromosome has fragmented and to introduce additional selective markers into the dicentric chromosome, whereby amplification of the pericentric heterochromatin will produce the heterochromatic chromosomes.", "The following discussion is with reference to the EC3/7 line and use of resulting cells.", "The same procedures can be applied to any other cells, particularly cell lines to create SATACs and euchromatic minichromosomes.", "3.6.5.1 Formation of De Novo Chromosomes De novo centromere formation in a transformed mouse LMTK-fibro-blast cell line [EC3/7] after cointegration of lambda constructs [lambda CM8 and lambda gtWESneo] carrying human and bacterial DNA [Hadlaczky et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 88: 8106-8110 and U.S. application Ser.", "No.", "08/375,271] has been shown.", "The integration of the “heterologous” engineered human, bacterial and phage DNA, and the subsequent amplification of mouse and heterologous DNA that led to the formation of a dicentric chromosome, occurred at the centromeric region of the short arm of a mouse chromosome.", "By G-banding, this chromosome was identified as mouse chromosome 7.Because of the presence of two functionally active centromeres on the same chromosome, regular breakages occur between the centromeres.", "Such specific chromosome breakages gave rise to the appearance [in approximately 10% of the cells] of a chromosome fragment carrying the neo-centromere.", "From the EC3/7 cell line [see, U.S. Pat.", "No.", "5,288,625, deposited at the European Collection of Animal Cell Culture (hereinafter ECACC) under accession no.", "90051001; see, also Hadlaczky et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 88: 8106-8110, and U.S. application Ser.", "No.", "08/375,271 and the corresponding published European application EP 0 473 253, two sublines [EC3/7C5 and EC3/7C6] were selected by repeated single-cell cloning.", "In these cell lines, the neo-centromere was found exclusively on a minichromosome [neo-minichromosome], while the formerly dicentric chromosome carried traces of “heterologous” DNA.", "It has now been discovered that integration of DNA encoding a selectable marker in the heterochromatic region of the centromere led to formation of the dicentric chromosome.", "3.6.5.2 The Neo-Minichromosome The chromosome breakage in the EC3/7 cells, which separates the neo-centromere from the mouse chromosome, occurred in the G-band positive “heterologous” DNA region.", "This is supported by the observation of traces of lambda and human DNA sequences at the broken end of the formerly dicentric chromosome.", "Comparing the G-band pattern of the chromosome fragment carrying the neo-centromere with that of the stable neo-minichromosome, it is apparent that the neo-minichromosome is an inverted duplicate of the chromosome fragment that bears the neo-centromere.", "This is supported by the observation that although the neo-minichromosome carries only one functional centromere, both ends of the minichromosome are heterochromatic, and mouse satellite DNA sequences were found in these heterochromatic regions by in situ hybridization.", "Mouse cells containing the minichromosome, which contains multiple repeats of the heterologous DNA, which in the exemplified embodiment is lambda DNA and the neomycin-resistance gene, can be used as recipient cells in cell transformation.", "Donor DNA, such as selected heterologous DNA containing lambda DNA linked to a second selectable marker, such as the gene encoding hygromycin phosphotransferase which confers hygromycin resistance [hyg], can be introduced into the mouse cells and integrated into the minichromosomes by homologous recombination of lambda DNA in the donor DNA with that in the minichromosomes.", "Integration is verified by in situ hybridization and Southern blot analyses.", "Transcription and translation of the heterologous DNA is confirmed by primer extension and immunoblot analyses.", "For example, DNA has been targeted into the lambda neo-minichromosome in EC3/7C5 cells using a lambda DNA-containing construct [pNem1ruc] that also contains DNA encoding hygromycin resistance and the Renilla luciferase gene linked to a promoter, such as the cytomegalovirus [CMV] early promoter, and the bacterial neomycin resistance-encoding DNA.", "Integration of the donor DNA into the chromosome in selected cells [designated PHN4] was confirmed by nucleic acid amplification [PCR] and in situ hybridization.", "The resulting engineered minichromosome that contains the heterologous DNA can then be transferred by cell fusion into a recipient cell line, such as Chinese hamster ovary cells [CHO] and correct expression of the heterologous DNA can be verified.", "Following production of the cells, metaphase chromosomes are obtained, such as by addition of colchicine, and the chromosomes purified by addition of AT and GC specific dyes on a dual laser beam based cell sorter.", "Preparative amounts of chromosomes [5×104-5×107 chromosomes/ml] at a purity of 95% or higher can be obtained.", "The resulting chromosomes are used for delivery to cells by methods such as microinjection and liposome-mediated transfer.", "Thus, the neo-minichromosome is stably maintained in cells, replicates autonomously, and permits the persistent long-term expression of the neo gene unde; non-selective culture conditions.", "It also contains megabases of heterologous known DNA [lambda NA in the exemplified embodiments] that serves as target sites for homologous recombination and integration of DNA of interest.", "The neo-minichromosome is, thus, a vector for genetic engineering of cells.", "The methods herein provide means to induce the events that lead to formation of the neo-minichromosome by introducing heterologous DNA with a selective marker (preferably a dominant selectable marker] into cells and culturing the cells under selective conditions.", "As a result, cells that contain a multicentric, e.g., dicentric chromosome, or fragments thereof, generated by amplification are produced.", "Cells with the dicentric chromosome can then be treated to destabilize the chromosomes with agents, such as BrdU and/or culturing under selective conditions, resulting in cells in which the dicentric chromosome has formed two chromosomes, a so-called minichromosome, and a formerly dicentric chromosome that has typically undergone amplification in the heterochromatin where the heterologous DNA has integrated to produce a SATAC or a sausage chromosome [discussed below].", "These cells can be fused with other cells to separate the minichromosome from the formerly dicentric chromosome into different cells so hat each type of MAC can be manipulated separately.", "3.6.5.3 Preparation of SATACs To prepare a SATAC, the starting materials are cells, preferably a stable cell line, such as a fibroblast cell line, and a DNA fragment that includes DNA that encodes a selective marker.", "The DNA fragment is introduced into the cell by methods of DNA transfer, including but not limited to direct uptake using calcium phosphate, electroporation, and lipid-mediated transfer.", "To insure integration of the DNA fragment in the heterochromatin, it is preferable to start with DNA that will be targeted to the pericentric heterochromatic region of the chromosome, such as lambda CM8 and vectors provided herein, such as pTEMPUD that include satellite DNA.", "After introduction of the DNA, the cells are grown under selective conditions.", "The resulting cells are examined and any that have multicentric, particularly dicentric, chromosomes, or heterochromatic chromosomes or sausage chromosomes or other such structure are selected.", "In particular, if a cell with a dicentric chromosome is selected, it can be grown under selective conditions, or, preferably, additional DNA encoding a second selectable marker is introduced, and the cells grown under conditions selective for the second marker.", "Cells with a structure, such as the sausage chromosome, can be selected and fused with a second cell line to eliminate other chromosomes that are not of interest.", "If desired, cells with other chromosomes can be selected and treated as described herein.", "If a cell with a sausage chromosome is selected, it can be treated with an agent, such as BrdU, that destabilizes the chromosome so that the heterochromatic arm forms a chromosome that is substantially heterochromatic [i.e., a megachromosome].", "Structures such as the gigachromsome in which the heterochromatic arm has amplified but not broken off from the euchromatic arm, will also be observed.", "The megachromosome is a stable chromosome.", "Further manipulation, such as fusions and growth in selective conditions and/or BrdU treatment or other such treatment, can lead to fragmentation of the egachromosome to form smaller chromosomes that have the amplicon as the basic repeating unit.", "The megachromosome can be further fragmented in vivo using a chromosome fragmentation vector, such as pTEMPUD to ultimately produce a chromosome that comprises a smaller stable replicable unit, about 15 Mb-60 Mb, containing one to four megareplicons.", "Thus, the stable chromosomes formed de novo that originate from the short arm of mouse chromosome 7 have been analyzed.", "This chromosome region shows a capacity for amplification of large chromosome segments, and promotes de novo chromosome formation.", "Large-scale amplification at the same chromosome region leads to the formation of dicentric and multicentric chromosomes, a minichromosome, the 150-200 Mb size lambda neo-chromosome, the “sausage” chromosome, the 500-1000 Mb gigachromosome, and the stable 250400 Mb megachromosome.", "A clear segmentation is observed along the arms of the megachromosome, and analyses show that the building units of this chromosome are amplicons of ˜30 Mb composed of mouse major satellite DNA with the integrated “foreign” DNA sequences at both ends.", "The ˜30 Mb amplicons are composed of two ˜15 Mb inverted doublets of ˜7.5 Mb mouse major satellite DNA blocks, which are separated from each other by a narrow band of non-satellite sequences.", "The wider non-satellite regions at the amplicon borders contain integrated, exogenous [heterologous] DNA, while the narrow bands of non-satellite DNA sequences within the amplicons are integral parts of the pericentric heterochromatin of mouse chromosomes.", "These results indicate that the ˜7.5 Mb blocks flanked by non-satellite DNA are the building units of the pericentric heterochromatin of mouse chromosomes, and the ˜15 Mb size pericentric regions of mouse chromosomes contain two ˜7.5 Mb units.", "Apart from the euchromatic terminal segments, the whole megachromosome is heterochromatic, and has structural homogeneity.", "Therefore, this large chromosome offers a unique possibility for obtaining information about the amplification process, and for analyzing some basic characteristics of the pericentric constitutive heterochromatin, as a vector for heterologous DNA, and as a target for further fragmentation.", "As shown herein, this phenomenon is generalizable and can be observed with other chromosomes.", "Also, although these de novo formed chromosome segments and chromosomes appear different, there are similarities that indicate that a similar amplification mechanism plays a role in their formation: (i) in each case, the amplification is initiated in the centromeric region of the mouse chromosomes and large (Mb size) amplicons are formed; (ii) mouse major satellite DNA sequences are constant constituents of the amplicons, either by providing the bulk of the heterochromatic amplicons [H-type amplification], or by bordering the euchromatic amplicons [E-type amplification]; (iii) formation of inverted segments can be demonstrated in the lambda neo-chromosome and megachromosome; (iv) chromosome arms and chromosomes formed by the amplification are stable and functional.", "The presence of inverted chromosome segments seems to be a common phenomenon in the chromosomes formed de novo at the centromeric region of mouse chromosome 7.During the formation of the neo-minichromosome, the event leading to the stabilization of the distal segment of mouse chromosome 7 that bears the neo-centromere may have been the formation of its inverted duplicate.", "Amplicons of the megachromosome are inverted doublets of-7.5 Mb mouse major satellite DNA blocks.", "3.6.5.4 Cell Lines Cell lines that contain MACs, such as the minichromosome, the .lambda.-neo chromosome, and the SATACs are provided herein or can be produced by the methods herein.", "Such cell lines provide a convenient source of these chromosomes and can be manipulated, such as by cell fusion or production of microcells for fusion with selected cell lines, to deliver the chromosome of interest into hybrid cell lines.", "Exemplary cell lines are described herein and some have been deposited with the ECACC.", "3.6.5.4.1 EC317C5 and EC37C6 Cell lines EC3/7C5 and EC3/7C6 were produced by single cell cloning of EC3/7.For exemplary purposes EC3/7C5 has been deposited with the ECACC.", "These cell lines contain a minichromosome and the formerly dicentric chromosome from EC3/7.The stable mini-chromosomes in cell lines EC31C5 and EC3/7C6 appear to be the same and they seem to be duplicated derivatives of the 10-15 Mb “broken-off” fragment of the dicentric chromosome.", "Their similar size in these independently generated cell lines might indicate that about 20-30 Mb is the minimal or close to the minimal physical size for a stable minichromosome.", "3.6.5.4.2 TF1004G19 Introduction of additional heterologous DNA, including DNA encoding a second selectable marker, hygromycin phosphotransferase, i.e., the hygromycin-resistance gene, and also a detectable marker, beta-galactosidase (i.e., encoded by the lacz gene), into the EC3/7C5 cell line and growth under selective conditions produced cells designated TF1004G19.In particular, this cell line was produced from the EC3/7C5 cell line by cotransfection with plasmids pH 132, which contains an anti-HIV ribozyme and hygromycin-resistance gene, pCH 110 [encodes, beta-galactosidase] and lambda phage [lambdac1875 Sam 7] DNA and selection with hygromycin B.", "Detailed analysis of the TF1004G 19 cell line by in situ hybridization with lambda phage and plasmid DNA sequences revealed the formation of the sausage chromosome.", "The formerly dicentric chromosome of the EC317C5 cell line translocated to the end of another acrocentric chromosome.", "The heterologous DNA integrated into the pericentric heterochromatin of the formerly dicentric chromosome and is amplified several times with megabases of mouse pericentric heterochromatic satellite DNA sequences forming the “sausage” chromosome.", "Subsequently the acrocentric mouse chromosome was substituted by a euchromatic telomere.", "In situ hybridization with biotin-labeled subfragments of the hygromycin-resistance and, beta-galactosidase genes resulted in a hybridization signal only in the heterochromatic arm of the sausage chromosome, indicating that in TF 1004G 19 transformant cells these genes are localized in the pericentric heterochromatin.", "A high level of gene expression, however, was detected.", "In general, heterochromatin has a silencing effect in Drosophila, yeast and on the HSV-tk gene introduced into satellite DNA at the mouse centromere.", "Thus, it was of interest to study the TF 1004G 19 transformed cell line to confirm that genes located in the heterochromatin were indeed expressed, contrary to recognized dogma.", "For this purpose, subclones of TF1004G19, containing a different sausage chromosome, were established by single cell cloning.", "Southern hybridization of DNA isolated from the subclones with subfragments of hygromycin phosphotransferase and lacZ genes showed a close correlation between the intensity of hybridization and the length of the sausage chromosome.", "This finding supports the conclusion that these genes are localized in the heterochromatic arm of the sausage chromosome.", "3.6.5.4.2.1 T1004G-19C5 TF1004G-19C5 is a mouse LMTK-fibroblast cell line containing neo-minichromosomes and stable “sausage” chromosomes.", "It is a subclone of TF1004G19 and was generated by single-cell cloning of the TF1004G19 cell line.", "It has been deposited with the ECACC as an exemplary cell line and exemplary source of a sausage chromosome.", "Subsequent fusion of this cell line with CHO K20 cells and selection with hygromycin and G418 and HAT (hypoxanthine, aminopteria, and thymidine medium, see Szybalski et al.", "(1962) Proc.", "Natl.", "Acad.", "Sci.", "48: 2026) resulted in hybrid cells (designated 19C5xHa4) that carry the sausage chromosome and/or the neo-minichromosome.", "BrdU treatment of the hybrid cells, followed by single cell cloning and selection with G418 and/or hygromycin produced various cells that carry chromosomes of interest, including G43 and G3D5.3.6.5.4.2.2 Other Subclones Cell lines GB43 and G3D5 were obtained by treating 19C5xHa4 cells with BrdU followed by growth in G418-containing selective medium and retreatment with BrdU.", "The two cell lines were isolated by single cell cloning of the selected cells.", "GB43 cells contain the neo-minichromosome only.", "G3D5, which has been deposited with the ECACC, carries the neo-minichromosome and the megachromosome.", "Single cell cloning of this cell line followed by growth of the subclones in G418- and hygromycin-containing medium yielded subclones such as the GHB42 cell line carrying the neo-minichromosome and the megachromosome.", "H1D3 is a mouse-hamster hybrid cell line carrying the megachromosome, but no neo-minichromosome, and was generated by treating 19C5xHa4 cells with rdU followed by growth in hygromycin-containing selective medium and single cell subcloning of selected cells.", "Fusion of this cell line with the CD4+ HeLa cell line that also carries DNA encoding an additional selection gene, the neomycin-resistance gene, produced cells [designated H1xHE41 cells) that carry the megachromosome as well as a human chromosome that carries CD4neo [H1D3 cells].", "Further BrdU treatment and single cell cloning produced cell lines, such as I B3, that include cells with a truncated megachromosome.", "3.6.5.5 DNA Constructs Used to Transform the Cells Heterologous DNA can be introduced into the cells by transfection or other suitable method at any stage during preparation of the chromosomes.", "In general, incorporation of such DNA into the MACs is assured through site-directed integration, such as may be accomplished by inclusion of lambda-DNA in the heterologous DNA (for the exemplified chromosomes), and also an additional selective marker gene.", "For example, cells containing a MAC, such as the minichromosome or a SATAC, can be cotransfected with a plasmid carrying the desired heterologous DNA, such as DNA encoding an HIV ribozyme, the cystic fibrosis gene, and DNA encoding a second selectable marker, such as hygromycin resistance.", "Selective pressure is then applied to the cells by exposing them to an agent that is harmful to cells that do not express the new selectable marker.", "In this manner, cells that include the heterologous DNA in the MAC are identified.", "Fusion with a second cell line can provide a means to produce cell lines that contain one particular type of chromosomal structure or MAC.", "Various vectors for this purpose can be readily constructed.", "The vectors preferably include DNA that is homologous to DNA contained within a MAC in order to target the DNA to the MAC for integration therein.", "The vectors also include a selectable marker gene and the selected heterologous gene(s) of interest.", "Based on the disclosure herein and the knowledge of the skilled artisan, one of skill can construct such vectors.", "Of particular interest herein is the vector pTEMPUD and derivatives thereof that can target DNA into the heterochromatic region of selected chromosomes.", "These vectors can also serve as fragmentation vectors.", "Heterologous genes of interest include any gene that encodes a therapeutic product and DNA encoding gene products of interest.", "These genes and DNA include, but are not limited to: the cystic fibrosis gene [CF], the cystic fibrosis transmembrane regulator (CFTR) gene [see, e.g., U.S. Pat.", "No.", "5,240,846; Rosenfeld et al.", "(1992) Cell 68: 143-155; Hyde et al.", "(1993) Nature 362: 250-255; Kerem et al.", "(1989) Science 245: 1073-1080; Riordan et al.", "(1989) Science 245: 1066-1072; Rommens et al.", "(1989) Science 245: 1059-1065; Osborne et al.", "(1991) Am.", "J. Hum.", "Genetics 48: 6089-6122; White et al.", "(1990) Nature 344: 665-667; Dean et al.", "(1990) Cell 61: 863-870; Erlich et al.", "(1991) Science 252: 1643; and U.S. Pat.", "Nos.", "5,453,357, 5,449,604, 5,434,086, and 5,240,846, which provides a retroviral vector encoding the normal CFTR gene].", "3.6.6 Isolation of Artificial Chromosomes The MACs provided herein can be isolated by any suitable method known to those of skill in the art.", "Also, a method is provided herein for effecting substantial purification, particularly of the SATACs.", "SATACs have been isolated by fluorescence-activated cell sorting [FACS].", "This method takes advantage of the nucleotide base content of the SATACs, which, by virtue of their heterochromatic DNA content, will differ from any other chromosomes in a cell.", "In particular, metaphase chromosomes are isolated (e.g., by addition of colchicine) and stained with base-specific dyes, such as Hoechst 33258 and chromomycin A3.Fluorescence-activated cell sorting will separate the SATACs from the genomic chromosomes.", "A dual-laser cell sorter FACStar Plus and FAXStar Vantage Becton Dickinson Immunocytometry System] in which two lasers were set to excite the dyes separately, allowed a bivariate analysis of the chromosomes by base-pair composition and size.", "Cells containing such SATACs can be similarly sorted.", "3.6.7 Introduction of Artificial Chromosomes into Cells, Tissues, Animals and Plants Suitable hosts for introduction of the MACs provided herein, include, but are not limited to, any animal or plant, cell or tissue thereof, including, but not limited to: mammals, birds, reptiles, amphibians, insects, fish, arachnids, tobacco, tomato, wheat, plants and algae.", "The MACs, if contained in cells, may be introduced by cell fusion or microcell fusion or, if the MACs have been isolated from cells, they may be introduced into host cells by any method known to those of skill in this art, including but not limited to: direct DNA transfer, electroporation, lipid-mediated transfer, e.g., lipofection and liposomes, microprojectile bombardment, microinjection in cells and embryos, protoplast regeneration for plants, and any other suitable method [see, e.g., Weissbach et al.", "(1988) Methods for Plant Molecular Biology, Academic Press, N.Y., Section VIII, pp.", "421-463; Grierson et al.", "(1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch.", "7-9; see, also U.S. Pat.", "Nos.", "5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat.", "No.", "5,470,708, which describes particle-mediated transformation of mammalian unattached cells].", "Other methods for introducing DNA into cells include nuclear microinjection, electroporation, and bacterial protoplast fusion with intact cells.", "Polycations, such as polybrene and polyomithine, may also be used.", "For various techniques for transforming mammalian cells, see e.g., Keown et al.", "Methods in Enzymology (1990) Vol.", "185, pp.", "527-537; and Mansour et al.", "(1988) Nature 336: 348-352.DNA may be introduced by direct DNA transformation; microinjection in cells or embryos, protoplast regeneration for plants, electroporation, microprojectile gun and other such methods [see, e.g., Weissbach et al.", "(1988) Methods for Plant Molecular Biology, Academic Press, N.Y., Section VIII, pp.", "421-463; Grierson et al.", "(1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch.", "7-9; see, also U.S. Pat.", "Nos.", "5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat.", "No.", "5,470,708, which describes particle-mediated transformation of mammalian unattached cells].", "For example, isolated, purified artificial chromosomes can be injected into an embryonic cell line such as a human kidney primary embryonic cell line [ATCC accession number CRL 1573] or embryonic stem cells [see, e.g., Hogan et al.", "(1994) Manipulating he Mouse Embryo, A: Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., see, especially, pages 255-264 and Appendix 3].", "Preferably the chromosomes are introduced by microinjection, using a system such as the Eppendorf automated microinjection system, and grown under selective conditions, such as in the presence of hygromycin B or neomycin.", "3.6.7.1.Methods for Introduction of Chromosomes into Hosts Depending on the host cell used, transformation is done using standard techniques appropriate to such cells.", "These methods include any, including those described herein, known to those of skill in the art.", "3.6.7.1.1 DNA Uptake For mammalian cells that do not have cell walls, the calcium phosphate precipitation method for introduction of exogenous DNA [see, e.g., Graham et al.", "(1978) Virology 52: 456457; Wigler et al.", "(1979) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 76: 1373-1376; and Current Protocols in Molecular Biology, Vol.", "1, Wiley Inter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)] is often preferred.", "DNA uptake can be accomplished by DNA alone or in the presence of polyethylene glycol [PEG-mediated gene transfer], which is a fusion agent, or by any variations of such methods known to those of skill in the art [see, e.g., U.S. Pat.", "No.", "4,684,611].", "Lipid-mediated carrier systems are also among the preferred methods for introduction of DNA into cells [see, e.g., Teifel et al.", "(1995) Biotechniques 19: 79-80; Albrecht et al.", "(1996) Ann.", "Hematol.", "72: 73-79; Holmen et al.", "(1995) In Vitro Cell Dev.", "Biol.", "Anim.", "31: 347-351; Remy et al.", "(I 994) Bioconjug.", "Chem.", "5: 647-654; Le Boic h et al.", "(1995) Tetrahedron Lett.", "36: 6681-6684; Loeffler et al.", "(1993) Meth.", "Enzymol.", "217: 599-618].", "Lipofection [see, e.g., Strauss (1996) Meth.", "Mol.", "Biol.", "54: 307-327] may also be used to introduce DNA into cells.", "This method is particularly well-suited for transfer of exogenous DNA into chicken cells (e.g., chicken blastodermal cells and primary chicken fibroblasts; see Brazolot et al.", "(1991) Mol.", "Repro.", "Dev.", "30: 304-312).", "In particular, DNA of interest can be introduced into chickens in operative linkage with promoters from genes, such as lysozyme and ovalbumin, that are expressed in the egg, thereby permitting expression of the heterologous DNA in the egg.", "Additional methods useful in the direct transfer of DNA into cells include particle gun electrofusion [see, e.g., U.S. Pat.", "Nos.", "4,955,378, 4,923,814, 4,476,004, 4,906,576 and 4,441,972] and virion-mediated gene transfer.", "A commonly used approach for gene transfer in land plants involves the direct introduction of purified DNA into protoplasts.", "The three basic methods for direct gene transfer into plant cells include: 1) polyethylene glycol [PEG]-mediated DNA uptake, 2) electroporation-mediated DNA uptake and 3) microinjection.", "In addition, plants may be transformed using ultrasound treatment [see, e.g., International PCT application publication No.", "WO 91/00358].", "3.6.7.1.2 Electroporation Electroporation involves providing high-voltage electrical pulses to a solution containing a mixture of protoplasts and foreign DNA to create reversible pores in the membranes of plant protoplasts as well as other cells.", "Electroporation is generally used for prokaryotes or other cells, such as plants that contain substantial cell-wall.", "barriers.", "Methods for effecting electroporation are well known [see, e.g., U.S. Pat.", "Nos.", "4,784,737, 5,501,967, 5,501,662, 5,019,034, 5,503,999; see, also Fromm et al.", "1985) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 82: 5824-5828].", "For example, electroporation is often used for transformation of plants [see, e.g., Ag Biotechnology News 7: 3 and 17 (September/October 1990)].", "In this technique, plant protoplasts are electroporated in the presence of the DNA of interest that also includes a phenotypic marker.", "Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.", "Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.", "Transformed plant cells will be identified by virtue of the expressed phenotypic marker.", "The exogenous DNA may be added to the protoplasts in any form such as, for example, naked linear, circular or supercoiled DNA, DNA encapsulated in liposomes, DNA in spheroplasts, DNA in other plant protoplasts, DNA complexed with salts, and other methods.", "3.6.7.1.3 Microcells The chromosomes can be transferred by preparing microcells containing an artificial chromosome and then fusing with selected target cells.", "Methods for such preparation and fusion of microcells are well known [see, e.g., U.S. Pat.", "Nos.", "5,240,840, 4, 806,476, 5,298,429, 5,396,767, Foumier (1981) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 78: 6349-6353; and Lambert et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 88: 5907-59].", "Microcell fusion, using microcells that contain an artificial chromosome, is a particularly useful method for introduction of MACs into avian cells, such as DT40 chicken pre-B cells [for a description of DT40 cell fusion, see, e.g., Dieken et al.", "(1996) Nature Genet.", "12: 174-182].", "3.6.7.2.Hosts Suitable hosts include any host known to be useful for introduction and expression of heterologous DNA.", "Of particular interest herein, animal and plant cells and tissues, including, but not limited to insect cells and larvae, plants, and animals, particularly transgenic animals, and animal cells.", "Other hosts include, but are not limited to mammals, birds, particularly fowl such as chickens, reptiles, amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots, dicots and algae, and any host into which introduction of heterologous DNA is desired.", "Such introduction can be effected using the MACs provided herein, or, if necessary by using the MACs provided herein to identify species-specific centromeres and/or functional chromosomal units and then using the resulting centromeres or chromosomal units as artificial chromosomes, or alternatively, using the methods exemplified herein for production of MACs to produce species-specific artificial chromosomes.", "3.6.7.2.1 Introduction of DNA into Embryos for Production of Transgenic Animals and Introduction of DNA into Animal Cells Transgenic animals can be produced by introducing exogenous genetic material into a pronucleus of a mammalian zygote by microinjection [see, e.g., U.S. Pat.", "Nos.", "4,873,191 and 5,354,674; see, also, International PCT application publication No.", "WO95/14769, which is based on U.S. application Ser.", "No.", "08/159,084).", "The zygote is capable of development into a mammal.", "The embryo or zygote is transplanted into a host female uterus and allowed to develop.", "Detailed protocols and examples are set forth below.", "Transgenic chickens can be produced by injection of dispersed blastodermal cells from Stage X chicken embryos into recipient embryos at a similar stage of development [see e.g., Etches et al.", "(1993) Poultry Sci.", "72: 882-889; Petitte et al.", "(1990) Development 108: 185-189].", "Heterologous DNA is first introduced into the donor blastodermal cells using methods such as, for example, lipofection [see, e.g., Brazolot et al.", "(1991) Mol.", "Repro.", "Dev.", "30: 304-312] or microcell fusion [see, e.g., Dieken et al.", "1996) Nature Genet.", "12: 174-182].", "The transfected donor cells are then injected into recipient chicken embryos [see e.g., Carsience et al.", "(1993) Development 117: 669-675].", "The recipient chicken embryos within the shell are candled and allowed to hatch to yield a germline chimeric chicken.", "DNA can be introduced into animal cells using any known procedure, including, but not limited to: direct uptake, incubation with polyethylene glycol [PEG], microinjection, electroporation, lipofection, cell fusion, microcell fusion, particle bombardment, including microprojectile bombardment [see, eg., U.S. Pat.", "No.", "5,470,708, which provides a method for transforming unattached mammalian cells via particle bombardment], and any other such method.", "For example, the transfer of plasmid DNA in liposomes directly to human cells in situ has been approved by the FDA for use in humans [see, eg., Nabel, et al.", "(1990) Science 249: 1285-1288 and U.S. Pat.", "No.", "5,461,032].", "3.6.7.2.2 Introduction of Heterologous DNA into Plants.", "Numerous methods for producing or developing transgenic plants are available to those of skill in the art.", "The method used is primarily a function of the species of plant.", "These methods include, but are not limited to: direct transfer of DNA by processes, such as PEG-induced DNA uptake, protoplast fusion, microinjection, electroporation, and microprojectile bombardment [see, e.g., Uchimiya et al.", "(1989) J. of Biotech.", "12: 1-20 for a review of such procedures, see, also, e.g., U.S. Pat.", "Nos.", "5,436,392 and 5,489,520 and many others].", "For purposes herein, when introducing a MAC, microinjection, protoplast fusion and particle gun bombardment are preferred.", "Plant species, including tobacco, rice, maize, rye, soybean, Brassica napus, cotton, lettuce, potato and tomato, have been used to produce transgenic plants.", "Tobacco and other species, such as petunias, often serve as experimental models in which the methods have been developed and the genes first introduced and expressed.", "DNA uptake can be accomplished by DNA alone or in the presence of PEG, which is a fusion agent, with plant protoplasts or by any variations of such methods known to those of skill in the art [see, e.g., U.S. Pat.", "No.", "4,684,611 to Schilperoot et al.].", "Electroporation, which involves high-voltage electrical pulses to a solution containing a mixture of protoplasts and foreign DNA to create reversible pores, has been used, for example, to successfully introduce foreign genes into rice and Brassica napus.", "Microinjection of DNA into plant cells, including cultured cells and cells in intact plant organs and embryoids in tissue culture and microprojectile bombardment [acceleration of small high density particles, which contain the DNA, to high velocity with a article gun apparatus, which forces the particles to penetrate plant cell walls and membranes] have also been used.", "All plant cells into which DNA can be introduced and that can be regenerated from the transformed cells can be used to produce transformed whole plants which contain the transferred artificial chromosome.", "The particular protocol and means for introduction of the DNA into the plant host may need to be adapted or refined to suit the particular plant species or cultivar.", "3.6.7.2.3 Insect Cells Insects are useful hosts for introduction of artificial chromosomes for numerous reasons, including, but not limited to: (a) amplification of genes encoding useful proteins can be accomplished in the artificial chromosome to obtain higher protein yields in insect cells; (b) insect cells support required post-translational modifications, such as glycosylation and phosphorylation, that can be required for protein biological functioning; (c) insect cells do not support mammalian viruses, and, thus, eliminate the problem of cross-contamination of products with such infectious agents; (d) this technology circumvents traditional recombinant baculovirus systems for production of nutritional, industrial or medicinal proteins in insect cell systems; (e) the low temperature optimum for insect cell growth (28° C.) permits reduced energy cost of production; (f) serum-free growth medium for insect cells permits lower production costs; (g) artificial chromosome-containing cells can be stored indefinitely at low temperature; and (h) insect larvae will be biological factories for production of nutritional, medicinal or industrial proteins by microinjection of fertilized insect eggs [see, eq., Joy et al.", "(1991) Current Science 66: 145-150, which provides a method for microinjecting heterologous DNA into Bombyx mori eggs].", "Either MACs or insect-specific artificial chromosomes [BUGACs] will be used to introduce genes into insects.", "It appears that MACs will function in insects to direct expression of heterologous DNA contained thereon.", "For example, a MAC containing the B. mori actin gene promoter fused to the lacZ gene has been generated by transfection of EC3/7C5 cells with a plasmid containing the fusion gene.", "Subsequent fusion of the B. mori cells with the transfected EC3/7C5 cells that survived selection yielded a MAC-containing insect-mouse hybrid cell line in which, beta-galactosidase expression was detectable.", "Insect host cells include, but are not limited to, hosts such as Spodoptera frugiperda [caterpillar], Aedes aegypti [mosquito], Aedes albopictus [mosquito], Drosphila melanogaster [fruitfly], Bombyx mori [silkworm], Manduca sexta [tomato horn worm] and Trichoplusia ni [cabbage looper].", "Efforts have been directed toward propagation of insect cells in culture.", "Such efforts have focused on the fall armyworm, Spodoptera frugiperda.", "Cell lines have been developed also from other insects such as the cabbage looper, Trichoplusia ni and the silkworm, Bombyx mori.", "It has also been suggested that analogous cell lines can be created using the tomato homworm, Manduca sexta.", "To introduce DNA into an insect, it should be introduced into the larvae, and allowed to proliferate, and then the hemolymph recovered from the larvae so that the proteins can be isolated therefrom.", "The preferred method herein for introduction of artificial chromosomes into insect cells is microinjection [see, e.g., Tamura et al.", "(1991) Bio Ind.", "8: 26-31; Nikolaev et al.", "(1989) Mol.", "Biol.", "(Moscow) 23: 1177-87; and methods exemplified and discussed erein].", "3.6.8 Applications for and Uses of Artificial chromosomes Artificial chromosomes provide convenient and useful vectors, and in some instances [e.g., in the case of very large heterologous genes] the only vectors, for introduction of heterologous genes into hosts.", "Virtually any gene of interest is amenable to introduction into a host via artificial chromosomes.", "Such genes include, but are not limited to, genes that encode receptors, cytokines, enzymes, proteases, hormones, growth factors, antibodies, tumor suppressor genes, therapeutic products and multigene pathways.", "The artificial chromosomes provided herein will be used in methods of protein and gene product production, particularly using insects as host cells for production of such products, and in cellular (e.g., mammalian cell) production systems in which the rtificial chromomsomes (particularly MACs) provide a reliable, stable and efficient means for optimizing the biomanufacturing of important compounds for medicine and industry.", "They are also intended for use in methods of gene therapy, and in for production of transgenic plants and animals [discussed above and below].", "3.6.8.1 Gene Therapy Any nucleic acid encoding a therapeutic gene product or product of a multigene pathway may be introduced into a host animal, such as a human, or into a target cell line for introduction into an animal, for therapeutic purposes.", "Such therapeutic purposes include, genetic therapy to cure or to provide gene products that are missing or defective, to deliver agents, such as anti-tumor agents, to targeted cells or to an animal, and to provide gene products that will confer resistance or reduce susceptibility to a pathogen or ameliorate symptoms of a disease or disorder.", "The following are some exemplary genes and gene products.", "Such exemplification is not intended to be limiting.", "3.6.8.1.1 Anti-HIV Ribozymes As exemplified below, DNA encoding anti-HIV ribozymes can be introduced and expressed in cells using MACs, including the euchromatin-based minichromosomes and the SATACs.", "These MACs can be used to make a transgenic mouse that expresses a ribozyme and, thus, serves as a model for testing the activity of such ribozymes or from which ribozyme-producing cell lines can be made.", "Also, introduction of a MAC that encodes an anti-HIV ribozyme into human cells will serve as treatment for HIV infection.", "Such systems further demonstrate the viability of using any disease-specific ribozyme to treat or ameliorate a particular disease.", "3.6.8.1.2 Tumor Suppressor Genes Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation.", "When the gene coding for a tumor suppressor protein is mutated or deleted, the resulting mutant protein or the complete lack of tumor suppressor protein expression may result in a failure to correctly regulate cellular proliferation.", "Consequently, abnormal cellular proliferation may take place, particularly if there is already existing damage to the cellular regulatory mechanism.", "A number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes.", "Examples of tumor suppression genes include, but are not limited to, the retinoblastoma susceptibility gene or RB gene, the p53 gene, the gene that is deleted in colon carcinoma [i.e., the DCC gene] and the neurofibromatosis type I [NF-1] tumor suppressor gene [see, e.g., U.S. Pat.", "No.", "5,496,731; Weinberg et al.", "(1991) 254: 1138-1146].", "Loss of function or inactivation of tumor suppressor genes may play a central role in the initiation and/or progression of a significant number of human cancers.", "3.6.8.1.2.1 The p53 Gene Somatic cell mutations of the p53 gene are said to be the most frequent of the gene mutations associated with human cancer [see, e.g., Weinberg et al.", "(1991) Science 254: 1138-1146].", "The normal or wild-type p53 gene is a negative regulator of cell growth, which, when damaged, favors cell transformation.", "The p53 expression product is found in the nucleus, where it may act in parallel or cooperatively with other gene products.", "Tumor cell lines in which p53 has been deleted have been successfully treated with wild-type p53 vector to reduce tumorigenicity [see, Baker et al.", "(1990) Science 249: 912-915].", "DNA encoding the p53 gene and plasmids containing this DNA are well known [see, e.g., U.S. Pat.", "No.", "5,260,191; see, also Chen et al.", "(1990) Science 250: 1576; Farrel et al.", "(1991) EMBO J.", "10: 2879-2887; plasmids containing the gene are available from the ATCC, and the sequence is in the GenBank Database, accession nos.", "X54156, X60020, M14695, M16494, K03199].", "3.6.8.1.3 The CFTR Gene Cystic fibrosis [CF] is an autosomal recessive disease that affects epithelia of the airways, sweat glands, pancreas, and other organs.", "It is a lethal genetic disease associated with a defect in chloride ion transport, and is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator [CFTR], a 1480 amino acid protein that has been associated with the expression of chloride conductance in a variety of eukaryotic cell types.", "Defects in CFTR destroy or reduce the ability of epithelial cells in the airways, sweat glands, pancreas and other tissues to transport chloride ions in response to cAMP-mediated agonists and impair activation of apical membrane channels by cAMP-dependent protein kinase A [PKA].", "Given the high incidence and devastating nature of this disease, development of effective CF treatments is imperative.", "The CFTR gene [˜250 kb] [600 kb] can be transferred into a MAC for use, for example, in gene therapy as follows.", "A CF-YAC [see Green et al.", "Science 250: 94-98] may be modified to include a selectable marker, such as a gene encoding a protein that confers resistance to puromycin or hygromycin, and lambda-DNA for use in site-specific integration into a neo-minichromosome or a SATAC.", "Such a modified CF-YAC can be introduced into MAC-containing cells, such as EC3/7C5 or 19C5xHa4 cells, by fusion with yeast protoplasts harboring the modified CF-YAC or microinjection of yeast nuclei harboring the modified CF-YAC into the cells.", "Stable transformants are then selected on the basis of antibiotic resistance.", "These transformants will carry the modified CF-YAC within the MAC contained in the cells.", "3.6.8.2 Animals, Birds, Fish and Plants that are Genetically Altered to Possess Desired Traits such as Resistance to Disease Artificial chromosomes are ideally suited for preparing animals, including vertebrates and invertebrates, including birds and fish as well as mammals, that possess certain desired traits, such as, for example, disease resistance, resistance to harsh environmental conditions, altered growth patterns, and enhanced physical characteristics.", "One example of the use of artificial chromosomes in generating disease-resistant organisms involves the preparation of multivalent vaccines.", "Such vaccines include genes encoding multiple antigens that can be carried in a MAC, or species-specific artificial chromosome, and either delivered to a host to induce immunity, or incorporated into embryos to produce transgenic animals and plants that are immune or less susceptible to certain diseases.", "Disease-resistant animals and plants may also be prepared in which resistance or decreased susceptibility to disease is conferred by introduction into the host organism or embryo of artificial chromosomes containing DNA encoding gene products (e.g., ribozymes and proteins that are toxic to certain pathogens) that destroy or attenuate pathogens or limit access of pathogens to the host.", "Animals and plants possessing desired traits that might, for example, enhance utility, processibility and commercial value of the organisms in areas such as the agricultural and ornamental plant industries may also be generated using artificial chromosomes in the same manner as described above for production of disease-resistant animals and plants.", "In such instances, the artificial chromosomes that are introduced into the organism or embryo contain DNA encoding gene products that serve to confer he desired trait in the organism.", "Birds, particularly fowl such as chickens, fish and crustaceans will serve as model hosts for production of genetically altered organisms using artificial chromosomes.", "3.6.83 Use of MACs and Other Artificial Chromosomes for Preparation and Screening of Libraries Since large fragments of DNA can be incorporated into each artificial chromosome, the chromosomes are well-suited for use as cloning vehicles that can accommodate entire genomes in the preparation of genomic DNA libraries, which then can be readily screened.", "For example, MACs may be used to prepare a genomic DNA library useful in the identification and isolation of functional centromeric DNA from different species of organisms.", "In such applications, the MAC used to prepare a genomic DNA library from particular organism is one that is not functional in cells of that organism.", "That is, the MAC does not stably replicate, segregate or provide for expression of genes contained within it in cells of the organism.", "Preferably, the MACs contain an indicator gene (e.g., the lacZ gene encoding beta-galactosidase or genes encoding products that confer resistance to antibiotics such as neomycin, puromycin, hygromycin) linked to a promoter that is capable of promoting transcription of the indicator gene in cells of the organism.", "Fragments of genomic DNA from the organism are incorporated into the MACs, and the MACs are transferred to cells from the organism.", "Cells that contain MACs that have incorporated functional centromeres contained within the genomic DNA fragments are identified by detection of expression of the marker gene.", "For example, DNA encoding tree growth factors can be introduced into trees.", "Libraries can be prepared, introduce large fragments into chromosomes, and introduce them all into trees, thereby insuring expression.", "3.6.8.4 Use of MACs and Other Artificial Chromosomes for Stable, High-Level Protein Production Cells containing the MACs and/or other artificial chromosomes provided herein are advantageously used for production of proteins, particularly several proteins from one cell line, such as multiple proteins involved in a biochemical pathway or multivalent vaccines.", "The genes encoding the proteins are introduced into the artificial chromosomes which are then introduced into cells.", "Alternatively, the heterologous gene(s) of interest are transferred into a production cell line that already contains artificial chromosomes in a manner that targets the gene(s) to the artificial chromosomes.", "The cells are cultured under conditions whereby the heterologous proteins are expressed.", "Because the proteins will be expressed at high levels in a stable permanent extra-genomic chromosomal system, selective conditions are not required.", "Any transfectable cells capable of serving as recombinant hosts adaptable to continuous propagation in a cell culture system [see, e.g., McLean (1993) Trends In Biotech.", "11: 232-238] are suitable for use in an artificial chromosome-based protein production system.", "Exemplary host cell lines include, but are not limited to, the following: Chinese hamster ovary (CHO) cells [see, e.g., Zang et al.", "(1995) Biotechnology 13: 389-392], HEK 293, Ltk−, COS-7, DG44, and BHK cells.", "CHO cells are particularly preferred host cells.", "Selection of host cell lines for use in artificial chromosome-based protein production systems is within the skill of the art, but often will depend on a variety of factors, including the properties of the heterologous protein to be produced, potential toxicity of the protein in the host cell, any requirements for post-translational modification (e.g., glycosylation, amination, phosphorylation) of the protein, transcription factors available in the cells, the type of promoter element(s) being used to drive expression of the heterologous gene, whether production will be completely intracellular or the heterologous protein will preferably be secreted from the cell, and the types of processing enzymes in the cell.", "The artificial chromosome-based system for heterologous protein production has many advantageous features.", "For example, as described above, because the heterologous DNA is located in an independent, extra-genomic artificial chromosome (as opposed to randomly inserted in an unknown area of the host cell genome or located as extrachromosomal element(s) providing only transient expression) it is stably maintained in an active transcription unit and is not subject to ejection via recombination or elimination during cell division.", "Accordingly, it is unnecessary to include a selection gene in the host cells and thus growth under selective conditions is also unnecessary.", "Furthermore, because the artificial chromosomes are capable of incorporating large segments of DNA, multiple copies of the heterologous gene and linked promoter element(s) can be retained in the chromosomes, thereby providing for high-level expression of the foreign protein(s).", "Alternatively, multiple copies of the gene can be linked to a single promoter element and several different genes may be linked in a fused polygene complex to a single promoter for expression of, for example, all the key proteins constituting a complete metabolic pathway [see, e.g., Beck von Bodman et al.", "(1995) Biotechnology 13: 587-591].", "Alternatively, multiple copies of a single gene can be operatively linked to a single promoter, or each or one or several copies may be linked to different promoters or multiple copies of the same promoter.", "Additionally, because artificial chromosomes have an almost unlimited capacity for integration and expression of foreign genes, they can be used not only for the expression of genes encoding end-products of interest, but also for the expression of genes associated with optimal maintenance and metabolic management of the host cell, e.g., genes encoding growth factors, as well as genes that may facilitate rapid synthesis of correct form of the desired heterologous protein product, e.g., genes encoding processing enzymes and transcription factors.", "The MACS are suitable for expression of any proteins or peptides, including proteins and peptides that require in vivo posttranslational modification for their biological activity.", "Such proteins include, but are not limited to antibody fragments, full-length antibodies, and multimeric antibodies, tumor suppressor proteins, naturally occurring or artificial antibodies and enzymes, heat shock proteins, and others.", "Thus, such cell-based “protein factories” employing MACs can generated using MACs constructed with multiple copies [theoretically an unlimited number or at least up to a number such that the resulting MAC is about up to the size of a genomic chromosome] of protein-encoding genes with appropriate promoters, or multiple genes driven by a single promoter, i.e., a fused gene complex [such as a complete metabolic pathway in plant expression system; see, e.g., Beck von Bodman (1995) Biotechnology 13: 587-591].", "Once such MAC is constructed, it can be transferred to a suitable cell culture system, such as a CHO cell line in protein-free culture medium [see, e.g., (1995) Biotechnology 13: 389-39] or other immortalized cell lines [see, e.g., (1993) TIBTECH 11: 232-238] where continuous production can be established.", "The ability of MACs to provide for high-level expression of heterologous proteins in host cells is demonstrated, for example, by analysis of the H1D3 and G3D5 cell lines described herein and deposited with the ECACC.", "Northern blot analysis of mRNA obtained from these cells reveals that expression of the hygromycin-resistance and beta-galactosidase genes in the cells correlates with the amplicon number of the megachromosome(s) contained therein.", "4.ENGINEERING APPROACHES 4.1.1 General Considerations In one aspect, this invention applies the technical field of molecular genetics to evolve the genomes of cells and organisms to acquire new and improved properties.", "Cells have a number of well-established uses in molecular biology.", "For example, cells are commonly used as hosts for manipulating DNA in processes such as transformation and recombination.", "Cells are also used for expression of recombinant proteins encoded by DNA transformed into the cells.", "Some types of cells are also used as progenitors for generation of transgenic animals and plants.", "Although all of these processes are now routine, in general, the genomes of the cells used in these processes have evolved little from the genomes of natural cells, and particularly not toward acquisition of new or improved properties for use in the above processes.", "The traditional approach to artificial or forced molecular evolution focuses on optimization of an individual gene having a discrete and selectable phenotype.", "The strategy is to clone a gene, identify a discrete function for the gene and an assay by which it can be selected, mutate selected positions in the gene (e.g., by error-prone PCR or cassette mutagenesis) and select variants of the gene for improvement in the known function of the gene.", "A variant having improved function can then be expressed in a desired cell type.", "This approach has a number of limitations.", "First, it is only applicable to genes that have been isolated and functionally characterized.", "Second, the approach is usually only applicable to genes that have a discrete function.", "In other words, multiple genes that cooperatively confer a single phenotype cannot usually be optimized in this manner.", "Probably, most genes do have explore a very limited number of the total number of permutations even for a single gene.", "For example, varying even ten positions in a protein with every possible amino acid would generate 2010 variants, which is more than can be accommodated by existing methods of transfection and screening.", "In view of these limitations, the traditional approach is inadequate for improving cellular genomes in many useful properties.", "For example, to improve a cell's capacity to express a recombinant protein might require modification in any or all of a substantial number of genes, known and unknown, having roles in transcription, translation, posttranslational modification, secretion or proteolytic degradation, among others.", "Attempting individually to optimize even all the known genes having such functions would be a virtually impossible task, let alone optimizing hitherto unknown genes which may contribute to expression in manners not yet understood.", "The present invention pro, des inter alia novel methods for evolving the genome of whole cells and organisms which overcome the difficulties and limitations of prior methods.", "This ability to evolve genes artificially is of fundamental importance.", "For example, cells have a number of well-established uses in molecular biology, medicine and industrial processes.", "For example, cells are commonly used as hosts for manipulating DNA in processes such as transformation and recombination.", "Cells are used for expression of recombinant proteins encoded by DNA transformed/transfected or otherwise introduced into the cells.", "Some types of cells are used as progenitors for generation of transgenic animals and plants.", "The genomes of the cells used in these processes had evolved little from the genomes of natural cells, and particularly not toward acquisition of new or improved properties for use in the above processes.", "Additional methods of recursively recombining nucleic acids in vivo and selecting resulting recombinants would be of use.", "The present invention provides a number of new and valuable methods and compositions for whole and partial genome evolution.", "Metabolic engineering is the manipulation of intermediary metabolism through the use of both classical genetics and genetic engineering techniques.", "Cellular engineering is generally a more inclusive term referring to the modification of cellular properties.", "Cameron et al.", "(Applied Biochem.", "Biotech.", "38: 105-140 (1993)) provide a summary of equivalent terms to describe this type of engineering, including “metabolic engineering”, which is most often used in the context of industrial microbiology and bioprocess engineering, “in vitro evolution” or “directed evolution”, most often used in the context of environmental microbiology, “molecular breeding”, most often used by Japanese researchers, “cellular engineering”, which is used to describe modifications of bacteria, animal, and plant cells, “rational strain development”, and “metabolic pathway evolution”.", "In this application, the terms “metabolic engineering” and “cellular engineering” are used preferentially for clarity; the term “evolved” genes is used as discussed below.", "Metabolic engineering can be divided into two basic categories: modification of genes endogenous to the host organism to alter metabolite flux and introduction of foreign genes into an organism.", "Such introduction can create new metabolic pathways leading to modified cell properties including but not limited to synthesis of known compounds not normally made by the host cell, production of novel compounds (e.g.", "polymers, antibiotics, etc.)", "and the ability to utilize new nutrient sources.", "Specific applications of metabolic engineering can include the production of specialty and novel chemicals, including antibiotics, extension of the range of substrates used for growth and product formation, the production of new catabolic activities in an organism for toxic chemical degradation, and modification of cell properties such as resistance to salt and other environmental factors.", "Bailey (Science 252: 1668-1674 (1991)) describes the application of metabolic engineering to the recruitment of heterologous genes for the improvement of a strain, with the caveat that such introduction can result in new compounds that may subsequently undergo further reactions, or that expression of a heterologous protein can result in proteolysis, improper folding, improper modification, or unsuitable intracellular location of the protein, or lack of access to required substrates.", "Bailey recommends careful configuration of a desired genetic change with minimal perturbation of the host.", "Liao (Curr.", "Opin.", "Biotech.", "4: 211-216 (1993)) reviews mathematical modeling and analysis of metabolic pathways, pointing out that in many cases the kinetic parameters of enzymes are unavailable or inaccurate.", "Stephanopoulos et al.", "(Trends.", "Biotechnol.", "11: 392-396 (1993)) describe attempts to improve productivity of cellular systems or effect radical alteration of the flux through primary metabolic pathways as having difficulty in that control architectures at key branch points have evolved to resist flux changes.", "They conclude that identification and characterization of these metabolic nodes is a prerequisite to rational metabolic engineering.", "Similarly, Stephanopoulos (Curr.", "Opin.", "Biotech.", "5: 196-200 (1994)) concludes that rather than modifying the “rate limiting step” in metabolic engineering, it is necessary to systematically elucidate the control architecture of bioreaction networks.", "The present invention is generally directed to the evolution of new metabolic pathways and the enhancement of bioprocessing through a process herein termed recursive sequence recombination.", "Recursive sequence recombination entails performing iterative cycles of recombination and screening or selection to “evolve” individual genes, whole plasmids or viruses, multigene clusters, or even whole genomes (Stemmer, Bio/Technolog 13: 549-553 (1995)).", "Such techniques do not require the extensive analysis and computation required by conventional methods for metabolic engineering.", "Recursive sequence recombination allows the recombination of large numbers of mutations in a minimum number of selection cycles, in contrast to traditional, pair wise recombination events.", "Thus, because metabolic and cellular engineering can pose the particular problem of the interaction of many gene products and regulatory mechanisms, recursive sequence recombination (RSR) techniques provide particular advantages in that they provide recombination between mutations in any or all of these, thereby providing a very fast way of exploring the manner in which different combinations of mutations can affect a desired result, whether that result is increased yield of a metabolite, altered catalytic activity or substrate specificity of an enzyme or an entire metabolic pathway, or altered response of a cell to its environment.", "4.1.2 The Evolutionary Importance of Recombination Strain improvement is the directed evolution of an organism to be more “fit” for a desired task.", "In nature, adaptation is facilitated by sexual recombination.", "Sexual recombination allows a population to exploit the genetic diversity within it, e.g., by consolidating useful mutations and discarding deleterious ones.", "In this way, adaptation and evolution can proceed in leaps.", "In the absence of a sexual cycle, members of a population must evolve independently by accumulating random mutations sequentially.", "Many useful mutations are lost while deleterious mutations can accumulate.", "Adaptation and evolution in this way proceeds slowly as compared to sexual evolution.", "Asexual Evolution is a Slow and Inefficient Process.", "Populations move as individuals rather than as groups.", "A diverse population is generated by the mutagenesis of a single parent resulting in a distribution of fit and unfit individuals.", "In the absence of a sexual cycle, each piece of genetic information of the surviving population remains in the individual mutants.", "Selection of the “fittest” results in many “fit” individuals being discarded along with the useful genetic information they carry.", "Asexual evolution proceeds one genetic event at a time and is thus limited by the intrinsic value of a single genetic event.", "Sexual evolution moves more quickly and efficiently.", "Mating within a population consolidates genetic information within the population and results in useful mutations being combined together.", "The combining of useful genetic information results in progeny that are much more fit than their parents.", "Sexual evolution thus proceeds much faster by multiple genetic events.", "Years of plant and animal breeding has demonstrated the power of employing sexual recombination to effect the rapid evolution of complex genomes towards a particular task.", "This general principle is further demonstrated by using DNA stochastic &/or non-stochastic mutagenesis to recombine DNA molecules in vitro to accelerate the rate of directed molecular evolution.", "The strain improvement efforts of the fermentation industry rely on the directed evolution of microorganisms by sequential random mutagenesis.", "Incorporation of recombination into this iterative process greatly accelerates the strain improvement process, which in turn increases the profitability of current fermentation processes and facilitates the development of new products.", "4.1.2.1 DNA Stochastic &/or Non-Stochastic Mutagenesis vs Natural Recombination DNA stochastic &/or non-stochastic mutagenesis includes the recursive recombination of DNA sequences.", "A significant difference between DNA stochastic &/or non-stochastic mutagenesis and natural sexual recombination is that DNA stochastic &/or non-stochastic mutagenesis can produce DNA sequences originating from multiple parental sequences while sexual recombination produces DNA sequences originating from only two parental sequences.", "The rate of evolution is in part limited by the number of useful mutations that a member of a population can accumulate between selection events.", "In sequential random mutagenesis, useful mutations are accumulated one per selection event.", "Many useful mutations are discarded each cycle in favor of the best performer, and neutral or deleterious mutations which survive are as difficult to lose as they were to gain and thus accumulate.", "In sexual evolution pairwise recombination allows mutations from two different parents to segregate and recombine in different combinations.", "Useful mutations can accumulate and deleterious mutations can be lost.", "Poolwise recombination, such as that effected by DNA stochastic &/or non-stochastic mutagenesis, has the same advantages as pairwise recombination but allows mutations from many parents to consolidate into a single progeny.", "Thus poolwise recombination provides a means for increasing the number of useful mutations that can accumulate each selection event.", "One can plot the potential number of mutations an individual can accumulate by each of these processes.", "Recombination is exponentially superior to sequential random mutagenesis, and this advantage increases exponentially with the number of parents that can recombine.", "Sexual recombination is thus more conservative.", "In nature, the pairwise nature of sexual recombination may provide important stability within a population by impeding the large changes in DNA sequence that can result from poolwise recombination.", "For the purposes of directed evolution, however, poolwise recombination is more efficient.", "The potential diversity that can be generated from a population is greater as a result of poolwise recombination as compared to that resulting from pairwise recombination.", "Further, poolwise recombination enables the combining of multiple beneficial mutations originating from multiple parental sequences.", "To demonstrate the importance of poolwise recombination vs pairwise recombination in the generation of molecular diversity consider the breeding of ten independent DNA sequences each containing only one unique mutation.", "There are 210=1024 different combinations of those ten mutations ranging from a single sequence having no mutations (the consensus) to that having all ten mutations.", "If this pool were recombined together by pairwise recombination, a population containing the consensus, the parents, and the 45 different combinations of any two of the mutations would result in 56 or ca.", "5% of the possible 1024 mutant combinations.", "Alternatively, if the pool were recombined together in a poolwise fashion, all 1024 would be theoretically generated, resulting in an approximately 20 fold increase in library diversity.", "When looking for a unique solution to a problem in molecular evolution, the more complex the library, the more complex the possible solution.", "Indeed, the most fit member of a stochastic &/or non-stochastic mutagenized library often contains several mutations originating from several independent starting sequences.", "4.1.2.2 DNA Stochastic &/or Non-Stochastic Mutagenesis Provides Recursive Pairwise Recombination In vitro DNA stochastic &/or non-stochastic mutagenesis results in the efficient production of combinatorial genetic libraries by catalyzing the recombination of multiple DNA sequences.", "While the result of DNA stochastic &/or non-stochastic mutagenesis is a population representing the poolwise recombination of multiple sequences, the process does not rely on the recombination of multiple DNA sequences simultaneously, but rather on their recursive pairwise recombination.", "The assembly of complete genes from a mixed pool of small gene fragments requires multiple annealing and elongation cycles, the thermal cycles of the primeness PCR reaction.", "During each thermal cycle many pairs of fragments anneal and are extended to form a combinatorial population of larger chimeric DNA fragments.", "After the first cycle of stochastic &/or non-stochastic mutagenesis, chimeric fragments contain sequence originating from predominantly two different parent genes, with all possible pairs of “parental” sequence theoretically represented.", "This is similar to the result of a single sexual cycle within a population.", "During the second cycle, these chimeric fragments anneal with each other or with other small fragments, resulting in chimeras originating from up to four of the different starting sequences, again with all possible combinations of the four parental sequences theoretically represented.", "This second cycle is analogous to the entire population resulting from a single sexual cross, both parents and offspring, inbreeding.", "Further cycles result in chimeras originating from 8, 16, 32, etc parental sequences and are analogous to further inbreedings of the preceding population.", "This could be considered similar to the diversity generated from a small population of birds that are isolated on an island, breeding with each other for many generations.", "The result mimics the outcome of “poolwise” recombination, but the path is via recursive pairwise recombination.", "For this reason, the DNA molecules generated from in vitro DNA stochastic &/or non-stochastic mutagenesis are not the “progeny” of the starting “parental” sequences, but rather the great, great great, greatn, (n=number of thermal cycles) grand progeny of the starting “ancestor” molecules.", "4.1.3 Definitions The term “cognate” refers to a gene sequence that is evolutionarily and functionally related between species.", "For example, in the human genome, the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these which functions in signaling T-cell activation through MHC class II-restricted antigen recognition.", "Screening is, in general, a two-step process in which one first determines which cells do and do not express a screening marker or phenotype (or a selected level of marker or phenotype), and then physically separates the cells having the desired property.", "Selection is a form of screening in which identification and physical separation are achieved simultaneously by expression of a selection marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa).", "Screening markers include luciferase, P-galactosidase, and green fluorescent protein.", "Selection markers include drug and toxin resistance genes.", "An exogenous DNA segment is one foreign (or heterologous) to the cell or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found.", "Exogenous DNA segments can be expressed to yield exogenous polypeptides.", "The term “gene” is used broadly to refer to any segment of DNA associated with a biological function.", "Thus, genes include coding sequences and/or the regulatory sequences required for their expression.", "Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins.", "The terms “identical” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.", "The phrase “substantially identical,” in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.", "Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.", "In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.", "For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.", "When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.", "The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.", "Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv.", "Appl Math.", "2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biot 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc.", "Natl.", "Acad.", "Sci.", "USA 85: 2444 (1988), by computerized implementations of algorithms GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, W 1.Another example of a useful alignment algorithm is PILEUP.", "PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity.", "It also plots a tree or dendogram showing the clustering relationships used to create the alignment.", "PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol.", "Evol.", "35: 351-360 (1987).", "The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153 (1989).", "The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.", "The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.", "This cluster is then aligned to the next most related sequence or cluster of aligned sequences.", "Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.", "The final alignment is achieved by a series of progressive, pairwise alignments.", "The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.", "For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.", "Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol.", "Biol.", "215: 403410 (1990).", "Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nim.nih.gov/).", "This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.", "T is referred to as the neighborhood word score threshold (Altschul et al, supra).", "These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.", "The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.", "Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0).", "For amino acid sequences, a scoring matrix is used to calculate the cumulative score.", "Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.", "The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.", "The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.", "For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff& Henikoff, Proc.", "Natl.", "Acad.", "Sci.", "USA 89: 10915 (1989)).", "In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc.", "Natl.", "Acad.", "Sci.", "USA 90: 5873-5787 (1993)).", "One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.", "For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.", "Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.", "Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.", "The term “naturally-occurring” is used to describe an object that can be found in nature.", "For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.", "Generally, the term naturally-occurring refers to an object as present in a non-pathological (undiseased) individual, such as would be typical for the species.", "Asexual recombination is recombination occurring without the fusion of gametes to form a zygote.", "A “mismatch repair deficient strain” can include any mutants in any organism impaired in the functions of mismatch repair.", "These include mutant gene products of mutS, mutT, muth, mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recj, etc.", "The impairment is achieved by genetic mutation, allelic replacement, selective inhibition by an added reagent such as a small compound or an expressed antisense RNA, or other techniques.", "Impairment can be of the genes noted, or of homologous genes in any organism.", "4.2.Strategies 4.2.1.Evolving a Cell to Acquire a Desired Function 4.2.1.1.Desired Function is Secretion of a Protein Optionally, the desired function is secretion of a protein, and the plurality of cells further comprises a construct encoding the protein.", "The protein is optionally inactive unless secreted, and further modified cells are optionally selected for protein function.", "Optionally, the protein is toxic to the plurality of cells, unless secreted.", "In this case, the modified or further modified cells which evolve toward acquisition of the desired function are screened by propagating the cells and recovering surviving cells.", "4.2.1.2.Desired Function is Enhanced Recombination In some methods, the desired function is enhanced recombination.", "In such methods, the library of fragments sometimes comprises a cluster of genes collectively conferring recombination capacity.", "Screening can be achieved using cells carrying a gene encoding a marker whose expression is prevented by a mutation removable by recombination.", "The cells are screened by their expression of the marker resulting from removal of the mutation by recombination.", "4.2.13.Desired Function is Improved Resistance in Plant Cells In some methods, the plurality of cells are plant cells and the desired property is improved resistance to a chemical or microbe.", "The modified or further modified cells (or whole plants) are exposed to the chemical or microbe and modified or further modified cells having evolved toward the acquisition of the desired function are selected by their capacity to survive the exposure.", "4.2.1.4.Desired Function is Predictiong Efficacy of a Drug 4.2.1.4.1.A Drug Treating a Viral Infection The invention further provides methods of predicting efficacy of a drug in treating a viral infection.", "Such methods entail recombining a nucleic acid segment from a virus, whose infection is inhibited by a drug, with at least a second nucleic acid segment from the virus, the second nucleic acid segment differing from the first nucleic acid segment in at least two nucleotides, to produce a library of recombinant nucleic acid segments.", "Host cells are then contacted with a collection of viruses having genomes including the recombinant nucleic acid segments in a media containing the drug, and progeny viruses resulting from infection of the host cells are collected.", "A recombinant DNA segment from a first progeny virus recombines with at least a recombinant DNA segment from a second progeny virus to produce a further library of recombinant nucleic acid segments.", "Host cells are contacted with a collection of viruses having genomes including the further library or recombinant nucleic acid segments, in media containing the drug, and further progeny viruses are produced by the host cells.", "The recombination and selection steps are repeated, as desired, until a further progeny virus has acquired a desired degree of resistance to the drug, whereby the degree of resistance acquired and the number of repetitions needed to acquire it provide a measure of the efficacy of the drug in treating the virus.", "Viruses are optionally adapted to grow on particular cell lines.", "4.2.1.4.2.A Drug Treating Infection by a Pathogenic Microorganism The invention further provides methods of predicting efficacy of a drug in treating an infection by a pathogenic microorganism.", "These methods entail delivering a library of DNA fragments into a plurality of microorganism cells, at least some of which undergo recombination with segments in the genome of the cells to produce modified microorganism cells.", "Modified microorganisms are propagated in a media containing the drug, and surviving microorganisms are recovered.", "DNA from surviving microorganisms is recombined with a further library of DNA fragments at least some of which undergo recombination with cognate segments in the DNA from the surviving microorganisms to produce further modified microorganisms cells.", "Further modified microorganisms are propagated in media containing the drug, and further surviving microorganisms are collected.", "The recombination and selection steps are repeated as needed, until a further surviving microorganism has acquired a desired degree of resistance to the drug.", "The degree of resistance acquired and the number of repetitions needed to acquire it provide a measure of the efficacy of the drug in killing the pathogenic microorganism.", "4.2.1.3.Method 4.2.1.3.1 Modify or Recombine Cells In one aspect, the invention provides methods of evolving a cell to acquire a desired function.", "Such methods entail, e.g., introducing a library of DNA fragments into a plurality of cells, whereby at least one of the fragments undergoes recombination with a segment in the genome or an episome of the cells to produce modified cells.", "Optionally, these modified cells are bred to increase the diversity of the resulting recombined cellular population.", "The modified cells, or the recombined cellular population are then screened for modified or recombined cells that have evolved toward acquisition of the desired function.", "DNA from the modified cells that have evolved toward the desired function is then optionally recombined with a further library of DNA fragments, at least one of which undergoes recombination with a segment in the genome or the episome of the modified cells to produce further modified cells.", "The further modified cells are then screened for further modified cells that have further evolved toward acquisition of the desired function.", "Steps of recombination and screening/selection are repeated as required until the further modified cells have acquired the desired function.", "In one preferred embodiment, modified cells are recursively recombined to increase diversity of the cells prior to performing any selection steps on any resulting cells.", "4.2.1.3.2 Coat with RecA In some methods, the library or further library of DNA fragments is coated with recA protein to stimulate recombination with the segment of the genome.", "The library of fragments is optionally denatured to produce single-stranded DNA, which are annealed to produce duplexes, some of which contain mismatches at points of variation in the fragments.", "Duplexes containing mismatches are optionally selected by affinity chromatography to immobilized MutS.", "4.2.1.33 Perform In Vivo Recombination The invention further provides methods for performing in vivo recombination.", "At least first and second segments from at least one gene are introduced into a cell, the segments differing from each other in at least two nucleotides, whereby the segments recombine to produce a library of chimeric genes.", "A chimeric gene is selected from the library having acquired a desired function.", "The invention further provides methods of evolving a cell to acquire a desired function.", "These methods entail providing a populating of different cells.", "The cells are cultured under conditions whereby DNA is exchanged between cells, forming cells with hybrid genomes.", "The cells are then screened or selected for cells that have evolved toward acquisition of a desired property.", "The DNA exchange and screening/selecting steps are repeated, as needed, with the screened/selected cells from one cycle forming the population of different cells in the next cycle, until a cell has acquired the desired property.", "Mechanisms of DNA exchange include conjugation, phage-mediated transduction, liposome delivery, protoplast fusion, and sexual recombination of the cells.", "Optionally, a library of DNA fragments can be transformed or electroporated into the cells.", "4.2.13.4 Protoplast-Mediated Exchange As noted, some methods of evolving a cell to acquire a desired property are effected by protoplast-mediated exchange of DNA between cells.", "Such methods entail forming protoplasts of a population of different cells.", "The protoplasts are then fused to form hybrid protoplasts, in which genomes from the protoplasts recombine to form hybrid genomes.", "The hybrid protoplasts are incubated under conditions promoting regeneration of cells.", "The regenerated cells can be recombined one or more times (i.e., via protoplasting or any other method than combines genomes of cells) to increase the diversity of any resulting cells.", "Preferably, regenerated cells are recombined several times, e.g., by protoplast fusion to generate a diverse population of cells.", "The next step is to select or screen to isolate regenerated cells that have evolved toward acquisition of the desired property.", "DNA exchange and selection/screening steps are repeated, as needed, with regenerated cells in one cycle being used to form protoplasts in the next cycle until the regenerated cells have acquired the desired property.", "Industrial microorganisms are a preferred class of organisms for conducting the above methods.", "Some methods further comprise a step of selecting or screening for fused protoplasts free from unfused protoplasts of parental cells.", "Some methods further comprise a step of selecting or screening for fused protoplasts with hybrid genomes free from cells with parental genomes.", "In some methods, protoplasts are provided by treating individual cells, mycelia or spores with an enzyme that degrades cell walls.", "In some methods, the strain is a mutant that is lacking capacity for intact cell wall synthesis, and protoplasts form spontaneously.", "In some methods, protoplasts are formed by treating growing cells with an inhibitor of cell wall formation to generate protoplasts.", "In some methods, the desired property is expression and/or secretion of a protein or secondary metabolite, such as an industrial enzyme, a therapeutic protein, a primary metabolite such as lactic acid or ethanol, or a secondary metabolite such as erythromycin cyclosporin A or taxol.", "In other methods it is the ability of the cell to convert compounds provided to the cell to different compounds.", "In yet other methods, the desired property is capacity for meiosis.", "In some methods, the desired property is compatibility to form a heterokaryon with another strain.", "The invention further provides methods of evolving a cell toward acquisition of a desired property.", "These methods entail providing a population of different cells.", "DNA is isolated from a first subpopulation of the different cells and encapsulated in liposomes.", "Protoplasts are formed from a second subpopulation of the different cells.", "Liposomes are fused with the protoplasts, whereby DNA from the liposomes is taken up by the protoplasts and recombines with the genomes of the protoplasts.", "The protoplasts are incubated under regenerating conditions.", "Regenerating or regenerated cells are then selected or screened for evolution toward the desired property.", "4.2.1.3.4 Reiterative Pooling and Breeding of higher Organisms The method also provides methods of reiterative pooling and breeding of higher organisms.", "In the methods, a library of diverse multicellular organisms are produced (e.g., plants, animals or the like).", "A pool of male gametes is provided along with a pool of female gametes.", "At least one of the male pool or the female pool comprises a plurality of different gametes derived from different strains of a species or different species.", "The male gametes are used to fertilize the female gametes.", "At least a portion of the resulting fertilized gametes grow into reproductively viable organisms.", "These reproductively viable organisms are crossed (e.g., by pairwise pooling and joining of the male and female gametes as before) to produce a library of diverse organisms.", "The library is then selected for a desired trait or property.", "The library of diverse organisms can comprise a plurality of plants such as Gramineae, Fetucoideae, Poacoideae, Agrostis, Phleum, Dactylis, Sorgum, Setaria, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Poa, Festuca, Stenotaphrum, Cynodon, Coix, Olyreae, Phareae, Compositae or Leguminosae.", "For example, the plants can be e.g., corn, rice, wheat, rye, oats, barley, pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, sweet pea, sorghum, millet, sunflower, canola or the like.", "Similarly, the library of diverse organisms can include a plurality of animals such as non-human mammals, fish, insects, or the like.", "Optionally, a plurality of selected library members can be crossed by pooling gametes from the selected members and repeatedly crossing any resulting additional reproductively viable organisms to produce a second library of diverse organisms (e.g., by split pair wise pooling and rejoining of the male and female gametes).", "Here again, the second library can be selected for a desired trait or property, with the resulting selected members forming the basis for additional poolwise breeding and selection.", "A feature of the invention is the libraries made by these (or any preceding) method.", "4.3.Origin of Cells 4.3.1 Embryonic Cells of an Animal In some methods, the plurality of cells are embryonic cells of an animal, and the method further comprises propagating the transformed cells to transgenic animals.", "The plurality of cells can be a plurality of industrial microorganisms that are enriched for microorganisms which are tolerant to desired process conditions (heat, light, radiation, selected pFL presence of detergents or other denaturants, presence of alcohols or other organic molecules, etc.).", "4.3.2 Artificial Chromosomes The invention further provides methods of evolving a cell toward acquisition of a desired property using artificial chromosomes.", "Such methods entail introducing a DNA fragment library cloned into an artificial chromosome into a population of cells.", "The cells are then cultured under conditions whereby sexual recombination occurs between the cells, and DNA fragments cloned into the artificial chromosome recombines by homologous recombination with corresponding segments of endogenous chromosomes of the populations of cells, and endogenous chromosomes recombine with each other.", "Cells can also be recombined via conjugation.", "Any resulting cells can be recombined via any method noted herein, as many times as desired, to generate a desired level of diversity in the resulting recombinant cells.", "In any case, after generating a diverse library of cells, the cells that have evolved toward acquisition of the desired property are screened and/or selected for a desired property.", "The method is then repeated with cells that have evolved toward the desired property in one cycle forming the population of different cells in the next cycle.", "Here again, multiple cycles of in vivo recombination are optionally performed prior to any additional selection or screening steps.", "The invention further provides methods of evolving a DNA segment cloned into an artificial chromosome for acquisition of a desired property.", "These methods entail providing a library of variants of the segment, each variant cloned into separate copies of an artificial chromosome.", "The copies of the artificial chromosome are introduced into a population of cells.", "The cells are cultured under conditions whereby sexual recombination occurs between cells and homologous recombination occurs between copies of the artificial chromosome bearing the variants.", "Variants are then screened or selected for evolution toward acquisition of the desired property.", "The invention further provides hyperrecombinogenic recA proteins.", "4.4.Method to Acquire a Biocatalytic Activity One aspect of the invention is a method of evolving a biocatalytic activity of a cell, comprising: (a) recombining at least a first and second DNA segment from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (b) screening at least one recombinant gene from the library that confers enhanced ability to catalyze the reaction of interest by the cell relative to a wild type form of the gene; (c) recombining at least a segment from at least one recombinant gene with a further DNA segment from at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (d) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the reaction of interest in the cell relative to a previous recombinant gene; (e) repeating (c) and (d), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the reaction of interest by the cell.", "4.4.1.Method to Evolve a Gene to Catalyze a RXN of Interest Another aspect of the invention is a method of evolving a gene to confer ability to catalyze a reaction of interest, the method comprising: (1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (2) screening at least one recombinant gene from the library that confers enhanced ability to catalyze a reaction of interest relative to a wild type form of the gene; (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze a reaction of interest relative to a previous recombinant gene; (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze a reaction of interest.", "4.4.2.Method to Generate a New Biocatalytic Activity in a Cell A further aspect of the invention is a method of generating a new biocatalytic activity in a cell, comprising: (1) recombining at least first and second DNA segments from at least one gene conferring ability to catalyze a first reaction related to a second reaction of interest, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (2) screening at least one recombinant gene from the library that confers a new ability to catalyze the second reaction of interest; (3) recombining at least a segment from at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (4) screening at least one further recombinant gene from the further library of recombinant genes that confers enhanced ability to catalyze the second reaction of interest in the cell relative to a previous recombinant gene; (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of enhanced ability to catalyze the second reaction of interest in the cell.", "4.4.3.Method to Modify a Metabolic Pathway Evolved by Recursive Sequence Recombination Another aspect of the invention is a modified form of a cell, wherein the modification comprises a metabolic pathway evolved by recursive sequence recombination.", "A further aspect of the invention is a method of optimizing expression of a gene product, the method comprising: (1) recombining at least first and second DNA segments from at least one gene conferring ability to produce the gene product, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (2) screening at least one recombinant gene from the library that confers optimized expression of the gene product relative to a wild type form of the gene; (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to produce the gene product relative to a previous recombinant gene; (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of optimized ability to express the gene product.", "4.4.4.Method to Evolve a Biosensor A further aspect of the invention is a method of evolving a biosensor for a compound A of interest, the method comprising: (1) recombining at least first and second DNA segments from at least one gene conferring ability to detect a related compound B, the segments differing from each other in at least two nucleotides, to produce a library of recombinant genes; (2) screening at least one recombinant gene from the library that confers optimized ability to detect compound A relative to a wild type form of the gene; (3) recombining at least a segment from the at least one recombinant gene with a further DNA segment from the at least one gene, the same or different from the first and second segments, to produce a further library of recombinant genes; (4) screening at least one further recombinant gene from the further library of recombinant genes that confers optimized ability to detect compound A relative to a previous recombinant gene; (5) repeating (3) and (4), as necessary, until the further recombinant gene confers a desired level of optimized ability to detect compound A.", "4.5.Fermentation of Micro-Organisms The fermentation of microorganisms for the production of natural products is the oldest and most sophisticated application of biocatalysis.", "Industrial microorganisms effect the multistep conversion of renewable feedstocks to high value chemical products in a single reactor and in so doing catalyze a multi-billion dollar industry.", "Fermentation products range from fine and commodity chemicals such as ethanol, lactic acid, amino acids and vitamins, to high value small molecule pharmaceuticals, protein pharmaceuticals, and industrial enzymes.", "(See, e.g., McCoy (1998) C&EN 13-19) for an introduction to biocatalysis.", "The methods herein allow biocatalysts to be improved at a faster pace than conventional methods.", "Whole genome stochastic &/or non-stochastic mutagenesis can at least double the rate of strain improvement for microorganisms used in fermentation as compared to traditional methods.", "This provides for a relative decrease in the cost of fermentation processes.", "New products can enter the market sooner, producers can increase profits as well as market share, and consumers gain access to more products of higher quality and at lower prices.", "Further, increased efficiency of production processes translates to less waste production and more frugal use of resources.", "Whole genome stochastic &/or non-stochastic mutagenesis provides a means of accumulating multiple useful mutation per cycle and thus eliminate the inherent limitation of current strain improvement programs (SIPs).", "One key to SIP is having an assay that can be dependably used to identify a few mutants out of thousands that have subtle increases in product yield.", "The limiting factor in many assay formats is the uniformity of cell growth.", "This variation is the source of baseline variability in subsequent assays.", "Inoculum size and culture environment (temperature/humidity) are sources of cell growth variation.", "Automation of all aspects of establishing initial cultures and state-of-the-art temperature and humidity controlled incubators are useful in reducing variability.", "Mutant cells or spores are separated on solid media to produce individual sporulating colonies.", "Using an automated colony picker (Q-bot, Genefix, U.K.), colonies are identified, picked, and 10,000 different mutants inoculated into 96 well microtitre dishes containing two 3 mm.", "glass balls/well.", "The Q-bot does not pick an entire colony but rather inserts a pin through the center of the colony and exits with a small sampling of cells (or mycelia) and spores.", "The time the pin is in the colony, the number of dips to inoculate the culture medium, and the time the pin is in that medium each effect inoculum size, and each can be controlled and optimized.", "The uniform process of the Q-bot decreases human handling error and increases the rate of establishing cultures (roughly 10,000/4 hours).", "These cultures are then shaken in a temperature and humidity controlled incubator.", "The glass balls act to promote uniform aeration of cells and the dispersal of mycelial fragments similar to the blades of a fermenter.", "1.Prescreen The ability to detect a subtle increase in the performance of a mutant over that of a parent strain relies on the sensitivity of the assay.", "The chance of finding the organisms having an improvement is increased by the number of individual mutants that can be screened by the assay.", "To increase the chances of identifying a pool of sufficient size a prescreen that increases the number of mutants processed by 10-fold can be used.", "The goal of the primary screen will be to quickly identify mutants having equal or better product titres than the parent strain(s) and to move only these mutants forward to liquid cell culture.", "The primary screen is an agar plate screen is analyzed by the Q-bot colony picker.", "Although assays can be fundamentally different, many result, e.g., in the production of colony halos.", "For example, antibiotic production is assayed on plates using an overlay of a sensitive indicator strain, such as B. subtilis.", "Antibiotic production is typically assayed as a zone of clearing (inhibited growth of the indicator organism) around the producing organism.", "Similarly, enzyme production can be assayed on plates containing the enzyme substrate, with activity being detected as a zone of substrate modification around the producing colony.", "Product titre is correlated with the ratio of halo area to colony area.", "The Q-bot or other automated system is instructed to only pick colonies having a halo ratio in the top 10% of the population i.e.", "10,000 mutants from the 100,000 entering the plate prescreen.", "This increases the number of improved clones in the secondary assay and eliminates the wasted effort of screening knock-out and low producers.", "This improves the “hit rate” of the secondary assay.", "4.6.Experimental Applications 4.6.1 Stochastic &/or Non-Stochastic Mutagenesis 4.6.1.1 General Techniques 4.6.1.1.1 Starting Materials Thus, a general method for recursive sequence recombination for the embodiments herein is to begin with a gene encoding an enzyme or enzyme subunit and to evolve that gene either for ability to act on a new substrate, or for enhanced catalytic properties with an old substrate, either alone or in combination with other genes in a multistep pathway.", "The term “gene” is used herein broadly to refer to any segment or sequence of DNA associated with a biological function.", "Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.", "The ability to use a new substrate can be assayed in some instances by the ability to grow on a substrate as a nutrient source.", "In other circumstances such ability can be assayed by decreased toxicity of a substrate for a host cell, hence allowing the host to grow in the presence of that substrate.", "Biosynthesis of new compounds, such as antibiotics, can be assayed similarly by growth of an indicator organism in the presence of the host expressing the evolved genes.", "For example, when an indicator organism used in an overlay of the host expressing the evolved gene(s), wherein the indicator organism is sensitive or expected to be sensitive to the desired antibiotic, growth of the indicator organism would be inhibited in a zone around the host cell or colony expressing the evolved gene(s).", "Another method of identifying new compounds is the use of standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc.", "Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools.", "Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”).", "In some instances, the starting material for recursive sequence recombination is a discrete gene, cluster of genes, or family of genes known or thought to be associated with metabolism of a particular class of substrates.", "One of the advantages of the instant invention is that structural information is not required to estimate which parts of a sequence should be mutated to produce a functional hybrid enzyme.", "In some embodiments of the invention, an initial screening of enzyme activities in a particular assay can be useful in identifying candidate enzymes as starting materials.", "For example, high throughput screening can be used to screen enzymes for dioxygenase type activities using aromatic acids as substrates.", "Dioxygenases typically transform indole-2-carboxylate and indole-3-carboxylate to colored products, including indigo (Eaton et. al.", "J. Bacteriol.", "177: 6983-6988 (1995)).", "DNA encoding enzymes that give some activity in the initial assay can then be recombined by the recursive techniques of the invention and rescreened.", "The use of such initial screening for candidate enzymes against a desired target molecule or analog of the target molecule can be especially useful to generate enzymes that catalyze reactions of interest such as catabolism of man-made pollutants.", "This type of high throughput screening can also be used during each round of recursive sequence recombination to identify mutants that possess the highest level of the desired activity.", "For example, penicillin G acylases have been isolated by looking for clones that allow a leucine auxotroph to hydrolyse penicillin G analogue phenylacetyl-L-leucine, thereby producing leucine and allowing cell growth (Martin, L. et al., FEMS Microbiology Lett.", "125: 287-292 (1995)).", "Positives from this selection are then screened by a more labour-intensive method for ability to hydrolyse penicillin G. This same selection on phenylacetyl-L-leucine can be used when evolving penicillin G acylase for greater activity by recursive sequence recombination.", "After each round of recombination the library of acylase genes is transformed into a leucine auxotroph.", "Those that grow fastest are picked as probably having the most active acylase.", "The acylases are then be tested against the real substrate, penicillin G, by a more laborious screen such as HPLC.", "Thus, even if there is no convenient high throughput screen for an enzyme or a metabolic pathway, it is often possible to find a rapid detection method that can approximately measure the desired phenotype, thereby reducing the numbers of colonies that must be screened more accurately.", "The starting material can also be a segment of such a gene or cluster that is recombined in isolation of its surrounding DNA, but is relinked to its surrounding DNA before screening/selection of recombination products.", "In other instances, the starting material for recombination is a larger segment of DNA that includes a coding sequence or other locus associated with metabolism of a particular substrate at an unknown location.", "For example, the starting material can be a chromosome, episome, YAC, cosmid, or phage P1 clone.", "In still other instances, the starting material is the whole genome of an organism that is known to have desirable metabolic properties, but for which no information localizing the genes associated with these characteristics is available.", "In general any type of cells can be used as a recipient of evolved genes.", "Cells of particular interest include many bacterial cell types, both gram-negative and gram-positive, such as Rhodococcus, Streptomycetes, Actinomycetes, Corynebacteria, Penicillium, Bacillus, Escherichia coli, Pseudomonas, Salmonella, and Erwinia.", "Cells of interest also include eukaryotic cells, particularly mammalian cells (e.g., mouse, hamster, primate, human), both cell lines and primary cultures.", "Such cells include stem cells, including embryonic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIHM), kidney, liver, muscle, and skin cells.", "Other eukaryotic cells of interest include plant cells, such as maize, rice, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi (Penicillium, Fusarium, Aspergillus, Podospora, Neurospora), insects, yeasts (Picchia and Saccharomyces).", "The choice of host will depend on a number of factors, depending on the intended use of the engineered host, including pathogenicity, substrate range, environmental hardiness, presence of key intermediates, ease of genetic manipulation, and likelihood of promiscuous transfer of genetic information to other organisms.", "Particularly advantageous hosts are E. coli, lactobacilli, Streptomycetes, Actinomycetes and filamentous fungi.", "The breeding procedure starts with at least two substrates, which generally show substantial sequence identity to each other (i.e., at least about 50%, 70%, 80% or 90% sequence identity) but differ from each other at certain positions.", "The difference can be any type of mutation, for example, substitutions, insertions and deletions.", "Often, different segments differ from each other in perhaps 5-20 positions.", "For recombination to generate increased diversity relative to the starting materials, the starting materials must differ from each other in at least two nucleotide positions.", "That is, if there are only two substrates, there should be at least two divergent positions.", "If there are three substrates, for example, one substrate can differ from the second as a single position, and the second can differ from the third at a different single position.", "The starting DNA segments can be natural variants of each other, for example, allelic or species variants.", "The segments can also be from nonallelic genes showing some degree of structural and is usually functional relatedness (e.g., different genes within a superfamily such as the immunoglobulin superfamily).", "The starting DNA segments can also be induced variants of each other.", "For example, one DNA segment can be produced by error-prone PCR replication of the other, or by substitution of a mutagenic cassette.", "Induced mutants can also be prepared by propagating one (or both) of the segments in a mutagenic strain.", "In these situations, strictly speaking, the second DNA segment is not a single segment but a large family of related segments.", "The different segments forming the starting materials are often the same length or substantially the same length.", "However, this need not be the case; for example; one segment can be a subsequence of another.", "The segments can be present as part of larger molecules, such as vectors, or can be in isolated form.", "The starting DNA segments are recombined by any of the recursive sequence recombination formats described above to generate a diverse library of recombinant DNA segments.", "Such a library can vary widely in size from having fewer than to more than 105, 107, or 109 members.", "In general, the starting segments and the recombinant libraries generated include full-length coding sequences and any essential regulatory sequences, such as a promoter and polyadenylation sequence, required for expression.", "However, if this is not the case, the recombinant DNA segments in the library can be inserted into a common vector providing the missing sequences before performing screening/selection.", "If the recursive sequence recombination format employed is an in vivo format, the library of recombinant DNA segments generated already exists in a cell, which is usually the cell type in which expression of the enzyme with altered substrate specificity is desired.", "If recursive sequence recombination is performed in vitro, the recombinant library is preferably introduced into the desired cell type before screening/selection.", "The members of the recombinant library can be linked to an episome or virus before introduction or can be introduced directly.", "In some embodiments of the is invention, the library is amplified in a first host, and is then recovered from that host and introduced to a second host more amenable to expression, selection, or screening, or any other desirable parameter.", "The manner in which the library is introduced into the cell type depends on the DNA-uptake characteristics of the cell type, e.g., having viral receptors, being capable of conjugation, or being naturally competent.", "If the cell type is insusceptible to natural and chemical-induced competence, but susceptible to electroporation, one would usually employ electroporation.", "If the cell type is insusceptible to electroporation as well, one can employ biolistics.", "The biolistic PDS-1000 Gene Gun (Biorad, Hercules, Calif.) uses helium pressure to accelerate DNA-coated gold or tungsten microcarriers toward target cells.", "The process is applicable to a wide range of tissues, including plants, bacteria, fungi, algae, intact animal tissues, tissue culture cells, and animal embryos.", "One can employ electronic pulse delivery, which is essentially a mild electroporation format for live tissues in animals and patients.", "Zhao, Advanced Drug Delivery Reviews 17: 257-262 (1995).", "After introduction of the library of recombinant DNA genes, the cells are optionally propagated to allow expression of genes to occur.", "4.6.1.1.2 Selection and Screening Screening is, in general, a two-step process in which one first determines which cells do and do not express a screening marker and then physically separates the cells having the desired property.", "Selection is a form of screening in which identification and physical separation are achieved simultaneously, for example, by expression of a selectable marker, which, in some genetic circumstances, allows cells expressing the marker to survive while other cells die (or vice versa).", "Screening markers include, for example, luciferase, β-galactosidase, and green fluorescent protein.", "Screening can also be done by observing such aspects of growth as colony size, halo formation, etc.", "Additionally, screening for production of a desired compound, such as a therapeutic drug or “designer chemical” can be accomplished by observing binding of cell products to a receptor or ligand, such as on a solid support or on a column.", "Such screening can additionally be accomplished by binding to antibodies, as in an ELISA.", "In some instances the screening process is preferably automated so as to allow screening of suitable numbers of colonies or cells.", "Some examples of automated screening devices include fluorescence activated cell sorting, especially in conjunction with cells immobilized in agarose (see Powell et. al.", "Bio/Technology 8: 333-337 (1990); Weaver et. al.", "Methods 2: 234-247 (1991)), automated ELISA assays, scintillation proximity assays (Hart, H. E. et al., Molecular Immunol.", "16: 265-267 (1979)) and the formation of fluorescent, coloured or UV absorbing compounds on agar plates or in microtitre wells (Krawiec, S., Devel.", "Indust.", "Microbiology 31: 103-114 (1990)).", "Selectable markers can include, for example, drug, toxin resistance, or nutrient synthesis genes.", "Selection is also done by such techniques as growth on a toxic substrate to select for hosts having the ability to detoxify a substrate, growth on a new nutrient source to select for hosts having the ability to utilize that nutrient source, competitive growth in culture based on ability to utilize a nutrient source, etc.", "In particular, uncloned but differentially expressed proteins (e.g., those induced in response to new compounds, such as biodegradable pollutants in the medium) can be screened by differential display (Appleyard et al.", "Mol.", "Gen. Gent.", "247: 338-342 (1995)).", "Hopwood (Phil Trans R. Soc.", "Lond B 324: 549-562) provides a review of screens for antibiotic production.", "Omura (Microbio.", "Rev.", "50: 259-279 (1986) and Nisbet (Ann Rev.", "Med.", "Chem.", "21: 149-157 (1986)) disclose screens for antimicrobial agents, including supersensitive bacteria, detection of beta-lactamase and D,D-carboxypeptidase inhibition, beta-lactamase induction, chromogenic substrates and monoclonal antibody screens.", "Antibiotic targets can also be used as screening targets in high throughput screening.", "Antifungals are typically screened by inhibition of fungal growth.", "Pharmacological agents can be identified as enzyme inhibitors using plates containing the enzyme and a chromogenic substrate, or by automated receptor assays.", "Hydrolytic enzymes (e.g., proteases, amylases) can be screened by including the substrate in an agar plate and scoring for a hydrolytic clear zone or by using a colorimetric indicator (Steele et al.", "Ann.", "Rev.", "Microbiol.", "45: 89-106 (1991)).", "This can be coupled with the use of stains to detect the effects of enzyme action (such as congo red to detect the extent of degradation of celluloses and hemicelluloses).", "Tagged substrates can also be used.", "For example, lipases and esterases can be screened using different lengths of fatty acids linked to umbelliferyl.", "The action of lipases or esterases removes this tag from the fatty acid, resulting in a quenching or enhancement of umbelliferyl fluorescence.", "These enzymes can be screened in microtiter plates by a robotic device.", "4.6.1.1.3 FACS Fluorescence activated cell sorting (FACS) methods are also a powerful tool for selection/screening.", "In some instances a fluorescent molecule is made within a cell (e.g., green fluorescent protein).", "The cells producing the protein can simply be sorted by FACS.", "Gel microdrop technology allows screening of cells encapsulated in agarose microdrops (Weaver et al.", "Methods 2: 234-247 (1991)).", "In this technique products secreted by the cell (such as antibodies or antigens) are immobilized with the cell that generated them.", "Sorting and collection of the drops containing the desired product thus also collects the cells that made the product, and provides a ready source for the cloning of the genes encoding the desired functions.", "Desired products can be detected by incubating the encapsulated cells with fluorescent antibodies (Powell et al.", "Bio/Technology 8: 333-337 (1990)).", "FACS sorting can also be used by this technique to assay resistance to toxic compounds and antibiotics by selecting droplets that contain multiple cells (i.e., the product of continued division in the presence of a cytotoxic compound; Goguen et al.", "Nature 363: 189-190 (1995)).", "This method can select for any enzyme that can change the fluorescence of a substrate that can be immobilized in the agarose droplet.", "4.6.1.1.4 Reporter Molecule In some embodiments of the invention, screening can be accomplished by assaying reactivity with a reporter molecule reactive with a desired feature of, for example, a gene product.", "Thus, specific functionalities such as antigenic domains can be screened with antibodies specific for those determinants.", "4.6.1.1.5 Cell-Cell Indictaor In other embodiments of the invention, screening is preferably done with a cell-cell indicator assay.", "In this assay format, separate library cells (Cell A, the cell being assayed) and reporter cells (Cell B, the assay cell) are used.", "Only one component of the system, the library cells, is allowed to evolve.", "The screening is generally carried out in a two-dimensional immobilized format, such as on plates.", "The products of the metabolic pathways encoded by these genes (in this case, usually secondary metabolites such as antibiotics, polyketides, carotenoids, etc.)", "diffuse out of the library cell to the reporter cell.", "The product of the library cell may affect the reporter cell in one of a number of ways.", "The assay system (indicator cell) can have a simple readout (e.g., green fluorescent protein, luciferase, β-galactosidase) which is induced by the library cell product but which does not affect the library cell.", "In these examples the desired product can be detected by calorimetric changes in the reporter cells adjacent to the library cell.", "4.6.1.1.6 Feedback Mechanism In other embodiments, indicator cells can in turn produce something that modifies the growth rate of the library cells via a feedback mechanism.", "Growth rate feedback can detect and accumulate very small differences.", "For example, if the library and reporter cells are competing for nutrients, library cells producing compounds to inhibit the growth of the reporter cells will have more available nutrients, and thus will have more opportunity for growth.", "This is a useful screen for antibiotics or a library of polyketide synthesis gene clusters where each of the library cells is expressing and exporting a different polyketide gene product.", "4.6.1.1.7 Secretion Another variation of this theme is that the reporter cell for an antibiotic selection can itself secrete a toxin or antibiotic that inhibits growth of the library cell.", "Production by the library cell of an antibiotic that is able to suppress growth of the reporter cell will thus allow uninhibited growth of the library cell.", "Conversely, if the library is being screened for production of a compound that stimulates the growth of the reporter cell (for example, in improving chemical syntheses, the library cell may supply nutrients such as amino acids to an auxotrophic reporter, or growth factors to a growth-factor-dependent reporter.", "The reporter cell in turn should produce a compound that stimulates the growth of the library cell.", "Interleukins, growth factors, and nutrients are possibilities.", "Further possibilities include competition based on ability to kill surrounding cells, positive feedback loops in which the desired product made by the evolved cell stimulates the indicator cell to produce a positive growth factor for cell A, thus indirectly selecting for increased product formation.", "In some embodiments of the invention it can be advantageous to use a different organism (or genetic background) for screening than the one that will be used in the final product.", "For example, markers can be added to DNA constructs used for recursive sequence recombination to make the microorganism dependent on the constructs during the improvement process, even though those markers may be undesirable in the final recombinant microorganism.", "Likewise, in some embodiments it is advantageous to use a different substrate for screening an evolved enzyme than the one that will be used in the final product.", "For example, Evnin et al.", "(Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 87: 6659-6663 (1990)) selected trypsin variants with altered substrate specificity by requiring that variant trypsin generate an essential amino acid for an arginine auxotroph by cleaving arginine-naphthylamide.", "This is thus a selection for arginine-specific trypsin, with the growth rate of the host being proportional to that of the enzyme activity.", "The pool of cells surviving screening and/or selection is enriched for recombinant genes conferring the desired phenotype (e.g.", "altered substrate specificity, altered biosynthetic ability, etc.).", "Further enrichment can be obtained, if desired, by performing a second round of screening and/or selection without generating additional diversity.", "The recombinant gene or pool of such genes surviving one round of screening/selection forms one or more of the substrates for a second round of recombination.", "Again, recombination can be performed in vivo or in vitro by any of the recursive sequence recombination formats described above.", "If recursive sequence recombination is performed in vitro, the recombinant gene or genes to form the substrate for recombination should be extracted from the cells in which screening/selection was performed.", "Optionally, a subsequence of such gene or genes can be excised for more targeted subsequent recombination.", "If the recombinant gene(s) are contained within episomes, their isolation presents no difficulties.", "If the recombinant genes are chromosomally integrated, they can be isolated by amplification primed from known sequences flanking the regions in which recombination has occurred.", "Alternatively, whole genomic DNA can be isolated, optionally amplified, and used as the substrate for recombination.", "Small samples of genomic DNA can be amplified by whole genome amplification with degenerate primers (Barrett et al.", "Nucleic Acids Research 23: 3488-3492 (1995)).", "These primers result in a large amount of random 3′ ends, which can undergo homologous recombination when reintroduced into cells.", "If the second round of recombination is to be performed in vivo, as is often the case, it can be performed in the cell surviving screening/selection, or the recombinant genes can be transferred to another cell type (e.g., a cell is type having a high frequency of mutation and/or recombination).", "In this situation, recombination can be effected by introducing additional DNA segment(s) into cells bearing the recombinant genes.", "In other methods, the cells can be induced to exchange genetic information with each other by, for example, electroporation.", "In some methods, the second round of recombination is performed by dividing a pool of cells surviving screening/selection in the first round into two subpopulations.", "DNA from one subpopulation is isolated and transfected into the other population, where the recombinant gene(s) from the two subpopulations recombine to form a further library of recombinant genes.", "In these methods, it is not necessary to isolate particular genes from the first subpopulation or to take steps to avoid random shearing of DNA during extraction.", "Rather, the whole genome of DNA sheared or otherwise cleaved into manageable sized fragments is transfected into the second subpopulation.", "This approach is particularly useful when several genes are being evolved simultaneously and/or the location and identity of such genes within chromosome are not known.", "The second round of recombination is sometimes performed exclusively among the recombinant molecules surviving selection.", "However, in other embodiments, additional substrates can be introduced.", "The additional substrates can be of the same form as the substrates used in the first round of recombination, i.e., additional natural or induced mutants of the gene or cluster of genes, forming the substrates for the first round.", "Alternatively, the additional substrate(s) in the second round of recombination can be exactly the same as the substrate(s) in the first round of replication.", "After the second round of recombination, recombinant genes conferring the desired phenotype are again selected.", "The selection process proceeds essentially as before.", "If a suicide vector bearing a selective marker was used in the first round of selection, the same vector can be used again.", "Again, a cell or pool of cells surviving selection is selected.", "If a pool of cells, the cells can be subject to further enrichment.", "4.6.1.2 General Methods 4.6.1.2.1 In Vitro In Vitro Formats one format for recursive sequence recombination in vitro is illustrated herein.", "The initial substrates for recombination are a pool of related sequences.", "The X's show where the sequences diverge.", "The sequences can be DNA or RNA and can be of various lengths depending on the size of the gene or DNA fragment to be recombined or stochastic &/or non-stochastic mutagenized.", "Preferably the sequences are from 50 bp to 100 kb.", "The pool of related substrates are converted into overlapping fragments, e.g., from about 5 bp to 5 kb or more, as shown herein.", "Often, the size of the fragments is from about 10 bp to 1000 bp, and sometimes the size of the DNA fragments is from about 100 bp to 500 bp.", "The conversion can be effected by a number of different methods, such as DNase1 or RNase digestion, random shearing or partial restriction enzyme digestion.", "Alternatively, the conversion of substrates to fragments can be effected by incomplete PCR amplification of substrates or PCR primed from a single primer.", "Alternatively, appropriate single-stranded fragments can be generated on a nucleic acid synthesizer.", "The concentration of nucleic acid fragments of a particular length and sequence is often less than 0.1% or 1% by weight of the total nucleic acid.", "The number of different specific nucleic acid fragments in the mixture is usually at least about 100, 500 or 1000.The mixed population of nucleic acid fragments are converted to at least partially single-stranded form.", "Conversion can be effected by heating to about 80C to 100C, more preferably from 90C to 96 C, to form single-stranded nucleic acid fragments and then reannealing.", "Conversion can also be effected by treatment with single-stranded DNA binding protein or recA protein.", "Single-stranded nucleic acid fragments having regions of sequence identity with other single-stranded nucleic acid fragments can then be reannealed by cooling to 4C to 75C, and preferably from 40C to 65C.", "Renaturation can be accelerated by the addition of polyethylene glycol (PEG), other volume-excluding reagents or salt.", "The salt concentration is preferably from 0 mM to 200 mM more preferably the salt concentration is from 10 mM to 100 mM.", "The salt may be KCl or NaCl.", "The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.", "The fragments that reanneal can be from different substrates as shown herein.", "The annealed nucleic acid fragments are incubated in the presence of a nucleic acid polymerase, such as Taq or Klenow, or proofreading polymerases, such as pfu or pwo, and dNTP's (i.e.", "dATP, dCTP, dGTP and dTTP).", "If regions of sequence identity are large, Taq polymerase can be used with an annealing temperature of between 45-65C.", "If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30T (Stemmer, Proc.", "Natl.", "Acad.", "Sci.", "USA (1994), supra).", "The polymerase can be added to the random nucleic acid fragments prior to annealing, simultaneously with annealing or after annealing.", "The process of denaturation, renaturation and incubation in the presence of polymerase of overlapping fragments to generate a collection of polynucleotides containing different permutations of fragments is sometimes referred to as stochastic &/or non-stochastic mutagenesis of the nucleic acid in vitro.", "This cycle is repeated for a desired number of times.", "Preferably the cycle is repeated from 2 to 100 times, more preferably the sequence is repeated from 10 to 40 times.", "The resulting nucleic acids are a family of double-stranded polynucleotides of from about 50 bp to about 100 kb, preferably from 500 bp to 50 kb, as shown herein.", "The population represents variants of the starting substrates showing substantial sequence identity thereto but also diverging at several positions.", "The population has many more members than the starting substrates.", "The population of fragments resulting from stochastic &/or non-stochastic mutagenesis is used to transform host cells, optionally after cloning into a vector.", "4.6.1.2.1.1 Full Length Sequences In a variation of in vitro stochastic &/or non-stochastic mutagenesis, subsequences of recombination substrates can be generated by amplifying the full-length sequences under conditions which produce a substantial fraction, typically at least 20 percent or more, of incompletely extended amplification products.", "The amplification products, including the incompletely extended amplification products are denatured and subjected to at least one additional cycle of reannealing and amplification.", "This variation, wherein at least one cycle of reannealing and amplification provides a substantial fraction of incompletely extended products, is termed “stuttering.” In the subsequent amplification round, the incompletely extended products anneal to and prime extension on different sequence-related template species.", "4.6.1.2.1.2 Overlapping Single Stranded DNA Fragments In a further variation, at least one cycle of amplification can be conducted using a collection of overlapping single-stranded DNA fragments of related sequence, and different lengths.", "Each fragment can hybridize to and prime polynucleotide chain extension of a second fragment from the collection, thus forming sequence-recombined polynucleotides.", "In a further variation, single-stranded DNA fragments of variable length can be generated from a single primer by Vent DNA polymerase on a first DNA template.", "The single stranded DNA fragments are used as primers for a second, Kunkel-type template, consisting of a uracil-containing circular single-stranded DNA.", "This results in multiple substitutions of the first template into the second (see Levichkin et al.", "Mol.", "Biology 29: 572-577 (1995)).", "4.6.1.2.1.3 Gene Clusters Gene clusters such as those involved in polyketide synthesis (or indeed any multi-enzyme pathways catalyzing is analogous metabolic reactions) can be recombined by recursive sequence recombination even if they lack DNA sequence homology.", "Homology can be introduced using synthetic oligonucleotides as PCR primers.", "In addition to the specific sequences for the gene being amplified, all of the primers used to amplify one type of enzyme (for example the acyl carrier protein in polyketide synthesis) are synthesized to contain an additional sequence of 2040 bases 51 to the gene (sequence A) and a different 2040 base sequence 31 to the gene (sequence B).", "The adjacent gene (in this case the keto-synthase) is amplified using a 51 primer which contains the complementary strand of sequence B (sequence B′), and a 31 primer containing a different 2040 base sequence (C).", "Similarly, primers for the next adjacent gene (keto-reductases) contain sequences C′ (complementary to C) and D. If 5 different polyketide gene clusters are being stochastic &/or non-stochastic mutagenized, all five acyl carrier proteins are flanked by sequences A and B following their PCR amplification.", "In this way, small regions of homology are introduced, making the gene clusters into site specific recombination cassettes.", "Subsequent to the initial amplification of individual genes, the amplified genes can then be mixed and subjected to primerless PCR.", "Sequence B at the 3′ end of all of the five acyl carrier protein genes can anneal with and prime DNA synthesis from sequence B1 at the 5′ end of all five keto reductase genes.", "In this way all possible combinations of genes within the cluster can be obtained.", "Oligonucleotides allow such recombinants to be obtained in the absence of sufficient sequence homology for recursive sequence recombination described above.", "Only homology of function is required to produce functional gene clusters.", "4.6.1.2.1.4 Multi Subunit Enzymes This method is also useful for exploring permutations of any other multi-subunit enzymes.", "An example of such enzymes composed of multiple polypeptides that have shown novel functions when the subunits are combined in novel ways are dioxygenases.", "Directed recombination between the four protein subunits of biphenyl and toluene dioxygenases produced functional dioxygenases with increased activity against trichloroethylene (Furukawa et. al.", "J. Bacteriol.", "176: 2121-2123 (1994)).", "This combination of subunits from the two dioxygenases could also have been produced by cassette-stochastic &/or non-stochastic mutagenesis of the dioxygenases as described above, followed by selection for degradation of trichloroethylene.", "In some polyketide synthases, the separate functions of the acyl carrier protein, keto-synthase, keto-reductase, etc.", "reside in a single polypeptide.", "In these cases domains within the single polypeptide may be stochastic &/or non-stochastic mutagenized, even if sufficient homology does not exist naturally, by introducing regions of homology as described above for entire genes.", "In this case, it may not be possible to introduce additional flanking sequences to the domains, due to the constraint of maintaining a continuous open reading frame.", "Instead, groups of oligonucleotides are synthesized that are homologous to the 3′ end of the first domain encoded by one of the genes to be stochastic &/or non-stochastic mutagenized, and the 5′ ends of the second domains encoded by all of the other genes to be stochastic &/or non-stochastic mutagenized together.", "This is repeated with all domains, thus providing sequences that allow recombination between protein domains while maintaining their order.", "4.6.1.2.1.5 Cassette-Based The cassette-based recombination method can be combined with recursive sequence recombination by including gene fragments (generated by DNase, physical shearing, DNA stuttering, etc.)", "for one or more of the genes.", "Thus, in addition to different combinations of entire genes within a cluster (e.g., for polyketide synthesis), individual genes can be stochastic &/or non-stochastic mutagenized at the same time (e.g., all acyl carrier protein genes can also be provided as fragmented DNA), allowing a more thorough search of sequence space.", "4.6.1.2.1.6 In Vitro Whole Genome Stochastic &/or Non-Stochastic Mutagenesis The stochastic &/or non-stochastic mutagenesis of large DNA sequences, such as eukaryotic chromosomes, is difficult by prior art in vitro stochastic &/or non-stochastic mutagenesis methods.", "A method for overcoming this limitation is described herein.", "The cells of related eukaryotic species are gently lysed and the intact chromosomes are liberated.", "The liberated chromosomes are then sorted by FACS or similar method (such as pulse field electrophoresis) with chromosomes of similar size being sequestered together.", "Each size fraction of the sorted chromosomes generally will represent a pool of analogous chromosomes, for example the Y chromosome of related mammals.", "The goal is to isolate intact chromosomes that have not been irreversibly damaged.", "The fragmentation and stochastic &/or non-stochastic mutagenesis of such large complex pieces of DNA employing DNA polymerases is difficult and would likely introduce an unacceptably high level of random mutations.", "An alternative approach that employs restriction enzymes and DNA ligase provides a feasible less destructive solution.", "A chromosomal fraction is digested with one or more restriction enzymes that recognize long DNA sequences (about 15-20 bp), such as the intron and intein encoded endonucleases (1-Ppo 1, I-Ceu 1, PI-Psp 1, PI-Tli 1, PI-Sce I (VDE).", "These enzymes each cut, at most, a few times within each chromosome, resulting in a combinatorial mixture of large fragments, each having overhanging single stranded termini that are complementary to other sites cleaved by the same enzyme.", "The digest is further modified by very short incubation with a single stranded exonuclease.", "The polarity of the nuclease chosen is dependent on the single stranded overhang resulting from the restriction enzyme chosen.", "5′-3′ exonuclease for 3′-overhangs, and 3′-5′-exonuclease for 5′overhangs.", "This digestion results in significantly long regions of ssDNA overhang on each dsDNA termini.", "The purpose of this incubation is to generate regions of DNA that define specific regions of DNA where recombination can occur.", "The fragments are then incubated under condition where the ends of the fragments anneal with other fragments having homologous ssDNA termini.", "Often, the two fragments annealing will have originated from different chromosomes and in the presence of DNA ligase are covalently linked to form a chimeric chromosome.", "This generates genetic diversity mimicking the crossing over of homologous chromosomes.", "The complete ligation reaction will contain a combinatorial mixture of all possible ligations of fragments having homologous overhanging termini.", "A subset of this population will be complete chimeric chromosomes.", "To screen the stochastic &/or non-stochastic mutagenized library, the chromosomes are delivered to a suitable host in a manner allowing for the uptake and expression of entire chromosomes.", "For example, YACs (yeast artificial chromosomes) can be delivered to eukaryotic cells by protoplast fusion.", "Thus, the reassemble library could be encapsulated in liposomes and fused with protoplasts of the appropriate host cell.", "The resulting transformants would be propagated and screened for the desired cellular improvements.", "Once an improved population was identified, the chromosomes would be isolated, stochastic &/or non-stochastic mutagenized, and screened recursively.", "4.6.1.2.1.7 Whole Genome Stochastic &/or Non-Stochastic Mutagenesis of Naturally Competent Microorganisms Natural competence is a phenomenon observed for some microbial species whereby individual cells take up DNA from the environment and incorporate it into their genome by homologous recombination.", "Bacillus subtilis and Acetinetobacter Spp.", "are known to be particularly efficient at this process.", "A method for the whole genome stochastic &/or non-stochastic mutagenesis of these and analogous organisms is described employing this process.", "One goal of whole genome stochastic &/or non-stochastic mutagenesis is the rapid accumulation of useful mutations from a population of individual strains into one superior strain.", "If the organisms to be evolved are naturally competent, then a split pooled strategy for the recursive transformation of naturally competent cells with DNA originating from the pool will effect this process.", "An example procedure is as follows.", "A population of naturally competent organisms that demonstrates a variety of useful traits (such as increased protein secretion) is identified.", "The strains are pooled, and the pool is split.", "One half of the pool is used as a source of gDNA, while the other is used to generate a pool of naturally competent cells.", "The competent cells are grown in the presence of the pooled gDNA to allow DNA uptake and recombination.", "Cells of one genotype uptake and incorporate gDNA from cells of a different type generating cells having chimeric genomes.", "The result is a population of cells representing a combinatorial mixture of the genetic variations originating in the original pool.", "These cells are pooled again and transformed with the same source of DNA again.", "This process is carried out recursively to increase the diversity of the genomes of cells resulting from transformation.", "Once sufficient diversity has been generated, the cell population is screened for new chimeric organisms demonstrating desired improvements.", "This process is enhanced by increasing the natural competence of the host organism.", "COMS is a protein that, when expressed in B. subtilis, enhances the efficiency of natural competence mediated transformation more than an order of magnitude.", "It was demonstrated that approximately 100% of the cells harboring the plasmid pCOMS uptake and recombine genomic DNA fragments into their genomes.", "In general, approximately 10% of the genome is recombined into any given transformed cell.", "This observation was demonstrated by the following.", "A strain of B. subtilis pCOMS auxotrophic for two nutritional markers was transformed with genomic DNA (gDNA) isolated from a prototrophic strain of the same organism.", "10% of the cells exposed to the DNA were prototrophic for one of the two nutrient markers.", "The average size of the DNA strand taken up by B. subtilis is approximately 50 kb or about 2% of the genome.", "Thus 1 of every ten cells had recombined a marker that was represented 1 in every fifty molecules of uptaken gDNA.", "Thus, most of the cells take up and recombine with approximately five 50 kb molecules or 10% of the genome.", "This method represents a powerful tool for rapidly and efficiently recombining whole microbial genomes.", "In the absence of pCOMS, only 0.3% of the cells prepared for natural competency uptake and integrate a specific marker.", "This suggested that about 15% of the cells actually underwent recombination with a single genomic fragment.", "Thus, a recursive transformation strategy as described above produces a whole genome stochastic &/or non-stochastic mutagenized library, even in the absence of pCOMS.", "In the absence of pCOMS, however, the complex genomes will represent a smaller, but still screenable percentage of the transformed or stochastic &/or non-stochastic mutagenized population.", "4.6.1.2.1.8 Congression Congression is the integration of two independent unlinked markers into a cell.", "0.3% of naturally competent B. subtilis cells integrate a single marker (described above).", "Of these, about 10% have taken up an additional marker.", "Thus, if one selects or screens for the integration of one specific marker, 10% of the resulting population will have integrated another specific marker.", "This provides a way of enriching for specific integration events.", "For example, if one is looking for the integration of a gene for which there is no easy screen or selection, it will exist as 0.3% of the cell population.", "If the population is first selected for a specific integration event, then the desired integration will be found in 10% of the population.", "This represents a significant (about 30-fold) enrichment for the desired event.", "This enrichment is defined as the “congression effect.” The congression effect is not influenced by the presence of pCOMS, thus the “pCOMS effect” is simply to increase the percentage of naturally competent cells that are truly naturally competent from about 15% in its absence to 100% in its presence.", "All competent cells still uptake about the same amount of DNA or about 10% of the Bacillus genome.", "The congression effect can be used in the following examples to enhance whole genome stochastic &/or non-stochastic mutagenesis as well, as the targeted integration of stochastic &/or non-stochastic mutagenized genes to the chromosome.", "4.6.1.2.1.9 B. subtilis Stochastic &/or Non-Stochastic Mutagenesis A population of B. subtilis cells having desired properties are identified, pooled and stochastic &/or non-stochastic mutagenized as described above with one exception: once the pooled population is split, half of the population is transformed with an antibiotic selection marker that is flanked by sequence that targets its integration and disruption of a specific nutritional gene, for example, one involved in amino biosynthesis.", "Transformants resistant to the drug are auxotrophic for that nutrient.", "The resistant population is pooled and grown under conditions rendering them naturally competent (or optionally first transformed with pCOMS).", "The competent cells are then transformed with gDNA isolated from the original pool, and prototrophs are selected.", "The prototrophic population will have undergone recombination with genomic fragments encoding a functional copy of the nutritional marker, and thus will be enriched for cells having undergone recombination at other genetic loci by the congression effect.", "4.6.1.2.1.10 Targeting of Genes and Gene Libraries to the Chromosome It is useful to be able to efficiently deliver genes or gene libraries directly to a specific location in a cells chromosome.", "As above, target cells are transformed with a positive selection marker flanked by sequences that target its homologous recombination into the chromosome.", "Selected cells harboring the marker are made naturally competent (with or without pCOMS, but preferably the former) and transformed with a mixture of two sets of DNA fragments.", "The first set contains a gene or a stochastic &/or non-stochastic mutagenized library of genes each flanked with sequence to target its integration to a specific chromosomal loci.", "The second set contains a positive selection marker (different from that first integrated into the cells) flanked by sequence that will target its integration and replacement of the first positive selection marker.", "Under optimal conditions, the mixture is such that the gene or gene library is in molar excess over the positive selection marker.", "Transformants are then selected for cells containing the new positive marker.", "These cells are enriched for cells having integrated a copy of the desired gene or gene library by the congression effect and can be directly screened for cells harboring the gene or gene variants of interest.", "This process was carried out using PCR fragments <10 kb, and it was found that, employing the congression effect, a population can be enriched such that 50% of the cells are congregants.", "Thus, one in two cells contained a gene or gene variant.", "Alternatively, the expression host can be absent of the first positive selection marker, and the competent cells are transformed with a mixture of the target genes and a limiting amount of the first positive selection marker fragment.", "Cells selected for the positive marker are screened for the desired properties in the targeted genes.", "The improved genes are amplified by the PCR, stochastic &/or non-stochastic mutagenized again, and then returned to the original host again with the first positive selection marker.", "This process is carried out recursively until the desired function of the genes are obtained.", "This process obviates the need to construct a primary host strain and the need for two positive markers.", "4.6.1.2.1.11 Conjugation-Mediated Genetic Exchange Conjugation can be employed in the evolution of cell genomes in several ways.", "Conjugative transfer of DNA occurs during contact between cells.", "See Guiney (1993) in: Bacterial Conjugation (Clewell, ed., Plenum Press, New York), pp.", "75-104; Reimmann & Haas in Bacterial Conjugation (Clewell, ed., Plenum Press, New York 1993), at pp.", "137-188 (incorporated by reference in their entirety for all purposes).", "Conjugation occurs between many types of gram negative bacteria, and some types of gram positive bacteria.", "Conjugative transfer is also known between bacteria and plant cells (Agrobacterium tumefaciens) or yeast.", "As discussed in U.S. Pat.", "No.", "5,837,458, the genes responsible for conjugative transfer can themselves be evolved to expand the range of cell types (e.g., from bacteria to mammals) between which such transfer can occur.", "Conjugative transfer is effected by an origin of transfer (oriT) and flanking genes (MOB A, B and C, and 15-25 genes, termed tra, encoding the structures and enzymes necessary for conjugation to occur.", "The transfer origin is defined as the site required in cis for DNA transfer.", "Tra genes include tra A, B, C, D, E, F, G, H, I, J, K, L, M, N, P, Q, R, S, T, U, V, W, X, Y, Z, virAB(allelesI-II), C, D, E, G, IHF, and FinOP.", "Tra genes can be expressed in cis or trans to oriT.", "Other cellular enzymes, including those of the RecBCD pathway, RecA, SSB protein, DNA gyrase, DNA poll, and DNA ligase, are also involved in conjugative transfer.", "RecE or recF pathways can substitute for RecBCD.", "One structural protein encoded by a tra gene is the sex pilus, a filament constructed of an aggregate of a single polypeptide protruding from the cell surface.", "The sex pilus binds to a polysaccharide on recipient cells and forms a conjugative bridge through which DNA can transfer.", "This process activates a site-specific nuclease encoded by a MOB gene, which specifically cleaves DNA to be transferred at oriT.", "The cleaved DNA is then threaded through the conjugation bridge by the action of other tra enzymes.", "Mobilizable vectors can exist in episomal form or integrated into the chromosome.", "Episomal mobilizable vectors can be used to exchange fragments inserted into the vectors between cells.", "Integrated mobilizable vectors can be used to mobilize adjacent genes from the chromosome.", "4.6.1.2.1.12 Use of Integrated Mobilized Vectors to Promote Exchange of Genomic DNA The F plasmid of E. coli integrates into the chromosome at high frequency and mobilizes genes unidirectional from the site of integration (Clewell, 1993, supra; Firth et al., in Escherichia coli and Salmonella Cellular and Molecular Biology 2, 23 77-2401 (1996); Frost et al., Microbiol.", "Rev.", "58, 162-210 (1994)).", "Other mobilizable vectors do not spontaneously integrate into a host chromosome at high efficiency, but can be induced to do so by growth under particular conditions (e.g., treatment with a mutagenic agent, growth at a nonpermissive temperature for plasmid replication).", "See Reimann & Haas in Bacterial Conjugation (ed.", "Clewell, Plenum Press, NY 1993), Ch.", "6.Of particular interest is the IncP group of conjugal plasmids which are typified by their broad host range (Clewell, 1993, supra.", "Donor “male” bacteria which bear a chromosomal insertion of a conjugal plasmid, such as the E. coli F factor can efficiently donate chromosomal DNA to recipient “female” enteric bacteria which lack F (F—).", "Conjugal transfer from donor to recipient is initiated at oriT.", "Transfer of the nicked single strand to the recipient occurs in a 5′ to 3′ direction by a rolling circle mechanisms which allows mobilization of tandem chromosomal copies.", "Upon entering the recipient, the donor strand is discontinuously replicated.", "The linear, single-stranded donor DNA strand is a potent substrate for initiation of recA-mediated homologous recombination within the recipient.", "Recombination between the donor strand and recipient chromosomes can result in the inheritance of donor traits.", "Accordingly, strains which bear a chromosomal copy of F are designated Hfr (for high frequency of recombination) (Low, 1996 in Escherichia coli and Salmonella Cellular and Molecular Biology Vol.", "2, pp.", "2402-2405; Sanderson, in Escherichia coli and Salmonella Cellular and Molecular Biology 2, 2406-2412 (1996)).", "The ability of strains with integrated mobilizable vector to transfer chromosomal DNA provides a rapid and efficient means of exchanging genetic material between a population of bacteria thereby allowing combination of positive mutations and dilution of negative mutations.", "Such stochastic &/or non-stochastic mutagenesis methods typically start with a population of strains with an integrated mobilizable vector encompassing at least some genetic diversity.", "The genetic diversity can be the result of natural variation, exposure to a mutagenic agent or introduction of a fragment library.", "The population of cells is cultured without selection to allow genetic exchange, recombination and expression of recombinant genes.", "The cells are then screened or selected for evolution toward a desired property.", "The population surviving selection screening can then be subject to a further round of stochastic &/or non-stochastic mutagenesis by IVR-mediated genetic exchange, or otherwise.", "The natural efficiency of Hfr and other strains with integrated mob vectors as recipients of conjugal transfer can be improved by several means.", "The relatively low recipient efficiency of natural BFR strains is attributable to the products of traS and traT genes of F (Clewell, 1993, supra; Firth et al., 1996, supra.-Frost et al., 1994, supra; Achtman et al., J. Mol.", "Biol.", "138, 779-795 (1980).", "These products are localized to the inner and outer membranes of F+ strains, respectively, where they serve to inhibit redundant matings between two strains which are both capable of donating DNA.", "The effects of traS and traT, and cognate genes in other conjugal plasmids, can be eliminated by use of knockout cells incapable of expressing these enzymes or reduced by propagating cells on a carbon-limited source.", "(Peters et al., J.", "Bacteriol., 178, 3037-3043 (1996)).", "In some methods, the starting population of cells has a mobilizable vector integrated at different genomic sites.", "Directional transfer from oriT typically results in more frequent inheritance of traits proximal to oriT.", "This is because mating pairs are fragile and tend to dissociate (particularly when in liquid medium) resulting in the interruption of transfer.", "In a population of cells having a mobilizable vector integrated at different sites, chromosomal exchange occurs in a more random fashion.", "Kits of Hfr strains are available from the E coli.", "Genetic Stock Center and the Salmonella Genetic Stock Centre (Frost et al., 1994, supra).", "Alternatively, a library of strains with oriT at random sites and orientations can be produced by insertion mutagenesis using a transposon which bears oriT.", "The use of a transposon bearing an oriT [e.g., the Tn5-oriT described by Yakobson E A, et al.", "J. Bacteriol.", "1984 October; 160(1): 451-453] provides a quick method of generating such a library.", "Transfer functions for mobilization from the transposon-borne oriT sites are provided by a helper vector in trans.", "It is possible to generate similar genetic constructs using other sequences known to one of skill as well.", "In one aspect, a recursive scheme for genomic stochastic &/or non-stochastic mutagenesis using Tn-oriT elements is provided.", "A prototrophic bacterial strain or set of related strains bearing a conjugal plasmid, such as the F fertility factor or a member of the IncP group of broad host range plasmids is mutagenized and screened for the desired properties.", "Individuals with the desired properties are mutagenized with a Tn-oriT element and screened for acquisition of an auxotrophy (e.g., by replica-plating to a minimal and complete media) resulting from insertion of the Tn-oriT element in any one of many biosynthetic gene scattered across the genome.", "The resulting auxotrophs are pooled and allowed to mate under conditions promoting male-to-male matings, e.g., during growth in close proximity on a filter membrane.", "Note that transfer functions are provided by the helper conjugal plasmid present in the original strain set.", "Recombinant transconjugants are selected on minimal medium and screened for further improvement.", "Optionally, strains bearing integrated mobilizable vectors are defective in mismatch repair gene(s).", "Inheritance of donor traits which arise from sequence heterologies increases in strains lacking the methyl-directed mismatch repair system.", "Optionally, the gene products which decrease recombination efficiency can be inhibited by small molecules.", "Intergenic conjugal transfer between species such as E. coli and Salmonella typhimurium, which are 20% divergent at the DNA level, is also possible if the recipient strain is muth, mutL or mutS (see Rayssiguier et al., Nature 342, 396401 (1989)).", "Such transfer can be used to obtain recombination at several points as shown by the following example.", "One example uses an S. typhimurium Hfr donor strain having markers thr557 at map position 0, pyrF2690 at 33 min, serA13 at 62 min and hfrK5 at 43 min.", "MutS+/−, F-E. coli.", "recipient strains had markers pyrD68 at 21 min aroC355 at 51 min, ilv3164 at 85 min and mutS215 at 59 min.", "The triauxotrophic S. typhimurium Hfr donor and isogenic mutS+/−triauxotrophic E. coli recipient were inoculated into 3 ml of Lb broth and shaken at 37C until fully grown.", "100 ul of the donor and each recipient were mixed in 10 ml fresh LB broth, and then deposited to a sterile Millipore 0.45 uM HA filter using a Nalgene 250 ml reusable filtration device.", "The donor and recipients alone were similarly diluted and deposited to check for reversion.", "The filters with cells were placed cell-side-up on the surface of an LB agar plate which was incubated overnight at 37C.", "The filters were removed with the aid of a sterile forceps and placed in a sterile 50 ml tube containing 5 ml of minimal salts broth.", "Vigorous vortexing was used to wash the cells from the filters.", "100 ul of mating mixtures, as well as donor and recipient controls were spread to LB for viable cell counts and minimal glucose supplemented with either two of the three recipient requirements for single recombinant counts, one of the three requirements for double recombinant counts, or none of the three requirements for triple recombinant counts.", "The plates were incubated for 48 hr at 37C after which colonies were counted.", "Frequencies are further enhanced by increasing the ratio of donor to recipient cells, or by repeatedly mating the original donor strains with the previously generated recombinant progeny.", "4.6.1.2.1.13 Introduction of Fragments by Conjugation Sobilizable vectors can also be used to transfer fragment libraries into cells to be evolved.", "This approach is particularly useful in situations in which the cells to be evolved cannot be efficiently transformed directly with the fragment library but can undergo conjugation with primary cells that can be transformed with the fragment library.", "DNA fragments to be introduced into host cells encompasses diversity relative to the host cell genome.", "The diversity can be the result of natural diversity or mutagenesis.", "The DNA fragment library is cloned into a mobilizable vector having an origin of transfer.", "Some such vectors also contain mob genes although alternatively these functions can also be provided in trans.", "The vector should be capable of efficient conjugal transfer between primary cells and the intended host cells.", "The vector should also confer a selectable phenotype.", "This 96 phenotype can be the same as the phenotype being evolved or can be conferred by a marker, such as a drug resistance marker.", "The vector should preferably allow self-elimination in the intended host cells thereby allowing selection for cells in which a cloned fragment has undergone genetic exchange with a homologous host segment rather than duplication.", "Such can be achieved by use of vector lacking an origin of replication functional in the intended host type or inclusion of a negative selection marker in the vector.", "One suitable vector is the broad host range conjugation plasmid described by Simon et al., Bio/Technology 1, 784-791 (1983); TrieuCuot et al., Gene 102, 99-104 (1991); Bierman et al., Gene 116, 43-49 (1992).", "These plasmids can be transformed into E. coli and then force-mated into bacteria that are difficult or impossible to transform by chemical or electrical induction of competence.", "These plasmids contain the origin of the IncP plasmid, oriT Mobilization functions are supplied in trans by chromosomally-integrated copies of the necessary genes.", "Conjugal transfer of DNA can in some cases be assisted by treatment of the recipient (if gram-positive) with sub-inhibitory concentrations of penicillins (Trieu-Cuot et al., 1993 FEMS Microbiol.", "Lett.", "109, 19-23).", "To increase diversity in populations, recursive conjugal mating prior to screening is performed.", "Cells that have undergone allelic exchange with library fragments can be screened or selected for evolution toward a desired phenotype.", "Subsequent rounds of recombination can be performed by repeating the conjugal transfer step.", "The library of fragments can be fresh or can be obtained from some (but not all) of the cells surviving a previous round of selection/screening.", "Conjugation-mediated stochastic &/or non-stochastic mutagenesis can be combined with other methods of stochastic &/or non-stochastic mutagenesis.", "4.6.1.2.1.14 Genetic Exchange Promoted by Transducing Prage in Cells Suseptible to Phage Phage transduction can include the transfer, from one cell to another, of nonviral genetic material within a viral coat (Masters, in Escherichia coli and Salmonella Cellular and Molecular Biology 2, 2421-2442 (1996).", "Perhaps the two best examples of generalized transducing phage are bacteriophages P I and P22 of E. coli and S. typhimurium, respectively.", "Generalized transducing bacteriophage particles are formed at a low frequency during lytic infection when viral-genome-sized, doubled-stranded fragments of host (which serves as donor) chromosomal DNA are packaged into phage heads.", "Promiscuous high transducing (HT) mutants of bacteriophage P22 which efficiently package DNA with little sequence specificity have been isolated.", "Infection of a susceptible host results in a lysate in which up to 50% of the phage are transducing particles.", "Adsorption of the generalized transducing particle to a susceptible recipient cell results in the injection of the donor chromosomal fragment.", "RecA-mediated homologous recombination following injection of the donor fragment can result in the inheritance of donor traits.", "Another type of phage which achieves quasi random insertion of DNA into the host chromosome is Mu.", "For an overview of Mu biology, see, Groisman (1991) in Methods in Enzymology v. 204.Mu can generate a variety of chromosomal rearrangements including deletions, inversions, duplications and transpositions.", "In addition, elements which combine the features of P22 and Mu are available, including Mud-P22, which contains the ends of the Mu genome in place of the P22 att site and int gene.", "See, Berg, supra.", "Generalized transducing phage can be used to exchange genetic material between a population of cells encompassing genetic diversity and susceptible to infection by the phage.", "Genetic diversity can be the result of natural variation between cells, induced mutation of cells or the introduction of fragment libraries into cells.", "DNA is then exchanged between cells by generalized transduction.", "If the phage does not cause lysis of cells, the entire population of cells can be propagated in the presence of phage.", "If the phage results in lytic infection, transduction is performed on a split pool basis.", "That is, the starting population of cells is divided into two.", "One subpopulation is used to prepare transducing phage.", "The transducing phage are then infected into the other subpopulation.", "Preferably, infection is performed at high multiplicity of phage per cell so that few cells remain uninfected.", "Cells surviving infection are propagated and screened or selected for evolution toward a desired property.", "The pool of cells surviving screening/selection can then be stochastic &/or non-stochastic mutagenized by a further round of generalized transduction or by other stochastic &/or non-stochastic mutagenesis methods.", "Recursive split pool tranduction is optionally performed prior to selection to increase the diversity of any population to be screened.", "The efficiency of the above methods can be increased by reducing infection of cells by infectious (nontransducing phage) and by reducing lysogen formation.", "The former can be achieved by inclusion of chelators of divalent cations, such as citrate and EDTA in culture media.", "Tail defective transducing phages can be used to allow only a single round of infection.", "Divalent cations are required for phage absorption and the inclusion of chelating agents therefore provides a means of preventing unwanted infection.", "Integration defective (int) derivatives of generalized transducing phage can be used to prevent lysogen formation.", "In a further variation, host cells with defects in mismatch repair gene(s) can be used to increase recombination between transduced DNA and genomic DNA.", "4.6.1.2.1.15 Use of Locked in Prophages to Facilitate DNA Stochastic &/or Non-Stochastic Mutagenesis The use of a hybrid, mobile genetic element (locked-in prophages) as a means to facilitate whole genome stochastic &/or non-stochastic mutagenesis of organisms using phage transduction as a means to transfer DNA from donor to recipient is a preferred embodiment.", "One such element (Mud-P22) based on the temperate Salmonella phage P22 has been described for use in genetic and physical mapping of mutations.", "See, Youderian et al.", "(1988) Genetics 118: 581-592, and Benson and Goldman (1992) J. Bacteriol.", "174(5): 1673-1681.Individual Mud-P22 insertions package specific regions of the Salmonella chromosome into phage P22 particles.", "Libraries of random Mud-P22 insertions can be readily isolated and induced to create pools of phage particles packaging random chromosomal DNA fragments.", "These phage particles can be used to infect new cells and transfer the DNA from the host into the recipient in the process of transduction.", "Alternatively, the packaged chromosomal DNA can be isolated and manipulated further by techniques such as DNA stochastic &/or non-stochastic mutagenesis or any other mutagenesis technique prior to being reintroduced into cells (especially recD cells for linear DNA) by transformation or electroporation, where they integrate into the chromosome.", "Either the intact transducing phage particles or isolated DNA can be subjected to a variety of mutagens prior to reintroduction into cells to enhance the mutation rate.", "Mutator cell fines such as mutD can also be used for phage growth.", "Either method can be used recursively in a process to create genes or strains with desired properties.", "E. coli cells carrying a cosmid clone of Salmonella LPS genes are infectable by P22 phage.", "It is possible to develop similar genetic elements using other combinations of transposable elements and bacteriophages or viruses as well.", "P22 is a lambdoid phage that packages its DNA into pstochastic &/or non-stochastic mutagenized phage particles (heads) by a “headful” mechanism.", "Packaging of phage DNA is initiated at a specific site (pac) and proceeds unidirectionally along a linear, double stranded normally concatameric molecule.", "When the phage head is full (about 43 kb), the DNA strand is cleaved, and packaging of the next phage head is initiated.", "Locked-in or excision-defective P22 prophages, however, initiate packaging at their pac site, and then proceed unidirectionally along the chromosome, packaging successive headfuls of chromosomal DNA (rather than phage DNA).", "When these transducing phages infect new Salmonella cells they inject the chromosomal DNA from the original host into the recipient cell, where it can recombine into the chromosome by homologous recombination creating a chimeric chromosome.", "Upon infection of recipient cells at a high multiplicity of infection, recombination can also occur between incoming transducing fragments prior to recombination into the chromosome.", "Integration of such locked-in P22 prophages at various sites in the chromosome allows flanking regions to be amplified and packaged into phage particles.", "The Mud-P22 mobile genetic element contains an excision-defective P22 prophage flanked by the ends of phage/transposon Mu.", "The entire Mud-P22 element can transpose to virtually any location in the chromosome or other episome (eg.", "F′, BAC clone) when the Mu A and B proteins are provided in trans.", "A number of embodiments for this type of genetic element are available.", "In one example, the locked in prophage are used as generalized transducing phage to transfer random fragments of a donor chromosome into a recipient.", "The Mud-P22 element acts as a transposon when Mu A and B transposase proteins are provided in trans and integrate copies of itself at random locations in the chromosome.", "In this way, a library of random chromosomal Mud-P22 insertions can be generated in a suitable host.", "When the Mud-P22 prophages in this library are induced, random fragments of chromosomal DNA will be packaged into phage particles.", "When these phages infect recipient cells, the chromosomal DNA is injected and can recombine into the chromosome of the recipient.", "These recipient cells are screened for a desired property and cells showing improvement are then propagated.", "The process can be repeated, since the Mud-P22 genetic element is not transferred to the recipient in this process.", "Infection at a high multiplicity allows for multiple chromosomal fragments to be injected and recombined into the recipient chromosome.", "Locked in prophages can also be used as specialized transducing phage.", "Individual insertions near a gene of interest can be isolated from a random insertion library by a variety of methods.", "Induction of these specific prophages results in packaging of flanking chromosomal DNA including the gene(s) of interest into phage particles.", "Infection of recipient cells with these phages and recombination of the packaged DNA into the chromosome creates chimeric genes that can be screened for desired properties.", "Infection at a high multiplicity of infection can allow recombination between incoming transducing fragments prior to recombination into the chromosome.", "These specialized transducing phage can also be used to isolate large quantities of high quality DNA containing specific genes of interest without any prior knowledge of the DNA sequence.", "Cloning of specific genes is not required.", "Insertion of such an element nearby a biosynthetic operon for example allows for large amounts of DNA from that operon to be isolated for use in DNA stochastic &/or non-stochastic mutagenesis (in vitro and/or in vivo), cloning, sequencing, or other uses as set forth herein.", "DNA isolated from similar insertions in other organisms containing homologous operons are optionally mixed for use in family stochastic &/or non-stochastic mutagenesis formats as described, herein, in which homologous genes from different organisms (or different chromosomal locations within a single species, or both).", "Alternatively, the transduced population is recursively transduced with pooled transducing phage or new transducing phage generated from the previously transduced cells.", "This can be carried out recursively to optimize the diversity of the genes prior to stochastic &/or non-stochastic mutagenesis.", "Phage isolated from insertions in a variety of strains or organisms containing homologous operons are optionally mixed and used to coinfect cells at a high MOI allowing for recombination between incoming transducing fragments prior to recombination into the chromosome.", "Locked in prophage are useful for mapping of genes, operons, and/or specific mutations with either desirable or undesirable phenotypes.", "Locked-in prophages can also provide a means to separate and map multiple mutations in a given host.", "If one is looking for beneficial mutations outside a gene or operon of interest, then an unmodified gene or operon can be transduced into a mutagenized or stochastic &/or non-stochastic mutagenized host then screened for the presence of desired secondary mutations.", "Alternatively, the gene/operon of interest can be readily moved from a mutagenized/stochastic &/or non-stochastic mutagenized host into a different background to screen/select for modifications in the gene/operon itself.", "It is also possible to develop similar genetic elements using other combinations of transposable elements and bacteriophages or viruses as well.", "Similar systems are set up in other organisms, e.g., that do not allow replication of P22 or P1.Broad host range phages and transposable elements are especially useful.", "Similar genetic elements are derived from other temperate phages that also package by a headful mechanism.", "In general, these are the phages that are capable of generalized transduction.", "Viruses infecting eukaryotic cells may be adapted for similar purposes.", "Examples of generalized transducing phages that are useful are described in: Green et al., “Isolation and preliminary characterization of lytic and lysogenic phages with wide host range within the streptomycetes”, J Gen Microbiol 131(9): 2459-2465 (1985); Studdard et al., “Genome structure in Streptomyces spp.", ": adjacent genes on the S. coelicolor A3(2) linkage map have cotransducible analogs in S. venezuelae”, J Bacteriol 169(8): 3814-3816 (1987); Wang et al., “High frequency generalized transduction by miniMu plasmid phage”, Genetics 116(2): 201-206, (1987); Welker, N. E., “Transduction in Bacillus stearothertnophilus”, J Bacteriol, 176(11): 3354-3359, (1988); Darzins et al., “Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa, J Bacteriol 171(7): 3909-3916 (1989); Hugouvieux-Cotte-Pattat et al., “Expanded linkage map of Erwinia chrysanthemi strain 3937”, Mol Microbiol 3(5): 573-581, (1989); Ichige et al.", "“Establishment of gene transfer systems for and construction of the genetic map of a marine Vibrio strain”, J Bacteriol 171(4):1825-1834 (1989); Murainatsu et al., “Two generalized transducing phages in Vibrio parahaemolyticus and Vibrio alginolyticus”, Microbiol Immunol (12): 1073-1084 (1991); Regue et al., “A generalized transducing bacteriophage for Serratia marcescens”, Res Microbiol 42(1): 23-27, (1991)-Kiesel et al, “Phage Acm 1-mediated transduction in the facultatively methanol-utilizing Acetobacter methanolicus MB 58/4”, J Gen Virol 74(9): 1741-1745 (1993); Blahova et al., “Transduction of imipenem resistance by the phage F-116 from a nosocomial strain of Pseudomonas aeruginosa isolated in Slovakia”, Acta Virol 38(5): 247-250 (1994); Kidambi et al., “Evidence for phage-mediated gene transfer among Pseudomonas aeruginosa strains on the phylloplane”, Appl Environ Microbiol 60: (2) 496-500 (1994); Weiss et al., “Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv.", "campestris”, J Bacteriol 176(11): 3354-3359 (1994); Matsumoto et al., “Clustering of the trp genes in Burkholderia (formerly Pseudomonas) cepacia”, FEMS Microbiol Lett 134(2-3): 265-271 (1995); Schicklmaier et al., “Frequency of generalized transducing phages in natural isolates of the Salmonella typhimurium complex”, Appl Environ Microbiol 61(4): 61(4): 1637-1640 (1995); Humphrey et al., “Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae”, J Bacteriol 179(2): 323-329 (1997); Willi et al, “Transduction of antibiotic resistance markers among Actinobacillus actinomycetemcomitans strains by temperate bacteriophages Aa phi 23”, Cell Mol Life Sci 53 (11-12): 904-910 (1997); Jensen et al., “Prevalence of broad-host-range lytic bacteriophages of Sphaerofilus natans, Escherichia coli, and Pseudomonas aeruginosa”, Appi Environ Microbiol 64(2): 575-580 (1998), and Nedelmann et al., “Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains”, Zentiviralaffil Bakteriol 287(1-2): 85-92 (1998).", "A Mud-PI/Tn-PI system comparable to Mud-P22 is developed using phage P 1.Phage P I has an advantage of packaging much larger (about 110 kb) fragments per headful.", "Phage P I is currently used to create bacterial artificial chromosomes or BAC's.", "P 1-based BAC vectors are designed along these principles so that cloned DNA is packaged into phage particles, rather than the current system, which requires DNA preparation from single-copy episomes.", "This combines the advantages of both systems in having the genes cloned in a stable single-copy format, while allowing for amplification and specific packaging of cloned DNA upon induction of the prophage.", "4.6.1.2.1.16 Random Placement of Genes or Improved Genes Throughout the Genome for Optimization of Gene Context The placement and orientation of genes in a host chromosome (the “context” of the gene in a chromosome) or episome has large effects on gene expression and activity.", "Random integration of plasmid or other episomal sequences into a host chromosome by non-homologous recombination, followed by selection or screening for the desired phenotype, is a preferred way of identifying optimal chromosomal positions for expression of a target.", "This strategy is illustrated herein.", "A variety of transposon mediated delivery systems can be employed to deliver genes of interest, either individual genes, genomic libraries, or a library of stochastic &/or non-stochastic mutagenized gene(s) randomly throughout the genome of a host.", "Thus, in one preferred embodiment, the improvement of a cellular function is achieved by cloning a gene of interest, for example a gene encoding a desired metabolic pathway, within a transposon delivery vehicle.", "Such transposon vehicles are available for both Gram-negative and Gram-positive bacteria.", "De Lorenzo and Timis (1994) Methods in Enzymology 235: 385404 describe the analysis and construction of stable phenotypes in gram-negative Bacteria with Tn5- and Tn 10-derived minitransposons.", "Kleckner et al.", "(1991) Methods in Enzymology 204, chapter 7 describe uses of transposons such as Tn 10, including for use in gram positive bacteria.", "Petit et al.", "(1990) Journal of Bacteriology, 172(12): 6736-6740 describe Tn 10 derived transposons active in Bacillus Subtilis.", "The transposon delivery vehicle is introduced into a cell population, which is then selected for recombinant cells that have incorporated the transposon into the genome.", "The selection is typically by any of a variety of drug resistant markers also carried within the transposon.", "The selected subpopulation is screened for cells having improved expression of the gene(s) of interest.", "Once cells harboring the genes of interest in the optimal location are isolated, the genes are amplified from within the genome using PCR, stochastic &/or non-stochastic mutagenized, and cloned back into a similar transposon delivery vehicle which contains a different selection marker within the transposon and lacks the transposon integrase gene.", "This stochastic &/or non-stochastic mutagenized library is then transformed back into the strain harboring the original transposon, and the cells are selected for the presence of the new resistance marker and the loss of the previous selection marker.", "Selected cells are enriched for those that have exchanged by homologous recombination the original transposon for the new transposon carrying members of the stochastic &/or non-stochastic mutagenized library.", "The surviving cells are then screened for further improvements in the expression of the desired phenotype.", "The genes from the improved cells are then amplified by the PCR and stochastic &/or non-stochastic mutagenized again.", "This process is carried out recursively, oscillating each cycle between the different selection markers.", "Once the gene(s) of interest are optimized to a desired level, the fragment can be amplified and again randomly distributed throughout the genome as described above to identify the optimal location of the improved genes.", "Alternatively, the gene(s) conferring a desired property may not be known.", "In this case the DNA fragments cloned within the transposon delivery vehicle could be a library of genomic fragments originating from a population of cells derived from one or more strains having the desired property(ies).", "The library is delivered to a population of cells derived from one or more strains having or lacking the desired property(ies) and cells incorporating the transposon are selected.", "The surviving cells are then screened for acquisition or improvement of the desired property.", "The fragments contained within the surviving cells are amplified by PCR and then cloned as a pool into a similar transposon delivery vector harboring a different selection marker from the first delivery vector.", "This library is then delivered to the pool of surviving cells, and the population having acquired the new selective marker is selected.", "The selected cells are then screened for further acquisition or improvement of the desired property.", "In this way the different possible combinations of genes conferring or improving a desired phenotype are explored in a combinatorial fashion.", "This process is carried out repetitively with each new cycle employing an additional selection marker.", "Alternatively, PCR fragments are cloned into a pool of transposon vectors having different selective markers.", "These are delivered to cells and selected for 1, 2, 3, or more markers.", "Alternatively, the amplified fragments from each improved cell are stochastic &/or non-stochastic mutagenized independently.", "The stochastic &/or non-stochastic mutagenized libraries are then cloned back into a transposon delivery vehicle similar to the original vector but containing a different selection marker and lacking the transposase gene.", "Selection is then for acquisition of the new marker and loss of the previous marker.", "Selected cells are enriched for those incorporating the stochastic &/or non-stochastic mutagenized variants of the amplified genes by homologous recombination.", "This process is carried out recursively, oscillating each cycle between the two selective markers.", "4.6.1.2.1.17 Improvement of Overexpressed Genes for a Desired Phenotype The improvement of a cellular property or phenotype is often enhanced by increasing the copy number or expression of gene(s) participating in the expression of that property.", "Genes that have such an effect on a desired property can also be improved by DNA stochastic &/or non-stochastic mutagenesis to have a similar effect.", "A genomic DNA library is cloned into an overexpression vector and transformed into a target cell population such that the genomic fragments are highly expressed in cells selected for the presence of the overexpression vector.", "The selected cells are then screened for improvement of a desired property.", "The overexpression vector from the improved cells are isolated and the cloned genomic fragments stochastic &/or non-stochastic mutagenized.", "The genomic fragment carried in the vector from each improved isolate is stochastic &/or non-stochastic mutagenized independently or with identified homologous genes (family stochastic &/or non-stochastic mutagenesis).", "The stochastic &/or non-stochastic mutagenized libraries are then delivered back to a population of cells and the selected transformants rescreened for further improvements in the desired property.", "This stochastic &/or non-stochastic mutagenesis/screening process is cycled recursively until the desired property has been optimized to the desired level.", "As stated above, gene dosage can greatly enhance a desired cellular property.", "One method of increasing gene copy number of unknown genes is using a method of random amplification (see also, Mavingui et. al.", "(1997) Nature Biotech, 15, 5 64).", "In this method, a genomic library is cloned into a suicide vector containing a selective marker that also at higher dosage provides an enhanced phenotype.", "An example of such a marker is the kanamycin resistance gene.", "At successively higher copy number, resistance to successively higher levels of kanamycin is achieved.", "The genomic library is delivered to a target cell by any of a variety of methods including transformation, transduction, conjugation, etc.", "Cells that have incorporated the vector into the chromosome by homologous recombination between the vector and chromosomal copies of the cloned genes can be selected by requiring expression of the selection marker under conditions where the vector does not replicate.", "This recombination event results in the duplication of the cloned DNA fragment in the host chromosome with a copy of the vector and selection marker separating the two copies.", "The population of surviving cells are screened for improvement of a desired cellular property resulting form the gene duplication event.", "Further gene duplication events resulting in additional copies of the original cloned DNA fragments can be generated by further propagating the cells under successively more stringent selective conditions i.e.", "increased concentrations of kanamycin.", "In this case selection requires increased copies of the selective marker, but increased copies of the desired gene fragment is also concomitant.", "Surviving cells are further screened for an improvement in the desired phenotype.", "The resulting population of cells likely resulted in the amplification of different genes since often many genes effect a given phenotype.", "To generate a library of the possible combinations of these genes, the original selected library showing phenotypic improvements are recombined, using the methods described herein, e.g., protoplast fusion, split pool transduction, transformation, conjugation, etc.", "The recombined cells are selected for increased expression of the selective marker.", "Survivors are enriched for cells having incorporated additional copies of the vector sequence by homologous recombination, and these cells will be enriched for those having combined duplications of different genes.", "In other words, the duplication from one cell of enhanced phenotype becomes combined with the duplication of another cell of enhanced phenotype.", "These survivors are screened for further improvements in the desired phenotype.", "This procedure is repeated recursively until the desired level of phenotypic expression is achieved.", "Alternatively, genes that have been identified or are suspected as being beneficial in increased copy number are cloned in tandem into appropriate plasmid vectors.", "These vectors are then transformed and propagated in an appropriate host organism.", "Plasmid-plasmid recombination between the cloned gene fragments result in further duplication of the genes.", "Resolution of the plasmid doublet can result in the uneven distribution of the gene copies, with some plasmids having additional gene copies and others having fewer gene copies.", "Cells carrying this distribution of plasmids are then screened for an improvement in the phenotype effected by the gene duplications.", "In summary, a method of selecting for increased copy number of a nucleic acid sequence by the above procedure is provided.", "In the method, a genomic library in a suicide vector comprising a dose-sensitive selectable marker is provided, as noted above.", "The genomic library is transduced into a population of target cells.", "The target cells are selected in a population of target cells for increasing doses of the selectable marker under conditions in which the suicide vector does not replicate episomally.", "A plurality of target cells are selected for the desired phenotype, recombined and reselected.", "The process is recursively repeated, if desired, until the desired phenotype is obtained.", "4.6.1.2.1.18 Strategies for Improving Genomic Stochastic &/or Non-Stochastic Mutagenesis via Transformation of Linear DNA Fragments Wild-type members of the Enterobacteriaceae (e.g., Escherichia coli) are typically resistant to genetic exchange following transformation of linear DNA molecules.", "This is due, at least in part, to the Exonuclease V (Exo V) activity of the RecBCD holoenzyme which rapidly degrades linear DNA molecules following transformation.", "Production of ExoV has been traced to the recD gene, which encodes the D subunit of the holoenzyme.", "As demonstrated by Russel et al.", "(1989) Journal of Bacteriology 2609-2613, homologous recombination between a transformed linear donor DNA molecule and the chromosome of recipient is readily detected in a strains bearing a loss of function mutation in a recD mutant.", "The use of recD strains provides a simple means for genomic stochastic &/or non-stochastic mutagenesis of the Enterobacteriaceae.", "For example, a bacterial strain or set of related strains bearing a recD null mutation (e.g., the E. coli recD1903::mini-Tet allele) is mutagenized and screened for the desired properties.", "In a split-pool fashion, chromosomal DNA prepared on one aliquot could be used to transform (e.g., via electroporation or chemically induced competence) the second aliquot.", "The resulting transformants are then screened for improvement, or recursively transformed prior to screening.", "The use of RecE/recT as described supra, can improve homologous recombination of linear DNA fragments.", "The RecBCD holoezyme plays an important role in initiation of RecA-dependent homologous recombination.", "Upon recognizing a dsDNA end, the RecBCD enzyme unwinds and degrades the DNA asymmetrically in a 5′ to 3′ direction until it encounters a chi (or ‘X’)-site (consensus 5′-GCTGGTGG-3) which attenuates the nuclease activity.", "This results in the generation of a ssDNA terminating near the c site with a 3′-ssDNA tail that is preferred for RecA loading and subsequent invasion of dsDNA for homologous recombination.", "Accordingly, preprocessing of transforming fragments with a 5′ to 3′ specific ssDNA Exonuclease, such as Lamda ( ) exonuclease (available, e.g., from Boeringer Mannheim) prior to transformation may serve to stimulate homologous recombination in recD-strain by providing ssDNA invasive end for RecA loading and subsequent strand invasion.", "The addition of DNA sequence encoding chi-sites (consensus 5′-GCTGGTGG-3′) to DNA fragments can serve to both attenuate Exonuclease V activity and stimulate homologous recombination, thereby obviating the need for a recD mutation (see also, Kowalczykowski, et al.", "(1994) “Biochemistry of homologous recombination in Escherichia coli,” Microbiol.", "Rev.", "58: 401465 and Jessen, et al.", "(1998) “Modification of bacterial artificial chromosomes through Chi-stimulated homologous recombination and its application in zebrafish transgenesis.” Proc.", "Natl.", "Acad.", "Sci.", "95: 5121-5126).", "Chi sites are optionally included in linkers ligated to the ends of transforming fragments or incorporated into the external primers used to generate DNA fragments to be transformed.", "The use of recombination-stimulatory sequences such as chi is a generally useful approach for evolution of a broad range of cell types by fragment transformation.", "Methods to inhibit or mutate analogs of Exo V or other nucleases (such as, Exonucleases I (endA 1), 111 (nth), IV (nfo), VII, and VIII of E. coli) is similarly useful.", "Inhibition or elimination of nucleases, or modification of ends of transforming DNA fragments to render them resistant to exonuclease activity has applications in evolution of a broad range of cell types.", "4.6.1.2.1.19 Stochastic &/or Non-Stochastic Mutagenesis to Optimize Unknown Interactions Many observed traits are the result of complex interactions of multiple genes or gene products.", "Most such interactions are still uncharacterized.", "Accordingly, it is often unclear which genes need to be optimized to achieve a desired trait, even if some of the genes contributing to the trait are known.", "This lack of characterization is not an issue during DNA stochastic &/or non-stochastic mutagenesis, which produces solutions that optimize whatever is selected for.", "An alternative approach, which has the potential to solve not only this problem, but also anticipated future rate limiting factors, is complementation by overexpression of unknown genomic sequences.", "A library of genomic DNA is first made as described, supra.", "This is transformed into the cell to be optimized and transformants are screened for increases in a desired property.", "Genomic fragments which result in an improved property are evolved by DNA stochastic &/or non-stochastic mutagenesis to further increase their beneficial effect.", "This approach requires no sequence information, nor any knowledge or assumptions about the nature of protein or pathway interactions, or even of what steps are rate-limiting; it relies only on detection of the desired phenotype.", "This sort of random cloning and subsequent evolution by DNA stochastic &/or non-stochastic mutagenesis of positively interacting genomic sequences is extremely powerful and generic.", "A variety of sources of genomic DNA are used, from isogenic strains to more distantly related species with potentially desirable properties.", "In addition, the technique is applicable to any cell for which the molecular biology basics of transformation and cloning vectors are available, and for any property which can be assayed (preferably in a high-throughput format).", "Alternatively, once optimized, the evolved DNA can be returned to the chromosome by homologous recombination or randomly by phage mediated site-specific recombination.", "4.6.1.2.1.20 Homologous Recombination within the Chromosome Homologous recombination within the chromosome is used to circumvent the limitations of plasmid based evolution and size restrictions.", "The strategy is similar to that described above for stochastic &/or non-stochastic mutagenesis genes within their chromosomal context, except that no in vitro stochastic &/or non-stochastic mutagenesis occurs.", "Instead, the parent strain is treated with mutagens such as ultraviolet light or nitrosoguanidine, and improved mutants are selected.", "The improved mutants are pooled and split.", "Half of the pool is used to generate random genomic fragments for cloning into a homologous recombination vector.", "Additional genomic fragments are optionally derived from related species with desirable properties.", "The cloned genomic fragments are homologously recombined into the genomes of the remaining half of the mutant pool, and variants with improved properties are selected.", "These are subjected to a further round of mutagenesis, selection and recombination.", "Again this process is entirely generic for the improvement of any whole cell biocatalyst for which a recombination vector and an assay can be developed.", "Here again, it should be noted that recombination can be performed recursively prior to screening.", "4.6.1.2.1.21 Methods for Recursive Sequence Recombination As shown herein, DNA Stochastic &/or non-stochastic mutagenesis provides most rapid technology for evolution of complex new functions.", "As shown herein, recombination in DNA stochastic &/or non-stochastic mutagenesis achieves accumulation of multiple beneficial mutations in a few cycles.", "In contrast, because of the high frequency of deleterious mutations relative to beneficial ones, iterative point mutation must build beneficial mutations one at a time, and consequently requires many cycles to reach the same point.", "As shown herein, rather than a simple linear sequence of mutation accumulation, DNA stochastic &/or non-stochastic mutagenesis is a parallel process where multiple problems may be solved independently, and then combined.", "4.6.1.2.2 In Vivo Formats 4.6.1.2.2.1 Plasmid-Plasmid Recombination The initial substrates for recombination are a collection of polynucleotides comprising variant forms of a gene.", "The variant forms usually show substantial sequence identity to each other sufficient to allow homologous recombination between substrates.", "The diversity between the polynucleotides can be natural (e.g., allelic or species variants), induced (e.g., error-prone PCR or error-prone recursive sequence recombination), or the result of in vitro recombination.", "Diversity can also result from resynthesizing genes encoding natural proteins with alternative codon usage.", "There should be at least sufficient diversity between substrates that recombination can generate more diverse products than there are starting materials.", "There must be at least two substrates differing in at least two positions.", "However, commonly a library of substrates of 103-108 members is employed.", "The degree of diversity depends on the length of the substrate being recombined and the extent of the functional change to be evolved.", "Diversity at between 0.1-25% of positions is typical.", "The diverse substrates are incorporated into plasmids.", "The plasmids are often standard cloning vectors, e.g., bacterial multicopy plasmids.", "However, in some methods to be described below, the plasmids include mobilization (MOB) functions.", "The substrates can be incorporated into the same or different plasmids.", "Often at least two different types of plasmid having different types of selectable markers are used to allow selection for cells containing at least two types of vector.", "Also, where different types of plasmid are employed, the different plasmids can come from two distinct incompatibility groups to S allow stable co-existence of two different plasmids within the cell.", "Nevertheless, plasmids from the same incompatibility group can still co-exist within the same cell for sufficient time to allow homologous recombination to occur.", "Plasmids containing diverse substrates are initially introduced into cells by any method (e.g., chemical transformation, natural competence, electroporation, biolistics, packaging into phage or viral systems).", "Often, the plasmids are present at or near saturating concentration (with respect to maximum transfection capacity) to increase the probability of more than one plasmid entering the same cell.", "The plasmids containing the various substrates can be transfected simultaneously or in multiple rounds.", "For example, in the latter approach cells can be transfected with a first aliquot of plasmid, transfectants selected and propagated, and then infected with a second aliquot of plasmid.", "Having introduced the plasmids into cells, recombination between substrates to generate recombinant genes occurs within cells containing multiple different plasmids merely by propagating the cells.", "However, cells that receive only one plasmid are unable to participate in recombination and the potential contribution of substrates on such plasmids to evolution is not fully exploited (although these plasmids may contribute to some extent if they are progagated in mutator cells).", "The rate of evolution can be increased by allowing all substrates to participate in recombination.", "Such can be achieved by subjecting transfected cells to electroporation.", "The conditions for electroporation are the same as those conventionally used for introducing exogenous DNA into cells (e.g., 1,000-2,500 volts, 400 uF and a 1-2 mM gap).", "Under these conditions, plasmids are exchanged between cells allowing all substrates to participate in recombination.", "In addition the products of recombination can undergo further rounds of recombination with each other or with the original substrate.", "The rate of evolution can also be increased by use of conjugative transfer.", "To exploit conjugative transfer, substrates can be cloned into plasmids having MOB genes, and tra genes are also provided in cis or in trans to the MOB genes.", "The effect of conjugative transfer is very similar to electroporation in that it allows plasmids to move between cells and allows recombination between any substrate and the products of previous recombination to occur, merely by propagating the culture.", "The rate of evolution can also be increased by fusing cells to induce exchange of plasmids or chromosomes.", "Fusion can be induced by chemical agents, such as PEG, or viral proteins, such as influenza virus hemagglutinin, HSV-1 gB and gD.", "The rate of evolution can also be increased by use of mutator host cells (e.g., Mut L, S, D, T, H in bacteria and Ataxia telangiectasia human cell lines).", "The time for which cells are propagated and recombination is allowed to occur, of course, varies with the cell type but is generally not critical, because even a small degree of recombination can substantially increase diversity relative to the starting materials.", "Cells bearing plasmids containing recombined genes are subject to screening or selection for a desired function.", "For example, if the substrate being evolved contains a drug resistance gene, one would select for drug resistance.", "Cells surviving screening or selection can be subjected to one or more rounds of screening/selection followed by recombination or can be subjected directly to an additional round of recombination.", "The next round of recombination can be achieved by several different formats independently of the previous round.", "For example, a further round of recombination can be effected simply by resuming the electroporation or conjugation-mediated intercellular transfer of plasmids described above.", "Alternatively, a fresh substrate or substrates, the same or different from previous substrates, can be transfected into cells surviving selection/screening.", "Optionally, the new substrates are included in plasmid vectors bearing a different selective marker and/or from a different incompatibility group than the original plasmids.", "As a further alternative, cells surviving selection/screening can be subdivided into two subpopulations, and plasmid DNA from one subpopulation transfected into the other, where the substrates from the plasmids from the two subpopulations undergo a further round of recombination.", "In either of the latter two options, the rate of evolution can be increased by employing DNA extraction, electroporation, conjugation or mutator cells, as described above.", "In a still further variation, DNA from cells surviving screening/selection can be extracted and subjected to in vitro recursive sequence recombination.", "After the second round of recombination, a second round of screening/selection is performed, preferably under conditions of increased stringency.", "If desired, further rounds of recombination and selection/screening can be performed using the same strategy as for the second round.", "With successive rounds of recombination and selection/screening, the surviving recombined substrates evolve toward acquisition of a desired phenotype.", "Typically, in this and other methods of recursive recombination, the final product of recombination that has acquired the desired phenotype differs from starting substrates at 0.1%-50% of positions and has evolved at a rate orders of magnitude in excess (e.g., by at least 10-fold, 100-fold, 1000-fold, or 10,000 fold) of the rate of naturally acquired mutation of about 1 mutation per 10−9 positions per generation (see Anderson et al.", "Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 93: 906-907 (1996)).", "The “final product” may be transferred to another host more desirable for utilization of the “stochastic &/or non-stochastic mutagenized” DNA.", "This is particularly advantageous in situations where the more desirable host is less efficient as a host for the many cycles of mutation/recombination due to the lack of molecular biology or genetic tools available for other organisms such as E. coli.", "4.6.1.2.2.2 Virus-Plasmid Recombination The strategy used for plasmid-plasmid recombination can also be used for virus-plasmid recombination; usually, phage-plasmid recombination.", "However, some additional comments particular to the use of viruses are appropriate.", "The initial substrates for recombination are cloned into both plasmid and viral vectors.", "It is usually not critical which substrate(s) are inserted into the viral vector and which into the plasmid, although usually the viral vector should contain different substrate(s) from the plasmid.", "As before, the plasmid (and the virus) typically contains a selective marker.", "The plasmid and viral vectors can both be introduced into cells by transfection as described above.", "However, a more efficient procedure is to transfect the cells with plasmid, select transfectants and infect the transfectants with virus.", "Because the efficiency of infection of many viruses approaches 100% of cells, most cells transfected and infected by this route contain both a plasmid and virus bearing different substrates.", "Homologous recombination occurs between plasmid and virus generating both recombined plasmids and recombined virus.", "For some viruses, such as filamentous phage, in which intracellular DNA exists in both double-stranded and single-stranded forms, both can participate in recombination.", "Provided that the virus is not one that rapidly kills cells, recombination can be augmented by use of electroporation or conjugation to transfer plasmids between cells.", "Recombination can also be augmented for some types of virus by allowing the progeny virus from one cell to reinfect other cells.", "For some types of virus, virus infected-cells show resistance to superinfection.", "However, such resistance can be overcome by infecting at high multiplicity and/or using mutant strains of the virus in which resistance to superinfection is reduced.", "The result of infecting plasmid-containing cells with virus depends on the nature of the virus.", "Some viruses, such as filamentous phage, stably exist with a plasmid in the cell and also extrude progeny phage from the cell.", "Other viruses, such as lambda having a cosmid genome, stably exist in a cell like plasmids without producing progeny virions.", "Other viruses, such as the T-phage and lytic lambda, undergo recombination with the plasmid but ultimately kill the host S cell and destroy plasmid DNA.", "For viruses that infect cells without killing the host, cells containing recombinant plasmids and virus can be screened/selected using the same approach as for plasmid-plasmid recombination.", "Progeny virus extruded by cells surviving selection/screening can also be collected and used as substrates in subsequent rounds of recombination.", "For viruses that kill their host cells, recombinant genes resulting from recombination reside only in the progeny virus.", "If the screening or selective assay requires expression of recombinant genes in a cell, the IS recombinant genes should be transferred from the progeny virus to another vector, e.g., a plasmid vector, and retransfected into cells before selection/screening is performed.", "For filamentous phage, the products of recombination are present in both cells surviving recombination and in phage extruded from these cells.", "The dual source of recombinant products provides some additional options relative to the plasmid-plasmid recombination.", "For example, DNA can be isolated from phage particles for use in a round of in vitro recombination.", "Alternatively, the progeny 2S phage can be used to transfect or infect cells surviving a previous round of screening/selection, or fresh cells transfected with fresh substrates for recombination.", "4.6.1.2.23 Virus-Virus Recombination The principles described for plasmid-plasmid and plasmid-viral recombination can be applied to virus-virus recombination with a few modifications.", "The initial substrates for recombination are cloned into a viral vector.", "Usually, the same vector is used for all substrates.", "Preferably, the virus is one that, naturally or as a result of mutation, does not kill cells.", "After insertion, some viral genomes can be packaged in vitro or using a packaging cell line.", "The packaged viruses are used to infect cells at high multiplicity such that there is a high probability that a cell will receive multiple viruses bearing different substrates.", "After the initial round of infection, subsequent steps depend on the nature of infection as discussed in the previous section.", "For example, if the viruses have phagemid genomes such as lambda cosmids or M13, F1 or Fd phagemids, the phagemids behave as plasmids within the cell and undergo recombination simply by propagating the cells.", "Recombination is particularly efficient between single-stranded forms of intracellular DNA.", "Recombination can be augmented by electroporation of cells.", "Following selection/screening, cosmids containing recombinant genes can be recovered from surviving cells, e.g., by heat induction of a cos-lysogenic host cell, or extraction of DNA by standard procedures, followed by repackaging cosmid DNA in vitro.", "If the viruses are filamentous phage, recombination of replicating form DNA occurs by propagating the culture of infected cells.", "Selection/screening identifies colonies of cells containing viral vectors having recombinant genes with improved properties, together with phage extruded from such cells.", "Subsequent options are essentially the same as for plasmid-viral recombination.", "4.6.1.2.2.4 Chromosome Recombination This format can be used to especially evolve chromosomal substrates.", "The format is particularly useful in situations in which many chromosomal genes contribute to a phenotype or one does not know the exact location of the chromosomal gene(s) to be evolved.", "The initial substrates for recombination are cloned into a plasmid vector.", "If the chromosomal gene(s) to be evolved are known, the substrates constitute a family of sequences showing a high degree of sequence identity but some divergence from the chromosomal gene.", "If the chromosomal genes to be evolved have not been located, the initial substrates usually constitute a library of DNA segments of which only a small number show sequence identity to the gene or gene(s) to be evolved.", "Divergence between plasmid-bome substrate and the chromosomal gene(s) can be induced by mutagenesis or by obtaining the plasmid-borne substrates from a different species than that of the cells bearing the chromosome.", "The plasmids bearing substrates for recombination are transfected into cells having chromosomal gene(s) to be evolved.", "Evolution can occur simply by propagating the culture, and can be accelerated by transferring plasmids between cells by conjugation or electroporation.", "Evolution can be further accelerated by use of mutator host cells or by seeding a culture of nonmutator host cells being evolved with mutator host cells and inducing intercellular transfer of plasmids by electroporation or conjugation.", "Preferably, mutator host cells used for seeding contain a negative selectable marker to facilitate isolation of a pure culture of the nonmutator cells being evolved.", "Selection/screening identifies cells bearing chromosomes and/or plasmids that have evolved toward acquisition of a desired function.", "Subsequent rounds of recombination and selection/screening proceed in similar fashion to those described for plasmid-plasmid recombination.", "For example, further recombination can be effected by propagating cells surviving recombination in combination with electroporation or conjugative transfer of plasmids.", "Alternatively, plasmids bearing additional substrates for recombination can be introduced into the surviving cells.", "Preferably, such plasmids are from a different incompatibility group and bear a different selective marker than the original plasmids to allow selection for cells containing at least two different plasmids.", "As a further alternative, plasmid and/or chromosomal DNA can be isolated from a subpopulation of surviving cells and transfected into a second subpopulation.", "Chromosomal DNA can be cloned into a plasmid vector before transfection.", "4.6.1.2.2.5 Virus-Chromosome Recombination As in the other methods described above, the virus is usually one that does not kill the cells, and is often a phage or phagemid.", "The procedure is substantially the same as for plasmid-chromosome recombination.", "Substrates for recombination are cloned into the vector.", "Vectors including the substrates can then be transfected into cells or in vitro packaged and introduced into cells by infection.", "Viral genomes recombine with host chromosomes merely by propagating a culture.", "Evolution can be accelerated by allowing intercellular transfer of viral genomes by electroporation, or reinfection of cells by progeny virions.", "Screening/selection identifies cells having chromosomes and/or viral genomes that have evolved toward acquisition of a desired function.", "There are several options for subsequent rounds of recombination.", "For example, viral genomes can be transferred between cells surviving selection/recombination by electroporation.", "Alternatively, viruses extruded from cells surviving selection/screening can be pooled and used to superinfect the cells at high multiplicity.", "Alternatively, fresh substrates for recombination can be introduced into the cells, either on plasmid or viral vectors.", "4.6.1.2.2.6 Poolwise Whole Genome Recombination Asexual evolution is a slow and inefficient process.", "Populations move as individuals rather than as a group.", "A diverse population is generated by mutagenesis of a single parent, resulting in a distribution of fit and unfit individuals.", "In the absence of a sexual cycle, each piece of genetic information for the surviving population remains in the individual mutants.", "Selection of the fittest results in many fit individuals being discarded, along with the genetically useful information they carry.", "Asexual evolution proceeds one genetic event at a time, and is thus limited by the intrinsic value of a single genetic event.", "Sexual evolution moves more quickly and efficiently.", "Mating within a population consolidates genetic information within the population and results in useful information being combined together.", "The combining of useful genetic information results in progeny that are much more fit than their parents.", "Sexual evolution thus proceeds much faster by multiple genetic events.", "These differences are further illustrated herein.", "In contrast to sexual evolution, DNA stochastic &/or non-stochastic mutagenesis is the recursive mutagenesis, recombination, and selection of DNA sequences.", "Sexual recombination in nature effects pairwise recombination and results in progeny that are genetic hybrids of two parents.", "In contrast, DNA stochastic &/or non-stochastic mutagenesis in vitro effects poolwise recombination, in which progeny are hybrids of multiple parental molecules.", "This is because DNA stochastic &/or non-stochastic mutagenesis effects many individual pairwise recombination events with each thermal cycle.", "After many cycles the result is a repetitively inbred population, with the “progeny” being the Fx (for X cycles of stochastic &/or non-stochastic mutagenesis) of the original parental molecules.", "These progeny are potentially descendants of many or all of the original parents.", "One can graph to show a plot of the potential number of mutations an individual can accumulate by sequential, pairwise and poolwise recombination.", "Poolwise recombination is an important feature to DNA stochastic &/or non-stochastic mutagenesis in that it provides a means of generating a greater proportion of the possible combinations of mutations from a single “breeding” experiment.", "In this way, the “genetic potential” of a population can be readily assessed by screening the progeny of a single DNA shufflmig experiment.", "For example, if a population consists of 10 single mutant parents, there are 210=1024 possible combinations of those mutations ranging from progeny having 0-10 mutations.", "Of these 1024, only 56 will result from a single pairwise cross (i.e those having 0, 1, and 2 mutations).", "In nature the multiparent combinations will eventually arise after multiple random sexual matings, assuming no selection is imparted to remove some mutations from the population.", "In this way, sex effects the consolidation and sampling of all useful mutant combinations possible within a population.", "For the purposes of directed evolution, having the greatest number of mutant combinations entering a screen or selection is desirable so that the best progeny (i.e., according to the selection criteria used in the selection screen) is identified in the shortest possible time.", "One challenge to in vivo and whole genome stochastic &/or non-stochastic mutagenesis is devising methods for effecting poolwise recombination or multiple repetitive pairwise recombination events.", "In crosses with a single pairwise cross per cycle before screening, the ability to screen the “genetic potential” of the starting population is limited.", "For this reason, the rate of in vivo and whole genome stochastic &/or non-stochastic mutagenesis mediated cellular evolution would be facilitated by effecting poolwise recombination.", "Two strategies for poolwise recombination are described below (protoplast fusion and transduction).", "4.6.1.2.2.7 Protoplast Fusion Protoplast fusion (discussed supra) mediated whole genome stochastic &/or non-stochastic mutagenesis is one format that can directly effect poolwise recombination.", "Whole gene stochastic &/or non-stochastic mutagenesis is the recursive recombination of whole genomes, in the form of one or more nucleic acid molecule(s) (fragments, chromosomes, episomes, etc), from a population of organisms, resulting in the production of new organisms having distributed genetic information from at least two of the starting population of organisms.", "The process of protoplast fusion is further illustrated in herein.", "Progeny resulting from the fusion of multiple parent protoplasts have been observed (Hopwood & Wright, 1978), however, these progeny are rare (10−4-10−6).", "The low frequency is attributed to the distribution of fusants arising from two, three, four, etc parents and the likelihood of the multiple recombination events (6 crossovers for a four parent cross) that would have to occur for multiparent progeny to arise.", "Thus, it is useful to enrich for the multiparent progeny.", "This can be accomplished, e.g., by repetitive fusion or enrichment for multiply fused protoplasts.", "The process of poolwise fusion and recombination is further illustrated herein.", "4.6.1.2.2.8 Repetitive Fusion Protoplasts of identified parental cells are prepared, fused and regenerated.", "Protoplasts of the regenerated progeny are then, without screening or enrichment, formed, fused and regenerated.", "This can be carried out for two, three, or more cycles before screening to increase the representation of multiparent progeny.", "The number of possible mutations/progeny doubles for each cycle.", "For example, if one cross produces predominantly progeny with 0, 1, and 2 mutations, a breeding of this population with itself will produce progeny with 0, 1, 2, 3, and 4 mutations, the third cross up to eight, etc.", "The representation of the multiparent progeny from these subsequent crosses will not be as high as the single and double parent progeny, but it will be detectable and much higher than from a single cross.", "The repetitive fusion prior to screening is analogous to many sexual crosses within a population, and the individual thermal cycles of in vitro DNA stochastic &/or non-stochastic mutagenesis described supra.", "A factor effecting the value of this approach is the starting size of the parental population.", "As the population grows, it becomes more likely that a multiparent fusion will arise from repetitive fusions.", "For example, if 4 parents are fused twice, the 4 parent progeny will make up approximately 0.2% of the total progeny.", "This is sufficient to find in a population of 3000 (95% confidence), but better representation is preferable.", "If ten parents are fused twice >20% of the progeny will be four parent offspring.", "4.6.1.2.2.9 Enrichment for Multiple Fused Protoplasts After the fusion of a population of protoplasts, the fusants are typically diluted into hypotonic medium, to dilute out the fusing agent (e.g., 50% PEG).", "The fused cells can be grown for a short period to regenerate cell walls or separated directly and are then separated on the basis of size.", "This is carried out, e.g., by cell sorting, using fight dispersion as an estimate of size, to isolate the largest fusants.", "Alternatively the fusants can be sorted by FACS on the basis of DNA content.", "The large fusants or those containing more DNA result from the fusion of multiple parents and are more likely to segregate to multiparent progeny.", "The enriched fusants are regenerated and screened directly or the progeny are fused recursively as above to further enrich the population for diverse mutant combinations.", "4.6.1.2.2.10 Transduction Transduction can theoretically effect poolwise recombination, if the transducing phage particles contain predominantly host genomic DNA rather than phage DNA.", "If phage DNA is overly represented, then most cells will receive at least one undesired phage genome.", "Phage particles generated from locked-in-prophage (supra) are useful for this purpose.", "A population of cells is infected with an appropriate transducing phage, and the lysate is collected and used to infect the same starting population.", "A high multiplicity of infection is employed to deliver multiple genomic fragments to each infected cell, thereby increasing the chance of producing recombinants containing mutations from more than two parent genomes.", "The resulting transductants are recovered under conditions where phage can not propagate e.g., in the presence of citrate.", "This population is then screened directly or infected again with phage, with the resulting transducing particles being used to transduce the first progeny.", "This would mimic recursive protoplast fusion, multiple sexual recombination, and in vitro DNA stochastic &/or non-stochastic mutagenesis.", "4.6.1.2.2.11 Methods for Whole Genome Stochastic &/or Non-Stochastic Mutagenesis by Blind Family Stochastic &/or NON-Stochastic Mutagenesis of Parsed Genomes and Recursive Cycles of Forced Integration and Excision by Homologous Recombination, and Screening for Improved Phenotypes In vitro methods have been developed to reassemble single genes and operons, as set forth, e.g., herein.", "“Family” stochastic &/or non-stochastic mutagenesis of homologous genes within species and from different species is also an effective methods for accelerating molecular evolution.", "This section describes additional methods for extending these methods such that they can be applied to whole genomes.", "In some cases, the genes that encode rate limiting steps in a biochemical process, or that contribute to a phenotype of interest are known.", "This method can be used to target family stochastic &/or non-stochastic mutagenized libraries to such loci, generating libraries of organisms with high quality family stochastic &/or non-stochastic mutagenized libraries of alleles at the locus of interest.", "An example of such a gene would be the evolution of a host chaperonin to more efficiently chaperone the folding of an overexpressed protein in E. coli.", "The goals of this process are to reassemble homologous genes from two or more species and to then integrate the stochastic &/or non-stochastic mutagenized genes into the chromosome of a target organism.", "Integration of multiple stochastic &/or non-stochastic mutagenized genes at multiple loci can be achieved using recursive cycles of integration (generating duplications), excision (leaving the improved allele in the chromosome) and transfer of additional evolved genes by serially applying the same procedure.", "In the first step, genes to be stochastic &/or non-stochastic mutagenized into suitable bacterial vectors are subcloned.", "These vectors can be plasmids, cosmids, BACS or the like.", "Thus, fragments from 100 bp to 100 kb can be handled.", "Homologous fragments are then “family stochastic &/or non-stochastic mutagenized” together (i.e.", "homologous fragments from different species or chromosomal locations are homologously recombined).", "As a simple case, homologs from two species (say, E. coli and Salmonella) are cloned, family stochastic &/or non-stochastic mutagenized in vitro and cloned into an allele replacement vector (e.g., a vector with a positively selectable marker, a negatively selectable marker and conditionally active origin of replication).", "The basic strategy for whole genome family stochastic &/or non-stochastic mutagenesis of parsed (subcloned) genomes is additionally set forth herein.", "The vectors are transfected into E. coli and selected, e.g., for drug resistance.", "Most drug resistant cells should arise by homologous recombination between a family stochastic &/or non-stochastic mutagenized insert and a chromosomal copy of the cloned insert.", "Colonies with improved phenotype are screened (e.g., by mass spectroscopy for enzyme activity or small molecule production, or a chromogenic screen, or the like, depending on the phenotype to be assayed).", "Negative selection (i.e.", "sue selection) is imposed to force excision of tandem duplication.", "Roughly half C, of the colonies should retain the improved phenotype.", "Importantly, this process regenerates a “clean” chromosome in which the wild type locus is replaced with a family stochastic &/or non-stochastic mutagenized fragment that encodes a beneficial allele.", "Since the chromosome is “clean” (i.e., has no vector sequences), other improved alleles can also be moved into this point on the chromosome by homologous recombination.", "Selection or screening for improved phenotype can occur either after step 3 or step 4.If selection or screening takes place after step 3, then the improved allele can be conveniently moved to other strains by, for example, P I transduction.", "One can then regenerate a strain containing the improved allele but lacking vector sequences by “negative selection” against the suc marker.", "In subsequent rounds, independently identified improved variants of the gene can be sequentially moved into the improved strain (e.g., by P I transduction of the drug marked tandem duplication above).", "Transductants are screened for further improvement in phenotype by virtue of receiving the transduced tandem duplication, which itself contains the family stochastic &/or non-stochastic mutagenized genetic material.", "Negative selection is again imposed and the process of stochastic &/or non-stochastic mutagenesis the improved strain is recursively repeated as desired.", "Although this process was described with reference to targeting a gene or genes of interest, it can be used “blindly,” making no assumptions about which locus is to be targeted.", "This procedure is set forth herein.", "For example, the whole genome of an organism of interest is cloned into manageable fragments (e.g., 10 kb for plasmid-based methods).", "Homologous fragments are then isolated from related species.", "Forced recombination with chromosomal homologs creates chimeras.", "4.6.1.2.2.12 Methods for High Throughput Family Stochastic &/or Non-Stochastic Mutagenesis of Genes For E.", "coli., cloning the genome in 10 kb fragments requires about 300 clones.", "The homologous fragments are isolated, e.g., from Salmonella.", "This gives roughly three hundred pairs of homologous fragments.", "Each pair is family stochastic &/or non-stochastic mutagenized and the stochastic &/or non-stochastic mutagenized fragments are cloned into an allele replacement vector.", "The inserts are integrated into the E. coli genome as described above.", "A global screen is made to identify variants with an improved phenotype.", "This serves as the basis collection of improvements that are to be stochastic &/or non-stochastic mutagenized to produce a desired strain.", "The stochastic &/or non-stochastic mutagenesis of these independently identified variants into one super strain is done as described above.", "Family stochastic &/or non-stochastic mutagenesis has been shown to be an efficient method for creating high quality libraries of genetic variants.", "Given a cloned gene from one species, it is of interest to quickly and rapidly isolate homologs from other species, and this process can be rate limiting.", "For example, if one wants to perform family stochastic &/or non-stochastic mutagenesis on an entire genome, one may need to construct hundreds to thousands of individual family stochastic &/or non-stochastic mutagenized libraries.", "In this embodiment, a gene of interest is optionally cloned into a vector in which ssDNA can be made.", "An example of such a vector is a phagemid vector with an Ml 3 origin of replication.", "Genomic DNA or cDNA from a species of interest is isolated, denatured, annealed to the phagemid, and then enzymatically manipulated to clone it.", "The cloned DNA is then used to family reassemble with the original gene of interest.", "PCR based formats are also available.", "These formats require no intermediate cloning steps, and are, therefore, of particular interest for high throughput applications.", "Alternatively, the gene of interest can be fished out using purified RecA protein.", "The gene of interest is PCR amplified using primers that are tagged with an affinity tag such as biotin, denatured, then coated with RecA protein (or an improved variant thereof).", "The coated ssDNA is then mixed with a gDNA plasmid library.", "Under the appropriate conditions, such as in the presence of non-hydrolyzable rATP analogs, RecA will catalyze the hybridization of the RecA coated gene (ssDNA) in the plasmid library.", "The heteroduplex is then affinity purified from the non-hybridizing plasmids of the gene library by adsorbtion of the labeled PCR products and its associated homologous DNA to an appropriate affinity matrix.", "The homologous DNA is used in a family stochastic &/or non-stochastic mutagenesis reaction for improvement of the desired function.", "Stochastic &/or non-stochastic mutagenesis the E. coli chaperonin gene DnaJ with other homologs is described below as an example.", "The example can be generalized to any other gene, including eukaryotic genes such as plant or animal genes (including mammalian genes), by following the format described.", "As a first step, the E. coli Dna1 gene is cloned into an M13 phagemid vector.", "ssDNA is then produced, preferably in a dut(−) ung(−) strain so that Kunkel site directed mutagenesis protocols can be applied.", "Genomic DNA is then isolated from a non-E. coli source, such as Salmonella and Yersinia Pestis.", "The bacterial genomic DNAs are denatured and reannealed to the phagemid ssDNA (e.g., about 1 microgram of ssDNA).", "The reannealed product is treated with an enzyme such as Mung Bean nuclease that degrades ssDNA as an exonuclease but not as an endonuclease (the nuclease does not degrade mismatched DNA that is embedded in a larger annealed fragment).", "The standard Kunkel site directed mutagenesis protocol is used to extend the fragment and the target cells are transformed with the resulting mutagenized DNA.", "In a first variation on the above, the procedure is adapted to the situation where the target gene or genes of interest are unknown.", "In this variation, the whole genome of the organism of interest is cloned in fragments (e.g., of about 10 kb each) into a phagemid.", "Single stranded phagemid DNA is then produced.", "Genomic DNA from the related species is denatured and annealed to the phagemids.", "Mung bean nuclease is used to trim away unhybridized DNA ends.", "Polymerase plus ligase is used to fill in the resulting gapped circles.", "These clones are transformed into a mismatch repair deficient strain.", "When the mismatched molecules are replicated in the bacteria, most colonies contain both the E. coli and the homologous fragment.", "The two homologous genes are then isolated from the colonies (e.g., either by standard plasmid purification or colony PCR) and stochastic &/or non-stochastic mutagenized.", "Another approach to generating chimeras that requires no in vitro stochastic &/or non-stochastic mutagenesis is simply to clone the Salmonella genome into an allele replacement vector, transform E. coli, and select for chromosomal integrants.", "Homologous recombination between Salmonella genes and E. coli homologs generate stochastic &/or non-stochastic mutagenized chimeras.", "A global screen is done to screen for improved phenotypes.", "Alternately, recursive transformation and recombination is performed to increase diversity prior to screening.", "If colonies with improved phenotypes are obtained, it is verified that the improvement is due to allele replacement by P 1 transduction into a fresh strain and counterscreening for improved phenotype.", "A collection of such improved alleles can then be combined into one strain using the methods for whole genome stochastic &/or non-stochastic mutagenesis by blind family stochastic &/or non-stochastic mutagenesis of parsed genomes as set forth herein.", "Additionally, once these loci are identified, it is likely that further rounds of stochastic &/or non-stochastic mutagenesis and screening will yield further improvements.", "This could be done by cloning the chimeric gene and then using the methods described in this disclosure to breed the gene with homologs from many different strains of bacteria.", "In general, the transformants contain clones of the homologue of the target gene (e.g., E. coli DnaJ in the example above).", "Mismatch repair in vivo results in a decrease in diversity of the gene.", "There are at least two solutions to this.", "First, transduction can be performed into a mismatch repair deficient strain.", "Alternatively or in addition, the Ml 3 template DNA can be selectively degraded, leaving the cloned homologue.", "This can be done using methods similar to the standard Eckstein site directed mutagenesis technique (General texts which describe general molecular biological techniques useful herein, including mutagenesis, include Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.", "), Vol.", "1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1998) (“Ausubel”)).", "This method relies on incorporation of alpha thiol modified dNTPs during synthesis of the new strand followed by selective degradation of the template and resynthesis of the template strand.", "In one embodiment, the template strand is grown in a dut(−) ung(−) strain so that uracil is incorporated into the phagemid DNA.", "After extension as noted above (and before transformation) the DNA is treated with uracil glycosylate and an apurinic site endonuclease such as Endo III or Endo IV.", "The treated DNA is then treated with a processive exonuclease that resects from the resulting gaps while leaving the other strand intact (as in Eckstein mutagenesis).", "The DNA is polymerized and ligated.", "Target cells are then transformed.", "This process enriches for clones encoding the homologue which is not derived from the target (i.e., in the example above, the non-E. coli.", "homologue).", "An analogous procedure is optionally performed in a PCR format.", "As applied to the DnaJ illustration above, Dnal DNA is amplified by PCR with primers that build 30-mer priming sites on each end.", "The PCR is denatured and annealed with an excess of Salmonella genomic DNA.", "The Salmonella DnaJ gene hybribidizes with the E. coli homologue.", "After treatment with Mung Bean nuclease, the resulting mismatched hybrid is PCR amplified with the flanking 30-mer primers.", "This PCR product can be used directly for family stochastic &/or non-stochastic mutagenesis.", "As genomics provides an increasing amount of sequence information, it is increasingly possible to directly PCR amplify homologs with designed primers.", "For example, given the sequence of the E. coli genome and of a related genome (i.e.", "Salmonella), each genome can be PCR amplified with designed primers in, e.g., 5 kb fragments.", "The homologous fragments can be put together in a pairwise fashion for stochastic &/or non-stochastic mutagenesis.", "For genome stochastic &/or non-stochastic mutagenesis, the stochastic &/or non-stochastic mutagenized products are cloned into the allele replacement vector and bred into the genome as described supra.", "4.6.1.2.2.13 Hyper-Recombinogenic RecA Clones The invention further provides hyper-recombinogenic RecA proteins (see, the examples below).", "It is fully expected that one of skill can make a variety of related recombinogenic proteins given the disclosed sequences.", "Standard molecular biological techniques can be used to make nucleic acids which comprise the given nucleic acids, e.g., by cloning the nucleic acids into any known vector.", "Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.", "(1989) Molecular Cloning—A Laboratory Manual (2nd ed.)", "Vol.", "1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel).", "Product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods.", "Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.", "), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Mich.)), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.", "It will be appreciated that conservative substitutions of the given sequences can be used to produce nucleic acids which encode hyperrecombinogenic clones.", "“Conservatively modified variations” of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.", "Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide.", "For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.", "Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.", "Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation.", "One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon, for methionine) can be modified to yield a functionally identical molecule by standard techniques.", "Accordingly, each “silent variation” of a nucleic acid which encodes a polypeptide is implicit in any described sequence.", "Furthermore, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the substitution of an amino acid with a chemically similar amino acid.", "Conservative substitution tables providing functionally similar amino acids are well known in the art.", "The following six groups each contain amino acids that are conservative substitutions for one another.", "1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).", "See also, Creighton (1984) Proteins W.H.", "Freeman and Company.", "Finally, the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as a non-functional sequence is a conservative modification of the basic nucleic acid.", "One of skill will appreciate that many conservative variations of the nucleic acid constructs disclosed yield a functionally identical construct.", "For example, due to the degeneracy of the genetic code, “silent substitutions” (ie., substitutions of a nucleic acid sequence which do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence which encodes an amino acid.", "Similarly, “conservative amino acid substitutions,” in one or a few amino acids in an amino acid sequence of a packaging or packageable construct are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct.", "Such conservatively substituted variations of each explicitly disclosed sequence are a feature of the present invention.", "Nucleic acids which hybridize under stringent conditions to the nucleic acids in the figures are a feature of the invention.", "“Stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters.", "An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier, New York.", "Generally, highly stringent hybridization and wash conditions are selected to be about 5C lower than the thermal melting point (T.) for the specific sequence at a defined ionic strength and ph.", "The T. is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.", "Very stringent conditions are selected to be equal to the T. for a particular probe.", "In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.", "Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.", "This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.", "Finally, preferred nucleic acids encode hyper-recombinogenic RecA proteins which are at least one order of magnitude (10 times) as active as a wild-type RecA protein in a standard assay for RecA activity.", "4.6.1.2.2.14 RecE/RecT Mediated Stochastic &/or Non-Stochastic Mutagenesis In Vivo Like recA, recE and recT (or their homologues, for example the lambda recombination proteins red and red) can stimulate homologous recombination in vivo.", "See, Muyrers et al.", "(1999) Nucleic Acids Res 27(6): 1555-7 and Zhang et al.", "(1998) Nat Genet (2): 123-8 Hyper-recombinogenic recE and recT are evolved by the same method as described for recA.", "Alternatively, variants with increased recombinogenicity are selected by their ability to cause recombination between a suicide vector (lacking an origin of replication) carrying a selectable marker, and a homologous region in either the chromosome or a stably-maintained episome.", "A plasmid containing recA and recE genes is stochastic &/or non-stochastic mutagenized (either using these genes as single starting points, or by family stochastic &/or non-stochastic mutagenesis (with for example red and red, or other homologous genes identified from available sequence databases).", "This stochastic &/or non-stochastic mutagenized library is then cloned into a vector with a selectable marker and transformed into an appropriate recombination-deficient strain.", "The library of cells would then be transformed with a second selectable marker, either borne on a suicide vector or as a linear DNA fragment with regions at its ends that are homologous to a target sequence (either in the plasmid or in the host chromosome).", "Integration of this marker by homologous recombination is a selectable event, dependent on the activity of the recE and recT gene products.", "The recE/recT genes are isolated from cells in which homologous recombination has occurred.", "The process is repeated several times to enrich for the most efficient variants before the next round of stochastic &/or non-stochastic mutagenesis is performed.", "In addition, cycles of recombination without selection can be performed to increase the diversity of a cell population prior to selection.", "Once hyper-recombinogenic recE/recT genes are isolated they are used as described for hyper-recombinogenic recA.", "For example they are expressed (constitutively or conditionally) in a host cell to facilitate homologous recombination between variant gene fragments and homologues within the host cell.", "They are alternatively introduced by microinjection, biolistics, lipofection or other means into a host cell at the same time as the variant genes.", "Hyper-recombinogenic recE/recT (either of bacterial/phage origin, or from plant homologues) are useful for facilitating homologous recombination in plants.", "They are, for example, cloned into the Agrobacterium cloning vector, where they are expressed upon entry into the plant, thereby stimulating homologous recombination in the recipient cell.", "In a preferred embodiment, recE/recT are used and or generated in mutS strains.", "4.6.1.2.2.15 Multi-Cyclic Recombination As noted, protoplast fusion is an efficient means of recombining two microbial genomes.", "The process reproducibly results in about 10% of a non-selected population being recombinant chimeric organisms.", "Protoplasts are cells that have been stripped of their cell walls by treatment in hypotonic medium with cell wall degrading enzymes.", "Protoplast fusion is the induced fusion of the membranes of two or more of these protoplasts by fusogenic agents such as polyethylene glycol.", "Fusion results in cytoplasmic mixing and places the genomes of the fused cells within the same membrane.", "Under these conditions recombination between the genomes is frequent.", "The fused protoplasts are regenerated, and, during cell division, single genomes segregate into each daughter cell.", "Typically, 10% of these daughter cells have genomes that originate partially from more than one of the original parental protoplast genomes.", "This result is similar to that of the crossing over of sister chromatids in eukaryotic cells during prophase of meiosis II.", "The percentage of daughter cells that are recombinant is just lower after protoplast fusion.", "While protoplast fusion does result in efficient recombination, the recombination predominantly occurs between two cells as in sexual recombination.", "In order to efficiently generate libraries of whole genome stochastic &/or non-stochastic mutagenized libraries, daughter cells having genetic information originating from multiple parents are made.", "In vitro DNA stochastic &/or non-stochastic mutagenesis results in the efficient poolwise recombination of multiple homologous DNA sequences.", "The stochastic &/or non-stochastic mutagenesis of full length genes from a mixed pool of small gene fragments requires multiple annealing and elongation cycles, the thermal cycles of the primerless PCR reaction.", "During each thermal cycle, many pairs of fragments anneal and are extended to form a combinatorial population of larger chimeric DNA fragments.", "After the first cycle of stochastic &/or non-stochastic mutagenesis, chimeric fragments contain sequences originating from two different parent genes.", "This is similar to the result of a single sexual cycle within a population, pairwise cross, or protoplast fusion.", "During the second cycle, these chimeric fragments can anneal with each other, or with other small fragments, resulting in chimeras originating from up to four different parental sequences.", "This second cycle is analogous to the entire progeny from a single sexual cross inbreeding with itself.", "Further cycles will result in chimeras originating from 8, 16, 32, etc parental sequences and are analogous to further inbreedings of the progeny population.", "The power of in vitro DNA stochastic &/or non-stochastic mutagenesis is that a large combinatorial library can be generated from a single pool of DNA fragments stochastic &/or non-stochastic mutagenized by these recursive pairwise “matings.” As described above, in vivo stochastic &/or non-stochastic mutagenesis strategies, such as protoplast fusion, result in a single pairwise mating reaction.", "Thus, to generate the level of diversity obtained by in vitro methods, in vivo methods are carried out recursively.", "That is, a pool of organisms is recombined and the progeny pooled, without selection, and then recombined again.", "This process is repeated for sufficient cycles to result in progeny having multiple parental sequences.", "Described below is a method used to reassemble four strains of Streptomyces coelicolor.", "From the initial four strains each containing a unique nutritional marker, three to four rounds of recursive pooled protoplast fusion was sufficient to generate a population of stochastic &/or non-stochastic mutagenized organisms containing all 16 possible combinations of the four markers.", "This represents a 106 fold improvement in the generation of four parent progeny as compared to a single pooled fusion of the four strains.", "Protoplasts were generated from several strains of S. coelicolor, pooled and fused.", "Mycelia were regenerated and allowed to sporulate.", "The spores were collected, allowed to grow into Mycelia, formed into protoplasts, pooled and fused and the process repeated for three to four rounds.", "the resulting spores were then subject to screening.", "The basic protocol for generating a whole genome stochastic &/or non-stochastic mutagenized library from four S. coelicolor strains, each having one of four distinct markers, was as follows.", "Four mycelial cultures, each of a strain having one of four different markers, were grown to early stationary phase.", "The mycelia from each were harvested by centrifugation and washed.", "Protoplasts from each culture were prepared as follows.", "Approximately 109 S. coelicolor spores were inoculated into 50 ml YEME with 0.5% Glycine in a 250 ml baffled flask.", "The spores were incubated at 30C for 36-40 hours in an orbital shaker.", "Mycelium were verified using a microscope.", "Some strains needed an additional day of growth.", "The culture was transferred into a 50 ml tube and centrifuged at 4,000 rpm for 10 min.", "The mycelium were twice washed with 10.3% sucrose and centriftiged at 4,000 rpm for 10 min.", "(mycelium can be stored at about 80C after wash).", "5 ml of lysozyme was added to the about 0.5 g of mycelium pellet.", "The pellet was suspended and incubated at 30C for 20-60 min., with gentle shaking every 10 min.", "The microscope was checked for protoplasting every 20 min.", "Once the majority were protoplasts, protoplasting was stopped by adding 10 ml of P buffer.", "The protoplasts were filtered through cotton and the protoplast spun down at 3,000 rpm for 7 min at room temperature.", "The supernatant was discarded and the protoplast gently resuspended, adding a suitable amount of P buffer according to the pellet size (usually about 500W).", "Ten-fold serial dilutions were made in P buffer, and the protoplasts counted at a 1012 dilution.", "Protoplasts were adjusted to 1010 protoplasts per ml.", "The protoplasts from each culture were quantitated by microscopy.", "108 protoplast from each culture were mixed in the same tube, washed, and then fused by the addition of 50% PEG.", "The fused protoplasts were diluted and plated regeneration medium and incubated until the colonies were sporulating (four days).", "Spores were harvested and washed.", "These spores represent a pool of all the recombinants and parents form the fusion.", "A sample of the pooled spores was then used to inoculate a single liquid culture.", "The culture was grown to early stationary phase, the myclelia harvested, and protoplasts prepared.", "108 protoplasts from this “mycelial library” were then fused with themselves by the addition of 50% PEG.", "The protoplast fusion/regeneration/harvesting/protoplast preparation steps were repeated two times.", "The spores resulting from the fourth round of fusion were considered the “whole genome stochastic &/or non-stochastic mutagenized library” and they were screened for the frequency of the 16 possible combinations of the four markers In particular, adding rounds of recombination prior to selection produced significant increases in the number of clones which incorporated all four of the relevant selectable markers, indicating that the population became increasingly diverse be recursive pooling and sporulation.", "The four strains of the four parent stochastic &/or non-stochastic mutagenesis were each auxotrophic for three and prototrophic for one of four possible nutritional markers: arginine (A), cystine (C), proline (P), and/or uracil (U).", "Spores from each fusion were plated in each of the 16 possible combinations of these four nutrients, and the percent of the population growing on a particulate medium was calculated as the ration of those colonies form a selective plate to those growing on a plate having all four nutrients (all variants grow on the medium having all four nutrients, thus the colonies from this plate thus represent the total viable population).", "The corrected percentages for each of the no, one, two, and three marker phenotypes were determined by subtracting the percentage of cells having additional markers that might grow on the medium having “unnecessary” nutrients.", "For example, the number of colonies growing on no additional nutrients (the prototroph) was subtracted from the number of colonies growing on any plate requiring nutrients.", "4.6.1.2.2.16 Whole Genome Stochastic &/or Non-Stochastic Mutagenesis Through Organized Heteroduplex Stochastic &/or Non-Stochastic Mutagenesis A new procedure to optimize phenotypes of interests by heteroduplex stochastic &/or non-stochastic mutagenesis of cosmids libraries of the organism of choice, is provided.", "This procedure does not require protoplast fusion and is applicable to bacteria for which well-established genetic systems are available, including cosmid cloning, transformation, in vitro packaging/transfection and plasmid transfer/mobilization.", "Microorganism that can be improved by these methods include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas spp., Rhizobium spp., Xanthomonas spp., and other gram-negative organisms.", "This method is also applicable to Gram-positive microorganisms.", "In step A, Chromosomal DNA of the organism to be improved is digested with suitable restriction enzymes and ligated into a cosmid.", "The cosmid used for cosmid-based heteroduplex guided whole genome stochastic &/or non-stochastic mutagenesis has at least two rare restriction enzyme recognition sites (e.g.", "Sfr and NotI) to be used for linearization in subsequent steps.", "Sufficient cosmids to represent the complete chromosome are purified and stored in 96-well microtiter dishes.", "In step B, small samples of the library are mutagenized in vitro using hydroxylamine or other mutagenic chemicals.", "In step C, a sample from each well of the mutagenized collection is used to transfect the target cells.", "In step D, the transfectants are assayed (as a pool from each mutagenized sample-well) for phenotypic improvements.", "Positives from this assay indicate that a cosmid from a particular well can confer phenotypic improvements and thus contain large genomic fragments that are suitable targets for heteroduplex mediated stochastic &/or non-stochastic mutagenesis.", "In step E, the transfected cells harboring a mutant library of the identified cosmid(s) are separated by plating on solid media and screened for independent mutants conferring an improved phenotype.", "In step F, DNA from positive cells is isolated and pooled by origin.", "In step G, the selected cosmid pools are divided so that one sample can be digested with Sfr and the other with NotI.", "These samples are pooled, denatured, reannealed, and religated.", "In step K target cells are transfected with the resulting heteroduplexes and propagated to allow “recombination” to occur between the strands of the heteroduplexes in vivo.", "The transfectants can be screened (the population will represent the pairwise recombinants) or, commonly, as represented by step 1, the recombined cosmids are further stochastic &/or non-stochastic mutagenized by recursive in vitro heteroduplex formation and in vivo recombination (to generate a complete combinatorial library of the possible mutations) prior to screening.", "An additional mutagenesis step could also be added for increased diversity during the stochastic &/or non-stochastic mutagenesis process.", "In step J, once several cosmids harboring different distributed loci have been improved, they are combined into the same host by chromosome integration.", "This organism can be used directly or subjected to a new round of heteroduplex guided whole genome stochastic &/or non-stochastic mutagenesis.", "4.7.Specialized Methods 4.7.1 Targeted Stochastic &/or Non-Stochastic Mutagenesis-Hot Spots In one aspect, targeted homologous genes are cloned into specific regions of the genome (e.g., by homologous recombination or other targeting procedures) which are known to be recombination “hot spots” (i.e., regions showing elevated levels of recombination compared to the average level of recombination observed across an entire genome), or known to be proximal to such hot spots.", "The resulting recombinant strains are mated recursively.", "During meiotic recombination, homologous recombinant genes recombine, thereby increasing the diversity of the genes.", "After several cycles of recombination by recursive mating, the resulting cells are screened.", "4.7.2 Stochastic &/or Non-Stochastic Mutagenesis Using Yeasts Yeasts are subspecies of fungi that grow as single cells.", "Yeasts are used for the production of fermented beverages and leavening, for production of ethanol as a fuel, low molecular weight compounds, and for the heterologous production of proteins and enzymes (see accompanying list of yeast strains and their uses).", "Commonly used strains of yeast include Saccharomyces cerevisiae, Pichia sp., Canidia sp.", "and Schizosaccharomyces pombe.", "Several types of vectors are available for cloning in yeast including integrative plasmid (Ylp), yeast replicating plasmid (YRp, such as the 2 circle based vectors), yeast episomal plasmid (YEp), yeast centromeric plasmid (YCp), or yeast artificial chromosome (YAC).", "Each vector can carry markers useful to select for the presence of the plasmid such as LUE2, URA3, and H1S3, or the absence of the plasmid such as URA3 (a gene that is toxic to cells grown in the presence of 5-fluoro orotic acid.", "Many yeasts have a sexual cycle and asexual (vegetative) cycles.", "The sexual cycle involves the recombination of the whole genome of the organism each time the cell passes through meiosis.", "For example, when diploid cells of S. cerevisiae are exposed to nitrogen and carbon limiting conditions, diploid cells undergo meiosis to form asci.", "Each ascus holds four haploid spores, two of mating type “a” and two of mating type “ ” Upon return to rich medium, haploid spores of opposite mating type mate to form diploid cells once again.", "Asiospores of opposite mating type can mate within the ascus, or if the ascus is degraded, for example with zymolase, the haploid cells are liberated and can mate with spores from other asci.", "This sexual cycle provides a format to reassemble endogenous genomes of yeast and/or exogenous fragment libraries inserted into yeast vectors.", "This process results in swapping or accumulation of hybrid genes, and for the stochastic &/or non-stochastic mutagenesis of homologous sequences shared by mating cells.", "Yeast strains having mutations in several known genes have properties useful for stochastic &/or non-stochastic mutagenesis.", "These properties include increasing the frequency of recombination and increasing the frequency of spontaneous mutations within a cell.", "These properties can be the result of mutation of a coding sequence or altered expression (usually overexpression) of a wildtype coding sequence.", "The HO nuclease effects the transposition of HMLa/ and HMRa/ to the MAT locus resulting in mating type switching.", "Mutants in the gene encoding this enzyme do not switch their mating type and can be employed to force crossing between strains of defined genotype, such as ones that harbor a library or have a desired phenotype and to prevent in breeding of starter strains.", "PMS 1, MLH 1, MSH2, MSH6 are involved in mismatch repair.", "Mutations in these genes all have a mutator phenotype (Chambers et al., Mol.", "Cell.", "Biol.", "16, 6110-6120 (1996)).", "Mutations in TOP3 DNA topoisomerase have a 6-fold enhancement of interchromosomal homologous recombination (Bailis et al., Molecular and Cellular Biology 12, 49884993 (1992)).", "The RAD50-57 genes confer resistance to radiation.", "Rad3 functions in excision of pyrimidine dimers.", "RAD52 functions in gene conversion.", "RAD50, MREI 1, XRS2 function in both homologous recombination and illegitimate recombination.", "HOP1, REDI function in early meiotic recombination (Mao-Draayer, Genetics 144, 71-86) Mutations in either HOPI or RED 1 reduce double stranded breaks at the HIS2 recombination hotspot.", "Strains deficient in these genes are useful for maintaining stability in hyper recombinogenic constructs such as tandem expression libraries carried on YACs.", "Mutations in HPR1 are hyperrecombinogenic.", "HDFI has DNA end binding activity and is involved in double stranded break repair and V(D)J recombination.", "Strains bearing this mutation are useful for transformation with random genomic fragments by either protoplast fusion or electroporation.", "Kar-1 is a dominant mutation that prevents karyogamy.", "Kar-1 mutants are useful for the directed transfer of single chromosomes from a donor to a recipient strain.", "This technique has been widely used in the transfer of YACs between strains, and is also useful in the transfer of evolved genes/chromosomes to other organisms (Markie, YA C Protocols, (Humana Press, Totowa, N.J., 1996).", "HOTI is an S. cerevisiae recombination hotspot within the promoter and enhancer region of the rDNA repeat sequences.", "This locus induces mitotic recombination at adjacent sequences—presumably due to its high level transcription.", "Genes and/or pathways inserted under the transcriptional control of this region undergo increased mitotic recombination.", "The regions surrounding the arg 4 and his 4 genes are also recombination hot spots, and genes cloned in these regions have an increased probability of undergoing recombination during meiosis.", "Homologous genes can be cloned in these regions and stochastic &/or non-stochastic mutagenized in vivo by recursively mating the recombinant strains.", "CDC2 encodes polymerase and is necessary for mitotic gene conversion.", "Overexpression of this gene can be used in a reassembler or mutator strain.", "A temperature sensitive mutation in CDC4 halts the cell cycle at G1 at the restrictive temperature and could be used to synchronize protoplasts for optimized fusion and subsequent recombination.", "As with filamentous fungi, the general goals of stochastic &/or non-stochastic mutagenesis yeast include improvement in yeast as a host organism for genetic manipulation, and as a production apparatus for various compounds.", "One desired property in either case is to improve the capacity of yeast to express and secrete a heterologous protein.", "The following example describes the use of stochastic &/or non-stochastic mutagenesis to evolve yeast to express and secrete increased amounts of RNase A. RNase A catalyzes the cleavage of the P-05′ bond of RNA specifically after pyrimidine nucleotides.", "The enzyme is a basic 124 amino acid polypeptide that has 8 half cystine residues, each required for catalysis.", "YEpWL-RNase A is a vector that effects the expression and secretion of RNaseA from the yeast S. cerevisiae, and yeast harboring this vector secrete 1-2 mg of recombinant RNase A per liter of culture medium (del Cardayre et al., Protein Engineering 8(3): 26, 1-273 (1995)).", "This overall yield is poor for a protein heterologously expressed in yeast and can be improved at least 10-100 fold by stochastic &/or non-stochastic mutagenesis.", "The expression of RNaseA is easily detected by several plate and microtitre plate assays (del Cardayre & Raines, Biochemistry 33, 6031-6037 1994)).", "Each of the described formats for whole genome stochastic &/or non-stochastic mutagenesis can be used to reassemble a strain of S. cerevisiae harboring YepWL-RNase A, and the resulting cells can be screened for the increased secretion of RNase A into the medium.", "The new strains are cycled recursively through the stochastic &/or non-stochastic mutagenesis format, until sufficiently high levels of RNase A secretion is observed.", "The use of RNase A is particularly useful since it not only requires proper folding and disulfide bond formation but also proper glycosylation.", "Thus numerous components of the expression, folding, and secretion systems can be optimized.", "The resulting strain is also evolved for improved secretion of other heterologous proteins.", "4.7.3 Reassemble to Increase Tolerance of Yeast to Ethanol Another goal of stochastic &/or non-stochastic mutagenesis yeast is to increase the tolerance of yeast to ethanol.", "Such is useful both for the commercial production of ethanol, and for the production of more alcoholic beers and wines.", "The yeast strain to be stochastic &/or non-stochastic mutagenized acquires genetic material by exchange or transformation with other strain(s) of yeast, which may or may not be know to have superior resistance to ethanol.", "The strain to be evolved is stochastic &/or non-stochastic mutagenized and shufflants are selected for capacity to survive exposure to ethanol.", "Increasing concentrations of ethanol can be used in successive rounds of stochastic &/or non-stochastic mutagenesis.", "The same principles can be used to reassemble baking yeasts for improved osmotolerance.", "4.7.4 Capacity to Grow under Desired Nutritional Conditions Another desired property of stochastic &/or non-stochastic mutagenesis yeast is capacity to grow under desired nutritional conditions.", "For example, it is useful to yeast to grow on cheap carbon sources such as methanol, starch, molasses, cellulose, cellobiose, or xylose depending on availability.", "The principles of stochastic &/or non-stochastic mutagenesis and selection are similar to those discussed for filamentous fungi.", "4.7.5 To Produce Secondary Metabolites Another desired property is capacity to produce secondary metabolites naturally produced by filamentous fungi or bacteria, Examples of such secondary metabolites are cyclosporin A, taxol, and cephalosporins.", "The yeast to be evolved undergoes genetic exchange or is transformed with DNA from organism(s) that produce the secondary metabolite.", "For example, fungi producing taxol include Taxomyces andreanae and Pestalotopis microspora (Stierle et al., Science 260, 214-216 (1993); Strobel et al., Microbiol.", "142, 435440 (1996)).", "DNA can also be obtained from trees that naturally produce taxol, such as Taxus brevifolia.", "DNA encoding one enzyme in the taxol pathway, taxadiene synthase, which it is believed catalyzes the committed step in taxol biosynthesis and may be rate limiting in overall taxol production, has been cloned (Wildung & Croteau, J Biol.", "Chem.", "271, 9201-4 (1996).", "The DNA is then stochastic &/or non-stochastic mutagenized, and shufflants are screened/selected for production of the secondary metabolite.", "For example, taxol production can be monitored using antibodies to taxol, by mass spectroscopy or UV spectrophotometry.", "Alternatively, production of intermediates in taxol synthesis or enzymes in the taxol synthetic pathway can be monitored.", "Concetti & Ripani, Biol.", "Chem.", "Hoppe Seyler 375, 419-23 (1994).", "Other examples of secondary metabolites are polyols, amino acids, polyketides, non-ribosomal polypeptides, ergosterol, carotenoids, terpinoids, sterols, vitamin E, and the like.", "4.7.6 Increase Ability to Separate in Ethanol Another desired property is to increase the flocculence of yeast to facilitate separation in preparation of ethanol.", "Yeast can be stochastic &/or non-stochastic mutagenized by any of the procedures noted above with selection for stochastic &/or non-stochastic mutagenized yeast forming the largest clumps.", "4.7.6.1 Exemplary Procedure for Yeast Protoplasting Protoplast preparation in yeast is reviewed by Morgan, in Protoplasts (Birkhauser Verlag, Basel, 1983).", "Fresh cells (˜108) are washed with buffer, for example 0.1 M potassium phosphate, then resuspended in this same buffer containing a reducing agent, such as 50 mM DTT, incubated for 1 h at 30° C. with gentle agitation, and then washed again with buffer to remove the reducing agent.", "These cells are then resuspended in buffer containing a cell wall degrading enzyme, such as Novozyme 234 (1 mg/mL), and any of a variety of osmotic stabilizers, such as sucrose, sorbitol, NaCl, KCl, MgSO4, MgCl2, or NH4Cl at any of a variety of concentrations.", "These suspensions are then incubated at 30° C. with gentle shaking (−60 rpm) until protoplasts are released.", "To generate protoplasts that are more likely to produce productive fusants several strategies are possible.", "Protoplast formation can be increased if the cell cycle of the protoplasts have been synchronized to be halted at G1.In the case of S. cerevisiae this can be accomplished by the addition of mating factors, either a or alpha (Curran & Carter, J. Gen. Microbiol.", "129, 1589-1591 (1983)).", "These peptides act as adenylate cyclase inhibitors which by decreasing the cellular level of cAMP arrest the cell cycle at G1.In addition, sex factors have been shown to induce the weakening of the cell wall in preparation for the sexual fusion of a and alpha cells (Crandall & Brock, Bacteriol.", "Rev.", "32, 139-163 (1968); Osumi et al., Arch.", "Microbiol 97, 27-38 (1974)).", "Thus in the preparation of protoplasts, cells can be treated with mating factors or other known inhibitors of adenylate cyclase, such as leflunomide or the killer toxin from K. lactis, to arrest them at G1 (Sugisaki et al., Nature 304, 464-466 (1983)).", "Then after fusing of the protoplasts (step 2), cAMP can be added to the regeneration medium to induce S-phase and DNA synthesis.", "Alternatively, yeast strains having a temperature sensitive mutation in the CDC4 gene can be used, such that cells could be synchronized and arrested at G1.After fusion cells are returned to the permissive temperature so that DNA synthesis and growth resumes.", "Once suitable protoplasts have been prepared, it is necessary to induce fusion by physical or chemical means.", "An equal number of protoplasts of each cell type is mixed in phosphate buffer (0.2 M, pH 5.8, 2×108 cells/mL) containing an osmotic stabilizer, for example 0.8 M NaCl, and PEG 6000 (33% w/v) and then incubated at 30° C. for 5 min while fusion occurs.", "Polyols, or other compounds that bind water, can be employed.", "The fusants are then washed and resuspended in the osmotically stabilized buffer lacking PEG, and transferred to osmotically stabilized regeneration medium on/in which the cells can be selected or screened for a desired property.", "4.7.7 Stochastic &/or Non-Stochastic Mutagenesis Using Artificial Chromosomes Yeast artificial chromosomes (Yacs) are yeast vectors into which very large DNA fragments (e.g., 50-2000 kb) can be cloned (see, e.g., Monaco & Larin, Trends.", "Biotech.", "12(7), 280-286 (1994); Ramsay, Mol Biotechnol 1(2), 181-201 1994; Huxley, Genet.", "Eng.", "16, 65-91 (1994); Jakobovits, Curr.", "Biol.", "4(8), 761-3 (1994); Lamb & Gearhart, Curr.", "Opin.", "Genet.", "Dev.", "5(3), 342-8 (1995); Montoliu et al., Reprod Fertil.", "Dev.", "6, 577-84 (1994)).", "These vectors have telomeres (Tel), a centromere (Cen), an autonomously replicating sequence (ARS), and can have genes for positive (e.g., TRPI) and negative (e.g., URA3) selection.", "YACs are maintained, replicated, and segregate as other yeast chromosomes through both meiosis and mitosis thereby providing a means to expose cloned DNA to true meiotic recombination.", "YACs provide a vehicle for the stochastic &/or non-stochastic mutagenesis of libraries of large DNA fragments in vivo.", "The substrates for stochastic &/or non-stochastic mutagenesis are typically large fragments from 20 kb to 2 Mb.", "The fragments can be random fragments or can be fragments known to encode a desirable property.", "For example, a fragment might include an operon of genes involved in production of antibiotics.", "Libraries can also include whole genomes or chromosomes.", "Viral genomes and some bacterial genomes can be cloned intact into a single YAC.", "In some libraries, fragments are obtained from a single organism.", "Other libraries include fragment variants, as where some libraries are obtained from different individuals or species.", "Fragment variants can also be generated by induced mutation.", "Typically, genes within fragments are expressed from naturally associated regulatory sequences within yeast.", "However, alternatively, individual genes can be linked to yeast regulatory elements to form an expression cassette, and a concatemer of such cassettes, each containing a different gene, can be inserted into a YAC.", "In some instances, fragments are incorporated into the yeast genome, and stochastic &/or non-stochastic mutagenesis is used to evolve improved yeast strains.", "In other instances, fragments remain as components of YACs throughout the stochastic &/or non-stochastic mutagenesis process, and after acquisition of a desired property, the YACs are transferred to a desired recipient cell.", "4.7.8 Stochastic &/or Non-Stochastic Mutagenesis of Genes for Bioremediation Modern industry generates many pollutants for which the environment can no longer be considered an infinite sink.", "Naturally occurring microorganisms are able to metabolize thousands of organic compounds, including many not found in nature (e.g xenobiotics).", "Bioremediation, the deliberate use of microorganisms for the biodegradation of man-made wastes, is an emerging technology that offers cost and practicality advantages over traditional methods of disposal.", "The success of bioremediation depends on the availability of organisms that are able to detoxify or mineralize pollutants.", "Microorganisms capable of degrading specific pollutants can be generated by genetic engineering and recursive sequence recombination.", "Although bioremediation is an aspect of pollution control, a more useful approach in the long term is one of prevention before industrial waste is pumped into the environment.", "Exposure of industrial waste streams to recursive sequence recombination-generated microorganisms capable of degrading the pollutants they contain would result in detoxification of mineralization of these pollutants before the waste stream enters the environment.", "Issues of releasing recombinant organisms can be avoided by containing them within bioreactors fitted to the industrial effluent pipes.", "This approach would also allow the microbial mixture used to be adjusted to best degrade the particular wastes being produced.", "Finally, this method would avoid the problems of adapting to the outside world and dealing with competition that face many laboratory microorganisms.", "In the wild, microorganisms have evolved new catabolic activities enabling them to exploit pollutants as nutrient sources for which there is no competition.", "However, pollutants that are present at low is concentrations in the environment may not provide a sufficient advantage to stimulate the evolution of catabolic enzymes.", "For a review of such naturally occurring evolution of biodegradative pathways and the manipulation of some of microorganisms by classical techniques, see Ramos et al., Bio/Technology “12: 1349-1355 (1994).", "Generation of new catabolic enzymes or pathways for bioremediation has thus relied upon deliberate transfer of specific genes between organisms (Wackett et al., supra), forced matings between bacteria with specific catabolic capabilities (Brenner et al.", "Biodegradation 5: 359-377 (1994)), or prolonged selection in a chemostat Some researchers have attempted to facilitate evolution via naturally occurring genetic mechanisms in their chemostat selections by including microorganisms with a variety of catabolic pathways (Kellogg et. al.", "Science 214: 1133-1135 (1981); Chakrabarty American Society of Micro.", "Biol.", "News 62: 130-137 (1996)).", "For a review of efforts in this area, see Cameron et al.", "Applied Biochem.", "Biotech 38: 105-140 (1993).", "Current efforts in improving organisms for bioremediation take a labor-intensive approach in which many parameters are optimized independently, including transcription efficiency from native and heterologous promoters, regulatory circuits and translational efficiency as well as improvement of protein stability and activity (Timmis et al.", "Ann.", "Rev.", "Microbiol.", "48: 525-527 (1994)).", "A recursive sequence recombination approach overcomes a number of limitations in the bioremediation capabilities of naturally occurring microorganisms.", "Both enzyme activity and specificity can be altered, simultaneously or sequentially, by the methods of the invention.", "For example, catabolic enzymes can be evolved to increase the rate at which they act on a substrate.", "Although knowledge of a rate-limiting step in a metabolic pathway is not required to practice the invention, rate-limiting proteins in pathways can be evolved to have increased expression and/or activity, the requirement for inducing substances can be eliminated, and enzymes can be evolved that catalyze novel reactions.", "Some examples of chemical targets for bioremediation include but are not limited to benzene, xylene, and toluene, camphor, naphthalene, halogenated hydrocarbons, polychlorinated biphenyls (PCBs), trichlorethylene, pesticides such as pentachlorophenyls (PCPs), and herbicides such as atrazine.", "4.7.8.1 Aromatic Hydrocarbons Preferably, when an enzyme is “evolved” to have a new catalytic function, that function is expressed, either constitutively or in response to the new substrate.", "Recursive sequence recombination subjects both structural and regulatory elements (including the structure of regulatory proteins) of a protein to recombinogenic mutagenesis simultaneously.", "Selection of mutants that are efficiently able to use the new substrate as a nutrient source will be sufficient to ensure that both the enzyme and its regulation are optimized, without detailed analysis of either protein structure or operon regulation.", "Examples of aromatic hydrocarbons include but are not limited to benzene, xylene, toluene, biphenyl, and polycyclic aromatic hydrocarbons such as pyrene and naphthalene.", "These compounds are metabolized via catechol intermediates.", "Degradation of catechol by Pseudomonas putida requires induction of the catabolic operon by cis, cis-muconate which acts on the CatR regulatory protein.", "The binding site for the CatR protein is G-N11-A, while the optimal sequence for the LysR class of activators (of which CatR is a member) is T-N11-A.", "Mutation of the G to a T in the CatR binding site enhances the expression of catechol metabolizing genes (Chakrabarty, American Society of Microbiology News 62: 130-137 (1996)).", "This demonstrates that the control of existing catabolic pathways is not optimized for the metabolism of specific xenobiotics.", "It is also an example of a type of mutant that would be expected from recursive sequence recombination of the operon followed by selection of bacteria that are better able to degrade the target compound.", "As an example of starting materials, dioxygenases are required for many pathways in which aromatic compounds are catabolized.", "Even small differences in dioxygenase sequence can lead to significant differences in substrate specificity (Furukawa et al.", "J. Bact.", "175: 5224-5232 (1993); Erickson et al.", "App.", "Environ.", "Micro.", "59: 3858-3862 (1993)).", "A hybrid enzyme made using sequences derived from two “parental” enzymes may possess catalytic activities that are intermediate between the parents (Erickson, ibid.", "), or may actually be better than either parent for a specific reaction (Furukawa et al.", "J. Bact.", "176: 2121-2123 (1994)).", "In one of these cases site directed mutagenesis was used to generate a single polypeptide with hybrid sequence (Erickson, ibid.", "); in the other, a four subunit enzyme was produced by expressing two subunits from each of two different dioxygenases (Furukawa, ibid.).", "Thus, sequences from one or more genes encoding dioxygenases can be used in the recursive sequence recombination techniques of the instant invention, to generate enzymes with new specificities.", "In addition, other features of the catabolic pathway can also be evolved using these techniques, simultaneously or sequentially, to optimize the metabolic pathway for an activity of interest.", "4.7.8.2 Halogenated Hydrocarbons Large quantities of halogenated hydrocarbons are produced annually for uses as solvents and biocides.", "These include, in the United States alone, over 5 million tons of both 1,2-dichloroethane and vinyl chloride used in PVC production in the U.S. alone.", "The compounds are largely not biodegradable by processes in single organisms, although in principle haloaromatic catabolic pathways can be constructed by combining genes from different microorganisms.", "Enzymes can be manipulated to change their substrate specificities.", "Recursive sequence recombination offers the possibility of tailoring enzyme specificity to new substrates without needing detailed structural analysis of the enzymes.", "As an example of possible starting materials for the methods of the instant invention, Wackett et al.", "(Nature 368: 627-629 (1994)) recently demonstrated that through classical techniques a recombinant Pseudomonas strain in which seven genes encoding two multi-component oxygenases are combined, generated a single host that can metabolize polyhalogenated compounds by sequential reductive and oxidative techniques to yield non-toxic products.", "These and/or related materials can be subjected to the techniques discussed above so as to evolve and optimize a biodegradative pathway in a single organism.", "Trichloroethylene is a significant groundwater contaminant.", "It is degraded by microorganisms in a cometabolic way (i.e., no energy or nutrients are derived).", "The enzyme must be induced by a different compound (e.g., Pseudomonas cepacia uses toluene4-monoxygenase, which requires induction by toluene, to destroy trichloroethylene).", "Furthermore, the degradation pathway involves formation of highly reactive epoxides that can inactivate the enzyme (Timmis et al.", "Ann.", "Rev.", "Microbiol.", "48: 525-557 (1994)).", "The recursive sequence recombination techniques of the invention could be used to mutate the enzyme and its regulatory region such that it is produced constitutively, and is less susceptible to epoxide inactivation.", "In some embodiments of the invention, selection of hosts constitutively producing the enzyme and less susceptible to the epoxides can be accomplished by demanding growth in the presence of increasing concentrations of trichloroethylene in the absence of inducing substances.", "4.7.8.3 Polychlorinated Biphenyls and Polycyclic Aromatic Hydrocarbons Polychlorinated Biphenyls (PCBs) an Polycyclic Aromatic Hydrocarbons (PAHs) PCBs and PAHs are families of structurally related compounds that are major pollutants at many Superfund sites.", "Bacteria transformed with plasmids encoding enzymes with broader substrate specificity have been used commercially.", "In nature, no known pathways have been generated in a single host that degrade the larger PAHs or more heavily chlorinated PCBs.", "Indeed, often the collaboration of anaerobic and aerobic bacteria are required for complete metabolism.", "Thus, likely sources for starting material for recursive sequence recombination include identified genes encoding PAH-degrading catabolic pathways on large (20-100KB) plasmids (Sanseverino et al.", "Applied Environ.", "Micro.", "59: 1931-1937 (1993); Simon et al.", "Gene 127: 31-37 (1993); Zylstra et al.", "Annals of the NY Acad.", "Sci.", "721: 386-398 (1994)); while biphenyl and PCB-metabolizing enzymes are encoded by chromosomal gene clusters, and in a number of cases have been cloned onto plasmids (Hayase et al.", "J. Bacteriol.", "172: 1160-1164 (1990); Furukawa et al.", "Gene 98: 21-28 (1992); Hofer et al.", "Gene 144: 9-16 (1994)).", "The materials can be subjected to the techniques discussed above so as to evolve a biodegradative pathway in a single organism.", "Substrate specificity in the PCB pathway largely results from enzymes involved in initial dioxygenation reactions, and can be significantly altered by mutations in those enzymes (Erickson et al.", "Applied Environ.", "Micro.", "59: 3858-38662 (1993); Furukawa et al.", "J. Bact.", "175: 5224-5232 (1993).", "Mineralization of PAHs and PCBs requires that the downstream pathway is able to metabolize the products of the initial reaction (Brenner et al.", "Biodegradation 5: 359-377 (1994)).", "In this case, recursive sequence recombination of the entire pathway with selection for bacteria able to use the PCE or PAH as the sole carbon source will allow production of novel PCB and PAH degrading bacteria.", "4.7.8.4 Herbicides A general method for evolving genes for the catabolism of insoluble herbicides is exemplified as follows for atrazine.", "Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine] is a moderately persistent herbicide which is frequently detected in ground and surface water at concentrations exceeding the 3 ppb health advisory level set by the EPA.", "Atrazine can be slowly metabolized by a Pseudomonas species (Mandelbaum et al.", "Appl.", "Environ.", "Micro.", "61: 1451-1457 (1995)).", "The enzymes catalyzing the first two steps in atrazine metabolism by Pseudomonas are encoded by genes AtzA and AtzB (de Souza et al.", "Appl.", "Environ.", "Micro.", "61: 3373-3378 (1995)).", "These genes have been cloned in a 6.8 kb fragment into pUC 18 (AtzAB-pUC).", "E. coli carrying this plasmid converts atrazine to much more soluble metabolites.", "It is thus possible to screen for enzyme activity by growing bacteria on plates containing atrazine.", "The herbicide forms an opaque precipitate in the plates, but cells containing AtzAB-pU18 secrete atrazine degrading enzymes, leading to a clear halo around those cells or colonies.", "Typically, the size of the halo and the rate of its formation can be used to assess the level of activity so that picking colonies with the largest halos allows selection of the more active or highly produced atrazine degrading enzymes.", "Thus, the plasmids carrying these genes can be subjected is to the recursive sequence recombination formats described above to optimize the catabolism of atrazine in E. coli or another host of choice, including Pseudomonas.", "After each round of recombination, screening of host colonies expressing the evolved genes can be done on agar plates containing atrazine to observe halo formation.", "This is a generally applicable method for screening enzymes that metabolize insoluble compounds to those that are soluble (e.g., polycyclicaromatic hydrocarbons).", "Additionally, catabolism of atrazine can provide a source of nitrogen for the cell; if no other nitrogen is available, cell growth will be limited by the rate at which the cells can catabolize nitrogen.", "Cells able to utilize atrazine as a nitrogen source can thus be selected from a background of non-utilizers or poor-utilizers.", "4.7.8.5 Heavy Metal Detoxification Bacteria are used commercially to detoxify arsenate waste generated by the mining of arsenopyrite gold ores.", "As well as mining effluent, industrial waste water is often contaminated with heavy metals (e.g., those used in the manufacture of electronic components and plastics).", "Thus, simply to be able to perform other bioremedial functions, microorganisms must be resistant to the levels of heavy metals present, including mercury, arsenate, chromate, cadmium, silver, etc.", "A strong selective pressure is the ability to metabolize a toxic compound to one less toxic.", "Heavy metals are toxic largely by virtue of their ability to denature proteins (Ford et al.", "Bioextraction and Biodeterioration of Metals, p. 1-23).", "Detoxification of heavy metal contamination can be effected in a number of ways including changing the solubility or bioavailability of the metal, changing its redox state (e.g.", "toxic mercuric chloride is detoxified by reduction to the much more volatile elemental mercury) and even by bioaccumulation of the metal by immobilized bacteria or is plants.", "The accumulation of metals to a sufficiently high concentration allows metal to be recycled; smelting burns off the organic part of the organism, leaving behind reusable accumulated metal.", "Resistances to a number of heavy metals (arsenate, cadmium, cobalt, chromium, copper, mercury, nickel, lead, silver, and zinc) are plasmid encoded in a number of species including Staphylococcus and Pseudomonas (Silver et al.", "Environ.", "Health Perspect.", "102: 107-113 (1994); Ji et al.", "J. Ind.", "Micro.", "14: 61-75 (1995).", "These genes also confer heavy metal resistance on other species as well (e.g., E. coli).", "The recursive sequence recombination techniques of the instant invention (RSR) can be used to increase microbial heavy metal tolerances, as well as to increase the extent to which cells will accumulate heavy metals.", "For example, the ability of E. coli to detoxify arsenate can be improved at least 100-fold by RSR.", "Cyanide is very efficiently used to extract gold from rock containing as little as 0.2 oz.", "per ton.", "This cyanide can be microbially neutralized and used as a nitrogen source by fungi or bacteria such as Pseudomonas fluorescens.", "A problem with microbial cyanide degradation is the presence of toxic heavy metals in the leachate.", "RSR can be used to increase the resistance of bioremedial microorganisms to toxic heavy metals, so that they will be able to survive the levels present in many industrial and Superfund sites.", "This will allow them to biodegrade organic pollutants including but not limited to aromatic hydrocarbons, halogenated hydrocarbons, and biocides.", "4.7.8.6 Microbial Mining “Bioleaching” is the process by which microbes convert insoluble metal deposits (usually metal sulfides or oxides) into soluble metal sulfates.", "Bioleaching is commercially important in the mining of arsenopyrite, but has additional potential in the detoxification and recovery of metals and acids from waste dumps.", "Naturally occurring bacteria capable of bioleaching are reviewed by Rawlings and Silver (Bio/Technology 13: 773-778 (1995)).", "These bacteria are typically divided into groups by their preferred temperatures for growth.", "The more important mesophiles are Thiobacillus and Leptospirillum species.", "Moderate thermophiles include Sulfobacillus species.", "Extreme thermophiles include Sulfolobus species.", "Many of these organisms are difficult to grow in commercial industrial settings, making their catabolic abilities attractive candidates for transfer to and optimization in other organisms such as Pseudomonas, Rhodococcus, T. ferrooxidans or E. coli.", "Genetic systems are available for at least one strain of T. ferrooxidans, allowing the manipulation of its genetic material on plasmids.", "The recursive sequence recombination methods described above can be used to optimize the catalytic abilities in native hosts or heterologous hosts for evolved bioleaching genes or pathways, such as the ability to convert metals from insoluble to soluble salts.", "In addition, leach rates of particular ores can be improved as a result of, for example, increased resistance to toxic compounds in the ore concentrate, increased specificity for certain substrates, ability to use different substrates as nutrient sources, and so on.", "4.7.8.7 Oil Desulfurization The presence of sulfur in fossil fuels has been correlated with corrosion of pipelines, pumping, and refining equipment, and with the premature breakdown of combustion engines.", "Sulfur also poisons many catalysts used in the refining of fossil fuels.", "The atmospheric emission of sulfur combustion products is known as acid rain.", "Microbial desulfurization is an appealing bioremediation application.", "Several bacteria have been is reported that are capable of catabolizing dibenzothiophene (DBT), which is the representative compound of the class of sulfur compounds found in fossil fuels.", "U.S. Pat.", "No.", "5,356,801 discloses the cloning of a DNA molecule from Rhodococcus rhodochrous capable of biocatalyzing the desulfurization of oil.", "Denome et al.", "(Gene 175: 6890-6901 (1995)) disclose the cloning of a 9.8 kb DNA fragment from Pseudomonas encoding the upper naphthalene catabolizing pathway which also degrades dibenzothiophene.", "Other genes have been identified that perform similar functions (disclosed in U.S. Pat.", "No.", "5,356,801).", "The activity of these enzymes is currently too low to be commercially viable, but the pathway could be increased in efficiency using the recursive sequence recombination techniques of the invention.", "The desired property of the genes of interest is their ability to desulfurize dibenzothiophene or its alkyl or aryl substituted analogues.", "In some embodiments of the invention, selection is preferably accomplished by coupling this pathway to one providing a nutrient to the bacteria.", "Thus, for example, desulfurization of dibenzothiophene results in formation of hydroxybiphenyl.", "This is a substrate for the biphenyl-catabolizing pathway which provides carbon and energy.", "Selection would thus be done by “stochastic &/or non-stochastic mutagenesis” the dibenzothiophene genes and transforming them into a host containing the biphenyl-catabolizing pathway.", "Increased dibenzothiophene desulfurization will result in increased nutrient availability and increased growth rate.", "Once the genes have been evolved they are easily separated from the biphenyl degrading genes.", "The latter are undesirable in the final product since the object is to desulfurize without decreasing the energy content of the oil.", "Alkyl or aryl substituted dibenzothiophenes can be detected by changes in fluorescence (Krawiec, S., Devel.", "Indus.", "Microbiology 31: 103-114 (1990)) or by detection of phenol groups formed as a result of desulfurization (Dacre, J. C. Anal.", "Chem.", "43: 589-591 (1971)).", "4.7.8.8 Organo-Nitro Compounds Organo-nitro compounds are used as explosives, dyes, drugs, polymers and antimicrobial agents.", "Biodegradation of these compounds occurs usually by way of reduction of the nitrate group, catalyzed by nitroreductases, a family of broadly-specific enzymes.", "Partial reduction of organo-nitro compounds often results in the formation of a compound more toxic than the original (Hassan et al.", "1979 Arch Bioch Biop.", "196: 385-395).", "Recursive sequence recombination of nitroreductases can produce enzymes that are more specific, and able to more completely reduce (and thus detoxify) their target compounds (examples of which include but are not limited to nitrotoluenes and nitrobenzenes).", "Nitro-reductases can be isolated from bacteria isolated from explosive-contaminated soils, such as Morganella morganii and Enterobacter cloacae (Bryant et.", "al., 1991.J.", "Biol.", "Chem.", "266: 41264130).", "A preferred selection method is to look for increased resistance to the organo-nitro compound of interest, since that will indicate that the enzyme is also able to reduce any toxic partial reduction products of the original compound.", "4.7.8.9 Alternative Substrates for Chemical Synthesis Metabolic engineering can be used to alter microorganisms that produce industrially useful chemicals, so that they will grow using alternate and more abundant sources of nutrients, including human-produced industrial wastes.", "This typically involves providing both a transport system to get the alternative substrate into the engineered cells and catabolic enzymes from the natural host organisms to the engineered cells.", "In some instances, enzymes can be secreted into the medium by engineered cells to degrade the alternate is substrate into a form that can more readily be taken up by the engineered cells; in other instances, a batch of engineered cells can be grown on one preferred substrate, then lysed to liberate hydrolytic enzymes for the alternate substrate into the medium, while a second inoculum of the same engineered host or a second host is added to utilize the hydrolyzate.", "The starting materials for recursive sequence recombination will typically be genes for utilization of a substrate or its transport.", "Examples of nutrient sources of interest include but are not limited to lactose, whey, galactose, mannitol, xylan, cellobiose, cellulose and sucrose, thus allowing cheaper production of compounds including but not limited to ethanol, tryptophan, rhamnolipid surfactants, xanthan gum, and polyhydroxylalkanoate.", "For a review of such substrates as desired target substances, see Cameron et al.", "(Appl.", "Biochem.", "Biotechnol.", "38: 105-140 (1993)).", "The recursive sequence recombination methods described above can be used to optimize the ability of native hosts or heterologous hosts to utilize a substrate of interest, to evolve more efficient transport systems, to increase or alter specificity for certain substrates, and so on.", "4.7.8.10 Modification of Cell Properties Although not strictly examples of manipulation of intermediary metabolism, recursive sequence recombination techniques can be used to improve or alter other aspects of cell properties, from growth rate to ability to secrete certain desired compounds to ability to tolerate increased temperature or other environmental stresses.", "Some examples of traits engineered by traditional methods include expression of heterologous proteins in bacteria, yeast, and other eukaryotic cells, antibiotic resistance, and phage resistance.", "Any of these traits is advantageously evolved by the recursive sequence recombination techniques of the instant invention.", "Examples include replacement of one nutrient uptake system (e.g.", "ammonia in Methylophilus methylotrophus) with another that is more energy efficient; expression of haemoglobin to improve growth under conditions of limiting oxygen; redirection of toxic metabolic end products to less toxic compounds; expression of genes conferring tolerance to salt, drought and toxic compounds and resistance to pathogens, antibiotics and bacteriophage, reviewed in Cameron et. al.", "Appl Biochem Biotechnol, 38: 105-140 (1993).", "The heterologous genes encoding these functions all have the potential for further optimization in their new hosts by existing recursive sequence recombination technology.", "Since these functions increase cell growth rates under the desired growth conditions, optimization of the genes by evolution simply involves recombining the DNA recursively and selecting the recombinants that grow faster with limiting oxygen, higher toxic compound concentration, or whatever is the appropriate growth condition for the parameter being improved.", "Since these functions increase cell growth rates under the desired growth conditions, optimization of the genes by “evolution” can simply involve “stochastic &/or non-stochastic mutagenesis” the DNA and selecting the recombinants that grow faster with limiting oxygen, higher toxic compound concentration or whatever restrictive condition is being overcome.", "Cultured mammalian cells also require essential amino acids to be present in the growth medium.", "This requirement could also be circumvented by expression of heterclogous metabolic pathways that synthesize these amino acids (Rees et al., Biotechnology 8: 629-633 (1990).", "Recursive sequence recombination would provide a mechanism for optimizing the expression of these genes in mammalian cells.", "Once again, a preferred selection would be for cells that can grow in the absence of added amino acids.", "Yet another candidate for improvement through the techniques of the invention is symbiotic nitrogen fixation.", "Genes involved in nodulation (nod, ndv), nitrogen reduction (nif, fix), host range determination (nod, hsp), bacteriocin production (tfx), surface polysaccharide synthesis (exo) and energy utilization (dct, hup) which have been identified (Paau, Biotech.", "Adv.", "9: 173-184 (1991)).", "The main function of recursive sequence recombination in this case is in improving the survival of strains that are already known to be better nitrogen fixers.", "These strains tend to be less good at competing with strains already present in the environment, even though they are better at nitrogen fixation.", "Targets for recursive sequence recombination such as nodulation and host range determination genes can be modified and selected for by their ability to grow on the new host.", "Similarly any bacteriocin or energy utilization genes that will improve the competitiveness of the strain will also result in greater growth rates.", "Selection can simply be performed by subjecting the target genes to recursive sequence recombination and forcing the inoculant to compete with wild type nitrogen fixing bacteria.", "The better the nitrogen fixing bacteria grow in the new host, the more copies of their recombined genes will be present for the next round of recombination.", "This growth rate differentiating selection is described above in detail.", "4.7.8.11 Biodetectors/Biosensors Bioluminescence or fluorescence genes can be used as reporters by fusing them to specific regulatory genes (Cameron et. al.", "Appl.", "Biochem Biotechnol, 38: 105-140 (1993)).", "A specific example is one in which the luciferase genes luxCDABE of Vibrio fischeri were fused to the regulatory region of the isopropylbenzene catabolism operon from Pseudomonas putida RE204.Transformation of this fusion construct into E. coli resulted in a strain which produced light in response to a variety of hydrophobic compound such as substituted benzenes, chlorinated solvents and naphthalene (Selifonova et.", "al., Appl Environ Microbiol 62: 778-783 (1996)).", "This type of construct is useful for the detection of pollutant levels, and has the added benefit of only measuring those pollutants that are bioavailable (and therefore potentially toxic).", "other signal molecules such as jellyfish green fluorescent protein could also be fused to genetic regulatory regions that respond to chemicals in the environment.", "This should allow a variety of molecules to be detected by their ability to induce expression of a protein or proteins which result in light, fluorescence or some other easily detected signal.", "Recursive sequence recombination can be used in several ways to modify this type of biodetection system.", "It can be used to increase the amplitude of the response, for example by increasing the fluorescence of the green fluorescent protein.", "Recursive sequence recombination could also be used to increase induced expression levels or catalytic activities of other signal-generating systems, for example of the luciferase genes.", "Recursive sequence recombination can also be used to alter the specificity of biosensors.", "The regulatory region, and transcriptional activators that interact with this region and with the chemicals that induce transcription can also be stochastic &/or non-stochastic mutagenized.", "This should generate regulatory systems in which transcription is activated by analogues of the normal inducer, so that biodetectors for different chemicals can be developed.", "In this case, selection would be for constructs that are activated by the (new) specific chemical to be detected.", "Screening could be done simply with fluorescence (or light) activated cell sorting, since the desired improvement is in light production.", "In addition to detection of environmental pollutants, biosensors can be developed that will respond to any chemical for which there are receptors, or for which receptors can be evolved by recursive sequence recombination, such as hormones, growth factors, metals and drugs.", "These receptors may be intracellular and direct activators of transcription, or they may be membrane bound receptors that activate transcription of the signal indirectly, for example by a phosphorylation cascade.", "They may also not act on transcription at all, but may produce a signal by some post-transcriptional modification of a component of the signal generating pathway.", "These receptors may also be generated by fusing domains responsible for binding different ligands with different signaling domains.", "Again, recursive sequence recombination can be used to increase the amplitude of the signal generated to optimize expression and functioning of chimeric receptors, and to alter the specificity of the chemicals detected by the receptor.", "4.8 Promoting Genetic Exchange Some methods of the invention effect recombination of cellular DNA by propagating cells under conditions inducing exchange of DNA between cells.", "DNA exchange can be promoted by generally applicable methods such as electroporation, biolistics, cell fusion, or in some instances, by conjugation, transduction, or agrobacterium mediated transfer and meiosis.", "For example, Agrobacterium can transform S. cerevisiae with T-DNA, which is incorporated into the yeast genome by both homologous recombination and a gap repair mechanism.", "(Piers et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 93(4), 1613-8 (1996)).", "In some methods, initial diversity between cells (i.e., before genome exchange) is induced by chemical or radiation-induced mutagenesis of a progenitor cell type, optionally followed by screening for a desired phenotype.", "In other methods, diversity is natural as where cells are obtained from different individuals, strains or species.", "In some stochastic &/or non-stochastic mutagenesis methods, induced exchange of DNA is used as the sole means of effecting recombination in each cycle of recombination.", "In other methods, induced exchange is used in combination with natural sexual recombination of an organism.", "In other methods, induced exchange and/or natural sexual recombination are used in combination with the introduction of a fragment library.", "Such a fragment library can be a whole genome, a whole chromosome, a group of functionally or genetically linked genes, a plasmid, a cosmid, a mitochondrial genome, a viral genome (replicative and nonreplicative) or specific or random fragments of any of these.", "The DNA can be linked to a vector or can be in free form.", "Some vectors contain sequences promoting homologous or nonhomologous recombination with the host genome.", "Some fragments contain double stranded breaks such as caused by shearing with glass beads, sonication, or chemical or enzymatic fragmentation, to stimulate recombination.", "In each case, DNA can be exchanged between cells after which it can undergo recombination to form hybrid genomes.", "Generally, cells are recursively subject to recombination to increase the diversity of the population prior to screening.", "Cells bearing hybrid genomes, e.g., generated after at least one, and usually several cycles of recombination are screened for a desired phenotype, and cells having this phenotype are isolated.", "These cells can additionally form starting materials for additional cycles of recombination in a recursive recombination/selection scheme.", "4.8.1 Protoplast Fusion One means of promoting exchange of DNA between cells is by fusion of cells, such as by protoplast fusion.", "A protoplast results from the removal from a cell of its cell wall, leaving a membrane-bound cell that depends on an isotonic or hypertonic medium for maintaining its integrity.", "If the cell wall is partially removed, the resulting cell is strictly referred to as a spheroplast and if it is completely removed, as a protoplast.", "However, here the term protoplast includes spheroplasts unless otherwise indicated.", "Protoplast fusion is described by Shaffner et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 77, 2163 (1980) and other exemplary procedures are described by Yoakum et al., U.S. Pat.", "No.", "4,608,339, Takahashi et al., U.S. Pat.", "No.", "4,677,066 and Sambrooke et al., at Ch.", "16.Protoplast fusion has been reported between strains, species, and genera (e.g., yeast and chicken erythrocyte).", "Protoplasts can be prepared for both bacterial and eukaryotic cells, including mammalian cells and plant cells, by several means including chemical treatment to strip cell walls.", "For example, cell walls can be stripped by digestion with a cell wall degrading enzyme such as lysozyme in a 10-20% sucrose, 50 mM EDTA buffer.", "Conversion of cells to spherical protoplasts can be monitored by phase-contrast microscopy.", "Protoplasts can also be prepared by propagation of cells in.", "media supplemented with an inhibitor of cell wall synthesis, or use of mutant strains lacking capacity for cell wall formation.", "Preferably, eukaryotic cells are synchronized in G I phase by arrest with inhibitors such as -factor, K. lactis killer toxin, leflonamide and adenylate cyclase inhibitors.", "Optionally, some but not all, protoplasts to be fused can be killed and/or have their DNA fragmented by treatment with ultraviolet irradiation, hydroxylamine or cupferon (Reeves et al., FFMS Microbiol.", "Lett.", "99, 193-198 (1992)).", "In this situation, killed protoplasts are referred to as donors, and viable protoplasts as acceptors.", "Using dead donors cells can be advantageous in subsequently recognizing fused cells with hybrid genomes, as described below.", "Further, breaking up DNA in donor cells is advantageous for stimulating recombination with acceptor DNA.", "Optionally, acceptor and/or fused cells can also be briefly, but nonlethally, exposed to UV irradiation further to stimulate recombination.", "Once formed, protoplasts can be stabilized in a variety of osmolytes and compounds such as sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sucrose, sorbitol in the presence of DTT.", "The combination of buffer, pH, reducing agent, and osmotic stabilizer can be optimized for different cell types.", "Protoplasts can be induced to fuse by treatment with a chemical such as PEG, calcium chloride or calcium propionate or electrofusion (Tsoneva, Acta Microbiologica Bulgaria 24, 53-59 (1989)).", "A method of cell fusion employing electric fields has also been described.", "See Chang U.S. Pat.", "No.", "4,970,154.Conditions can be optimized for different strains.", "The fused cells are heterokaryons containing genomes from two or more component protoplasts.", "Fused cells can be enriched from unfused parental cells by sucrose gradient sedimentation or cell sorting.", "The two nuclei in the heterokaryons can fuse (karyogamy) and homologous recombination can occur between the genomes.", "The chromosomes can also segregate asymmetrically resulting in regenerated protoplasts that have lost or gained whole chromosomes.", "The frequency of recombination can be increased by treatment with ultraviolet irradiation or by use of strains overexpressing recA or other recombination genes, or the yeast rad genes, and cognate variants thereof in other species, or by the inhibition of gene products of MutS, MutL, or MutD.", "Overexpression can be either the result of introduction of exogenous recombination genes or the result of selecting strains, which as a result of natural variation or induced mutation, overexpress endogenous recombination genes.", "The fused protoplasts are propagated under conditions allowing regeneration of cell walls, recombination and segregation of recombinant genomes into progeny cells from the heterokaryon and expression of recombinant genes.", "This process can be reiteratively repeated to increase the diversity of any set of protoplasts or cells.", "After, or occasionally before or during, recovery of fused cells, the cells are screened or selected for evolution toward a desired property.", "Thereafter a subsequent round of recombination can be performed by preparing protoplasts from the cells surviving selection/screening in a previous round.", "The protoplasts are fused, recombination occurs in fused protoplasts, and cells are regenerated from the fused protoplasts.", "This process can again be reiteratively repeated to increase the diversity of the starting population.", "Protoplasts, regenerated or regenerating cells are subject to further selection or screening.", "Subsequent rounds of recombination can be performed on a split pool basis as described above.", "That is, a first subpopulation of cells surviving selection/screening from a previous round are used for protoplast formation.", "A second subpopulation of cells surviving selection/screening from a previous round are used as a source for DNA library preparation.", "The DNA library from the second subpopulation of cells is then transformed into the protoplasts from the first subpopulation.", "The library undergoes recombination with the genomes of the protoplasts to form recombinant genomes.", "This process can be repeated several times in the absence of a selection event to increase the diversity of the cell population.", "Cells are regenerated from protoplasts, and selection/screening is applied to regenerating or regenerated cells.", "In a further variation, a fresh library of nucleic acid fragments is introduced into protoplasts surviving selection/screening from a previous round.", "Protoplast formation of donor and recipient strains, heterokaryon formation, karyogamy, recombination, and segregation of recombinant genomes into separate cells.", "Optionally, the recombinant genomes, if having a sexual cycle, can undergo further recombination with each other as a result of meiosis and mating.", "Recursive cycles of protoplast fusion, or recursive mating/meiosis is often used to increase the diversity of a cell population.", "After achieving a sufficiently diverse population via one of these forms of recombination, cells are screened or selected for a desired property.", "Cells surviving selection/screening can then used as the starting materials in a further cycle of protoplasting or other recombination methods as noted herein.", "4.8.2 Parasexual Reproduction Parasexual reproduction provides a further means for stochastic &/or non-stochastic mutagenesis genetic material between cells.", "This process allows recombination of parental DNA without involvement of mating types or gametes.", "Parasexual fusion occurs by hyphal fusion giving rise to a common cytoplasm containing different nuclei.", "The two nuclei can divide independently in the resulting heterokaryon but occasionally fuse.", "Fusion is followed by haploidization, which can involve loss of chromosomes and mitotic crossing over between homolgous chromosomes.", "Protoplast fusion is a form of parasexual reproduction.", "4.8.3 Selection for Hybrid Strains 4.8.3.1 Identifying cells Formed by the Fusion of Components of Parental Cells from Two or more Distinct Subpopulations The invention provides selection strategies to identify cells formed by fusion of components from parental cells from two or more distinct subpopulations.", "Selection for hybrid cells is usually performed before selecting or screening for cells that have evolved (as a result of genetic exchange) to acquisition of a desired property.", "A basic premise of most such selection schemes is that two initial subpopulations have two distinct markers.", "Cells with hybrid genomes can thus be identified by selection for both markers.", "4.8.3.2 Method Where One Subpopulation has a Marker In one such scheme, at least one subpopulation of cells bears a selective marker attached to its cell membrane.", "Examples of suitable membrane markers include biotin, fluorescein and rhodamine.", "The markers can be linked to amide or thiol groups or through more specific derivatization chemistries, such as iodo-acetates, iodoacetamides, maleimides.", "For example, a marker can be attached as follows.", "Cells or protoplasts are washed with a buffer (e.g., PBS), which does not interfere with the chemical coupling of a chemically active ligand which reacts with amino groups of lysines or N-terminal amino groups of membrane proteins.", "The ligand is either amine reactive itself (e.g., isothiocyanates, succinimidyl esters, sulfonyl chlorides) or is activated by a heterobifunctional linker (e.g.", "EMCS, SLAB, SPDP, SMB) to become amine reactive.", "The ligand is a molecule which is easily bound by protein derivatized magnetic beads or other capturing solid supports.", "For example, the ligand can be succinimidyl activated biotin (Molecular Probes Inc.: B-1606, B-2603, S-1515, S-1582).", "This linker is reacted with amino groups of proteins residing in and on the surface of a cell.", "The cells are then washed to remove excess labeling agent before contacting with cells from the second subpopulation bearing a second selective marker.", "The second subpopulation of cells can also bear a membrane marker, albeit a different membrane marker from the first subpopulation.", "Alternatively, the second subpopulation can bear a genetic marker.", "The genetic marker can confer a selective property such as drug resistance or a screenable property, such as expression of green fluorescent protein.", "After fusion of first and second subpopulations of cells and recovery, cells are screened or selected for the presence of markers on both parental subpopulations.", "For example, fusants are enriched for one population by adsorbtion to specific beads and these are then sorted by FACS for those expressing a marker.", "Cells surviving both screens for both markers are those having undergone protoplast fusion, and are therefore more likely to have recombined genomes.", "Usually, the markers are screened or selected separately.", "Membrane-bound markers, such as biotin, can be screened by affinity enrichment for the cell membrane marker (e.g., by panning fused cells on an affinity matrix).", "For example, for a biotin membrane label, cells can be affinity purified using streptavidin-coated magnetic beads (Dynal).", "These beads are washed several times to remove the non-fused host cells.", "Alternatively, cells can be panned against an antibody to the membrane marker.", "In a further variation, if the membrane marker is fluorescent, cells bearing the marker can be identified by FACS.", "Screens for genetic markers depend on the nature of the markers, and include capacity to grow on drug-treated media or FACS selection for green fluorescent protein.", "If first and second cell populations have fluorescent markers of different wavelengths, both markers can be screened simultaneously by FACS sorting.", "In a further selection scheme for hybrid cells, first and second populations of cells to be fused express different subunits of a heteromultimeric enzyme.", "Usually, the heteromultimeric enzyme has two different subunits, but heteromultimeric enzymes having three, four or more different subunits can be used.", "If an enzyme has more than two different subunits, each subunit can be expressed in a different subpopulation of cells (e.g., three subunits in three subpopulations), or more than one subunit can be expressed in the same subpopulation of cells (e.g., one subunit in one subpopulation, two subunits in a second subpopulation).", "In the case where more than two subunits are used, selection for the poolwise recombination of more than two protoplasts can be achieved.", "Hybrid cells representing a combination of genomes of first, second or more subpopulation component cells can then be recognized by an assay for intact enzyme.", "Such an assay can be a binding assay, but is more typically a functional assay (e.g., capacity to metabolize a substrate of the enzyme).", "Enzymatic activity can be detected for example by processing of a substrate to a product with a fluorescent or otherwise easily detectable absorbance or emission spectrum.", "The individual subunits of a heteromultimeric enzyme used in such an assay preferably have no enzymic activity in dissociated form, or at least have significantly less activity in dissociated form than associated form.", "Preferably, the cells used for fusion lack an endogenous form of the heteromultimeric enzyme, or at least have significantly less endogenous activity than results from heteromultimeric enzyme formed by fusion of cells.", "Penicillin acylase enzymes, cephalosporin acylase and penicillin acyltransferase are examples of suitable heteromultimeric enzymes.", "These enzymes are encoded by a single gene, which is translated as a proenzyme and cleaved by posttranslational autocatalytic proteolysis to remove a spacer endopeptide and generate two subunits, which associate to form the active heterodimeric enzyme.", "Neither subunit is active in the absence of the other subunit.", "However, activity can be reconstituted if these separated gene portions are expressed in the same cell by co-transformation.", "Other enzymes that can be used have subunits that are encoded by distinct genes (e.g., faoA and faoB genes encode 3-oxoacyl-CoA thiolase of Pseudoumonas fragi (Biochem.", "J.", "328, 815-820 (1997)).", "An exemplary enzyme is penicillin G acylase from Escherichia coli, which has two subunits encoded by a single gene.", "Fragments of the gene encoding the two subunits operably linked to appropriate expression regulation sequences are transfected into first and second subpopulations of cells, which lack endogenous penicillin acylase activity.", "A cell formed by fusion of component cells from the first and second subpopulations expresses the two subunits, which assemble to form functional enzyme, e.g., penicillin acylase.", "Fused cells can then be selected on agar plates containing penicillin G, which is degraded by penicillin acylase.", "In another variation, fused cells are identified by complementation.", "of auxotrophic mutants.", "Parental subpopulations of cells can be selected for known auxotrophic mutations.", "Alternatively, auxotrophic mutations in a starting population of cells can be generated spontaneously by exposure to a mutagenic agent.", "Cells with auxotrophic mutations are selected by replica plating on minimal and complete media.", "Lesions resulting in auxotrophy are expected to be scattered throughout the genome, in genes for amino acid, nucleotide, and vitamin biosynthetic pathways.", "After fusion of parental cells, cells resulting from fusion can be identified by their capacity to grow on minimal media.", "These cells can then be screened or selected for evolution toward a desired property.", "Further steps of mutagenesis generating fresh auxotrophic mutations can be incorporated in subsequent cycles of recombination and screening/selection.", "In variations of the above method, de novo generation of auxotrophic mutations in each round of stochastic &/or non-stochastic mutagenesis can be avoided by reusing the same auxotrophs.", "For example, auxotrophs can be generated by transposon mutagenesis using a transposon bearing selective marker.", "Auxotrophs are identified by a screen such as replica plating.", "Auxotrophs are pooled, and a generalized transducing phage lysate is prepared by growth of phage on a population of auxotrophic cells.", "A separate population of auxtrophic cells is subjected to genetic exchange, and complementation is used to selected cells that have undergone genetic exchange and recombination.", "These cells are then screened or selected for acquisition of a desired property.", "Cells surviving screening or selection then have auxotrophic markers regenerated by introduction of the transducing transposon library.", "The newly generated auxotrophic cells can then be subject to further genetic exchange and screening/selection.", "In a further variation, auxotrophic mutations are generated by homologous recombination with a targeting vector comprising a selective marker flanked by regions of homology with a biosynthetic region of the genome of cells to be evolved.", "Recombination between the vector and the genome inserts the positive selection marker into the genome causing an auxotrophic mutation.", "The vector is in linear form before introduction of cells.", "Optionally, the frequency of introduction of the vector can be increased by capping its ends with self-complementarity oligonucleotides annealed in a hair pin formation.", "Genetic exchange and screening/selection proceed as described above.", "In each round, targeting vectors are reintroduced regenerating the same population of auxotrophic markers.", "In another variation, fused cells are identified by screening for a genomic marker present on one subpopulation of parental cells and an episomal marker present on a second subpopulation of cells.", "For example, a first subpopulation of yeast containing mitochondria can be used to complement a second subpopulation of yeast having a petite phenotype (i.e., lacking mitochondria).", "In a further variation, genetic exchange is performed between two subpopulations of cells, one of which is dead.", "Cells are preferably killed by brief exposure to DNA fragmenting agents such as hydroxylamine, cupferon, or irradiation.", "Viable cells are then screened for a marker present on the dead parental subpopulation.", "4.8.4 Liposome Mediated Transfers 4.8.4.1 Nucleic Acid Fragment Libraries are Introduced into Protoplasts 4.8.4.1.1 The Nucleic Acids are Encapsulated in Liposomes to Help Uptake by Protoplasts In the methods noted above, in which nucleic acid fragment libraries are introduced into protoplasts, the nucleic acids are sometimes encapsulated in liposomes to facilitate uptake by protoplasts.", "Liposome-mediated uptake of DNA by protoplasts is described in Redford et al., Mol.", "Gen. Genet.", "184, 567-569 (1981).", "Liposomes can efficiently deliver large volumes of DNA to protoplasts (see Deshayes et al., FMBO J.", "4, 2731-2737 (1985)).", "See also, Philippot and Schuber (eds) (1995) Liposomes as Tools in Basic Research and Industry CRC press, Boca Raton, e.g., Chapter 9, Remy et al.", "“Gene Transfer with Cationic Amphiphiles.” Further, the DNA can be delivered as linear fragments, which are often more recombinogenic that whole genomes.", "In some methods, fragments are mutated prior to encapsulation in liposomes.", "In some methods, fragments are combined with RecA and homologs, or nucleases (e.g., restriction endonucleases) before encapsulation in liposomes to promote recombination.", "Alternatively, protoplasts can be treated with lethal doses of nicking reagents and then fused.", "Cells which survive are those which are repaired by recombination with other genomic fragments, thereby providing a selection mechanism to select for recombinant (and therefore desirably diverse) protoplasts.", "4.9 Shuffing Using Filamentous Fungi Filamentous fungi are particularly suited to performing the stochastic &/or non-stochastic mutagenesis methods described above.", "Filamentous fungi are divided into four main classifications based on their structures for sexual reproduction: Phycomycetes, Ascomycetes, Basidiomycetes and the Fungi Imperfecti.", "Phycomycetes (e-g., Rhizopus, Mucor) form sexual spores in sporangium.", "The spores can be uni or multinucleate and often lack septated hyphae (coenocytic).", "Ascomycetes (e.g., Aspergillus, Neurospora, Penicillum) produce sexual spores in an ascus as a result of meiotic division.", "Asci typically contain 4 meiotic products, but some contain 8 as a result of additional mitotic division.", "Basidiomycetes include mushrooms, and smuts and form sexual spores on the surface of a basidium.", "In holobasidiomycetes, such as mushrooms, the basidium is undivided.", "In hemibasidiomycetes, such as ruts (Uredinales) and smut fungi (Ustilaginales), the basidium is divided.", "Fungi imperfecti, which include most human pathogens, have no known sexual stage.", "Fungi can reproduce by asexual, sexual or parasexual means.", "Asexual reproduction, involves vegetative growth of mycelia, nuclear division and cell division without involvement of gametes and without nuclear fusion.", "Cell division can occur by sporulation, budding or fragmentation of hyphae.", "4.9.1 Evolve fungi from Stochastic &/or Non-Stochastic Mutagenesis to Become Useful Hosts for Genetic Engineering of Unrelated Genes 4.9.2 To Improve the Capacity of Fungi to Make Specific Compounds One general goal of stochastic &/or non-stochastic mutagenesis is to evolve fungi to become useful hosts for genetic engineering, in particular for the stochastic &/or non-stochastic mutagenesis of unrelated genes.", "A. nidulans and neurospora are generally the fungal organisms of choice to serve as a hosts for such manipulations because of their sexual cycles and wellestablished use in classical and molecular genetics.", "Another general goal is to improve the capacity of fungi to make specific compounds (e.g.", "antibacterials (penicillins, cephalosporins), antifungals (e.g.", "echinocandins, aureobasidins), and wood-degrading enzymes).", "There is some overlap between these general goals, and thus, some desired properties are useful for achieving both goals.", "4.93 Mutator Strain Another desired property is the production of a mutator strain of fungi.", "Such a fungus can be produced by stochastic &/or non-stochastic mutagenesis a fungal strain containing a marker gene with one or more mutations that impair or prevent expression of a functional product.", "Shufflants are propagated under conditions that select for expression of the positive marker (while allowing a small amount of residual growth without expression).", "Shufflants growing fastest are selected to form the starting materials for the next round of stochastic &/or non-stochastic mutagenesis.", "4.9.4 Expanded Host Range so able to Form Heterokaryons with More Strains Another desired property is to expand the host range of a fungus so it can form heterokaryons with fungi from other vegetative compatibility groups.", "Incompatability between species results from the interactions of specific alleles at different incompatability loci (such as the “het” loci).", "If two strains undergo hyphal anastomosis, a lethal cytoplasmic incomparability reaction may occur if the strains differ at these loci.", "Strains must carry identical loci to be entirely compatible.", "Several of these loci have been identified in various species, and the incompatibility effect is somewhat additive (hence, “partial incompatibility” can occur).", "Some tolerant and het-negative mutants have been described for these organisms (e.g.", "Dales & Croft, J. Gen. Microbiol.", "0.136, 1717-1724 (1990)).", "Further, a tolerance gene (tol) has been reported, which suppresses mating-type heterokaryon incompatibility.", "Stochastic &/or non-stochastic mutagenesis is performed between protoplasts of strains from different incompatibility groups.", "A preferred format uses a five acceptor strain and a UV-irradiated dead acceptor strain.", "The UV irradiation serves to introduce mutations into DNA inactivating het genes.", "The two strains should bear different genetic markers.", "Protoplasts of the strain are fused, cells are regenerated and screened for complementation of markers.", "Subsequent rounds of stochastic &/or non-stochastic mutagenesis and selection can be performed in the same manner by fusing the cells surviving screening with protoplasts of a fresh population of donor cells.", "Similar to other procedures noted herein, the cells resulting from regeneration of the protoplasts are optionally refused by protoplasting and regenerated into cells one or more times prior to any selection step to increase the diversity of the resulting population of cells to be screened.", "4.9.5 Ability to Outbreed without Self-Breeding Another desired property is the introduction of multiple-allelomorph heterothallism into Ascomyceles and Fungi imperfecti, which do not normally exhibit this property.", "This mating system allows outbreeding without self-breeding.", "Such a mating system can be introduced by stochastic &/or non-stochastic mutagenesis Ascomycetes and Fungi imperfecti with DNA from Gasteromycetes or Hymenomycetes, which have such a system.", "4.9.6 Spontaneous Formation of Protoplasts Another desired property is spontaneous formation of protoplasts to facilitate use of a fungal strain as a stochastic &/or non-stochastic mutagenesis host.", "Here, the fungus to be evolved is typically mutagenized.", "Spores of the fungus to be evolved are briefly treated with a cell-wall degrading agent for a time insufficient for complete protoplast formation, and are mixed with protoplasts from other strain(s) of fungi.", "Protoplasts formed by fusion of the two different subpopulations are identified by genetic or other selection/or screening as described above.", "These protoplasts are used to regenerate mycelia and then spores, which form the starting material for the next round of stochastic &/or non-stochastic mutagenesis.", "In the next round, at least some of the surviving spores are treated with cell-wall removing enzyme but for a shorter time than the previous round.", "After treatment, the partially stripped cells are labeled with a first label.", "These cells are then mixed with protoplasts, which may derive from other cells surviving selection in a previous round, or from a fresh strain of fungi.", "These protoplasts are physically labeled with a second label.", "After incubating the cells under conditions for protoplast fusion fusants with both labels are selected.", "These fusants are used to generate mycelia and spores for the next round of stochastic &/or non-stochastic mutagenesis, and so forth.", "Eventually, progeny that spontaneously form protoplasts (i.e., without addition of cell wall degrading agent) are identified.", "As with other procedures noted herein, cells or protoplasts can be reiteratively fused and regenerated prior to performing any selection step to increase the diversity of the resulting cells or protoplasts to be screened.", "Similarly, selected cells or protoplasts can be reiteratively fused and regenerated for one or several cycles without imposing selection on the resulting cellular or protoplast populations, thereby increasing the diversity of cells or protoplasts which are eventually screened.", "This process of performing multiple cycles of recombination interspersed with selection steps can be reiteratively repeated as desired.", "4.9.7 Acquisition or Improvement of Genes Encoding In Biosynthetic Pathways; Transporter Proteins; And Metabolic Flux Another desired property is the acquisition and/or improvement of genes encoding enzymes in biosynthetic pathways, genes encoding transporter proteins, and genes encoding proteins involved in metabolic flux control.", "In this situation, genes of the pathway can be introduced into the fungus to be evolved either by genetic exchange with another strain of fungus possessing the pathway or by introduction of a fragment library from an organism possessing the pathway.", "Genetic material of these fungi can then be subjected to further stochastic &/or non-stochastic mutagenesis and screening/selection by the various procedures discussed in this application.", "Shufflant strains of fungi are selected/screened for production of the compound produced by the metabolic pathway or precursors thereof.", "4.9.8 Increased Stability to Extreme Conditions Another desired property is increasing the stability of fungi to extreme conditions such as heat.", "In this situation, genes conferring stability can be acquired by exchanging DNA with or transforming DNA from a strain that already has such properties.", "Alternatively, the strain to be evolved can be subjected to random mutagenesis.", "Genetic material of the fungus to be evolved can be stochastic &/or non-stochastic mutagenized by any of the procedures described in this application, with shufflants being selected by surviving exposure to extreme conditions.", "4.9.9 Growth Under Altered Nutritional Requirements Another desired property is capacity of a fungus to grow under altered nutritional requirements (e.g., growth on particular carbon or nitrogen sources).", "Altering nutritional requirements is particularly valuable, e.g., for natural isolates of fungi that produce valuable commercial products but have esoteric and therefore expensive nutritional requirement.", "The strain to be evolved undergoes genetic exchange and/or transformation with DNA from a strain that has the desired nutritional requirements.", "The fungus to be evolved can then optionally be subjected to further stochastic &/or non-stochastic mutagenesis as described in this application and with recombinant strains being selected for capacity to grow in the desired nutritional circumstances.", "Optionally, the nutritional circumstances can be varied in successive rounds of stochastic &/or non-stochastic mutagenesis starting at close to the natural requirements of the fungus to be evolved and in subsequent rounds approaching the desired nutritional requirements.", "4.9.10 Natural Competance to Take Up a Plasmid Bearing a Selective Marker Another desired property is acquisition of natural competence in a fungus.", "The procedure for acquisition of natural competence by stochastic &/or non-stochastic mutagenesis is generally described in PCT/US97/04494.The fungus to be evolved typically undergoes genetic exchange or transformation with DNA from a bacterial strain or fungal strain that already has this property.", "Cells with recombinant genomes are then selected by capacity to take up a plasmid bearing a selective marker.", "Further rounds of recombination and selection can be performed using any of the procedures described above.", "4.9.11 Reduced or Increased Secretion of Proteases and DNAses Another desired property is reduced or increased secretion of proteases and DNase.", "In this situation, the fungus to be evolved can acquire DNA by exchange or transformation from another strain known to have the desired property.", "Alternatively, the fungus to be evolved can be subject to random mutagenesis.", "The fungus to be evolved is stochastic &/or non-stochastic mutagenized as above.", "The presence of such enzymes, or lack thereof, can be assayed by contacting the culture media from individual isolates with a fluorescent molecule tethered to a support via a peptide or DNA linkage.", "Cleavage of the linkage releases detectable fluorescence to the media.", "4.9.12 Altered Transporters to Use Secondary Components Another desired property is producing fungi with altered transporters (e.g., MDR).", "Such altered transporters are useful, for example, in fungi that have been evolved to produce new secondary metabolites, to allow entry of precursors required for synthesis of the new secondary metabolites into a cell, or to allow efflux of the secondary metabolite from the cell.", "Transporters can be evolved by introduction of a library of transporter variants into fungal cells and allowing the cells to recombine by sexual or parasexual recombination.", "To evolve a transporter with capacity to transport a precursor into the cells, cells are propagated in the present of precursor, and cells are then screened for production of metabolite.", "To evolve a transporter with capacity to export a metabolite, cells are propagated under conditions supporting production of the metabolite, and screened for export of metabolite to culture medium.", "A general method of fungal stochastic &/or non-stochastic mutagenesis is shown herein.", "Spores from a frozen stock, a lyophilized stock, or fresh from an agar plate are used to inoculate suitable liquid medium (1).", "Spores are germinated resulting in hyphal growth (2).", "Mycelia are harvested, and washed by filtration and/or centrifugation.", "Optionally the sample is pretreated with DTT to enhance protoplast formation (3).", "Protoplasting is performed in an osmotically stabling medium (e.g., 1 m NaCl/20 mM MgSO4, pH 5.8) by the addition of cell wall-degrading enzyme (e.g., Novozyme 234) (4).", "Cell wall degrading enzyme is removed by repeated washing with osmotically stabilizing solution (5).", "Protoplasts can be separated from mycelia, debris and spores by filtration through miracloth, and density centrifugation (6).", "Protoplasts are harvested by centrifugation and resuspended to the appropriate concentration.", "This step may lead to some protoplast fusion (7).", "Fusion can be stimulated by addition of PEG (e.g., PEG 3350), and/or repeated centrifugation and resuspension with or without PEG.", "Electrofusion can also be performed (8).", "Fused protoplasts can optionally be enriched from unfused protoplasts by sucrose gradient sedimentation (or other methods of screening described above).", "Fused protoplasts can optionally be treated with ultraviolet irradiation to stimulate recombination (9).", "Protoplasts are cultured on osmotically stabilized agar plates to regenerate cell walls and form mycelia (10).", "The mycelia are used to generate spores (11), which are used as the starting material in the next round of stochastic &/or non-stochastic mutagenesis (12).", "Selection for a desired property can be performed either on regenerated mycelia or spores derived therefrom.", "In an alternative method, protoplasts are formed by inhibition of one or more enzymes required for cell wall synthesis.", "The inhibitor should be fungistatic rather than fungicidal under the conditions of use.", "Examples of inhibitors include antifungal compounds described by (e.g., Georgopapadakou & Walsh, Antimicrob.", "Ag.", "Chemother.", "40, 279-291 (1996); Lyman & Walsh, Drugs 44, 9-35 (1992)).", "Other examples include chitin synthase inhibitors (polyoxin or nikkomycin compounds) and/or glucan synthase inhibitors (e.g.", "echinocandins, papulocandins, pneumocandins).", "Inhibitors should be applied in osmotically stabilized medium.", "Cells stripped of their cell walls can be fused or otherwise employed as donors or hosts in genetic transformation/strain development programs.", "In a further variation, protoplasts are prepared using strains of fungi, which are genetically deficient or compromised in their ability to synthesize intact cell walls.", "Such mutants are generally referred to as fragile, osmotic-remedial, or cell wall-less, and are obtainable from strain depositories.", "Examples of such strains include Neurospora crassa os mutants (Selitrennikoff, Antimicrob.", "Agents.", "Chemother.", "23, 757-765 (1983)).", "Some such mutations are temperature-sensitive.", "Temperature-sensitive strains can be propagated at the permissive temperature for purposes of selection and amplification and at a nonpermissive temperature for purposes of protoplast formation and fusion.", "A temperature sensitive strain Neurospora crassa os strain has been described which propagates as protoplasts when growth in osmotically stabilizing medium containing sorbose and polyoxin at nonpermissive temperature but generates whole cells on transfer to medium containing sorbitol at a permissive temperature.", "See U.S. Pat.", "No.", "4,873,196.Other suitable strains can be produced by targeted mutagenesis of genes involved in chitin synthesis, glucan synthesis and other cell wall-related processes.", "Examples of such genes include CHT1, CHT2 and CALI (or CSD2) of Saccharomyces cerevisiae and Candida spp.", "(Georgopapadakou & Walsh 1996); ETGI/FKSI/CNDI/CWH53/PB RI and homologs in S. cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, ChvAlNdvA Agrobacterium and Rhizobium.", "Other examples are M4, orlB, orlC, MD, tsE, and bimG of Aspergillus nidulans (Borgia, J. Bacteriol.", "174, 377-389 (1992)).", "Strains of A. nidulans containing OrlA1 or tse1 mutations lyse at restrictive temperatures.", "Lysis of these strains may be prevented by osmotic stabilization, and the mutations may be complemented by the addition of N-acetylglucosimine (GlcNac).", "BimG11 mutations are ts for a type I protein phosphatase (germlines of strains carrying this mutation lack chitin, and condia swell and lyse).", "Other suitable genes are chsA, chsB, chsC, chsD and chsE of Aspergillus fumigatus; chs1 and chs2 of Neurospora crassa; Phycomyces blakesleeanus MM and chs 1, 2 and 3 of S. cerevisiae.", "Chs 1 is a non-essential repair enzyme; chs2 is involved in septum formation and chs3 is involved in cell wall maturation and bud ring formation.", "Other useful strains include S. cerevisiae CLY (cell lysis) mutants such as ts strains (Paravicini et al., Mol.", "Cell Biol 12, 4896-4905 (1992)), and the CLY 15 strain which harbors a PKC 1 gene deletion.", "Other useful strains include strain VY 1160 containing a ts mutation in srb (encoding actin) (Schade et al.", "Acta Histochem.", "Suppl.", "41, 193-200 (1991)), and a strain with an ses mutation which results in increased sensitivity to cell-wall digesting enzymes isolated from snail gut (Metha & Gregory, Appl.", "Environ.", "Microbiol 41, 992-999 (1981)).", "Useful strains of C albicans include those with mutations in chs1, chs2, or chs3 (encoding chitin synthetases), such as osmotic remedial conditional lethal mutants described by Payton & de Tiani, Curr.", "Genet.", "17, 293-296 (1990); C. utilis mutants with increased sensitivity to cell-wall digesting enzymes isolated from snail gut (Metha & Gregory, 1981, supra); and X crassa mutants os-1, os-2, os-3, os-4, os-5, and os-6.See, Selitrennikoff, Antimicrob.", "Agents Chemother.", "23, 757-765 (1983).", "Such mutants grow and divide without a cell wall at 37 C, but at 22 C produce a cell wall.", "Targeted mutagenesis can be achieved by transforming cells with a positive-negative selection vector containing homologous regions flanking a segment to be targeted, a positive selection marker between the homologous regions and a negative selection marker outside the homologous regions (see Capecchi, U.S. Pat.", "No.", "5,627,059).", "In a variation, the negative selection marker can be an antisense transcript of the positive selection marker (see U.S. Pat.", "No.", "5,527,674).", "Other suitable cells can be selected by random mutagenesis or stochastic &/or non-stochastic mutagenesis procedures in combination with selection.", "For example, a first subpopulation of cells are mutagenized, allowed to recover from mutagenesis, subjected to incomplete degradation of cell walls and then contacted with protoplasts of a second subpopulation of cells.", "Hybrids cells bearing markers from both subpopulations are identified (as described above) and used as the starting materials in a subsequent round of stochastic &/or non-stochastic mutagenesis.", "This selection scheme selects both for cells with capacity for spontaneous protoplast formation and for cells with enhanced recombinogenicity.", "In a further variation, cells having capacity for spontaneous protoplast formation can be crossed with cells having enhanced recombinogenicity evolved using other methods of the invention.", "The hybrid cells are particularly suitable hosts for whole genome stochastic &/or non-stochastic mutagenesis.", "Cells with mutations in enzymes involved in cell wall synthesis or maintenance can undergo fusion simply as a result of propagating the cells in osmotic-protected culture due to spontaneous protoplast formation.", "If the mutation is conditional, cells are shifted to a nonpermissive condition.", "Protoplast formation and fusion can be accelerated by addition of promoting agents, such as PEG or an electric field (See Philipova & Venkov, Yeast 6, 205-212 (1990); Tsoneva et al., FFMS Microbiol Lett.", "51, 61-65 (1989)).", "4.10 Process of Sexual Reproduction Sexual reproduction provides a mechanism for stochastic &/or non-stochastic mutagenesis genetic material between cells.", "A sexual reproductive cycle is characterized by an alteration of a haploid phase and a diploid phase.", "Diploidy occurs when two haploid gamete nuclei fuse (karyogamy).", "The gamete nuclei can come from the same parental strains (self-fertile), such as in the homothallic fungi.", "In heterothallic fungi, the parental strains come from strains of different mating type.", "A diploid cell converts to haploidy via meiosis, which essentially consists of two divisions of the nucleus accompanied by one division of the chromosomes.", "The products of one meiosis are a tetrad (4 haploid nuclei).", "In some cases, a mitotic division occurs after meiosis, giving rise to eight product cells.", "The arrangement of the resultant cells (usually enclosed in spores) resembles that of the parental strains.", "The length of the haploid and diploid stages differs in various fungi: for example, the Basidiomycetes and many of the Ascomycetes have a mostly haploid life cycle (that is, meiosis occurs immediately after karyogamy), whereas others (e.g., Saccharomyces cerevisiae) are diploid for most of their life cycle (karyogamy occurs soon after meiosis).", "Sexual reproduction can occur between cells in the same strain (selfing) or between cells from different strains (outcrossing).", "Sexual dimorphism (dioecism) is the separate production of male and female organs on different mycelia.", "This is a rare phenomenon among the fungi, although a few examples are known.", "Heterothallism (one locus-two alleles) allows for outcrossing between crosscompatable strains which are self-incompatable.", "The simplest form is the two allele-one locus system of mating types/factors, illustrated by the following organisms: A and a in Neurospora; a and in Saccharomyces, plus and minus in Schizzosaccharomyces and Zygomycetes; 1 and 2 in Ustilago.", "Multiple-allelomorph heterothallism is exhibited by some of the higher Basidiomycetes (e.g.", "Gasteromycetes and Hymenomycetes), which are heterothallic and have several mating types determined by multiple alleles.", "Heterothallism.", "In these organisms is either bipolar with one mating type factor, or tetrapolar with two unlinked factors, A and B.", "Stable, fertile heterokaryon formation depends on the presence of different A factors and, in the case of tetrapolar organisms, of different B factors as well.", "This system is effective in the promotion of outbreeding and the prevention of self-breeding.", "The number of different mating factors may be very large (i.e.", "thousands) (Kothe, FEMS Microbiol Rev.", "18, 65-87 (1996)), and non-parental mating factors may arise by recombination.", "4.10.1 Introducing Sexual Cycles 4.10.2 Meiosis 4.10.2.1 Heterokaryon-A Cell or Hypha Containing Two or More Nuclei of Different Genetic Constitutions One desired property is the introduction of meiotic apparatus into fungi presently lacking a sexual cycle (see Sharon et al., Mol.", "Gen. Genet.", "251, 60-68 (1996)).", "A scheme for introducing a sexual cycle into the fungi P. chrysogenum (a fungus imperfecti) is shown herein.", "Subpopulations of protoplasts are formed from.4.nidulans (which has a sexual cycle) and P. chrysogenum, which does not.", "The two strains preferably bear different markers.", "The A. nidulans protoplasts are killed by treatment with UV or hydroxylamine.", "The two subpopulations are fused to form heterokaryons.", "In some heterokaryons, nuclei fuse, and some recombination occurs.", "Fused cells are cultured under conditions to generate new cell walls and then to allow sexual recombination to occur.", "Cells with recombinant genomes are then selected (e.g., by selecting for complementation of auxotrophic markers present on the respective parent strains).", "Cells with hybrid genomes are more likely to have acquired the genes necessary for a sexual cycle.", "Protoplasts of cells can then be crossed with killed protoplasts of a further population of cells known to have a sexual cycle (the same or different as the previous round) in the same manner, followed by selection for cells with hybrid genomes.", "4.10.2.2 Vegetative Compatibility Between Classes of Fungi Within the above four classes, fungi are also classified by vegetative compatibility group.", "Fungi within a vegetative compatibility group can form heterokaryons with each other.", "Thus, for exchange of genetic material between different strains of fungi, the fungi are usually prepared from the same vegetative compatibility group.", "However, some genetic exchange can occur between fungi from different incompatibility groups as a result of parasexual reproduction (see Timberlake et al., U.S. Pat.", "No.", "5,605,820).", "Further, as discussed elsewhere, the natural vegetative compatibility group of fungi can be expanded as a result of stochastic &/or non-stochastic mutagenesis.", "Several isolates of Aspergillus nidulans, A. flavus, A. fumigatus, Penicillium chrysogenum, P. notatum, Cephalosporium chrysogenum, Neurospora crassa, Aureobasidium pullulans have been karyotyped.", "Genome sizes generally range between 20 and 50 Mb among the Aspergilli.", "Differences in karyotypes often exist between similar strains and are also caused by transformation with exogenous DNA.", "Filamentous fungal genes contain introns, usually 50-100 bp in size, with similar consensus 5′ and 3′ splice sequences.", "Promotion and termination signals are often cross-recognizable, enabling the expression of a gene/pathway from one fungus (e.g.", "A. nidulans) in another (e.g.", "P. chrysogenum).", "The major components of the fungal cell wall are chitin (or chitosan), beta-glucan, and mannoproteins.", "Chitin and beta-glucan form the scaffolding, mannoproteins are interstitial components which dictate the wall's porosity, antigenicity and adhesion.", "Chitin synthetase catalyzes the polymerization of beta-(1,4)-linked N-acetylglucosamine (GIcNAc) residues, forming linear strands running antiparallel; beta-(1,3)-glucan synthetase catalyze the homopolymerization of glucose.", "4.11 Evolution 4.11.1 Artificially Evolving Cells to Acquire a New or Improved Property by Stochastic &/or Non-Stochastic Mutagenesis The invention provides a number of strategies for evolving metabolic and bioprocessing pathways through the technique of recursive sequence recombination.", "One strategy entails evolving genes that confer the ability to use a particular substrate of interest as a nutrient source in one species to confer either more efficient use of that substrate in that species, or comparable or more efficient use of that substrate in a second species.", "Another strategy entails evolving genes that confer the ability to detoxify a compound of interest in one or more species of organisms.", "Another strategy entails evolving new metabolic pathways by evolving an enzyme or metabolic pathway for biosynthesis or degradation of a compound A related to a compound B for the ability to biosynthesize or degrade compound B, either in the host of origin or a new host.", "A further strategy entails evolving a gene or metabolic pathway for more efficient or optimized expression of a particular metabolite or gene product.", "A further strategy entails evolving a host/vector system for expression of a desired heterologous product.", "These strategies may involve using all the genes in a multi-step pathway, one or several genes, genes from different organisms, or one or more fragments of a gene.", "The strategies generally entail evolution of gene(s) or segment(s) thereof to allow retention of function in a heterologous cell or improvement of function in a homologous or heterologous cell.", "Evolution is effected generally by a process termed recursive sequence recombination.", "Recursive sequence recombination can be achieved in many different formats and permutations of formats, as described in further detail below.", "These formats share some common principles.", "Recursive sequence recombination entails successive cycles of recombination to generate molecular diversity, i.e., the creation of a family of nucleic acid molecules showing substantial sequence identity to each other but differing in the presence of mutations.", "Each recombination cycle is followed by at least one cycle of screening or selection for molecules having a desired characteristic.", "The molecule(s) selected in one round form the starting materials for generating diversity in the next round.", "In any given cycle, recombination can occur in vivo or in vitro.", "Furthermore, diversity resulting from recombination can be augmented in any cycle by applying prior methods of mutagenesis (e.g., error-prone PCR or cassette mutagenesis, passage through bacterial mutator strains, treatment with chemical mutagens) to either the substrates for or products of recombination.", "4.11.2 Basic Approach 4.11.2.1 Successive Cycles of Recombination and Screening/Selection The invention provides methods for artificially evolving cells to acquire a new or improved property by recursive sequence recombination.", "Briefly, recursive sequence recombination entails successive cycles of recombination to generate molecular diversity and screening/selection to take advantage of that molecular diversity.", "That is, a family of nucleic acid molecules is created showing substantial sequence and/or structural identity but differing as to the presence of mutations.", "These sequences are then recombined in any of the described formats so as to optimize the diversity of mutant combinations represented in the resulting recombined library.", "Typically, any resulting recombinant nucleic acids or genomes are recursively recombined for one or more cycles of recombination to increase the diversity of resulting products.", "After this recursive recombination procedure, the final resulting products are screened and/or selected for a desired trait or property.", "Alternatively, each recombination cycle can followed by at least one cycle of screening or selection for molecules having a desired characteristic.", "In this embodiment, the molecule(s) selected in one round form the starting materials for generating diversity in the next round.", "The cells to be evolved can be bacteria, archaebacteria, or eukaryotic cells and can constitute a homogeneous cell line or mixed culture.", "Suitable cells for evolution include the bacterial and eukaryotic, cell lines commonly used in genetic engineering, protein expression, or the industrial production or conversion of proteins, enzymes, primary metabolites, secondary metabolites, fine, specialty or commodity chemicals.", "Suitable mammalian cells include those from, e.g., mouse, rat, hamster, primate, and human, both cell fines and primary cultures.", "Such cells include stem cells, including embryonic stem cells and hemopoietic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIHM), kidney, liver, muscle, and skin cells.", "Other eukaryotic cells of interest include plant cells, such as maize, rice, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi (penicillium, aspergillus, podospora, neurospora, saccharomyces), insect (e.g., baculo lepidoptera), yeast (picchia and saccharomyces, Schizosaccharomyces pombe).", "Also of interest are many bacterial cell types, both gram-negative and gram-positive, such as Bacillus subtilis, B. licehniformis, B. cereus, Escherichia coli, Streptomyces, Pseudomonas, Salmonella, Actinomycetes, Lactobacillius, Acelonitcbacter, Deinococcus, and Erwinia.", "The complete genome sequences of E. coli and Bacillus subtilis are described by Blattner et al., Science 277, 1454-1462 (1997); Kunst et al., Nature 390, 249-256 (1997).", "4.11.2.1.1 Goal is to Achieve Variaton Evolution commences by generating a population of variant cells.", "Typically, the cells in the population are of the same type but represent variants of a progenitor cell.", "in some instances, the variation is natural as when different cells are obtained from different individuals within a species, from different species or from different genera.", "in other instances, variation is induced by mutagenesis of a progenitor cell.", "Mutagenesis can be effected by subjecting the cell to mutagenic agents, or if the cell is a mutator cell (e.g., has mutations in genes involved in DNA replication, recombination and/or repair which favor introduction of mutations) simply by propagating the mutator cells.", "Mutator cells can be generated from successive selections for simple phenotypic changes (e.g., acquisition of rifampicin-resistance, then nalidixic acid resistance then lac− to lac+ (see Mao et al., J Bacteriol 179, 417-422 (1997)), or mutator cells can be generated by exposure to specific inhibitors of cellular factors that result in the mutator phenotype.", "These could be inhibitors of mutS, mutL, mutD, recD, mutY, mutM, dam, uvrD and the like.", "More generally, mutations are induced in cell populations using any available mutation technique.", "Common mechanisms for inducing mutations include, but are not limited to, the use of strains comprising mutations such as those involved in mismatch repair.", "e.g.", "mutations in mutS, mutT, mutL and mutH; exposure to UV light; Chemical mutagenesis, e.g.", "use of inhibitors of MMR, DNA damage inducible genes, or SOS inducers; overproduction/underproduction/mutation of any component of the homologous recombination complex/pathway, e.g.", "RecA, ssb, etc.", "overproduction/underproduction/mutation of genes 9 involved in DNA synthesis/homeostasis; overproduction/underproduction/mutation of recombination-stimulating genes from bacteria, phage (e.g.", "Lambda Red function), or other organisms; addition of chi sites into/flanking the donor DNA fragments; coating the DNA fragments with RecA/ssb and the like.", "In other instances, variation is the result of transferring a library of DNA fragments into the cells (e.g., by conjugation, protoplast fusion, liposome fusion, transformation, transduction or natural competence).", "At least one, and usually many of the fragments in the library, show some, but not complete, sequence or structural identity with a cognate or allelic gene within the cells sufficient to allow homologous recombination to occur.", "For example, in one embodiment, homologous integration of a plasmid carrying a stochastic &/or non-stochastic mutagenized gene or metabolic pathway leads to insertion of the plasmid-borne sequences adjacent to the genomic copy.", "Optionally, a counter-selectable marker strategy is used to select for recombinants in which recombination occurred between the homologous sequences, leading to elimination of the counter-selectable marker.", "A variety of selectable and counter selectable markers are amply illustrated in the art.", "For a fist of useful markers, see, Berg and Berg (1996), Transposable element tools for microbial genetics, Escherichia coli and Salmonella Neidhardt.", "Washington, D.C., ASM Press.", "2: 2588-2612; La Rossa, ibid., 2527-2587.This strategy can be recursively repeated to maximize sequence diversity of targeted genes prior to screening/selection for a desired trait or property.", "The library of fragments can derive from one or more sources.", "One source of fragments is a genomic library of fragments from a different species, cell type, organism or individual from the cells being transfected.", "In this situation, many of the fragments in the library have a cognate or allelic gene in the cells being transformed but differ from that gene due to the presence of naturally occurring species variation, polymorphisms, mutations, and the presence of multiple copies of some homologous genes in the genome.", "Alternatively, the library can be derived from DNA from the same cell type as is being transformed after that DNA has been subject to induced mutation, by conventional methods, such as radiation, error-prone PCR, growth in a mutator organism, transposon mutagenesis, or cassette mutagenesis.", "Alternatively, the library can derive from a genomic library of fragments generated from the pooled genomic DNA of a population of cells having the desired characteristics.", "Alternatively, the library can derive from a genomic library of fragments generated from the pooled genomic DNA of a population of cells having desired characteristics.", "In any of these situations, the genomic library can be a complete genomic library or subgenome & library deriving, for example, from a selected chromosome, or part of a chromosome or an episomal element within a cell.", "As well as, or instead of these sources of DNA fragments, the library can contain fragments representing natural or selected variants of selected genes of known function (i.e., focused libraries).", "The number of fragments in a library can vary from a single fragment to about 1010 with libraries having from 103 to 108 fragments being common.", "The fragments should be sufficiently long that they can undergo homologous recombination and sufficiently short that they can be introduced into a cell, and if necessary, manipulated before introduction.", "Fragment sizes can range from about 10 b to about 20 mb.", "Fragments can be double- or single-stranded.", "The fragments can be introduced into cells as whole genomes or as components of viruses, plasmids, YACS, HACs or BACs or can be introduced as they are, in which case all or most of the fragments lack an origin of replication.", "Use of viral fragments with single-stranded genomes offer the advantage of delivering fragments in single stranded form, which promotes recombination.", "The fragments can also be joined to a selective marker before introduction.", "Inclusion of fragments in a vector having an origin of replication affords a longer period of time after introduction into the cell in which fragments can undergo recombination with a cognate gene before being degraded or selected against and lost from the cell, thereby increasing the proportion of cells with recombinant genomes.", "Optionally, the vector is a suicide vector capable of a longer existence than an isolated DNA fragment but not capable of permanent retention in the cell line.", "Such a vector can transiently express a marker for a sufficient time to screen for or select a cell bearing the vector (e.g., because cells transduced by the vector are the target cell type to be screened in subsequent selection assays), but is then degraded or otherwise rendered incapable of expressing the marker.", "The use of such vectors can be advantageous in performing optional subsequent rounds of recombination to be discussed below.", "For example, some suicide vectors express a long-lived toxin which is neutralized by a short-lived molecule expressed from the same vector.", "Expression of the toxin alone will not allow vector to be established.", "Jense & Gerdes, Mol.", "Microbiol, 17, 205-210 (1995); Bernard et al., Gene 162, 159-160.Alternatively, a vector can be rendered suicidal by incorporation of a defective origin of replication (e.g.", "a temperature-sensitive origin of replication) or by omission of an origin of replication.", "Vectors can also be rendered suicidal by inclusion of negative selection markers, such as ura3 in yeast or sacB in many bacteria.", "These genes become toxic only in the presence of specific compounds.", "Such vectors can be selected to have a wide range of stabilities.", "A list of conditional replication defects for vectors which can be used, e.g., to render the vector replication defective is found, e.g., in Berg and Berg (1996), “Transposable element tools for microbial genetics” Escherichia coli and Salmonella Neidhardt.", "Washington, D.C., ASM Press.", "2: 2588-2612.Similarly, a fist of counter selectable markers, generally applicable to vector selection is also found in Berg and Berg, id.", "See also, LaRossa (1996), “Mutant selections linking physiology, inhibitors, and genotypes” Escherichia coli and Salmonella F. C. Neidhardt.", "Washington, D.C., ASM Press.", "2: 2527-2587.After introduction into cells, the fragments can recombine with DNA present in the genome, or episomes of the cells by homologous, nonhomologous or site-specific recombination.", "For present purposes, homologous recombination makes the most significant contribution to evolution of the cells because this form of recombination amplifies the existing diversity between the DNA of the cells being transfected and the DNA fragments.", "For example, if a DNA fragment being transfected differs from a cognate or allelic gene at two positions, there are four possible recombination products, and each of these recombination products can be formed in different cells in the transformed population.", "Thus, homologous recombination of the fragment doubles the initial diversity in this gene.", "When many fragments recombine with corresponding cognate or allelic genes, the diversity of recombination products with respect to starting products increases exponentially with the number of mutations.", "Recombination results in modified cells having modified genomes and/or episomes.", "Recursive recombination prior to selection further increases diversity of resulting modified cells.", "The variant cells, whether the result of natural variation, mutagenesis, or recombination are screened or selected to identify a subset of cells that have evolved toward acquisition of a new or improved property.", "The nature of the screen, of course, depends on the property and several examples will be discussed below.", "Typically, recombination is repeated before initial screening.", "Optionally, however, the screening can also be repeated before performing subsequent cycles of recombination.", "Stringency can be increased in repeated cycles of screening.", "The subpopulation of cells surviving screening are optionally subjected to a further round of recombination.", "In some instances, the further round of recombination is effected by propagating the cells under conditions allowing exchange of DNA between cells.", "For example, protoplasts can be formed from the cells, allowed to fuse, and regenerated.", "Cells with recombinant genomes are propagated from the fused protoplasts.", "Alternatively, exchange of DNA can be promoted by propagation of cells or protoplasts in an electric field.", "For cells having a conjugative transfer apparatus, exchange of DNA can be promoted simply by propagating the cells.", "4.11.2.1.2 Using Two Separate Pools: Amplify First Pool and Add to Second Pool In other methods, the further round of recombination is performed by a split and pool approach.", "That is, the surviving cells are divided into two pools.", "DNA is isolated from one pool, and if necessary amplified, and then transformed into the other pool.", "Accordingly, DNA fragments from the first pool constitute a further library of fragments and recombine with cognate fragments in the second pool resulting in further diversity.", "As shown, a pool of mutant bacteria with improvements in a desired phenotype is obtained and split.", "Genes are obtained from one half, e.g., by PCR, by cloning of random genomic fragments, by infection with a transducing phage and harvesting transducing particles, or by the introduction of an origin of transfer (OriT) randomly into the relevant chromosome to create a donor population of cells capable of transferring random fragments by conjugation to an acceptor population.", "These genes are then stochastic &/or non-stochastic mutagenized (in vitro by known methods or in vivo as taught herein), or simply cloned into an allele replacement vector (e.g., one carrying selectable and counter-selectable markers).", "The gene pool is then transformed into the other half of the original mutant pool and recombinants are selected and screened for further improvements in phenotype.", "These best variants are used as the starting point for the next cycle.", "Alternatively, recursive recombination by any of the methods noted can be performed prior to screening, thereby increasing the diversity of the population of cells to be screened.", "4.11.2.13 Surviving Cells are Transfected into Fresh DNA In other methods, some or all of the cells surviving screening are transfected with a fresh library of DNA fragments, which can be the same or different from the library used in the first round of recombination.", "In this situation, the genes in the fresh library undergo recombination with cognate genes in the surviving cells.", "If genes are introduced as components of a vector, compatibility of this vector with any vector used in a previous round of transfection should be considered.", "If the vector used in a previous round was a suicide vector, there is no problem of incompatibility.", "If, however, the vector used in a previous round was not a suicide vector, a vector having a different incompatibility origin should be used in the subsequent round.", "In all of these formats, further recombination generates additional diversity in the DNA component of the cells resulting in further modified cells.", "The further modified cells are subjected to another round of screening/selection according to the same principles as the first round.", "Screening/selection identifies a subpopulation of further modified cells that have further evolved toward acquisition of the property.", "This subpopulation of cells can be subjected to further rounds of recombination and screening according to the same principles, optionally with the stringency of screening being increased at each round.", "Eventually, cells are identified that have acquired the desired property.", "4.11.3 Variations 4.11.3.1 Coating with RecA to Enrich Diversity of Homologous Recombination The frequency of homologous recombination between library fragments and cognate endogenous genes can be increased by coating the fragments with a recombinogenic protein before introduction into cells.", "See Pati et al., Molecular Biology of Cancer 1, 1 (1996); Sena & Zarling, Nature Genetics 3, 365 (1996); Revet et al., J. Mol.", "Biol.", "232, 779-791 (1993); Kowalczkowski & Zarling in Gene Targeting (CRC 1995), Ch.", "7.The recombinogenic protein promotes homologous pairing and/or strand exchange.", "The best characterized recA protein is from E. coli and is available from Pharmacia (Piscataway, N.J.).", "In addition to the wild-type protein, a number of mutant recA-like proteins have been identified (e.g., recA803).", "Further, many organisms have recA-like recombinases with strand-transfer activities (e.g., Ogawa et al., Cold Spring Harbor Symposium on Quantitative Biology 18, 567-576 (1993); Johnson & Symington, Mol.", "Cell.", "Biol.", "15, 4843-4850 (1995); Fugisawa et al., Nucl.", "Acids Res.", "13, 7473 (1985); Hsieh et al., Cell 44, 885 (1986); Hsieh et al., J. Biol.", "Chem.", "264, 5089 (1989); Fishel et al., Proc.", "Nail.", "Acad.", "Sci.", "USA 85, 3683 (1988); Cassuto et al., Mol.", "Gen. Genet.", "208, 10 (1987); Ganea et al., Mol.", "Cell Biol.", "7, 3124 (1987); Moore et al., J. Biol.", "Chem.", "19, 11108 (1990); Keene et al., Nucl.", "Acids Res.", "12, 3057 (1984); Kimiec, Cold Spring Harbor Symp.", "48, 675 (1984); Kimeic, Cell 44, 545 (1986); Kolodner et al., Proc.", "Nad.", "Acad.", "Sci.", "USA 84, 5560 (1987); Sugino et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 85, 3683 (1985).", "Halbrook et al., J. Biol.", "Chem.", "264, 21403 (1989); Eisen et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 85, 7481 (1988); McCarthy et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 85, 5854 (1988).", "Lowenhaupt et al., J. Biol.", "Chem.", "264, 20568 (1989).", "Examples of such recombinase proteins include recA, recA803, uvsX, (Roca, A. I., Crit.", "Rev.", "Biochem.", "Molec.", "Biol.", "25, 415 (1990)), sepI (Kolodner et al., Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 84, 5560 (1987); Tishkoff et al., Molec.", "Cell.", "Biol 11, 2593), RuvC (Dunderdale et al., Nature 354, 506 (1991)), DS72, KEMI, XRATI (Dykstra et al., Molec.", "Cell.", "Biol 11, 25 83 (1991)), STP/DSTI (Clark et al., Molec.", "Cell.", "Biol 11, 2576 (1991)), HPP-I (Moore et al., Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 88, 9067 (1991)), other eukaryotic recombinases (Bishop et al., Cell 69, 439 (1992); Shinohara et al., Cell 69, 457.RecA protein forms a nucleoprotein filament when it coats a single-stranded DNA.", "In this nucleoprotein filament, one monomer of recA protein is bound to about 3 nucleotides.", "This property of recA to coat single-stranded DNA is essentially sequence independent, although particular sequences favor initial loading of recA onto a polynucleotide (e.g., nucleation sequences).", "The nucleoprotein filament(s) can be formed on essentially any DNA to be stochastic &/or non-stochastic mutagenized and can form complexes with both single-stranded and double-stranded DNA in prokaryotic and eukaryotic cells.", "Before contacting with recA or other recombinase, fragments are often denatured, e.g., by heat-treatment.", "RecA protein is then added at a concentration of about 1-10 gM.", "After incubation, the recA-coated single-stranded DNA is introduced into recipient cells by conventional methods, such as chemical transformation or electroporation.", "In general, it can be desirable to coat the DNA with a RecA homolog isolated from the organism into which the coated DNA is being delivered.", "Recombination involves several cellular factors and the host RecA equivalent generally interacts better with other host factors than less closely related RecA molecules.", "The fragments undergo homologous recombination with cognate endogenous genes.", "Because of the increased frequency of recombination due to recombinase coating, the fragments need not be introduced as components of vectors.", "Fragments are sometimes coated with other nucleic acid binding proteins that promote recombination, protect nucleic acids from degradation, or target nucleic acids to the nucleus.", "Examples of such proteins includes Agrobacterium virE2 (Duffenberger et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 86, 9154-9158 (1989)).", "Alternatively, the recipient strains are deficient in RecD activity.", "Single stranded ends can also be generated by 3′-5′ exonuclease activity or restriction enzymes producing 5′ overhangs.", "4.11.3.2 Affinity Chromatography with MutS to Enrich for Fragments Having at Least One Mismatch The E. coli mismatch repair protein MutS can be used in affinity chromatography to enrich for fragments of double-stranded DNA containing at least one base of mismatch.", "The MutS protein recognizes the bubble formed by the individual strands about the point of the mismatch.", "See, e.g., Hsu & Chang, WO 9320233.The strategy of affinity enriching for partially mismatched duplexes can be incorporated into the present methods to increase the diversity between an incoming library of fragments and corresponding cognate or allelic genes in recipient cells.", "MutS is used to increase diversity.", "The DNA substrates for enrichment are substantially similar to each other but differ at a few sites.", "For example, the DNA substrates can represent complete or partial genomes (e.g., a chromosome library) from different individuals with the differences being due to polymorphisms.", "The substrates can also represent induced mutants of a wild type sequence.", "The DNA substrates are pooled, restriction digested, and denatured to produce fragments of single-stranded DNA.", "The single-stranded DNA is then allowed to reanneal.", "Some single-stranded fragments reanneal with a perfectly matched complementary strand to generate perfectly matched duplexes.", "Other single-stranded fragments anneal to generate mismatched duplexes.", "The mismatched duplexes are enriched from perfectly matched duplexes by MutS chromatography (e.g., with MutS immobilized to beads).", "The mismatched duplexes recovered by chromatography are introduced into recipient cells for recombination with cognate endogenous genes as described above.", "MutS affinity chromatography increases the proportion of fragments differing from each other and the cognate endogenous gene.", "Thus, recombination between the incoming fragments and endogenous genes results in greater diversity.", "A second strategy for MutS enrichment.", "In this strategy, the substrates for MutS enrichment represent variants of a relatively short segment, for example, a gene or cluster of genes, in which most of the different variants differ at no more than a single nucleotide.", "The goal of MutS enrichment is to produce substrates for recombination that contain more variations than sequences occurring in nature.", "This is achieved by fragmenting the substrates at random to produce overlapping fragments.", "The fragments are denatured and reannealed as in the first strategy.", "Reannealing generates some mismatched duplexes which can be separated from perfectly matched duplexes by MutS affinity chromatography.", "As before, MutS chromatography enriches for duplexes bearing at least a single mismatch.", "The mismatched duplexes are then stochastic &/or non-stochastic mutagenized into longer fragments.", "This is accomplished by cycles of denaturation, reannealing, and chain extension of partially annealed duplexes.", "After several such cycles, fragments of the same length as the original substrates are achieved, except that these fragments differ from each other at multiple sites.", "These fragments are then introduced into cells where they undergo recombination with cognate endogenous genes.", "4.1133 Suicide Vector Enriches Mutations for Cells that have Integrated the Vector into the Host Chromosome The invention further provides methods of enriching for cells bearing modified genes relative to the starting cells.", "This can be achieved by introducing a DNA fragment library (e.g., a single specific segment or a whole or partial genomic library) in a suicide vector (i.e., lacking a functional replication origin in the recipient cell type) containing both positive and negative selection markers.", "Optionally, multiple fragment libraries from different sources (e.g., B. sublilis, B. licheniformis and B. cereus) can be cloned into different vectors bearing different selection markers.", "Suitable positive selection markers include neoR, kanamycinR, hyg, hisD, gpt, ble, tetR.", "Suitable negative selection markers include hsv-tk, hprt, gpt, SacB ura3 and cytosine deaminase.", "A variety of examples of conditional replication vectors, mutations affecting vector replication, limited host range vectors, and counterselectable markers are found in Berg and Berg, supra, and LaRossa, ibid.", "and the references therein.", "In one example, a plasmid with R6K and fl origins of replication, a positively selectable marker (beta-lactamase), and a counterselectable marker (B. subtilis sacB) was used.", "M 13 transduction of plasmids containing cloned genes were efficiently recombined into the chromosomal copy of that gene in a rep mutant E. coli strain.", "Another strategy for applying negative selection is to include a wild type rpsL gene (encoding ribosomal protein S12) in a vector for use in cells having a mutant rpsL gene conferring streptomycin resistance.", "The mutant form of rpsL is recessive in cells having wild type rpsL.", "Thus, selection for Sm resistance selects against cells having a wild type copy of rpsL.", "See Skorupski & Taylor, Gene 169, 47-52 (1996).", "Alternatively, vectors bearing only a positive selection marker can be used with one round of selection for cells expressing the marker, and a subsequent round of screening for cells that have lost the marker (e.g., screening for drug sensitivity).", "The screen for cells that have lost the positive selection marker is equivalent to screening against expression of a negative selection marker.", "For example, Bacillus can be transformed with a vector bearing a CAT gene and a sequence to be integrated.", "See Harwood & Cutting, Molecular Biological Methods for Bacillus, at pp.", "31-33.Selection for chloramphenicol resistance isolates cells that have taken up vector.", "After a suitable period to allow recombination, selection for CAT sensitivity isolates cells which have lost the CAT gene.", "About 50% of such cells will have undergone recombination with the sequence to be integrated.", "Suicide vectors bearing a positive selection marker and optionally, a negative selection marker and a DNA fragment can integrate into host chromosomal DNA by a single crossover at a site in chromosomal DNA homologous to the fragment.", "Recombination generates an integrated vector flanked by direct repeats of the homologous sequence.", "In some cells, subsequent recombination between the repeats results in excision of the vector and either acquisition of a desired mutation from the vector by the genome or restoration of the genome to wild type.", "In the present methods, after transfer of the gene library cloned in a suitable vector, positive selection is applied for expression of the positive selection marker.", "Because nonintegrated copies of the suicide vector are rapidly eliminated from cells, this selection enriches for cells that have integrated the vector into the host chromosome.", "The cells surviving positive selection can then be propagated and subjected to negative selection, or screened for loss of the positive selection marker.", "Negative selection selects against cells expressing the negative selection marker.", "Thus, cells that have retained the integrated vector express the negative marker and are selectively eliminated.", "The cells surviving both rounds of selection are those that initially integrated and then eliminated the vector.", "These cells are enriched for cells having genes modified by homologous recombination with the vector.", "This process diversifies by a single exchange of genetic information.", "However, if the process is repeated either with the same vectors or with a library of fragments generated by PCR of pooled DNA from the enriched recombinant population, resulting in the diversity of targeted genes being enhanced exponentially each round of recombination.", "This process can be repeated recursively, with selection being performed as desired.", "4.11.3.4 Exploiting Known Information such as Map Location or Function In general, the above methods do not require knowledge of the number of genes to be optimized, their map location or their function.", "However, in some instances, where this information is available for one or more gene, it can be exploited.", "For example, if the property to be acquired by evolution is enhanced recombination of cells, one gene likely to be important is recA, even though many other genes, known and unknown, may make additional contributions.", "In this situation, the recA gene can be evolved, at least in part, separately from other candidate genes.", "The recA gene can be evolved by any of the methods of recursive recombination described in Section V. Briefly, this approach entails obtaining, diverse forms of a recA gene, allowing the forms to recombine, selecting recombinants having improved properties, and subjecting the recombinants to further cycles of recombination and selection.", "At any point in the individualized improvement of recA, the diverse forms of recA can be pooled with fragments encoding other genes in a library to be used in the general methods described herein.", "In this way, the library is seeded to contain a higher proportion of variants in a gene known to be important to the property sought to be acquired than would otherwise be the case.", "In one example, a plasmid is constructed carrying a non-functional (mutated) version of a chromosomal gene such as URA3, where the wild-type gene confers sensitivity to a drug (in this case 5-fluoro orotic acid).", "The plasmid also carries a selectable marker (resistance to another drug such as kanamycin), and a library of recA variants.", "Transformation of the plasmid into the cell results in expression of the recA variants, some of which will catalyze homologous recombination at an increased rate.", "Those cells in which homologous recombination occurred are resistant to the selectable drug on the plasmid, and to 5-fluoro orotic acid because of the disruption of the chromosomal copy of this gene.", "The recA variants which give the highest rates of homologous recombination are the most highly represented in a pool of homologous recombinants.", "The mutant recA genes can be isolated from this pool by PCR, re-stochastic &/or non-stochastic mutagenized, cloned back into the plasmid and the process repeated.", "Other sequences can be inserted in place of recA to evolve other components of the homologous recombination system.", "4.11.3.5 Using Own Harvest of Cells so No Impurities In some stochastic &/or non-stochastic mutagenesis methods, DNA substrates are isolated from natural sources and are not easily manipulated by DNA modifying or polymerizing enzymes due to recacitrant impurities, which poison enzymatic reactions.", "Such difficulties can be avoided by processing DNA substrates through a harvesting strain.", "The harvesting strain is typically a cell type with natural competence and a capacity for homologous recombination between sequences with substantial diversity (e.g., sequences exhibiting only 75% sequence identity).", "The harvesting strain bears a vector encoding a negative selection marker flanked by two segments respectively complementary to two segments flanking a gene or other region of interest in the DNA from a target organism.", "The harvesting strain is contacted with fragments of DNA from the target organism.", "Fragments are taken up by natural competence, or other methods described herein, and a fragment of interest from the target organism recombines with the vector of the harvesting strain causing loss of the negative selection marker.", "Selection against the negative marker allows isolation of cells that have taken up the fragment of interest.", "Stochastic &/or non-stochastic mutagenesis can be carried out in the harvester strain (e.g., a RecE/T strain) or vector can be isolated from the harvester strain for in vitro stochastic &/or non-stochastic mutagenesis or transfer to a different cell type for in vivo stochastic &/or non-stochastic mutagenesis.", "Alternatively, the vector can be transferred to a different cell type by conjugation, protoplast fusion or electrofusion.", "An example of a suitable harvester strain is Acinelobacter calcoaceticus mutS.", "Melnikov and Youngman, (1999) Nucl Acid Res 27(4): 1056-1062.This strain is naturally competent and takes up DNA in a nonsequence-specific manner.", "Also, because of the mutS mutation, this strain is capable of homologous recombination of sequences showing only 75% sequence identity.", "4.12 Further Applications 4.12.1 Improved Recombinancy One goal of whole cell evolution is to generate cells having improved capacity for recombination.", "Such cells are useful for a variety of purposes in molecular genetics including the in vivo formats of recursive sequence recombination described in Section V. Almost thirty genes (e.g., recA, recB, recC, recD, recE, recF, recG, recO, recQ, recR, recT, ruvA, ruvB, ruvC, sbcB, ssb, topA, gyrA and B, lig, polA, uvrD, E, recL, mutD, mutH, mutL, mutT, mutU, helD) and DNA sites (e.g., chi, recN, sbcC) involved in genetic recombination have been identified in E. coli, and cognate forms of several of these genes have been found in other organisms (e.g., rad51, rad55-rad57, Dmcl in yeast (see Kowalczykowski et al., Microbiol.", "Rev.", "58, 401465 (1994); Kowalczkowski & Zarling, supra) and human homologs of Rad51 and Dmcl have been identified (see Sandier et al., Nucl.", "Acids Res.", "24, 2125-2132 (1996)).", "At least some of the E. coli genes, including recA are functional in mammalian cells, and can be targeted to the nucleus as a fusion with SV40 large T antigen nuclear targeting sequence (Reiss et al., Proc.", "Mad.", "Acad.", "Sci.", "USA, 93, 3094-3098 (1996)).", "Further, mutations in mismatch repair genes, such as mutL, mutS, mutH mutT relax homology requirements and allow recombination between more diverged sequences (Rayssiguier et al., Nature 342, 396-401 (1989)).", "The extent of recombination between divergent strains can be enhanced by impairing mismatch repair genes and stimulating SOS genes.", "Such can be achieved by use of appropriate mutant strains and/or growth under conditions of metabolic stress, which have been found to stimulate SOS and inhibit mismatch repair genes.", "Vulic et al., Proc.", "Mad.", "Acad.", "Sci.", "USA 94 (1997).", "In addition, this can be achieved by impairing the products of mismatch repair genes by exposure to selective inhibitors.", "Starting substrates for recombination are selected according to the general principles described above.", "That is, the substrates can be whole genomes or fractions thereof containing recombination genes or sites.", "Large libraries of essentially random fragments can be seeded with collections of fragments constituting variants of one or more known recombination genes, such as recA.", "Alternatively, libraries can be formed by mixing variant forms of the various known recombination genes and sites.", "4.12.2 Expression of GFP Indicates Cell is Capable of Homologous Recombination The library of fragments is introduced into the recipient cells to be improved and recombination occurs, generating modified cells.", "The recipient cells preferably contain a marker gene whose expression has been disabled in a manner that can be corrected by recombination.", "For example, the cells can contain two copies of a marker gene bearing mutations at different sites, which copies can recombine to generate the wild type gene.", "A suitable marker gene is green fluorescent protein.", "A vector can be constructed encoding one copy of GFP having stop codons near the N-terminus, and another copy of GFP having stop codons near the C-terminus of the protein.", "The distance between the stop codons at the respective ends of the molecule is 500 bp and about 25% of recombination events result in active GFP.", "Expression of GFP in a cell signals that a cell is capable of homologous recombination to recombine in between the stop codons to generate a contiguous coding sequence.", "By screening for cells expressing GFP, one enriches for cells having the highest capacity for recombination.", "The same type of screen can be used following subsequent rounds of recombination.", "However, unless the selection marker used in previous round(s) was present on a suicide vector, subsequent round(s) should employ a second disabled screening marker within a second vector bearing a different origin of replication or a different positive selection marker to vectors used in the previous rounds.", "4.123 Increased Genome Copy Number so more Chromosomes per Bacterial Cell to Make Evolution Quicker The majority of bacterial cells in stationary phase cultures grown in rich media contain two, four or eight genomes.", "In minimal medium the cells contain one or two genomes.", "The number of genomes per bacterial cell thus depends on the growth rate of the cell as it enters stationary phase.", "This is because rapidly growing cells contain multiple replication forks, resulting in several genomes in the cells after termination.", "The number of genomes is strain dependent, although all strains tested have more than one chromosome in stationary phase.", "The number of genomes in stationary phase cells decreases with time.", "This appears to be due to fragmentation and degradation of entire chromosomes, similar to apoptosis in mammalian cells.", "This fragmentation of genomes in cells containing multiple genome copies results in massive recombination and mutagenesis.", "Useful mutants may find ways to use energy sources that will allow them to continue growing.", "Multigenome or gene-redundant cells are much more resistant to mutagenesis and can be improved for a selected trait faster.", "Some cell types, such as Deinococcus radians (Daly and Minton J Bacteriol 177, 5495-5505 (1995)) exhibit polyploidy throughout the cell cycle.", "This cell type is highly radiation resistant due to the presence of many copies of the genome.", "High frequency recombination between the genomes allows rapid removal of mutations induced by a variety of DNA damaging agents.", "A goal of the present methods is to evolve other cell types to have increased genome copy number akin to that of Deinoccocus radians.", "Preferably, the increased copy number is maintained through all or most of its cell cycle in all or most growth conditions.", "The presence of multiple genome copies in such cells results in a higher frequency of homologous recombination in these cells, both between copies of a gene in different genomes within the cell, and between a genome within the cell and a transfected fragment.", "The increased frequency of recombination allows the cells to be evolved more quickly to acquire other useful characteristics.", "Starting substrates for recombination can be a diverse library of genes only a few of which are relevant to genomic copy number, a focused library formed from variants of gene(s) known or suspected to have a role in genomic copy number or a combination of the two.", "As a general rule one would expect increased copy number would be achieved by evolution of genes involved in replication and cell septation such that cell septation is inhibited without impairing replication.", "Genes involved in replication include tus, xerc, xerD, dif, gyrA, gyrB, parE, parc, dif, TerA, TerB, TerC, TerD, TerE, TerF, and genes influencing chromosome partitioning and gene copy number include minD, mukA (toIC), mukB, mukC, mukD, spoOJ, spoIIIE (Wake & Errington, Annu.", "Rev.", "Genet.", "29, 41-67 (1995)).", "A useful source of substrates is the genome of a cell type such as Deinoccocus radians known to have the desired phenotype of multigenomic copy number.", "As well as, or instead of, the above substrates, fragments encoding protein or antisense RNA inhibitors to genes known to be involved in cell septation can also be used.", "In nature, the existence of multiple genomic copies in a cell type would usually not be advantageous due to the greater nutritional requirements needed to maintain this copy number.", "However, artificial conditions can be devised to select for high copy number.", "Modified cells having recombinant genomes are grown in rich media (in which conditions, multicopy number should not be a disadvantage) and exposed to a mutagen, such as ultraviolet or gamma irradiation or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, which induces DNA breaks amenable to repair by recombination.", "These conditions select for cells having multicopy number due to the greater efficiency with which mutations can be excised.", "Modified cells surviving exposure to mutagen are enriched for cells with multiple genome copies.", "If desired, selected cells can be individually analyzed for genome copy number (e.g., by quantitative hybridization with appropriate controls).", "Some or all of the collection of cells surviving selection provide the substrates for the next round of recombination.", "In addition, individual cells can be sorted using a cell sorter for those cells containing more DNA, e.g., using DNA specific fluorescent compounds or sorting for increased size using light dispersion.", "Eventually cells are evolved that have at least 2, 4, 6, 8 or 10 copies of the genome throughout the cell cycle.", "In a similar manner, protoplasts can also be recombined.", "4.12.4 Evolve Secretion Pathways for Better Efficiency 4.12.5 Evolve to Manufacture Drugs or Chemicals The protein (or metabolite) secretion pathways of bacterial and eukaryotic cells can be evolved to export desired molecules more efficiently, such as for the manufacturing of protein pharmaceuticals, small molecule drugs or specialty chemicals.", "Improvements in efficiency are particularly desirable for proteins requiring multisubunit assembly (such as antibodies) or extensive posttranslational modification before secretion.", "The efficiency of secretion may depend on a number of genetic sequences including a signal peptide coding sequence, sequences encoding protein(s) that cleave or otherwise recognize the coding sequence, and the coding sequence of the protein being secreted.", "The latter may affect folding of the protein and the ease with which it can integrate into and traverse membranes.", "The bacterial secretion pathway in E. coli include the SecA, SecB, SecE, SecD and SecF genes.", "In Bacillus subtilis, the major genes are secA, secD, secE, secF, secY, ffh, ftsy together with five signal peptidase genes (sipS, sipT, sipU, sipV and sipW) (Kunst et al, supra).", "For proteins requiring posttranslational modification, evolution of genes effecting such modification may contribute to improved secretion.", "Likewise genes with expression products having a role in assembly of multisubunit proteins (e.g., chaperonins) may also contribute to improved secretion.", "Selection of substrates for recombination follows the general principles discussed above.", "In this case, the focused libraries referred to above comprise variants of the known secretion genes.", "For evolution of prokaryotic cells to express eukaryotic proteins, the initial substrates for recombination are often obtained at least in part from eukaryotic sources.", "Incoming fragments can undergo recombination both with chromosomal DNA in recipient cells and with the screening marker construct present in such cells.", "(see below).", "The latter form of recombination is important for evolution of the signal coding sequence incorporated in the screening marker construct.", "Improved secretion can be screened by the inclusion of marker construct in the cells being evolved.", "The marker construct encodes a marker gene, operably linked to expression sequences, and usually operably linked to a signal peptide coding sequence.", "The marker gene is sometimes expressed as a fusion protein with a recombinant protein of interest.", "This approach is useful when one wants to evolve the recombinant protein coding sequence together with secretion genes.", "4.12.6 Evolve so Product is Toxic to Cell Unless Secreted In one variation, the marker gene encodes a product that is toxic to the cell containing the construct unless ne product is secreted.", "Suitable toxin proteins include diphtheria toxin and ricin toxin.", "Propagation of modified cells bearing such a construct selects for cells that have evolved to improve secretion of the toxin.", "Alternatively, the marker gene can encode a ligand to a known receptor, and cells bearing the ligand can be detected by FACS using labeled receptor.", "Optionally, such a ligand can be operably linked to a phospholipid anchoring sequence that binds the ligand to the cell membrane surface following secretion.", "In a further variation, secreted marker protein can be maintained in proximity with the cell secreting it by distributing individual cells into agar drops.", "This is done, e.g., by droplet formation of a cell suspension.", "Secreted protein is confined within the agar matrix and can be detected by e.g., FACS.", "In another variation, a protein of interest is expressed as a fusion protein together with beta-lactamase or alkaline phosphatase.", "These enzymes metabolize commercially available chromogenic substrates (e.g., X-gal), but do so only after secretion into the periplasm.", "Appearance of colored substrate in a colony of cells therefore indicates capacity to secrete the fusion protein and the intensity of color is related to the efficiency of secretion.", "The cells identified by these screening and selection methods have the capacity to secrete increased amounts of protein.", "This capacity may be attributable to increased secretion and increased expression, or from increased secretion alone.", "4.12.7 Evolve to Acquire Increased Expression of Recombinant Protein Expression Cells can also be evolved to acquire increased expression of a recombinant protein.", "The level of expression is, of course, highly dependent on the construct from which the recombinant protein is expressed and the regulatory sequences, such as the promoter, enhancer(s) and transcription termination site contained therein.", "Expression can also be affected by a large number of host genes having roles in transcription, posttranslational modification and translation.", "In addition, host genes involved in synthesis of ribonucleotide and amino acid monomers for transcription and translation may have indirect effects on efficiency of expression.", "Selection of substrates for recombination follows the general principles discussed above.", "In this case, focused libraries comprise variants of genes known to have roles in expression.", "For evolution of prokaryotic cells to express eukaryotic proteins, the initial substrates for recombination are often obtained, at least in part, from eukaryotic sources; that is eukaryotic genes encoding proteins such as chaperonins involved in secretion and/assembly of proteins.", "Incoming fragments can undergo recombination both with chromosomal DNA in recipient cells and with the screening marker construct present in such cells (see below).", "Screening for improved expression can be effected by including a reporter construct in the cells being evolved.", "The reporter construct expresses (and usually secretes) a reporter protein, such as GFP, which is easily detected and nontoxic.", "The reporter protein can be expressed alone or together with a protein of interest as a fusion protein.", "If the reporter gene is secreted, the screening effectively selects for cells having either improved secretion or improved expression, or both.", "4.12.8 Evovle Plant Cells to Acquire Resistance A further application of recursive sequence recombination is the evolution of plant cells, and transgenic plants derived from the same, to acquire resistance to pathogenic diseases (fungi, viruses and bacteria), insects, chemicals (such as salt, selenium, pollutants, pesticides, herbicides, or the like), including, e.g., atrazine or glyphosate, or to modify chemical composition, yield or the like.", "The substrates for recombination can again be whole genomic libraries, fractions thereof or focused libraries containing variants of gene(s) known or suspected to confer resistance to one of the above agents.", "Frequently, library fragments are obtained from a different species to the plant being evolved.", "The DNA fragments are introduced into plant tissues, cultured plant cells, plant microspores, or plant protoplasts by standard methods including electroporation (From et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 82, 5824 (1985), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al., Molecular Biology of Plant Tumors, (Academic Press, New York, 1982) pp.", "549-560; Howell, U.S. Pat.", "No.", "4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)), use of pollen as vector (WO 85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned.", "The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al., Science 233, 496498 (1984); Fraley et al., Proc.", "Natl.", "Acad.", "Sci.", "USA 80, 4803 (1983)).", "Diversity can also be generated by genetic exchange between plant protoplasts according to the same principles described below for fungal protoplasts.", "Procedures for formation and fusion of plant protoplasts are described by Takahashi et al., U.S. Pat.", "No.", "4,677,066; Akagi et al., U.S. Pat.", "No.", "5,360,725; Shimamoto et al., U.S. Pat.", "No.", "5,250,433; Cheney et al., U.S. Pat.", "No.", "5,426,040.4.12.9 Plant Genome Stochastic &/or Non-Stochastic Mutagenesis Plant genome stochastic &/or non-stochastic mutagenesis allows recursive cycles to be used for the introduction and recombination of genes or pathways that confer improved properties to desired plant species.", "Any plant species, including weeds and wild cultivars, showing a desired trait, such as herbicide resistance, salt tolerance, pest resistance, or temperature tolerance, can be used as the source of DNA that is introduced into the crop or horticultural host plant species.", "Genomic DNA prepared from the source plant is fragmented (e.g.", "by DNase1, restriction enzymes, or mechanically) and cloned into a vector suitable for making plant genomic libraries, such as pGA482 (An.", "G., 1995, Methods Mol.", "Biol.", "44: 47-58).", "This vector contains the A. tumefaciens left and right borders needed for gene transfer to plant cells and antibiotic markers for selection in E. coli, Agrobacterium, and plant cells.", "A multicloning site is provided for insertion of the genomic, fragments.", "A cos sequence is present for the efficient packaging of DNA into bacteriophage lambda heads for transfection of the primary library into E. coli.", "The vector accepts DNA fragments of 2540 kb.", "The primary library can also be directly electroporated into an A. tumefaciens or A. rhizogenes strain that is used to infect and transform host plant cells (Main, G D et al., 1995, Methods Mol.", "Biol.", "44: 405-412).", "Alternatively, DNA can be introduced by electroporation or PEG-mediated uptake into protoplasts of the recipient plant species (Bilang et al.", "(1994) Plant Mol.", "Biol.", "Manual, Kluwer Academic Publishers, A1: 1-16) or by particle bombardment of cells or tissues (Christou, ibid, A2: 1-15).", "If necessary, antibiotic markers in the T-DNA region can be eliminated, as long as selection for the trait is possible, so that the final plant products contain no antibiotic genes.", "Stably transformed whole cells acquiring the trait are selected on solid or liquid media containing the agent to which the introduced DNA confers resistance or tolerance.", "If the trait in question cannot be selected for directly, transformed cells can be selected with antibiotics and allowed to form callus or regenerated to whole plants and then screened for the desired property.", "The second and further cycles consist of isolating genomic DNA from each transgenic line and introducing it into one or more of the other transgenic fines.", "In each round, transformed cells are selected or screened for incremental improvement.", "To speed the process of using multiple cycles of transformation, plant regeneration can be deferred until the last round.", "Callus tissue generated from the protoplasts or transformed tissues can serve as a source of genomic DNA and new host cells.", "After the final round, fertile plants are regenerated and the progeny are selected for homozygosity of the inserted DNAs.", "Ultimately, a new plant is created that carries multiple inserts which additively or synergistically combine to confer high levels of the desired trait.", "Alternatively, microspores can be isolated as homozygotes generated from spontaneous diploids.", "In addition, the introduced DNA that confers the desired trait can be traced because it is flanked by known sequences in the vector.", "Either PCR or plasmid rescue is used to isolate the sequences and characterize them in more detail.", "Long PCR (Foord, O S and Rose, E A, 1995, PCR Primer: A Laboratory Manual, CSBL Press, pp 63-77) of the full 2540 kb insert is achieved with the proper reagents and techniques using as primers the T-DNA border sequences.", "If the vector is modified to contain the E. coli origin of replication and an antibiotic marker between the T-DNA borders, a rare cutting restriction enzyme, such as NotI or SfiI, that cuts only at the ends of the inserted DNA is used to create fragments containing the source plant DNA that are then self-ligated and transformed into E. coli where they replicate as plasmids.", "The total DNA or subfragment of it that is responsible for the transferred trait can be subjected to in vitro evolution by DNA stochastic &/or non-stochastic mutagenesis.", "The stochastic &/or non-stochastic mutagenized library can be reiteratively recombined by any method herein and then introduced into host plant cells and screened for improvement of the trait.", "In this way, single and multigene traits can be transferred from one species to another and optimized for higher expression or activity leading to whole organism improvement.", "This entire process can also be reiteratively repeated.", "Alternatively, the cells can be transformed microspores with the regenerated haploid plants being screened directly for improved traits as noted below.", "4.12.10 Plant Cell is Put into Contact with Agent to See which Cells Survive.", "After a suitable period of incubation to allow recombination to occur and for expression of recombinant genes, the plant cells are contacted with the agent to which resistance is to be acquired, and surviving plant cells are collected.", "Some or all of these plant cells can be subject to a further round of recombination and screening.", "Eventually, plant cells having the required degree of resistance are obtained.", "These cells can then be cultured into transgenic plants.", "Plant regeneration from cultured protoplasts is described in Evans et al., “Protoplast Isolation and Culture,” Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York, 1983); Davey, “Recent Developments in the Culture and Regeneration of Plant Protoplasts,” Protoplasts, (1983) pp.", "12-29, (Birkhauser, Basal 1983); Dale, “Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops,” Protoplasts (1983) pp.", "31-41, (Birkhauser, Basel 1983); Binding, “Regeneration of Plants,” Plant Protoplasts, pp.", "21-73, (CRC Press, Boca Raton, 1985).", "4.12.11 Start in Bacterial Cell Since Faster Evolution and Transform into Plant In a variation of the above method, one or more preliminary rounds of recombination and screening can be performed in bacterial cells according to the same general strategy as described for plant cells.", "More rapid evolution can be achieved in bacterial cells due to their greater growth rate and the greater efficiency with which DNA can be introduced into such cells.", "After one or more rounds of recombination/screening, a DNA fragment library is recovered from bacteria and transformed into the plant cells.", "The library can either be a complete library or a focused library.", "A focused library can be produced by amplification from primers specific for plant sequences, particularly plant sequences known or suspected to have a role in conferring resistance.", "4.12.12 Microspore Manipulation Microspores are haploid (In) male spores that develop into pollen grains.", "Anthers contain a large numbers of microspores in early-uninucleate to first-mitosis stages.", "Microspores have been successfully induced to develop into plants for most species, such as, e.g., rice (Chen, CC 1977 In Vitro.", "13: 484-489), tobacco (Atanassov, I. et al.", "1998 Plant Mol.", "Biol.", "38: 1169-1178), Tradescantia (Savage J R K and Papworth D G. 1998 Mutat Res.", "422: 313-322), Arabidopsis (Park S K et al.", "1998 Development.", "125: 3789-3799), sugar beet (Majewska-Sawka A and Rodrigues-Garcia N E 1996 J Cell Sci.", "109: 859-866), Barley (Olsen F L 1991 Hereditas 115: 255-266) and oilseed rape (Boutillier K A et al.", "1994 Plant Mol.", "Biol.", "26: 1711-1723).", "The plants derived from microspores are predominantly haploid or diploid (infrequently polyploid and aneuploid).", "The diploid plants are homozygous and fertile and can be generated in a relatively short time.", "Microspores obtained from F1 hybrid plants represent great diversity, thus being an excellent model for studying recombination.", "In addition, microspores can be transformed with T-DNA introduced by agrobacterium or other available means and then regenerated into individual plants.", "Furthermore, protoplasts can be made from microspores and they can be fused similar to what occur in fungi and bacteria.", "Microspores, due to their complex ploidy and regenerating ability, provide a tool for plant whole genome stochastic &/or non-stochastic mutagenesis.", "For example, if pollens from 4 parents are collected 4 and pooled, and then used to randomly pollinate the parents, the progenies should have 24=16 possible combinations.", "Assuming this plant has 7 chromosomes, microspores collected from the 16 progenies will represent 27×16=2048 possible chromosomal combinations.", "This number is even greater if meiotic processes occur.", "When diploid, homozygous embryos are generated from these microspores, in many cases, they are screened for desired phenotypes, such as herbicide- or disease-resistant.", "In addition, for plant oil composition these embryos can be dissected into two halves: one for analysis the other for regeneration into a viable plant.", "Protoplasts generated from microspores (especially the haploid ones) are pooled and fused.", "Microspores obtained from plants generated by protoplast fusion are pooled and fused again, increasing the genetic diversity of the resulting microspores.", "Microspores can be subjected to mutagenesis in various ways, such as by chemical mutagenesis, radiation-induced mutagenesis and, e.g., t-DNA transformation, prior to fusion or regeneration.", "New mutations which are generated can be recombined through the recursive processes described above and herein.", "4.12.13 Acquistion of Salt Tolerance DNA from a salt tolerant plant is isolated and used to create a genomic library.", "Protoplasts made from the recipient species are transformed/transfected with the genomic library (e.g., by electroporation, agrobacterium, etc.).", "Cells are selected on media with a normally inhibitory level of NaCl.", "Only the cells with newly acquired salt tolerance will grow into callus tissue.", "The best lines are chosen and genomic libraries are made from their pooled DNA.", "These libraries are transformed into protoplasts made from the first round transformed calli.", "Again, cells are selected on increased salt concentrations.", "After the desired level of salt tolerance is achieved, the callus tissue can be induced to regenerate whole plants.", "Progeny of these plants are typically analyzed for homozygosity of the inserts to ensure stability of the acquired trait.", "At the indicated steps, plant regeneration or isolation and stochastic &/or non-stochastic mutagenesis of the introduced genes can be added to the overall protocol.", "4.13 Evolve Transgenic Animals 4.13.1 Optimize Transgene One goal of transgenesis is to produce transgenic animals, such as mice, rabbits, sheep, pigs, goats, and cattle, secreting a recombinant protein in the milk.", "A transgene for this purpose typically comprises in operable linkage a promoter and an enhancer from a milk-protein gene (e.g., alpha, beta, or gamma casein, beta-lactoglobulin, acid whey protein or alpha-lactalbumin), a signal sequence, a recombinant protein coding sequence and a transcription termination site.", "Optionally, a transgene can encode multiple chains of a multichain protein, such as an immunoglobulin, in which case, the two chains are usually individually operably linked to sets of regulatory sequences.", "Transgenes can be optimized for expression and secretion by recursive sequence recombination.", "Suitable substrates for recombination include regulatory sequences such as promoters and enhancers from milk-protein genes from different species or individual animals.", "Cycles of recombination can be performed in vitro or in vivo by any of the formats discussed.", "Screening is performed in vivo on cultures of mammary-gland derived cells, such as HCII or MacT, transfected with transgenes and reporter constructs such as those discussed above.", "After several cycles of recombination and screening, transgenes resulting in the highest levels of expression and secretion are extracted from the mammary gland tissue culture cells and used to transfect embryonic cells, such as zygotes and embryonic stem cells, which are matured into transgenic animals.", "4.13.2 Optimize Whole Animal by Transforming into Embryonic Cells Gene of Desired Trait 4.13.2.1 Growth Hormone In this approach, libraries of incoming fragments are transformed into embryonic cells, such as ES cells or zygotes.", "The fragments can be variants of a gene known to confer a desired property, such as growth hormone.", "Alternatively, the fragments can be partial or complete genomic libraries including many genes.", "Fragments are usually introduced into zygotes by microinjection as described by Gordon et al., Methods Enzymol.", "10 1, 414 (1984); Hogan et al., Manipulation of the Mouse Embryo: A Laboratory Manual (C.S.H.L.", "N.Y., 1986) (mouse embryo),- and Hammer et al., Nature 315, 680 (1985) (rabbit and porcine embryos); Gandolfi et al., J Reprod.", "Fert.", "81, 23-28 (1987); Rexroad et al., J Anim.", "Sci.", "66, 947-953 (1988) (ovine embryos) and Eyestone et al., J Reprod.", "Fert.", "85, 715-720 (1989); Camous et al., J Reprod.", "Fert.", "72, 779-785 (1984); and Heyman et al., Theriogenology 27, 5968 (1987) (bovine embryos).", "Zygotes are then matured and introduced into recipient female animals which gestate the embryo and give birth to a transgenic offspring.", "Alternatively, transgenes can be introduced into embryonic stem cells (ES).", "These cells are obtained from preimplantation embryos cultured in vitro.", "Bradley et al., Nature 309, 255-258 (1984).", "Transgenes can be introduced into such cells by electroporation or microinjection.", "Transformed ES cells are combined with blastocysts from a non-human animal.", "The ES cells colonize the embryo and in some embryos form the germ line of the resulting chimeric animal.", "See Jaenisch, Science, 240, 1468-1474 (1988).", "Regardless whether zygotes or ES are used, screening is performed on whole animals for a desired property, such as increased size and/or growth rate.", "DNA is extracted from animals having evolved toward acquisition of the desired property.", "This DNA is then used to transfect further embryonic cells.", "These cells can also be obtained from animals that have acquired toward the desired property in a split and pool approach.", "That is, DNA from one subset of such animals is transformed into embryonic cells prepared from another subset of the animals.", "Alternatively, the DNA from animals that have evolved toward acquisition of the desired property can be transfected into fresh embryonic cells.", "In either alternative, transfected cells are matured into transgenic animals, and the animals subjected to a further round of screening for the desired property.", "Initially, a library is prepared of variants of a growth hormone gene.", "The variants can be natural or induced.", "The library is coated with recA protein and transferred into fertilized fish eggs.", "The fish eggs then mature into fish of different sizes.", "The growth hormone gene fragment of genomic DNA from large fish is then amplified by PCR and used in the next round of recombination.", "Alternatively, fish -IFN involved to enhance resistance to viral infections as described below.", "4.13.2.2 Evolution of Improved Hormones for Expansion in Transgenic Animals 4.13.3 To Create Animals with Improved Traits Evolution of improved hormones for expression in transgenic animals (e.g., Fish) to create animals with improved traits.", "Hormones and cytokines are key regulators of size, body weight, viral resistance and many other commercially important traits.", "DNA stochastic &/or non-stochastic mutagenesis is used to rapidly evolve the genes for these proteins using in vitro assays.", "This was demonstrated with the evolution of the human alpha interferon genes to have potent antiviral activity on murine cells.", "Large improvements in activity were achieved in two cycles of family stochastic &/or non-stochastic mutagenesis of the human IFN genes.", "In general, a method of increasing resistance to virus infection in cells can be performed by first introducing a stochastic &/or non-stochastic mutagenized library comprising at least one stochastic &/or non-stochastic mutagenized interferon gene into animal cells to create an initial library of animal cells or animals.", "The initial library is then challenged with the virus.", "Animal cells or animals are selected from the initial library which are resistant to the virus and a plurality of transgenes from a plurality of animal cells or animals which are resistant to the virus are recovered.", "The plurality of transgenes is recovered to produce an evolved library of animal cells or animals which is again challenged with the virus.", "Cells or animals are selected from the evolved library the which are resistant to the virus.", "For example, genes evolved with in vitro assays are introduced into the germplasm of animals or plants to create improved strains.", "One limitation of this procedure is that in vitro assays are often only crude predictors of in vivo activity.", "However, with improving methods for the production of transgenic plants and animals, one can now marry whole organism breeding with molecular breeding.", "The approach is to introduce stochastic &/or non-stochastic mutagenized libraries of hormone genes into the species of interest.", "This can be done with a single gene per transgenic or with pools of genes per transgenic.", "Progeny are then screened for the phenotype of interest.", "In this case, stochastic &/or non-stochastic mutagenized libraries of interferon genes (alpha IFN for example) are introduced into transgenic fish.", "The library of transgenic fish are challenged with a virus.", "The most resistant fish are identified (i.e.", "either survivors of a lethal challenge; or those that are deemed most ‘healthy’ after the challenge).", "The IFN transgenes are recovered by PCR and stochastic &/or non-stochastic mutagenized in either a poolwise or a pairwise fashion.", "This generates an evolved library of IFN genes.", "A second library of transgenic fish is created and the process is repeated.", "In this way, IFN is evolved for improved antiviral activity in a whole organism assay.", "This procedure is general and can be applied to any trait that is affected by a gene or gene family of interest and which can be quantitatively measured.", "Fish interferon sequence data is available for the Japanese flatfish (Paralichthys olivaceus) as mRNA sequence (Tamai et al (1993) “Cloning and expression of flatfish (Paralichthys olivaceus) interferon cDNA.” Biochem.", "Biophs.", "Act 1174, 182-186; Y see also, Tami et al.", "(1993) “Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes.” Cytotechnology 1993; 11 (2): 121-131).", "This sequence can be used to clone out IFN genes from this species.", "This sequence can also be used as a probe to clone homologous interferons from additional species of fish.", "As well, additional sequence information can be utilized to clone out more species of fish interferons.", "Once a library of interferons has been cloned, these can be family stochastic &/or non-stochastic mutagenized to generate a library of variants.", "In one embodiment, BHK-21 (A fibroblast cell line from hamster) can be transfected with the stochastic &/or non-stochastic mutagenized WN-expression plasmids.", "Active recombinant IFN is produced and then purified by WGA agarose affinity chromatography (Tamai, et al.", "1993 Biochim Ciophys Acta.", "supra).", "The antiviral activity of IFN can be measured on fish cells challenged by rhabdovirus.", "Tami et al.", "(1993) “Purification and characterization of interferon-like antiviral protein derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by oncogenes.” Cytotechnolgy 1993; 11 (2): 121-131).", "4.13.4 Whole Genome Stochastic &/or Non-Stochastic Mutagenesis in Higher Organisms Poolwise Recursive Breeding The present invention provides a procedure for generating large combinatorial libraries of higher eukaryotes, plants, fish, domesticated animals, etc.", "In addition to the procedures outlined above, poolwise combination of male and female gametes can also be used to generate large diverse molecular libraries.", "In one aspect, the process includes recursive poolwise matings for several generations without any deliberate screening.", "This is similar to classical breeding, except that pools of organisms, rather than pairs of organisms, are mated, thereby accelerating the generation of genetic diversity.", "This method is similar to recursive fusion of a diverse population of bacterial protoplasts resulting in the generation of multiparent progeny harboring genetic information from all of the starting population of bacteria.", "The process described here is to perform analogous artificial or natural matings of large populations of natural isolates, imparting a split pool mating strategy.", "Before mating, all of the male gametes i.e.", "pollen, sperm, etc., are isolated from the starting population and pooled.", "These are then used to “self” fertilize a mixed pool of the female gametes from the same population.", "The process is repeated with the subsequent progeny for several generations, with the final progeny being a combinatorial organism library with each member having genetic information originating from many if not all of the starting “parents.” This process generates large diverse organism libraries on which many selections and or screens can be imparted, and it does not require sophisticated in vitro manipulation of genes.", "However, it results in the creation of useful new strains (perhaps well diluted in the population) in a much shorter time frame than such organisms could be generated using a classical targeted breeding approach.", "These libraries are generated relatively quickly (e.g., typically in less than three years for most plants of commercial interest, with six cycles or less of recursive breeding being sufficient to generate desired diversity).", "An additional benefit of these methods is that the resulting libraries provide organismal diversity in areas, such as agriculture, aquaculture, and animal husbandry, that are currently genetically homogeneous.", "Examples of these methods for several organisms are described below.", "4.13.5 Plants Plants A population of plants, for example all of the different corn strains in a commercial seed/germplasm collection, are grown and the pollen from the entire population is harvested and pooled.", "This mixed pollen population is then used to “self” fertilize the same population.", "Self pollination is prevented, so that the fertilization is combinatorial.", "The cross results in all pairwise crosses possible within the population, and the resulting seeds result in many of the possible outcomes of each of these pairwise crosses.", "The seeds from the fertilized plants are then harvested, pooled, planted, and the pollen is again harvested, pooled, and used to “self fertilize the population.", "After only several generations, the resulting population is a very diverse combinatorial library of corn.", "The seeds from this library are harvested and screened for desirable traits, e.g., salt tolerance, growth rate, productivity, yield, disease resistance, etc.", "Essentially any plant collection can be modified by this approach.", "Important commercial crops include both monocots and dicots.", "Monocots include plants in the grass family (Gramineae), such as plants in the sub families Fetucoideae and Poacoideae, which together include several hundred genera including plants in the genera Agrostis, Phleum, Dactylis, Sorgum, Setaria, Zea (e.g., corn), Oryza (e.g., rice), Triticum (e.g., wheat), Secale (e.g., rye), Avena (e.g., oats), Hordeum (e.g., barley), Saccharum, Poa, Festuca, Stenotaphrum, Cynodon, Coix, the Olyreae, Phareae and many others.", "Plants in the family Gramineae are a particularly preferred target plants for the methods of the invention.", "Additional preferred targets include other commercially important crops, e.g., from the families Compositae (the largest family of vascular plants, including at least 1,000 genera, including important commercial crops such as sunflower), and Leguminosae or “pea family,” which includes several hundred genera, including many commercially valuable crops such as pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea.", "Common crops applicable to the methods of the invention include Zea mays, rice, soybean, sorghum, wheat, oats, barley, millet, sunflower, and canola.", "This process can also be carried out using pollen from different species or more divergent strains (e.g., crossing the ancient grasses with corn).", "Different plant species can be forced to cross.", "Only a few plants from an initial cross would have to result in order to make the process viable.", "These few progeny, e.g., from a cross between soy bean and corn, would generate pollen and eggs, each of which would represent a different meiotic outcome from the recombination of the two genomes.", "The pollen would be harvested and used to “self pollinate the original progeny.", "This process would then be carried out recursively.", "This would generate a large family stochastic &/or non-stochastic mutagenized library of two or more species, which could be subsequently screened.", "4.13.6 Fish Fish The natural tendency of fish to lay their eggs outside of the body and to have a male cover those eggs with sperm provides another opportunity for a split pooled breeding strategy.", "The eggs from many different fish, e.g., salmon from different fisheries about the world, can be harvested, pooled, and then fertilized with similarly collected and pooled salmon sperm.", "The fertilization will result in all of the possible pairwise matings of the starting population.", "The resulting progeny is then grown and again the sperm and eggs are harvested, and pooled, with each egg and sperm representing a different meiotic outcome of the different crosses.", "The pooled sperm are then used to fertilize the pooled eggs and the process is carried out recursively.", "After several generations the resulting progeny can then be subjected to selections and screens for desired properties, such as size, disease resistance, etc.", "4.13.7 Animals Animals The advent of in vitro fertilization and surrogate motherhood provides a means of whole genome stochastic &/or non-stochastic mutagenesis in animals such as mammals.", "As with fish, the eggs and the sperm from a population, for example from all slaughter cows, are collected and pooled.", "The pooled eggs are then in vitro fertilized with the pooled sperm.", "The resulting embryos are then returned to surrogate mothers for development.", "As above, this process is repeated recursively until a large diverse population is generated that can be screened for desirable traits.", "A technically feasible approach would be similar to that used for plants.", "In this case, sperm from the males of the starting population is collected and pooled, and then this pooled sample is used to artificially inseminate multiple females from each of the starting populations.", "Only one (or a few) sperm would succeed in each animal, but these should be different for each fertilization.", "The process is reiterated by harvesting the sperm from all of the male progeny, pooling it, and using it to fertilize all of the female progeny.", "The process is carried out recursively for several generations to generate the organism library, which can then be screened.", "4.14 Predictive Tool in Looking for Drugs Recursive sequence recombination can be used to simulate natural evolution of pathogenic microorganisms in response to exposure to a drug under test.", "Using recursive sequence recombination, evolution proceeds at a faster rate than in natural evolution.", "One measure of the rate of evolution is the number of cycles of recombination and screening required until the microorganism acquires a defined level of resistance to the drug.", "The information from this analysis is of value in comparing the relative merits of different drugs and in particular, in predicting their long term efficacy on repeated administration.", "The pathogenic microorganisms used in this analysis include the bacteria that are a common source of human infections, such as chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streplocci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lymes disease bacteria.", "Evolution is effected by transforming an isolate of bacteria that is sensitive to a drug under test with a library of DNA fragments.", "The fragments can be a mutated version of the genome of the bacteria being evolved.", "If the target of the drug is a known protein or nucleic acid, a focused library containing variants of the corresponding gene can be used.", "Alternatively, the library can come from other kinds of bacteria, especially bacteria typically found inhabiting human tissues, thereby simulating the source material available for recombination in vivo.", "The library can also come from bacteria known to be resistant to the drug.", "After transformation and propagation of bacteria for an appropriate period to allow for recombination to occur and recombinant genes to be expressed, the bacteria are screened by exposing them to the drug under test and then collecting survivors.", "Surviving bacteria are subject to further rounds of recombination.", "The subsequent round can be effected by a split and pool approach in which DNA from one subset of surviving bacteria is introduced into a second subset of bacteria.", "Alternatively, a fresh library of DNA fragments can be introduced into surviving bacteria.", "Subsequent round(s) of selection can be performed at increasing concentrations of drug, thereby increasing the stringency of selection.", "4.14.1 Biosynthesis Metabolic engineering can be used to alter organisms to optimize the production of practically any metabolic intermediate, including antibiotics, vitamins, amino acids such as phenylalanine and aromatic amino acids, ethanol, butanol, polymers such as xanthan gum and bacterial cellulose, peptides, and lipids.", "When such compounds are already produced by a host, the recursive sequence recombination techniques described above can be used to optimize production of the desired metabolic intermediate, including such features as increasing enzyme substrate specificity and turnover number, altering metabolic fluxes to reduce the concentrations of toxic substrates or intermediates, increasing resistance of the host to such toxic compounds, eliminating, reducing or altering the need for inducers of gene expression/activity, increasing the production of enzymes necessary for metabolism, etc.", "Enzymes can also be evolved for improved activity in solvents other than water.", "This is useful because intermediates in chemical syntheses are often protected by blocking groups which dramatically affect the solubility of the compound in aqueous solvents.", "Many compounds can be produced by a combination of pure chemical and enzymically catalyzed reactions.", "Performing enzymic reactions on almost insoluble substrates is clearly very inefficient, so the availability of enzymes that are active in other solvents will be of great use.", "One example of such a scheme is the evolution of a para-nitrobenzyl esterase to remove protecting groups from an intermediate in loracarbef synthesis (Moore, J. C. and Arnold, F. H. Nature Biotechnology 14: 458467 (1996)).", "In this case alternating rounds of error-prone PCR and colony screening for production of a fluorescent reporter from a substrate analogue were used to generate a mutant esterase that was 16-fold more active than the parent molecule in 30%.", "dimethylformamide.", "No individual mutation was found to contribute more than a 2-fold increase in activity, but it was the combination of a number of mutations which led to the overall increase.", "Structural analysis of the mutant protein showed that the amino acid changes were distributed throughout the length of the protein in a manner that could not have been rationally predicted.", "Sequential rounds of error-prone PCR have the problem that after each round all but one mutant is discarded, with a concomitant loss of information contained in all the other beneficial mutations.", "Recursive sequence recombination avoids this problem, and would thus be ideally suited to evolving enzymes for catalysis in other solvents, as well as in conditions where salt concentrations or pH were different from the original enzyme optimas.", "In addition, the yield of almost any metabolic pathway can be increased, whether consisting entirely of genes endogenous to the host organisms or all or partly heterologous genes.", "Optimization of the expression levels of the enzymes in a pathway is more complex than simply maximizing expression.", "In some cases regulation, rather than constitutive expression of an enzyme may be advantageous for cell growth and therefore for product yield, as seen for production of phenylalanine (Backman et al.", "Ann.", "NY Acad.", "Sci.", "589: 16-24 (1990)) and 2-keto-L-gluconic acid (Anderson et al.", "U.S. Pat.", "No.", "5,032,514).", "In addition, it is often advantageous for industrial purposes to express proteins in organisms other than their original hosts.", "New host strains may be preferable for a variety of reasons, including ease of cloning and transformation, pathogenicity, ability to survive in particular environments and a knowledge of the physiology and genetics of the organisms.", "However, proteins expressed in heterologous organisms often show markedly reduced activity for a variety of reasons including inability to fold properly in the new host (Sarthy et al.", "Appl.", "Environ.", "Micro.", "53: 1996-2000 (1987)).", "Such difficulties can indeed be overcome by the recursive sequence recombination strategies of the instant invention.", "4.14.2 Antibiotics The range of natural small molecule antibiotics includes but is not limited to peptides, peptidolactones, thiopeptides, beta-lactams, glycopeptides, lantibiotics, microcins, polyketide-derived antibiotics (anthracyclins, tetracyclins, macrolides, avennectins, polyethers and ansamycins), chloramphenicol, aminoglycosides, aminocyclitols, polyoxins, agrocins and isoprenoids.", "There are at least three ways in which recursive sequence recombination techniques of the instant invention can be used to facilitate novel drug synthesis, or to improve biosynthesis of existing antibiotics.", "First, antibiotic synthesis enzymes can be “evolved” together with transport systems that allow entry of compounds used as antibiotic precursors to improve uptake and incorporation of function-altering artificial side chain precursors.", "For example, penicillin V is produced by feeding Penicillium the artificial side chain precursor phenoxyacetic acid, and LY146032 by feeding Streptomyces roseosporus decanoic acid (Hopwood, Phil.", "Trans.", "R. Soc.", "Lond.", "B 324: 549-562 (1989)).", "Poor precursor uptake and poor incorporation by the synthesizing enzyme often lead to inefficient formation of the desired product.", "Recursive sequence recombination of these two systems can increase the yield of desired product.", "Furthermore, a combinatorial approach can be taken in which an enzyme is stochastic &/or non-stochastic mutagenized for novel catalytic activity/substrate recognition (perhaps by including randomizing oligonucleotides in key positions such as the active site).", "A number of different substrates (for example, analogues of side chains that are normally incorporated into the antibiotic) can then be tested in combination with all the different enzymes and tested for biological activity.", "In this embodiment, plates are made containing different potential antibiotic precursors (such as the side chain analogues).", "The microorganisms containing the stochastic &/or non-stochastic mutagenized library (the library strain) are replicated onto those plates, together with a competing, antibiotic sensitive, microorganism (the indicator strain).", "Library cells that are able to incorporate the new side chain to produce an effective antibiotic will thus be able to compete with the indicator strain, and will be selected for.", "Second, the expression of heterologous genes transferred from one antibiotic synthesizing organism to another can be optimized.", "The newly introduced enzyme(s) act on secondary metabolites in the host cell, transforming them into new compounds with novel properties.", "Using traditional methods, introduction of foreign genes into antibiotic synthesizing hosts has already resulted in the production of novel hybrid antibiotics.", "Examples include mederrhodin, dihydrogranatirhodin, 6-deoxyerythromycin A, isovalerylspiramycin and other hybrid macrolides (Cameron et. al.", "Appl.", "Biochem.", "Biotechnol.", "38: 105-140 (1993)).", "The recursive sequence recombination techniques of the instant invention can be used to optimize expression of the foreign genes, to stabilize the enzyme in the new host cell, and to increase the activity of the introduced enzyme against its new substrates in the new host cell.", "In some embodiments of the invention, the host genome may also be so optimized.", "Third, the substrate specificity of an enzyme involved in secondary metabolism can be altered so that it will act on and modify a new compound or so that its so activity is changed and it acts at a different subset of positions of its normal substrate.", "Recursive sequence recombination can be used to alter the substrate specificities of enzymes.", "Furthermore, in addition to recursive sequence recombination of individual enzymes being a strategy to generate novel antibiotics, recursive sequence recombination of entire pathways, by altering enzyme ratios, will alter metabolite fluxes and may result, not only in increased antibiotic synthesis, but also in the synthesis of different antibiotics.", "This can be deduced from the observation that expression of different genes from the same cluster in a foreign host leads to different products being formed (see p. 80 in Hutchinson et.", "al., (1991) Ann NY Acad Sci, 646: 78-93).", "Recursive sequence recombination of the introduced gene clusters may result in a variety of expression levels of different proteins within the cluster (because it produces different combinations of, in this case regulatory, mutations).", "This in turn may lead to a variety of different end products.", "Thus, “evolution” of an existing antibiotic synthesizing pathway could be used to generate novel antibiotics either by modifying the rates or substrate specificities of enzymes in that pathway.", "Additionally, antibiotics can also be produced in vitro by the action of a purified enzyme on a precursor.", "For example isopenicillin N synthase catalyses the cyclization of many analogues of its normal substrate (d-(L-a-aminoadipyl)L-cysteinyl-D-valine) (Hutchinson, Med.", "Res.", "Rev.", "8: 557-567 (1988)).", "Many of these products are active as antibiotics.", "A wide variety of substrate analogues can be tested for incorporation by secondary metabolite synthesizing enzymes without concern for the initial efficiency of the reaction.", "Recursive sequence recombination can be used subsequently to increase the rate of reaction with a promising new substrate.", "Thus, organisms already producing a desired antibiotic can be evolved with the recursive sequence recombination techniques described above to maximize production of that antibiotic.", "Additionally, new antibiotics can be evolved by manipulation of genetic material from the host by the recursive sequence recombination techniques described above.", "Genes for antibiotic production can be transferred to a preferred host after cycles of recursive sequence recombination or can be evolved in the preferred host as described above.", "Antibiotic genes are generally clustered and are often positively regulated, making them especially attractive candidates for the recursive sequence recombination techniques of the instant invention.", "Additionally, some genes of related pathways show cross-hybridization, making them preferred candidates for the generation of new pathways for new antibiotics by the recursive sequence recombination techniques of the invention.", "Furthermore, increases in secondary metabolite production including enhancement of substrate fluxes (by increasing the rate of a rate limiting enzyme, deregulation of the pathway by suppression of negative control elements or over expression of activators and the relief of feedback controls by mutation of the regulated enzyme to a feedback-insensitive deregulated protein) can be achieved by recursive sequence recombination without exhaustive analysis of the regulatory mechanisms governing expression of the relevant gene clusters.", "The host chosen for expression of evolved genes is preferably resistant to the antibiotic produced, although in some instances production methods can be designed so as to sacrifice host cells when the amount of antibiotic produced is commercially significant yet lethal to the host.", "Similarly, bioreactors can be designed so that the growth medium is continually replenished, thereby “drawing off” antibiotic produced and sparing the lives of the producing cells.", "Preferably, the mechanism of resistance is not the degradation of the antibiotic produced.", "Numerous screening methods for increased antibiotic expression are known in the art, as discussed above, including screening for organisms that are more resistant to the antibiotic that they produce.", "This may result from linkage between expression of the antibiotic synthesis and antibiotic resistance genes (Chater, Bio/Technology 8: 115-121 (1990)).", "Another screening method is to fuse a reporter gene (e.g.", "xylE from the Pseudomonas TOL plasmid) to the antibiotic production genes.", "Antibiotic synthesis gene expression can then be measured by looking for expression of the reporter (e.g.", "xylE encodes a catechol dioxygenase which produces yellow muconic semialdehyde when colonies are sprayed with catechol (Zukowski et al.", "Proc.", "Natl.", "Acad.", "Sci.", "U.S.A. 80: 1101-1105 (1983)).", "The wide variety of cloned antibiotic genes provides a wealth of starting materials for the recursive sequence recombination techniques of the instant invention.", "For example, genes have been cloned from Streptomyces cattleya which direct cephamycin C synthesis in the non-antibiotic producer Streptomyces lividans (Chen et al.", "Bio/Technology 6: 1222-1224 (1988)).", "Clustered genes for penicillin biosynthesis (-(L—aminoadipyl)-L-cysteinyl-D-valine synthetase; isopenicillin N synthetase and acyl coenzyme A:6-aminopenicillanic acid acyltransferase) have been cloned from Penicillium chrysogenum.", "Transfer of these genes into Neurospora crassa and Aspergillus niger result in the synthesis of active penicillin V (Smith et al.", "Bio/Technology 8: 39-41 (1990)).", "For a review of cloned genes involved in Cephalosporin C, Penicillins G and V and Cephamycin C biosynthesis, see Piepersberg, Crit.", "Rev.", "Biotechnol.", "14: 251-285 (1994).", "For a review of cloned clusters of antibiotic-producing genes, see Chater Bio/Technology 8: 115-121 (1990).", "Other examples of antibiotic synthesis genes transferred to industrial producing strains, or over expression of genes, include tylosin, cephamycin C, cephalosporin C, LL-E33288 complex (an antitumor and antibacterial agent), doxorubicin, spiramycin and other macrolide antibiotics, reviewed in Cameron et al.", "Appl.", "Biochem.", "Biotechnol.", "38: 105-140 (1993).", "4.14.3 Biosythsesis to Replace Chemical Synthesis of Antibiotics Some antibiotics are currently made by chemical modifications of biologically produced starting compounds.", "Complete biosynthesis of the desired molecules may currently be impractical because of the lack of an enzyme with the required enzymatic activity and substrate specificity.", "For example, 7-aminodeacetooxycephalosporanic acid (7-ADCA) is a precursor for semi-synthetically produced cephalosporins.", "7-ADCA is made by a chemical ring expansion from penicillin V followed by enzymatic deacylation of the phenoxyacetal group.", "Cephalosporins could in principle be produced biologically from their corresponding penicillins (e.g., cephalosporin V or G from penicillin V or G) using penicillin N expandase, but other penicillins (such as penicillin V or G) are not used as substrates by known expandases.", "The recursive sequence recombination techniques of the invention can be used to alter the enzyme so that it will use penicillin V as a substrate.", "Similarly, penicillin transacylase could be so modified to accept cephalosporins or cephamycins as substrates.", "In yet another example, penicillin amidase expressed in E. coli is a key enzyme in the production of penicillin G derivatives.", "The enzyme is generated from a precursor peptide and tends to accumulate as insoluble aggregates in the periplasm unless non-metabolizable sugars are present in the medium (Scherrer et al., Appl.", "Microbiol.", "Biotechnol.", "42: 85-91 (1994)).", "Evolution of this enzyme through the methods of the instant invention could be used to generate an enzyme that folds better, leading to a higher level of active enzyme expression.", "In yet another example, Penicillin G acylase covalently linked to agarose is used in the synthesis of penicillin G derivatives.", "The enzyme can be stabilized for increased activity, longevity and/or thermal stability by chemical modification (Fernandez-Lafuente et. al.", "Enzyme Microb.", "Technol.", "14: 489495 (1992).", "Increased thermal stability is an especially attractive application of the recursive sequence recombination techniques of the instant invention, which can obviate the need for the chemical modification of such enzymes.", "Selection for thermostability can be performed in vivo in E. coli or in thermophiles at higher temperatures.", "In general, thermostability is a good first step in enhancing general stabilization of enzymes.", "Random mutagenesis and selection can also be used to adapt enzymes to function in non-aqueous solvents (Arnold Curr Opin Biotechnol, 4: 450455 (1993); Chen et. al.", "Proc.", "Natl.", "Acad.", "Sci.", "U.S.A., 90: 5618-5622 (1993)).", "Recursive sequence recombination represents a more powerful (since recombinogenic) method of generating mutant enzymes that are stable and active in non-aqueous environments.", "Additional screening can be done on the basis of enzyme stability in solvents.", "4.14.4 Polyketides Polyketides include antibiotics such as tetracycline and erythromycin, anti-cancer agents such as daunomycin, immunosuppressants such as FK506 and rapamycin and veterinary products such as monesin and avermectin.", "Polyketide synthases (PKS's) are multifunctional enzymes that control the chain length, choice of chain-building units and reductive cycle that generates the huge variation in naturally occurring polyketides.", "Polyketides are built up by sequential transfers of “extender units” (fatty acyl CoA groups) onto the appropriate starter unit (examples are acetate, coumarate, propionate and malonamide).", "The PKS's determine the number of condensation reactions and the type of extender groups added and may also fold and cyclize the polyketide precursor.", "PKS's reduce specific -keto groups and may dehydrate the resultant-hydroxyls to form double bonds.", "Modifications of the nature or number of building blocks used, positions at which -keto groups are reduced, the extent of reduction and different positions of possible cyclizations, result in formation of different final products.", "Polyketide research is currently focused on modification and inhibitor studies, site directed mutagenesis and 3-D structure elucidation to lay the groundwork for rational changes in enzymes that will lead to new polyketide products.", "Recently, McDaniel et al.", "(Science 262: 1546-1550 (1995)) have developed a Streptomyces host-vector system for efficient construction and expression of recombinant PKSs.", "Hutchinson (Bio/Technolo 12: 375-308 (1994)) reviewed targeted mutation of specific biosynthetic genes and suggested that microbial isolates can be screened by DNA hybridization for genes associated with known pharmacologically active agents so as to provide new metabolites or increased yields of metabolites already being produced.", "In particular, that review focuses on polyketide synthase and pathways to aminoglycoside and oligopeptide antibiotics.", "The recursive sequence recombination techniques of the instant invention can be used to generate modified enzymes that produce novel polyketides without such detailed analytical effort.", "The availability of the PKS genes on plasmids and the existence of E. coli-Streptomyces shuttle vectors (Wehmeier Gene 165: 149-150 (1995)) makes the process of recursive sequence recombination especially attractive by the techniques described above.", "Techniques for selection of antibiotic producing organisms can be used as described above; additionally, in some embodiments screening for a particular desired polyketide activity or compound is preferable.", "4.14.5 Isoprenoids Isoprenoids result from cyclization of farnesyl pyrophosphate by sesquiterpene synthases.", "The diversity of isoprenoids is generated not by the backbone, but by control of cyclization.", "Cloned examples of isoprenoid synthesis genes include trichodiene synthase from Fusarium sprorotrichioides, pentalene synthase from Streptomyces, aristolochene synthase from Penicillium roquefortii, and epi-aristolochene synthase from N. tabacum (Cane, D. E. (1995).", "Isoprenoid antibiotics, pages 633-655, in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann).", "Recursive sequence recombination of sesquiterpene synthases will be of use both in allowing expression of these enzymes in heterologous hosts (such as plants and industrial microbial strains) and in alteration of enzymes to change the cyclized product made.", "A large number of isoprenoids are active as antiviral, antibacterial, antifungal, herbicidal, insecticidal or cytostatic agents.", "Antibacterial and antifungal isoprenoids could thus be preferably screened for using the indicator cell type system described above, with the producing cell competing with bacteria or fungi for nutrients.", "Antiviral isoprenoids could be screened for preferably by their ability to confer resistance to viral attack on the producing cell.", "4.14.6 Bioactive Peptide Derivatlves Examples of bioactive non-ribosomally synthesized peptides include the antibiotics cyclosporin, pepstatin, actinomycin, gramicidin, depsipeptides, vancomycin, etc.", "These peptide derivatives are synthesized by complex enzymes rather than ribosomes.", "Again, increasing the yield of such non-ribosomally synthesized peptide antibiotics has thus far been done by genetic identification of biosynthetic “bottlenecks” and over expression of specific enzymes (See, for example, p. 133-135 in “Genetics and Biochemistry of Antibiotic Production” edited by Vining, L. C. & Stuttard, C., published by Butterworth-Heinemann).", "Recursive sequence recombination of the enzyme clusters can be used to improve the yields of existing bioactive non-ribosomally made peptides in both natural and heterologous hosts.", "Like polyketide synthases, peptide synthases are modular and multifunctional enzymes catalyzing condensation reactions between activated building blocks (in this case amino acids) followed by modifications of those building blocks (see Kleinkauf, H. and von Dohren, H. Eur.", "J. Biochem.", "236: 335-351 (1996)).", "Thus, as for polyketide synthases, recursive sequence recombination can also be used to alter peptide synthases: modifying the specificity of the amino acid recognized by each binding site on the enzyme and altering the activity or substrate specificities of sites that modify these amino acids to produce novel compounds with antibiotic activity.", "other peptide antibiotics are made ribosomally and then post-translationally modified.", "Examples of this type of antibiotics are lantibiotics (produced by gram positive bacteria such Staphylococcus, Streptomyces, Bacillus, and Actinoplanes) and microcins (produced by Enterobacteriaceae).", "Modifications of the original peptide include (in lantibiotics) dehydration of serine and threonine, condensation of dehydroamino acids with cysteine, or simple N- and C-terminal blocking (microcins).", "For ribosomally made antibiotics both the peptide-encoding sequence and the modifying enzymes may have their expression levels modified by recursive sequence recombination.", "Again, this will lead to both increased levels of antibiotic synthesis, and by modulation of the levels of the modifying enzymes (and the sequence of the ribosomally synthesized peptide itself) novel antibiotics.", "Screening can be done as for other antibiotics as described above, including competition with a sensitive (or even initially insensitive) microbial species.", "Use of competing bacteria that have resistances to the antibiotic being produced will select strongly either for greatly elevated levels of that antibiotic (so that it swamps out the resistance mechanism) or for novel derivatives of that antibiotic that are not neutralized by the resistance mechanism.", "4.14.7 Polymers Several examples of metabolic engineering to produce biopolymers have been reported, including the production of the biodegradable plastic polyhydroxybutarate (PHB), and the polysaccharide xanthan gum.", "For a review, see Cameron et al.", "Applied Biochem.", "Biotech.", "38: 105-140 (1993).", "Genes for these pathways have been cloned, making them excellent candidates for the recursive sequence recombination techniques described above.", "Expression of such evolved genes in a commercially viable host such as E. coli is an especially attractive application of this technology.", "Examples of starting materials for recursive sequence recombination include but are not limited to genes from bacteria such as Alcaligenes, Zoogloea, Ihizobium, Bacillus, and Azobacter, which produce polyhydroxyalkanoates (PHAs) such as polyhyroxybutyrate (PHB) intracellularly as energy reserve materials in response to stress.", "Genes from Alcaligenes eutrophus that encode enzymes catalyzing the conversion of acetoacetyl CoA to PHB have been transferred both to E. coli and to the plant Arabidopsis thaliana (Poirier et al.", "Science 256: 520-523 (1992)).", "Two of these genes (phbB and phbC, encoding acetoacetyl-CoA reductase and PHB synthase respectively) allow production of PHE in Arabidopsis.", "The plants producing the plastic are stunted, probably because of adverse interactions between the new metabolic pathway and the plants' original metabolism (i.e., depletion of substrate from the mevalonate pathway).", "Improved production of PHB in plants has been attempted by localization of the pathway enzymes to organelles such as plastids.", "Other strategies such as regulation of tissue specificity, expression timing and cellular localization have been suggested to solve the deleterious effects of PHB expression in plants.", "The recursive sequence recombination techniques of the invention can be used to modify such heterologous genes as well as specific cloned interacting pathways (e.g., mevalonate), and to optimize PHB synthesis in industrial microbial strains, for example to remove the requirement for stresses (such as nitrogen limitation) in growth conditions.", "Additionally, other microbial polyesters are made by different bacteria in which additional monomers are incorporated into the polymer (Peoples et al.", "in Novel Biodegradable Microbial Polymers, E A Dawes, ed., pp 191-202 (1990)).", "Recursive sequence recombination of these genes or pathways singly or in combination into a heterologous host will allow the production of a variety of polymers with differing properties, including variation of the monomer subunit ratios in the polymer.", "Another polymer whose synthesis may be manipulated by recursive sequence recombination is cellulose.", "The genes for cellulose biosynthesis have been cloned from Agrobacterium tumefaciens (Matthysse, A. G. et. al.", "J. Bacteriol.", "177: 1069-1075 (1995)).", "Recursive sequence recombination of this biosynthetic pathway could be used either to increase synthesis of cellulose, or to produce mutants in which alternative sugars are incorporated into the polymer.", "4.14.8 Carotenoids Carotenoids are a family of over 600 terpenoids produced in the general isoprenoid biosynthetic pathway by bacteria, fungi and plants (for a review, see Armstrong, J. Bact.", "176: 4795-4802 (1994)).", "These pigments protect organisms against photooxidative damage as well as functioning as anti-tumor agents, free radical-scavenging anti-oxidants, and enhancers of the immune response.", "Additionally, they are used commercially in pigmentation of cultured fish and shellfish.", "Examples of carotenoids include but are not limited to myxobacton, spheroidene, spheroidenone, lutein, astaxanthin, violaxanthin, 4-ketorulene, myxoxanthrophyll, echinenone, lycopene, zeaxanthin and its mono- and di-glucosides, alpha-, beta-, gamma- and sigma-carotene, beta-cryptoxanthin monoglucoside and neoxanthin.", "Carotenoid synthesis is catalyzed by relatively small numbers of clustered genes: 11 different genes within 12 kb of DNA from Myxococcus xanthus (Botella et al.", "Eur.", "J. Biochem.", "233: 238-248 (1995)) and 8 genes within 9 kb of DNA from Rhodobacter sphaeroides (Lang et. al.", "J. Bact.", "177: 2064-2073 (1995)).", "In some microorganisms, such as Thermus thermophilus, these genes are plasmid-borne (Tabata et al.", "FEBS Letts 341: 251-255 (1994)).", "These features make carotenoid synthetic pathways especially attractive candidates for recursive sequence recombination.", "Transfer of some carotenoid genes into heterologous organisms results in expression.", "For example, genes from Erwina uredovora and Haematococcus pluvialis will function together in E. coli (Kajiwara et al.", "Plant Mol.", "Biol.", "29: 343-352 (1995)).", "E. herbicola genes will function in R. sphaeroides (Hunter et al.", "J. Bact.", "176: 3692-3697 (1994)).", "However, some other genes do not; for example, R. capsulatus genes do not direct carotenoid synthesis in E. coli (Marrs, J. Bact.", "146: 1003-1012 (1981)).", "In an embodiment of the invention, the recursive sequence recombination techniques of the invention can be used to generate variants in the regulatory and/or structural elements of genes in the carotenoid synthesis pathway, allowing increased expression in heterologous hosts.", "Indeed, traditional techniques have been used to increase carotenoid production by increasing expression of a rate limiting enzyme in Thermus thermophilus (Hoshino et al.", "Appl.", "Environ.", "Micro.", "59: 3150-3153 (1993)).", "Furthermore, mutation of regulatory genes can cause constitutive expression of carotenoid synthesis in actinomycetes, where carotenoid photoinducibility is otherwise unstable and lost at a relatively high frequency in some species (Kato et al.", "Mol.", "Gen. Genet.", "247: 387-390 (1995)).", "These are both mutations that can be obtained by recursive sequence recombination.", "The recursive sequence recombination techniques of the invention as described above can be used to evolve one or more carotenoid synthesis genes in a desired host without the need for analysis of regulatory mechanisms.", "Since carotenoids are colored, a calorimetric assay in microtiter plates, or even on growth media plates, can be used for screening for increased production.", "In addition to increasing expression of carotenoids, carotenogenic biosynthetic pathways have the potential to produce a wide diversity of carotenoids, as the enzymes involved appear to be specific for the type of reaction they will catalyze, but not for the substrate that they modify.", "For example, two enzymes from the marine bacterium Agrobacterium aurantiacum (CrtW and CrtZ) synthesize six different ketocarotenoids from beta-carotene (Misawa et al.", "J. Bact.", "177: 6576-6584 (1995)).", "This relaxed substrate specificity means that a diversity of substrates can be transformed into an even greater diversity of products.", "Introduction of foreign carotenoid genes into a cell can lead to novel and functional carotenoid-protein complexes, for example in photosynthetic complexes (Hunter et al.", "J. Bact.", "176: 3692-3697 (1994)).", "Thus, the deliberate recombination of enzymes through the recursive sequence recombination techniques of the invention is likely to generate novel compounds.", "Screening for such compounds can be accomplished, for example, by the cell competition/survival techniques discussed above and by a calorimetric assay for pigmented compounds.", "Another method of identifying new compounds is to use standard analytical techniques such as mass spectroscopy, nuclear magnetic resonance, high performance liquid chromatography, etc.", "Recombinant microorganisms can be pooled and extracts or media supernatants assayed from these pools.", "Any positive pool can then be subdivided and the procedure repeated until the single positive is identified (“sib-selection”).", "4.14.9 Indigo Biosyntesis Many dyes, i.e.", "agents for imparting color, are specialty chemicals with significant markets.", "As an example, indigo is currently produced chemically.", "However, nine genes have been combined in E. coli to allow the synthesis of indigo from glucose via the tryptophan/indole pathway (Murdock et al.", "Bio/Technology 11: 381-386 (1993)).", "A number of manipulations were performed to optimize indigo synthesis: cloning of nine genes, modification of the fermentation medium and directed changes in two operons to increase reaction rates and catalytic activities of several enzymes.", "Nevertheless, bacterially produced indigo is not currently an economic proposition.", "The recursive sequence recombination techniques of the instant invention could be used to optimize indigo synthesizing enzyme expression levels and catalytic activities, leading to increased indigo production, thereby making the process commercially viable and reducing the environmental impact of indigo manufacture.", "Screening for increased indigo production can be done by calorimetric assays of cultures in microtiter plates.", "4.14.10 Amino Acids Amino acids of particular commercial importance include but are not limited to phenylalanine, monosodium glutamate, glycine, lysine, threonine, tryptophan and methionine.", "Backman et al.", "(Ann.", "NY Acad.", "Sci.", "589: 16-24 (1990)) disclosed the enhanced production of phenylalanine in E. coli via a systematic and downstream strategy covering organism selection, optimization of biosynthetic capacity, and development of fermentation and recovery processes.", "As described in Simpson et al.", "(Biochem Soc Trans, 23: 381-387 (1995)), current work in the field of amino acid production is focused on understanding the regulation of these pathways in great molecular detail.", "The recursive sequence recombination techniques of the instant invention would obviate the need for this analysis to obtain bacterial strains with higher secreted amino acid yields.", "Amino acid production could be optimized for expression using recursive sequence recombination of the amino acid synthesis and secretion genes as well as enzymes at the regulatory phosphoenolpyruvate branchpoint, from such organisms as Serratia marcescens, Bacillus, and the Corynebacterium-Brevibacterium group.", "In some embodiments of the invention, screening for enhanced production is preferably done in microtiter wells, using chemical tests well known in the art that are specific for the desired amino acid.", "Screening/selection for amino acid synthesis can also be done by using auxotrophic reporter cells that are themselves unable to synthesize the amino acid in question.", "If these reporter cells also produce a compound that stimulates the growth of the amino acid producer (this could be a growth factor, or even a different amino acid), then library cells that produce more amino acid will in turn receive more growth stimulant and will therefore grow more rapidly.", "4.14.11 Vitamin C Synthesis L-Ascorbic acid (vitamin C) is a commercially important vitamin with a world production of over 35,000 tons in 1984.Most vitamin C is currently manufactured chemically by the Reichstein process, although recently bacteria have been engineered that are able to transform glucose to 2,5-keto-gluconic acid, and that product to 2-keto-L-idonic acid, the precursor to L-ascorbic acid (Boudrant, Enzyme Microb.", "Technol.", "12: 322-329 (1990)).", "The efficiencies of these enzymatic steps in bacteria are currently low.", "Using the recursive sequence recombination techniques of the instant invention, the genes can be genetically engineered to create one or more operons followed by expression optimization of such a hybrid L-ascorbic acid synthetic pathway to result in commercially viable microbial vitamin C biosynthesis.", "In some embodiments, screening for enhanced L-ascorbic acid production is preferably done in microtiter plates, using assays well known in the art.", "4.15 Test for Resistance to Drugs 4.15.1 Find Drugs that Induce Resistance Slowly A similar strategy can be used to simulate viral acquisition of drug resistance.", "The object is to identify drugs for which resistance can be acquired only slowly, if at all.", "The viruses to be evolved are those that cause infections in humans for which at least modestly effective drugs are available.", "Substrates for recombination can come from induced mutants, natural variants of the same viral strain or different viruses.", "If the target of the drug is known (e.g., nucleotide analogs which inhibit the reverse transcriptase gene of HIV), focused libraries containing variants of the target gene can be produced.", "Recombination of a viral genome with a library of fragments is usually performed in vitro.", "However, in situations in which the library of fragments constitutes variants of viral genomes or fragments that can be encompassed in such genomes, recombination can also be performed in vivo, e.g., by transfecting cells with multiple substrate copies (see Section V).", "For screening, recombinant viral genomes are introduced into host cells susceptible to infection by the virus and the cells are exposed to a drug effective against the virus (initially at low concentration).", "The cells can be spun to remove any noninfected virus.", "After a period of infection, progeny viruses can be collected from the culture medium, the progeny viruses being enriched for viruses that have acquired at least partial resistance to the drug.", "Alternatively, virally infected cells can be plated in a soft agar lawn and resistant viruses isolated from plaques.", "Plaque size provides some indication of the degree of viral resistance.", "Progeny viruses surviving screening are subject to additional rounds of recombination and screening at increased stringency until a predetermined level of drug resistance has been acquired.", "The predetermined level of drug resistance may reflect the maximum dosage of a drug practical to administer to a patient without intolerable side effects.", "The analysis is particularly valuable for investigating acquisition of resistance to various combination of drugs, such as the growing list of approved anti-HIV drugs (e.g., AZT, ddI, ddC, d4T, TIBO 82150, nevaripine, 3TC, crixivan and ritonavir).", "4.15.2 Method to Evolve Yeast Strains Fragments are cloned into a YAC vector, and the resulting YAC library is transformed into competent yeast cells.", "Transformants containing a YAC are identified by selecting for a positive selection marker present on the YAC.", "The cells are allowed to recover and are then pooled.", "Thereafter, the cells are induced to sporulate by transferring the cells from rich medium, to nitrogen and carbon limiting medium.", "In the course of sporulation, cells undergo meiosis.", "Spores are then induced to mate by return to rich media.", "Optionally, asci are lysed to liberate spores, so that the spores can mate with other spores originating from other asci.", "Mating results in recombination between YACs bearing different inserts, and between YACs and natural yeast chromosomes.", "The latter can be promoted by irradiating spores with ultra violet light.", "Recombination can give rise to new phenotypes either as a result of genes expressed by fragments on the YACs or as a result of recombination with host genes, or both.", "After induction of recombination between YACs and natural yeast chromosomes, YACs are often eliminated by selecting against a negative selection marker on the YACs.", "For example, YACs containing the marker URA3 can be selected against by propagation on media containing 5-fluro orotic acid.", "Any exogenous or altered genetic material that remains is contained within natural yeast chromosomes.", "Optionally, further rounds of recombination between natural yeast chromosomes can be performed after elimination of YACs.", "Optionally, the same or different library of YACs can be transformed into the cells, and the above steps repeated.", "By recursively repeating this process, the diversity of the population is increased prior to screening.", "After elimination of YACs, yeast are then screened or selected for a desired property.", "The property can be a new property conferred by transferred fragments, such as production of an antibiotic.", "The property can also be an improved property of the yeast such as improved capacity to express or secrete an exogenous protein, improved recombinogenicity, improved stability to temperature or solvents, or other property required of commercial or research strains of yeast.", "Yeast strains surviving selection/screening are then subject to a further round of recombination.", "Recombination can be exclusively between the chromosomes of yeast surviving selection/screening.", "Alternatively, a library of fragments can be introduced into the yeast cells and recombined with endogenous yeast chromosomes as before.", "This library of fragments can be the same or different from the library used in the previous round of transformation.", "For example, the YACs could contain a library of genomic DNA isolated from a pool of the improved strains obtained in the earlier steps.", "YACs are eliminated as before, followed by additional rounds of recombination and/or transformation with further YAC libraries.", "Recombination is followed by another round of selection/screening, as above.", "Further rounds of recombination/screening can be performed as needed until a yeast strain has evolved to acquire the desired property.", "An exemplary scheme for evolving yeast by introduction of a YAC library is yeast containing an endogenous diploid genome and a YAC library of fragments representing variants of a sequence.", "The library is transformed into the cells to yield 100-1000 colonies per μg DNA.", "Most transformed yeast cells now harbor a single YAC as well as endogenous chromosomes.", "Meiosis is induced by growth on nitrogen and carbon limiting medium.", "In the course of meiosis the YACs recombine with other chromosomes in the same cell.", "Haploid spores resulting from meiosis mate and regenerated diploid forms.", "The diploid forms now harbor recombinant chromosomes, parts of which come from endogenous chromosomes and parts from YACs.", "Optionally, the YACs can now be cured from the cells by selecting against a negative selection marker present on the YACS.", "Irrespective whether YACS are selected against, cells are then screened or selected for a desired property.", "Cells surviving selection/screening are transformed with another YAC library to start another stochastic &/or non-stochastic mutagenesis cycle.", "4.153 Evolve YACs for Transfer into Recipient Strain These methods are based in part on the fact that multiple YACs can be harbored in the same yeast cell, and YAC-YAC recombination is known to occur (Green & Olson, Science 250, 94-98 1990)).", "Inter-YAC recombination provides a format for which families of homologous genes harbored on fragments of >20 kb can be stochastic &/or non-stochastic mutagenized in vivo.", "The starting population of DNA fragments show sequence similarity with each other but differ as a result of for example, induced, allelic or species diversity.", "Often DNA fragments are known or suspected to encode multiple genes that function in a common pathway.", "The fragments are cloned into a Yac and transformed into yeast, typically with positive selection for transformants.", "The transformants are induced to sporulate, as a result of which chromosomes undergo meiosis.", "The cells are then mated.", "Most of the resulting diploid cells now carry two YACs each having a different insert.", "These are again induced to sporulate and mated.", "The resulting cells harbor YACs of recombined sequence.", "The cells can then be screened or selected for a desired property.", "Typically, such selection occurs in the yeast strain used for stochastic &/or non-stochastic mutagenesis.", "However, if fragments being stochastic &/or non-stochastic mutagenized are not expressed in yeast, YACs can be isolated and transferred to an appropriate cell type in which they are expressed for screening.", "Examples of such properties include the synthesis or degradation of a desired compound, increased secretion of a desired gene product, or other detectable phenotype.", "Preferably, the YAC library is transformed into haploid a and haploid a cells.", "These cells are then induced to mate with each other, i.e., they are pooled and induced to mate by growth on rich medium.", "The diploid cells, each carrying two YACs, are then transferred to sporulation medium.", "During sporulation, the cells undergo meiosis, and homologous chromosomes recombine.", "In this case, the genes harbored in the YACs will recombine, diversifying their sequences.", "The resulting haploid acospores are then liberated from the asci by enzymatic degradation of the asci wall or other available means and the pooled liberated haploid acospores are induced to mate by transfer to rich medium.", "This process is repeated for several cycles to increase the diversity of the DNA cloned into the YACs.", "The resulting population of yeast cells, preferably in the haploid state, are either screened for improved properties, or the diversified DNA is delivered to another host cell or organism for screening.", "Cells surviving selection/screening are subjected to successive cycles of pooling, sporulation, mating and selection/screening until the desired phenotype has been observed.", "Recombination can be achieved simply by transferring cells from rich medium to carbon and nitrogen limited medium to induce sporulation, and then returning the spores to rich media to induce mating.", "Asci can be lysed to stimulate mating of spores originating from different asci.", "After YACs have been evolved to encode a desired property they can be transferred to other cell types.", "Transfer can be by protoplast fusion, or retransformation with isolated DNA.", "For example, transfer of YACs from yeast to mammalian cells is discussed by Monaco & Larin, Trends in Biotechnology 12, 280-286 (1994); Montoliu et al., Reprod.", "Fertil.", "Dev.", "6, 577-84 (1994); Lamb et al., Curr.", "Opin.", "Genet.", "Dev.", "5, 342-8 (1995).", "An exemplary scheme for stochastic &/or non-stochastic mutagenesis a YAC fragment library in yeast is shown herein.", "A library of YAC fragments representing genetic variants are transformed into yeast that have diploid endogenous chromosomes.", "The transformed yeast continue to have diploid endogenous chromosomes, plus a single YAC.", "The yeast are induced to undergo meiosis and sporulate.", "The spores contain haploid genomes and are selected for those which contain a YAC, using the YAC selective marker.", "The spores are induced to mate generating diploid cells.", "The diploid cells now contain two YACs bearing different inserts as well as diploid endogenous chromosomes.", "The cells are again induced to undergo meiosis and sporulate.", "during meiosis, recombination occurs between the YAC inserts, and recombinant YACs are segregated to ascoytes.", "Some ascoytes thus contain haploid endogenous chromosomes plus a YAC chromosome with a recombinant insert.", "The ascoytes mature to spores, which can mate again generating diploid cells.", "Some diploid cells now possess a diploid complement of endogenous chromosomes plus two recombinant YACs.", "These cells can then be taken through further cycles of meiosis, sporulation and mating.", "In each cycle, further recombination occurs between YAC inserts and further recombinant forms of inserts are generated.", "After one or several cycles of recombination has occurred, cells can be tested for acquisition of a desired property.", "Further cycles of recombination, followed by selection, can then be performed in similar fashion.", "4.15.4 In Vivo Stochastic &/or Non-Stochastic Mutagenesis of Genes by The Recursive Mating of Yeast Cells Harboring Homologous Genes in Identical LOCI A goal of DNA stochastic &/or non-stochastic mutagenesis is to mimic and expand the combinatorial capabilities of sexual recombination.", "In vitro DNA stochastic &/or non-stochastic mutagenesis succeeds in this process.", "However, by changing the mechanism of recombination and altering the conditions under which recombination occurs, naturally in vitro recombination methods may jeopardize intrinsic information in a DNA sequence that renders it “evolvable.” Stochastic &/or non-stochastic mutagenesis in vivo by employing the natural crossing over mechanisms that occur during meiosis may access inherent natural sequence information and provide a means of creating higher quality stochastic &/or non-stochastic mutagenized libraries.", "Described here is a method for the in vivo stochastic &/or non-stochastic mutagenesis of DNA that utilizes the natural mechanisms of meiotic recombination and provides an alternative method for DNA stochastic &/or non-stochastic mutagenesis.", "The basic strategy is to clone genes to be stochastic &/or non-stochastic mutagenized into identical loci within the haploid genome of yeast.", "The haploid cells are then recursively induced to mate and to sporulate.", "The process subjects the cloned genes to recursive recombination during recursive cycles of meiosis.", "The resulting stochastic &/or non-stochastic mutagenized genes are then screened in in situ or isolated and screened under different conditions.", "For example, if one wished to reassemble a family of five lipase genes, the following provides a means of doing so in vivo.", "The open reading frame of each lipase is amplified by the PCR such that each ORF is flanked by identical 3′ and 5′ sequences.", "The 5′ flanking sequence is identical to a region within the 5′ coding sequence of the S. cerevisiae ura 3 gene and the 3′ flanking sequence is identical to a region within the 3′ of the ura 3 gene.", "The flanking sequences are chosen such that homologous recombination of the PCR product with the ura 3 gene results in the incorporation of the lipase gene and the disruption of the ura 3 ORF.", "Both S. cerevisiae a and haploid cells are then transformed with each of the PCR amplified lipase ORFs, and cells having incorporated a lipase gene into the ura 3 locus are selected by growth on 5 fluoro orotic acid (5FOA is lethal to cells expressing functional URA3).", "The result is 10 cell types, two different mating types each harboring one of the five lipase genes in the disrupted ura 3 locus.", "These cells are then pooled and grown under conditions where mating between the a and cells are favored, e.g.", "in rich medium.", "Mating results in a combinatorial mixture of diploid cells having all 32 possible combinations of lipase genes in the two ura 3 loci.", "The cells are then induced to sporulate by growth under carbon and nitrogen limited conditions.", "During sporulation the diploid cells undergo meiosis to form four (two a and two) haploid ascospores housed in an ascus.", "During meiosis II of the sporulation process sister chromatids align and crossover.", "The lipase genes cloned into the ura 3 loci will also align and recombine.", "Thus the resulting haploid ascospores will represent a library of cells each harboring a different possible chimeric lipase gene, each a unique result of the meiotic recombination of the two lipase genes in the original diploid cell.", "The walls of asci are degraded by treatment with zymolase to liberate and allow the mixing of the individual ascospores.", "This mixture is then grown under conditions that promote the mating of the a and haploid cells.", "It is important to liberate the individual ascospores, since mating will otherwise occur between the ascospores within an ascus.", "Mixing of the haploid cells allows recombination between more than two lipase genes, enabling “poolwise recombination.” Mating brings together new combinations of chimeric genes that can then undergo recombination upon sporulation.", "The cells are recursively cycled through sporulation, ascospore mixing, and mating until sufficient diversity has been generated by the recursive pairwise recombination of the five lipase genes.", "The individual chimeric lipase genes either can be screened directly in the haploid yeast cells or transferred to an appropriate expression host.", "The process is described above for lipases and yeast; however, any sexual organisms into which genes can be directed can be employed, and any genes, of course, could be substituted for lipases.", "This process is analogous to the method of stochastic &/or non-stochastic mutagenesis whole genomes by recursive pairwise mating.", "The diversity, however, in the whole genome case is distributed throughout the host genome rather than localized to specific loci.", "4.15.5 Using YACs to Clone Unlinked Genes but Functionally Important Genes from One Species into Another Stochastic &/or non-stochastic mutagenesis of YACs is particularly amenable to transfer of unlinked but functionally related genes from one species to another, particularly where such genes have not been identified.", "Such is the case for several commercially important natural products, such as taxol.", "Transfer of the genes in the metabolic pathway to a different organism is often desirable because organisms naturally producing such compounds are not well suited for mass culturing.", "Clusters of such genes can be isolated by cloning a total genomic library of DNA from an organisms producing a useful compound into a YAC library.", "The YAC library is then transformed into yeast.", "The yeast is sporulated and mated such that recombination occurs between YACs and/or between YACs and natural yeast chromosomes.", "Selection/screening is then performed for expression of the desired collection of genes.", "If the genes encode a biosynthetic pathway, expression can be detected from the appearance of product of the pathway.", "Production of individual enzymes in the pathway, or intermediates of the final expression product or capacity of cells to metabolize such intermediates indicates partial acquisition of the synthetic pathway.", "The original library or a different library can be introduced into cells surviving/selection screening, and further rounds of recombination and selection/screening can be performed until the end product of the desired metabolic pathway is produced.", "4.15.6 YAC-YAC Stochastic &/or Non-Stochastic Mutagenesis If a phenotype of interest can be isolated to a single stretch of genomic DNA less than 2 megabases in length, it can be cloned into a YAC and replicated in S. cerevisiae.", "The cloning of similar stretches of DNA from related hosts into an identical YAC results in a population of yeast cells each harboring a YAC having a homologous insert effecting a desired phenotype.", "The recursive breeding of these yeast cells allows the homologous regions of these YACs to recombine during meiosis, allowing genes, pathways, and clusters to recombine during each cycle of meiosis.", "After several cycles of mating and segregation, the YAC inserts are well stochastic &/or non-stochastic mutagenized.", "The now very diverse yeast library could then be screened for phenotypic improvements resulting from the stochastic &/or non-stochastic mutagenesis of the YAC inserts.", "4.15.7 Yac-Chromosome Stochastic &/or Non-Stochastic Mutagenesis “Mitotic” recombination occurs during cell division and results from the recombination of genes during replication.", "This type of recombination is not limited to that between sister chromatids and can be enhanced by agents that induce recombination machinery, such as nicking chemicals and ultraviolet irradiation.", "Since it is often difficult to directly mate across a species barrier, it is possible to induce the recombination of homologous genes originating from different species by providing the target genes to a desired host organism as a YAC library.", "The genes harbored in this library are then induced to recombine with homologous genes on the host chromosome by enhanced mitotic recombination.", "This process is carried out recursively to generate a library of diverse organisms and then screened for those having the desired phenotypic improvements.", "The improved subpopulation is then mated recursively as above to identify new strains having accumulated multiple useful genetic alterations.", "4.15.8 Accumulation of Multiple YACs Harboring Useful Genes The accumulation of multiple unlinked genes that are required for the acquisition or improvement of a given phenotype can be accomplished by the stochastic &/or non-stochastic mutagenesis of YAC libraries.", "Genomic DNA from organisms having desired phenotypes, such as ethanol tolerance, thermotolerance, and the ability to ferment pentose sugars are pooled, fragmented and cloned into several different YAC vectors, each having a different selective marker (his, ura, ade, etc).", "S. cerevisiae are transformed with these libraries, and selected for their presence (using selective media i.e uracil dropout media for the YAC containing the ura3 selective marker) and then screened for having acquired or improved a desired phenotype.", "Surviving cells are pooled, mated recursively, and selected for the accumulation of multiple YACs (by propagation in medium with multiple nutritional dropouts).", "Cells that acquire multiple YACs harboring useful genomic inserts are identified by further screening.", "Optimized strains can be used directly, however, due to the burden a YAC may pose to a cell, the relevant YAC inserts can be minimized, subcloned, and recombined into the host chromosome, to generate a more stable production strain.", "4.15.9 Choice of Host SSF Organism One example use for the present invention is to create an improved yeast for the production of ethanol from lignocellulosic biomass.", "Specifically, a yeast strain with improved ethanol tolerance and thermostability/thermotolerance is desirable.", "Parent yeast strains known for good behavior in a Simultaneous Saccharification and Fermentation (SSF) process are identified.", "These strains are combined with others known to possess ethanol,” tolerance and/or thermostability.", "S. cerevisiae is highly amenable to development for optimized SSF processes.", "It inherently possesses several traits for this use, including the ability to import and ferment a variety of sugars such as sucrose, glucose, galactose, maltose and maltriose.", "Also, yeast has the capability to flocculate, enabling recovery of the yeast biomass at the end of a fermentation cycle, and allowing its re-use in subsequent bioprocesses.", "This is an important property in that it optimizes the use of nutrients in the growth medium.", "S. cerevisiae is also highly amenable to laboratory manipulation, has highly characterized genetics and possesses a sexual reproductive cycle.", "S. cerevisiae may be grown under either aerobic or anaerobic conditions, in contrast to some other potential SSF organisms that are strict anaerobes (e.g.", "Clostridium spp.", "), making them very difficult to handle in the laboratory.", "S. cerevisiae are also “generally regarded as safe” (“GRAS”), and, due to its widespread use for the production of important comestibles for the general public (e.g.", "beer, wine, bread, etc), is generally familiar and well known.", "S. cerevisiae is commonly used in fermentative processes, and the familiarity in its handling by fermentation experts eases the introduction of novel improved yeast strains into the industrial setting.", "S. cerevisiae strains that previously have been identified as particularly good SSF organisms, for example, S. cerevisiae D5A.", "(ATCC200062) (South C R and Lynd L R. (1994) Appl.", "Biochem.", "Biotechnol.", "45/46: 467-481; Ranatunga T D et al.", "(1997) Biotechnol.", "Lett.", "19: 1125-1127) can be used for starting materials.", "In addition, other industrially used S. cerevisiae strains are optionally used as host strains, particularly those showing desirable fermentative characteristics, such as S. cerevisiae Y567 (ATCC24858) (Sitton O C et al.", "(1979) Process Biochem.", "14(9): 7-10; Sitton O C et al.", "(1981) Adv.", "Biotechnol.", "2: 231-237; McMurrough I et al.", "(1971) Folia Microbiol.", "16: 346-349) and S. cerevisiae ACA 174 (ATCC 60868) (Benitez T et al.", "(1983) Appl.", "Environ.", "Microbiol.", "45: 1429-1436; Chem.", "Eng.", "J.", "50: B17-B22, 1992), which have been shown to have desirable traits for large-scale fermentation.", "4.15.10 Choice of Ethanol Tolerant Strains Many strains of S. cerevisiae have been isolated from high-ethanol environments, and have survived in the ethanol-rich environment by adaptive evolution.", "For example, strains from Sherry wine aging (“Flor” strains) have evolved highly functional mitochondria to enable their survival in a high-ethanol environment.", "It has been shown that transfer of these wine yeast mitochondria to other strains increases the recipient's resistance to high ethanol concentration, as well as thermotolerance (Jimenez, J. and Benitez, T (1988) Curr.", "Genet.", "13: 461469).", "There are several flor strains deposited in the ATCC, for example S. cerevisiae MY9] (ATCC 201301), MY138 (ATCC 201302), C5 (ATCC 201298), ET7 (ATCC 201299), LA6 (ATCC 201300), OSB21 (ATCC 201303), F23 (S. globosus ATCC 90920).", "Also, several flor strains of S. uvarum and Torulaspora pretoriensis have been deposited.", "Other ethanol-tolerant wine strains include S. cerevisiae ACA 174 (ATCC 60868), 15% ethanol, and S. cerevisiae A54 (ATCC 90921), isolated from wine containing 18% (v/v) ethanol, and NRCC 202036 (ATCC 46534), also a wine yeast.", "Other S. cerevisiae ethanologens that additionally exhibit enhanced ethanol tolerance include ATCC 24858, ATCC 24858, G 3706 (ATCC 42594), NRRL Y-265 (ATCC 60593), and ATCC 24845-ATCC 24860.A strain of S. pastorianus (S. carlsbergensis ATCC 2345) has high ethanol-tolerance (13% v/v).", "S. cerevisiae Sa28 (ATCC 26603), from Jamaican cane juice sample, produces high levels of alcohol from molasses, is sugar tolerant, and produces ethanol from wood acid hydrolyzate.", "Several of the listed strains, as well as additional strains can be used as starting materials for breeding ethanol tolerance.", "4.15.11 Choice of Temperature Tolerant Strains A few temperature tolerant strains have been reported, including the highly flocculent strain S. pastorianus SA 23 (S. carlsbergensis ATCC 26602), which produces ethanol at elevated temperatures, and S. cerevisiae Kyokai 7 (S. sake, ATCC 26422), a sake yeast tolerant to brief heat and oxidative stress.", "Ballesteros et al ((1991) Appl.", "Biochem.", "Biotechnol.", "28/29: 307-315) examined 27 strains of yeast for their ability to grow and ferment glucose in the 32-45° C. temperature range, including Saccharomyces, Kluyveromyces and Candida spp.", "Of these, the best thermotolerant clones were Kluyveromyces marxianus LG and Kluyveromyces fragilis 2671 (Ballesteros et al (1993) Appl.", "Biochem.", "Biotechnol.", "39/40: 201-211).", "S. cerevisiae-pretoriensis FDHI was somewhat thermotolerant, however was poor in ethanol tolerance.", "Recursive recombination of this strain with others that display ethanol tolerance can be used to acquire the thermotolerant characteristics of the strain in progeny which also display ethanol tolerance.", "Candida acidothernophilum (Issatchenlaa orientalis, ATCC 203 8 1) is a good SSF strain that also exhibits improved performance in ethanol production from lignocellulosic biomass at higher SSF temperatures than S. cerevisiae D5A (Kadam, K L, Schmidt, S L (1997) Appl.", "Microbial.", "Biotechnol.", "48: 709-713).", "This strain can also be a genetic contributor to an improved SSF strain.", "4.15.12 Stochastic &/or Non-Stochastic Mutagenesis of Strains In those instances where strains are highly related, a recursive mating strategy may be pursued.", "For example, a population of haploid S. cerevisiae (a and) are mutagenized and screened for improved EtOH or thermal tolerance.", "The improved haploid subpopulation are mixed together and mated as a pool and induced to sporulate.", "The resulting haploid spores are fred by degrading the asci wall and mixed.", "The freed spores are then induced to mate and sporulate recursively.", "This process is repeated a sufficient number of times to generate all possible mutant combinations.", "The whole genome stochastic &/or non-stochastic mutagenized population (haploid) is then screened for further EtOH or thermal tolerance.", "When strains are not sufficiently related for recursive mating, formats based on protoplast fusion may be employed.", "Recursive and poolwise protoplast fusion can be performed to generate chimeric populations of diverse parental strains.", "The resultant pool of progeny is selected and screened to identify improved ethanol and thermal tolerant strains.", "Alternatively, a YAC-based Whole Genome Stochastic &/or non-stochastic mutagenesis format can be used.", "In this format, YACs are used to shuttle large chromosomal fragments between strains.", "As detailed earlier, recombination occurs between YACs or between YACs, and the host chromosomes.", "Genomic DNA from organisms having desired phenotypes are pooled, fragmented and cloned into several different YAC vectors, each having a different selective marker (his, ura, ade, etc).", "S. cerevisiae are transformed with these libraries, and selected for their presence (using selective media, i.e.", "uracil dropout media for the YAC containing the Ura 3 selective marker) and then screened for having acquired or improved a desired phenotype.", "Surviving cells are pooled, mated recursively (as above), and selected for the accumulation of multiple YACs (by propagation in medium with multiple nutritional dropouts).", "Cells that acquire multiple YACs harboring useful genomic inserts are identified by further screening (see below).", "4.15.13 Selection for Improved Strains Having produced large libraries of novel strains by mutagenesis and recombination, a first task is to isolate those strains that possess improvements in the desired phenotypes.", "Identification of the organism libraries is facilitated where the desired key traits are selectable phenotypes.", "For example, ethanol has different effects on the growth rate of a yeast population, viability, and fermentation rate.", "Inhibition of cell growth and viability increases with ethanol concentration, but high fermentative capacity is only inhibited at higher ethanol concentrations.", "Hence, selection of growing cells in ethanol is a viable approach to isolate ethanol-tolerant strains.", "Subsequently, the selected strains may be analyzed for their fermentative capacity to produce ethanol.", "Provided that growth and media conditions are the same for all strains (parents and progeny), a hierarchy of ethanol tolerance may be constructed.", "Simple selection schemes for identification of thermal tolerant and ethanol tolerant strains are available and, in this case, are based on those previously designed to identify potentially useful SSF strains.", "Selection of ethanol tolerance is performed by exposing the population to ethanol, then plating the population and looking for growth.", "Colonies capable of growing after exposure to ethanol can be re-exposed to a higher concentration of ethanol and the cycle repeated until the most tolerant strains are selected.", "In order to discern strains possessing heritable ethanol tolerance from with temporarily acquired adaptations, these cycles may be punctuated with cycles of growth in the absence of selection (e.g.", "no ethanol).", "Alternatively, the mixed population can be grown directly at increasing concentrations of ethanol, and the most tolerant strains enriched (Aguilera and Benitez, 1986, Arch Microbiol 4: 33744).", "For example this enrichment could be carried out in a chemostat or turbidostat.", "Similar selections can be developed for thermal tolerance, in which strains are identified by their ability to grow after a heat treatment, or directly for growth at elevated temperatures (Ballesteros et al., 1991, Applied Biochem and Biotech, 28: 307-315).", "The best strains identified by these selections will be assayed more thoroughly in subsequent screens for ethanol, thermal tolerance or other properties of interest.", "In one aspect, organisms having increased ethanol tolerance are selected for.", "A population of natural S. cerevisiae isolates are mutagenized.", "This population is then grown under fermentor conditions under low initial ethanol concentrations.", "Once the culture has reached saturation, the culture is diluted into fresh medium having a slightly higher ethanol content.", "This process of successive dilution into medium of incrementally increasing ethanol concentration is continued until a threshold of ethanol tolerance is reached.", "The surviving mutant population having the highest ethanol tolerance are then pooled and their genomes recombined by any method noted herein.", "Enrichment could also be achieved by a continuous culture in a chemostat or turbidostat in which temperature or ethanol concentrations are progressively elevated.", "The resulting stochastic &/or non-stochastic mutagenized population are then exposed once again to the enrichment strategy but at a higher starting medium ethanol concentration.", "This strategy is optionally applied for the enrichment of thermotolerant cells and for the enrichment of cells having combined thermo- and ethanol tolerance.", "4.15.14 Screening for Improved Strains Strains showing viability in initial selections are assayed more quantitatively for improvements in the desired properties before being restochastic &/or non-stochastic mutagenized with other strains.", "Progeny resulting from mutagenesis of a strain, or those pre-selected for their ethanol tolerance and/or thermostability, can be plated on non-selective agar.", "Colonies can be picked robotically into microtiter dishes and grown.", "Cultures are replicated to fresh microtiter plates, and the replicates are incubated under the appropriate stress condition(s).", "The growth or metabolic activity of individual clones may be monitored and ranked.", "Indicators of viability can range from the size of growing colonies on solid media, density of growing cultures, or color change of a metabolic activity indicator added to liquid media.", "Strains that show the greatest viability are then mixed and stochastic &/or non-stochastic mutagenized, and the resulting progeny are rescreened under more stringent conditions.", "4.15.15 Development of a Yeast Strain Capable of Converting Cellulose to Monomeric Sugars Once a strain of yeast exhibiting thermotolerance and ethanol tolerance is developed, the degradation of cellulose to monomeric sugars is provided by the inclusion to the host strain of an efficient cellulase degradation pathway.", "Additional desirable characteristic can be useful to enhance the production of ethanol by the host.", "For example, inclusion of heterologous enzymes and pathways that broaden the substrate sugar range may be performed.", "“Tuning” of the strain can be accomplished by the addition of various other traits, or the restoration of certain endogenous traits that are desirable, but lost during the recombination procedures.", "4.15.16 Conferring of Cellulase Activity A vast number of cellulases and cellulase degradation systems have been characterized from fungi, bacteria and yeast (see reviews by Beguin, P and Aubert, J-P (1994) FEMS Microbial.", "Rev.", "13: 25-58; Ohima, K. et al.", "(1997) Biotechnol.", "Genet.", "Eng.", "Rev.", "14: 365414).", "An enzymatic pathway required for efficient saccharification of cellulose involves the synergistic action of endoglucanases (endo-1,4-D-glucanases, EC 3.2.1.4), exocellobiohydrolases (exo-1,4-D-glucanases, EC 3.2.1.91), and -glucosidases (cellobiases, 1,4-D-glucanases EC 3.2.1.21).", "The heterologous production of cellulase enzymes in the ethanologen would enable the saccharification of cellulose, producing monomeric sugars that may be used by the organism for ethanol production.", "There are several advantages to the heterologous expression of a functional cellulase pathway in the ethanologen.", "For example, the SSF process would eliminate the need for a separate bioprocess step for saccharification, and would ameliorate end-product inhibition of cellulase enzymes by accumulated intermediate and product sugars.", "Naturally occurring cellulase pathways are inserted into the ethanologen, or one may choose to use custom improved “hybrid” cellulase pathways, employing the coordinate action of cellulases derived from different natural sources, including thermophiles.", "Several cellulases from non-Saccharomyces have been produced and secreted from this organism successfully, including bacterial, fungal, and yeast enzymes, for example T. reesei CBH I ((Shoemaker (1994), in “The Cellulase System of Trichoderma reesei: Trichoderma strain improvement and Expression of Trichoderma cellulases in Yeast,” Online, Pinner, UK, 593-600).", "It is possible to employ straightforward metabolic engineering techniques to engender cellulase activity in Saccharomyces.", "Also, yeast have been forced to acquire elements of cellulose degradation pathways by protoplast fusion (e.g.", "intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati, a cellobiase-producing yeast, have been created (Pina A, et. al.", "(1986) Appl.", "Environ.", "Microbial.", "51: 995-1003).", "In general, any cellulase component enzyme that derives from a closely related yeast organism could be transferred by protoplast fusion.", "Cellobiases produced by a somewhat broader range of yeast may be accessed by whole genome stochastic &/or non-stochastic mutagenesis in one of its many formats (e.g.", "whole, fragmented, YAC-based).", "Optimally, the cellulase enzymes to be used should exhibit good synergy, an appropriate level of expression and secretion from the host, good specific activity (i.e.", "resistance to host degradation factors and enzyme modification) and stability in the desired SSF environment.", "An example of a hybrid cellulose degradation pathway having excellent synergy includes the following enzymes: CBH I exocellobiohydrolase of Trichoderma reesei, the Acidothermus cellulolyticus E1 endoglucanase, and the Thermomonospera fusca E3 exocellulase (Baker, et. al.", "(1998) Appl.", "Biochem.", "Biotechnol.", "70-72: 395403).", "It is suggested here that these enzymes (or improved mutants thereof) be considered for use in the SSF organism, along with a cellobiase (-glucosidase), such as that from Candida peltata.", "Other possible cellulase systems to be considered should possess particularly good activity against crystalline cellulose, such as the T. reesei cellulase system (Teeri, T T, et. al.", "(1998) Biochem.", "Soc, Trans.", "26: 173-178), or possess particularly good thermostability characteristics (e.g.", "cellulase systems from thermophilic organisms, such as Thermomonospora fusca (Zhang, S., et. al.", "(1995) Biochem.", "34: 3 3 86-3 3 5).", "A rational approach to the cloning of cellulases in the ethanologenic yeast host could be used.", "For example, known cellulase genes are cloned into expression cassettes utilizing S. cerevisiae promoter sequences, and the resultant linear fragments of DNA may be transformed into the recipient host by placing short yeast sequences at the termini to encourage site-specific integration into the genome.", "This is preferred to plasmidic transformation for reasons of genetic stability and maintenance of the transforming DNA.", "If an entire cellulose degradative pathway were introduced, a selection could be implemented in an agar-plate-based format, and a large number of clones could be assayed for cellulase activity in a short period of time.", "For example, selection for an exocellulase may be accessible by providing a soluble oligocellulose substrate or carboxymethylcellulose (CMC) as a sole carbon source to the host, otherwise unable to grow on agar containing this sole carbon source.", "Clones producing active cellulase pathways would grow by virtue of their ability to produce glucose.", "Alternatively, if the different cellulases were to be introduced sequentially, it would be useful to first introduce a cellobiase, enabling a selection using commercially available cellobiose as a sole carbon source.", "Several strains of S. cerevisiae that are able to grow on cellobiose have been created by introduction of a cellobiase gene (e.g.", "Rajoka M I, et. al.", "(1998) Floia Microbiol.", "(Praha) 43, 129-135; Skory, C D, et. al.", "(1996) Curr.", "Genet.", "30, 417422; D'Auria, S, et. al.", "(1996) Appl.", "Biochem.", "Biotechnol.", "61, 157-166; Adam, A C, et. al.", "(1995) Yeast 11, 395-406; Adam, AC (1991) Cuff.", "Genet.", "20, 5-8).", "Subsequent transformation of this organism with CBHI exocellulase can be selected for by growth on a cellulose substrate such as carboxymethylcellulose (CMC).", "Finally, addition of an endoglucanase creates a yeast strain with improved crystalline degradation capacity.", "4.15.17 Conferring of Pentose Sugar Utilization Inclusion of pentose sugar utilization pathways is an important facet to a potentially useful SSF organism.", "The successful expression of xylose sugar utilization pathways for ethanol production has been reported in Saccharomyces (e.g.", "Chen, Z D and Ho, N W Y (1993) Appl.", "Biochem.", "Biotechnol.", "39/40 135-147).", "It would also be useful to accomplish L-arabinose substrate utilization for ethanol production in the Saccharomyces host.", "Yeast strains that utilize L-arabinose include some Candida and Pichia spp.", "(McMillan J D and Boynton B L (1994) Appl.", "Biochem.", "Biotechnol.", "4546: 569-584; Dien B S, et al.", "(1996) Appl.", "Biochem.", "Biotechnol.", "57-58: 233-242).", "Genes necessary for arabinose fermentation in E. coli could also be introduced by rational means (e.g.", "as has been performed previously in Z. mobilis (Deanda K, et. al.", "(1996) Appl.", "Environ.", "Microbial.", "62: 4465-4470)).", "4.15.18 Conferring of Other Useful Activites Several other traits that are important for optimization of an SSF strain have been shown to be transferable to S. cerevisiae.", "Like thermal tolerance, cellulase activity and pentose sugar utilization, these traits may not normally be exhibited by Saccharomyces (or the particular strain of Saccharomyces being used as a host), and may be added by genetic means.", "For example, expression of human muscle acylphosphatase in S. cerevisiae has been suggested to increase ethanol production (Rougei, G., et. al.", "(1996) Biotechnol.", "App.", "Biochem.", "23: 273-278).", "It can occur that evolved stress-tolerant SSF strain acquire some undesirable mutations in the course of the evolution strategy.", "Indeed, this is a pervasive problem in strain improvement strategies that rely on mutagenesis techniques, and can result in highly unstable or fragile production strains.", "It is possible to restore some of these desirable traits by rational methods such as cloning of specific genes that have been knocked out or negatively influenced in the previous rounds of strain improvement.", "The advantage to this approach is specificity—the offending gene may be targeted directly.", "The disadvantage is that it may be time-consuming and repetitious if several genes have been compromised, and it only addresses problems that have been characterized.", "A preferred (and more traditional) approach to the removal of undesirable/deleterious mutations is to back-cross the evolved strain to a desirable parent strain (e.g.", "the original “host” SSF strain).", "This strategy has been employed successfully throughout strain improvement where accessible (i.e.", "for organisms that have sexual cycles of reproduction).", "When lacking the advantage of a sexual process, it has been accomplished by using other methods, such as parasexual recombination or protoplast fusion.", "For example, the ability to flocculate was conferred on a non-flocculating strain of S. cerevisiae by protoplast fusion with a flocculation competent S. cerevisiae (Watari, J., et. al (1990) Agric.", "Biol.", "Chem.", "54: 1677-1681).", "4.16 Method of In Vivo and In Vitro DNA Shuffling 4.16.1 Applications Disclosed is a method of producing random polynucleotides by introducing two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.", "Also provided are vector and expression vehicles including such polynucleotides, polypeptides expressed by the hybrid polynucleotides and a method for screening for hybrid polypeptides.", "4.16.2 Experimental Applications This invention relates generally to recombination and more specifically to a method for preparing polynucleotides encoding a polypeptide by a method of in vivo re-assortment of polynucleotide sequences containing regions of partial homology, assembling the polynucleotides to form at least one polynucleotide and screening the polynucleotides for the production of polypeptide(s) having a useful property.", "4.16.3 History An exceedingly large number of possibilities exist for purposeful and random combinations of amino acids within a protein to produce useful hybrid proteins and their corresponding biological molecules encoding for these hybrid proteins, i.e., DNA, RNA.", "Accordingly, there is a need to produce and screen a wide variety of such hybrid proteins for a useful utility, particularly widely varying random proteins.", "The complexity of an active sequence of a biological macromolecule (e.g., proteins, DNA) has been called its information content (“IC”), which has been defined as the resistance of the active protein to amino acid sequence variation (calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function.", "Proteins that are more sensitive to random mutagenesis have a high information content.", "Molecular biology developments, such as molecular libraries, have allowed the identification of quite a large number of variable bases, and even provide ways to select functional sequences from random libraries.", "In such libraries, most residues can be varied (although typically not all at the same time) depending on compensating changes in the context.", "Thus, while a 100 amino acid protein can contain only 2,000 different mutations, 20100 sequence combinations are possible.", "Information density is the IC per unit length of a sequence.", "Active sites of enzymes tend to have a high information density.", "By contrast, flexible linkers of information in enzymes have a low information density.", "Current methods in widespread use for creating alternative proteins in a library format are error-prone polymerase chain reactions and cassette mutagenesis, in which the specific region to be optimized is replaced with a synthetically mutagenized oligonucleotide.", "In both cases, a substantial number of mutant sites are generated around certain sites in the original sequence.", "4.163.1 Error-Prone PCR Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence.", "In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture.", "The published error-prone PCR protocols suffer from a low processivity of the polymerase.", "Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene.", "This inability limits the practical application of error-prone PCR.", "Some computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the large-scale block changes that are required for continued and dramatic sequence evolution.", "Further, the published error-prone PCR protocols do not allow for amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application.", "In addition, repeated cycles of error-prone PCR can lead to an accumulation of neutral mutations with undesired results, such as affecting a protein's immunogenicity but not its binding affinity.", "4.16.3.2 Oligonucleotide-Directed Mutagenesis In oligonucleotide-directed mutagenesis, a short sequence is replaced with a synthetically mutagenized oligonucleotide.", "This approach does not generate combinations of distant mutations and is thus not combinatorial.", "The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization.", "Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping them into families, arbitrarily choosing a single family, and reducing it to a consensus motif.", "Such motif is resynthesized and reinserted into a single gene followed by additional selection.", "This step process constitutes a statistical bottleneck, is labor intensive, and is not practical for many rounds of mutagenesis.", "Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine tuning, but rapidly become too limiting when they are applied for multiple cycles.", "Another limitation of error-prone PCR is that the rate of down-mutations grows with the information content of the sequence.", "As the information content, library size, and mutagenesis rate increase, the balance of down-mutations to up-mutations will statistically prevent the selection of further improvements (statistical ceiling).", "4.16.3.3 Cassette Mutagenesis In cassette mutagenesis, a sequence block of a single template is typically replaced by a (partially) randomized sequence.", "Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (ie., library size).", "This eliminates other sequence families which are not currently best, but which may have greater long term potential.", "Also, mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round.", "Thus, such an approach is tedious and impractical for many rounds of mutagenesis.", "Thus, error-prone PCR and cassette mutagenesis are best suited, and have been widely used, for fine-tuning areas of comparatively low information content.", "One apparent exception is the selection of an RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection.", "In nature, the evolution of most organisms occurs by natural selection and sexual reproduction.", "Sexual reproduction ensures mixing and combining of the genes in the offspring of the selected individuals.", "During meiosis, homologous chromosomes from the parents line up with one another and cross-over part way along their length, thus randomly swapping genetic material.", "Such swapping or shuffling of the DNA allows organisms to evolve more rapidly.", "In recombination, because the inserted sequences were of proven utility in a homologous environment, the inserted sequences are likely to still have substantial information content once they are inserted into the new sequence.", "4.16.3.4 Applied Molecular Evolution The term Applied Molecular Evolution (“AME”) means the application of an evolutionary design algorithm to a specific, useful goal.", "While many different library formats for AME have been reported for polynucleotides, peptides and proteins (phage, lad and polysomes), none of these formats have provided for recombination by random crossovers to deliberately create a combinatorial library.", "Theoretically there are 2,000 different single mutants of a 100 amino acid protein.", "However, a protein of 100 amino acids has 20100 possible sequence combinations, a number which is too large to exhaustively explore by conventional methods.", "It would be advantageous to develop a system which would allow generation and screening of all of these possible combination mutations.", "4.16.3.5 Reported In Vivo Recombination Systems Some workers in the art have utilized an in vivo site specific recombination system to generate hybrids of combine light chain antibody genes with heavy chain antibody genes for expression in a phage system.", "However, their system relies on specific sites of recombination and is limited accordingly.", "Simultaneous mutagenesis of antibody CDR regions in single chain antibodies (scFv) by overlapping extension and PCR have been reported.", "Others have described a method for generating a large population of multiple hybrids using random in vivo recombination.", "This method requires the recombination of two different libraries of plasmids, each library having a different selectable marker.", "The method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).", "In vivo recombination between two homologous, but truncated, insect-toxin genes on a plasmid has been reported as a method of producing a hybrid gene.", "The in vivo recombination of substantially mismatched DNA sequences in a host cell having defective mismatch repair enzymes, resulting in hybrid molecule formation has been reported.", "4.16.4 Strategies In one aspect this invention provides a method that utilizes the natural property of cells to recombine molecules and/or to mediate reductive processes that reduce the complexity of sequences and extent of repeated or consecutive sequences possessing regions of homology.", "It is an object of the present invention to provide a method for generating hybrid polynucleotides encoding biologically active hybrid polypeptides with enhanced activities.", "In accomplishing these and other objects, there has been provided, in accordance with one aspect of the invention, a method for introducing polynucleotides into a suitable host cell and growing the host cell under conditions which produce a hybrid polynucleotide.", "In another aspect of the invention, the invention provides a method for screening for biologically active hybrid polypeptides encoded by hybrid polynucleotides.", "The present method allows for the identification of biologically active hybrid polypeptides with enhanced biological activities.", "Other objects, features and advantages of the present invention will become apparent from the following detailed description.", "It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.", "4.16.5 Possible Uses The invention described herein is directed to the use of repeated cycles of reductive reassortment, recombination and selection which allow for the directed molecular evolution of highly complex linear sequences, such as DNA, RNA or proteins thorough recombination.", "In vivo shuffling of molecules can be performed utilizing the natural property of cells to recombine multimers.", "While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules.", "The formation of the chiasma requires the recognition of homologous sequences.", "4.16.5.1 Production of a Hybrid Polynucleotide In a preferred embodiment, the invention relates to a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide.", "The present invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell.", "The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide.", "The term “hybrid polynucleotide”, as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences.", "Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules.", "In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.", "The invention provides a means for generating hybrid polynucleotides which may encode biologically active hybrid polypeptides.", "In one aspect, the original polynucleotides encode biologically active polypeptides.", "The method of the invention produces new hybrid polypeptides by utilizing cellular processes which integrate the sequence of the original polynucleotides such that the resulting hybrid polynucleotide encodes a polypeptide demonstrating activities derived from the original biologically active polypeptides.", "For example, the original polynucleotides may encode a particular enzyme from different microorganisms.", "An enzyme encoded by a first polynucleotide from one organism may, for example, function effectively under a particular environmental condition, e.g.", "high salinity.", "An enzyme encoded by a second polynucleotide from a different organism may function effectively under a different environmental condition, such as extremely high temperatures.", "A hybrid polynucleotide containing sequences from the first and second original polynucleotides may encode an enzyme which exhibits characteristics of both enzymes encoded by the original polynucleotides.", "Thus, the enzyme encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the enzymes encoded by the first and second polynucleotides, e.g., high salinity and extreme temperatures.", "4.16.5.1.1 Encoded Enzymes Enzymes encoded by the original polynucleotides of the invention include, but are not limited to; oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.", "A hybrid polypeptide resulting from the method of the invention may exhibit specialized enzyme activity not displayed in the original enzymes.", "For example, following recombination and/or reductive reassortment of polynucleotides encoding hydrolase activities, the resulting hybrid polypeptide encoded by a hybrid polynucleotide can be screened for specialized hydrolase activities obtained from each of the original enzymes, i.e.", "the type of bond on which the hydrolase acts and the temperature at which the hydrolase functions.", "Thus, for example, the hydrolase may be screened to ascertain those chemical functionalities which distinguish the hybrid hydrolase from the original hydrolyases, such as: (a) amide (peptide bonds), i.e.", "proteases; (b) ester bonds, i.e.", "esterases and lipases; (c) acetals, ie., glycosidases and, for example, the temperature, pH or salt concentration at which the hybrid polypeptide functions.", "4.16.5.1.2 Sources of the Original Polynucleotides Sources of the original polynucleotides may be isolated from individual organisms (“isolates”), collections of organisms that have been grown in defined media (“enrichment cultures”), or, most preferably, uncultivated organisms (“environmental samples”).", "The use of a culture-independent approach to derive polynucleotides encoding novel bioactivities from environmental samples is most preferable since it allows one to access untapped resources of biodiversity.", "“Environmental libraries” are generated from environmental samples and represent the collective genomes of naturally occurring organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts.", "Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture.", "Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample.", "This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample which may be under-represented by several orders of magnitude compared to the dominant species.", "For example, gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest.", "Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries.", "Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell.", "The host cell is grown under conditions which promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities.", "The microorganisms from which the polynucleotide may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae and protozoa.", "Polynucleotides may be isolated from environmental samples in which case the nucleic acid may be recovered without culturing of an organism or recovered from one or more cultured organisms.", "In one aspect, such microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.", "Polynucleotides encoding enzymes isolated from extremophilic microorganisms are particularly preferred.", "Such enzymes may function at temperatures above 1001C in terrestrial hot springs and deep sea thermal vents, at temperatures below 0° C. in arctic waters, in the saturated salt environment of the Dead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge.", "For example, several esterases and lipases cloned and expressed from extremophilic organisms show high activity throughout a wide range of temperatures and pHs.", "4.16.5.13 Suitable Host Cells Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell.", "A suitable host cell is any cell which is capable of promoting recombination and/or reductive reassortment.", "The selected polynucleotides are preferably already in a vector which includes appropriate control sequences.", "The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bacterial cell.", "Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).", "As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera SJ9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells.", "The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.", "4.16.5.1.3.1 Mammalian Cell Culture Systems With particular references to various mammalian cell culture systems that can be employed to express recombinant protein, examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines.", "Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences.", "DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.", "Host cells containing the polynucleotides of interest can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes.", "The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.", "The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity.", "4.16.5.1.4 Generation of Polynucleotides Encoding Biochemical Pathways In another aspect, it is envisioned the method of the present invention can be used to generate novel polynucleotides encoding biochemical pathways from one or more operons or gene clusters or portions thereof.", "For example, bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes.", "The genes are clustered, in structures referred to as “gene clusters,” on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster.", "Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.", "An example of a biochemical pathway encoded by gene clusters are polyketides.", "Polyketides are molecules which are an extremely rich source of bioactivities, including antibiotics (such as tetracyclines and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin).", "Many polyketides (produced by polyketide synthases) are valuable as therapeutic agents.", "Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of an enormous variety of carbon chains differing in length and patterns of functionality and cyclization.", "Polyketide synthase genes fall into gene clusters and at least one type (designated type 1) of polyketide synthases have large size genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins.", "The ability to select and combine desired components from a library of polyketides, or fragments thereof, and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing.", "The method of the present invention makes it possible to facilitate the production of novel polyketide synthases through intermolecular recombination.", "4.16.5.1.5 Gene Cluster DNA Preferably, gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related array activity from the ligated gene clusters.", "Use of vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E. coli.", "This f-factor of E. coli is a plasmid which affect high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples.", "Once ligated into an appropriate vector, two or more vectors containing different polyketide synthase gene clusters can be introduced into a suitable host cell.", "Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster.", "The novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.", "Therefore, in a preferred embodiment, the present invention relates to a method for producing a biologically active hybrid polypeptide and screening such a polypeptide for enhanced activity by: introducing at least a first polynucleotide in operable linkage and a second polynucleotide in operable linkage, said at least first polynucleotide and second polynucleotide sharing at least one region of partial sequence homology, into a suitable host cell; growing the host cell under conditions which promote sequence reorganization resulting in a hybrid polynucleotide in operable linkage; expressing a hybrid polypeptide encoded by the hybrid polynucleotide; screening the hybrid polypeptide under conditions which promote identification of enhanced biological activity; and isolating the a polynucleotide encoding the hybrid polypeptide.", "Methods for screening for various enzyme activities are known to those of skill in the art and discussed throughout the present specification.", "Such methods may be employed when isolating the polypeptides and polynucleotides of the present invention.", "The term “isolated” means that material is removed from its original environment (e.g., the natural environment if it is naturally occurring).", "For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system, is isolated.", "As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship.", "A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.", "For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.", "Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join'two protein coding regions, contiguous and in reading frame.", "4.16.5.1.6 Expression Vectors As representative examples of expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g.", "vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus and yeast) Thus, for example, the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide.", "Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences.", "Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.", "The following vectors are provided by way of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).", "However, any other plasmid or other vector may be used as long as they are replicable and viable in the host.", "Low copy number or high copy number vectors may be employed with the present invention.", "A preferred type of vector for use in the present invention contains an f-factor origin replication.", "The f-factor (or fertility factor) in E. coli is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself.", "A particularly preferred embodiment is to use cloning vectors, referred to as “fosmids” or bacterial artificial chromosome (BAC) vectors.", "These are derived from E. coli f-factor which is able to stably integrate large segments of genomic DNA.", "When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.” Another preferred type of vector for use in the present invention is a cosmid vector.", "Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA.", "Cloning into cosmid vectors is described in detail in Sambrook, et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.4.16.5.1.6.1 Expression Control Sequence The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct RNA synthesis.", "Particular named bacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, PL and trp.", "Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-1.Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.", "The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.", "The vector may also include appropriate sequences for amplifying expression.", "Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.", "4.16.5.1.6.2 Selectable Marker Genes In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.", "Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPI gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.", "Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), -factor, acid phosphatase, or heat shock proteins, among others.", "The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.", "The cloning strategy permits expression via both vector driven and endogenous promoters; vector promotion may be important with expression of genes whose endogenous promoter will not function in E. coli.", "4.16.5.1.7 Insertion Into a Vector or Plasmid The DNA isolated or derived from microorganisms can preferably be inserted into a vector or a plasmid prior to probing for selected DNA.", "Such vectors or plasmids are preferably those containing expression regulatory sequences, including promoters, enhancers and the like.", "Such polynucleotides can be part of a vector and/or a composition and still be isolated, in that such vector or composition is not part of its natural environment.", "Particularly preferred phage or plasmid and methods for introduction and packaging into them are described in detail in the protocol set forth herein.", "The selection of the cloning vector depends upon the approach taken, for example, the vector can be any cloning vector with an adequate capacity for multiply repeated copies of a sequence, or multiple sequences that can be successfully transformed and selected in a host cell.", "One example of such a vector is described in “Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts”, Alting-Mecs M A, Short J M, Gene, 1993 Dec. 27, 137: 1, 93-100.Propagation/maintenance can be by an antibiotic resistance carried by the cloning vector.", "After a period of growth, the naturally abbreviated molecules are recovered and identified by size fractionation on a gel or column, or amplified directly.", "The cloning vector utilized may contain a selectable gene that is disrupted by the insertion of the lengthy construct.", "As reductive reassortment progresses, the number of repeated units is reduced and the interrupted gene is again expressed and hence selection for the processed construct can be applied.", "The vector may be an expression/selection vector which will allow for the selection of an expressed product possessing desirable biologically properties.", "The insert may be positioned downstream of a functional promotor and the desirable property screened by appropriate means.", "4.16.5.1.8 Reductive Reassortment In vivo reassortment is focused on “inter-molecular” processes collectively referred to as “recombination” which in bacteria, is generally viewed as a “RecA-dependent” phenomenon.", "The present invention can rely on recombination processes of a host cell to recombine and re-assort sequences, or the cells ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion.", "This process of “reductive reassortment” occurs by an “intra-molecular”, RecA-independent process.", "Therefore, in another aspect of the present invention, novel polynucleotides can be generated by the process of reductive reassortment.", "The method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropriate host cell.", "The reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units.", "The reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel molecular species.", "Various treatments may be applied to enhance the rate of reassortment.", "These could include treatment with ultra-violet light, or DNA damaging chemicals, and/or the use of host cell lines displaying enhanced levels of “genetic instability”.", "Thus the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution.", "4.16.5.1.9 Repeated or “Quasi-Repeated” Sequences Repeated or “quasi-repeated” sequences play a role in genetic instability.", "In the present invention, “quasi-repeats” are repeats that are not restricted to their original unit structure.", "Quasi-repeated units can be presented as an array of sequences in a construct; consecutive units of similar sequences.", "Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level.", "The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi-repeated sequences.", "The quasi-repeated units provide a practically limitless repertoire of templates upon which slippage events can occur.", "The constructs containing the quasi-repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units.", "When the quasi-repeated sequences are all ligated in the same orientation, for instance head to tail or vice versa, the cell cannot distinguish individual units.", "Consequently, the reductive process can occur throughout the sequences.", "In contrast, when for example, the units are presented head to head, rather than head to tail, the inversion delineates the endpoints of the adjacent unit so that deletion formation will favor the loss of discrete units.", "Thus, it is preferable with the present method that the sequences are in the same orientation.", "Random orientation of quasi-repeated sequences will result in the loss of reassortment efficiency, while consistent orientation of the sequences will offer the highest efficiency.", "However, while having fewer of the contiguous sequences in the same orientation decreases the efficiency, it may still provide sufficient elasticity for the effective recovery of novel molecules.", "Constructs can be made with the quasi-repeated sequences in the same orientation to allow higher efficiency.", "4.16.5.1.10 Assembly of Sequences in a Head to Tail Orientation Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following: a) Primers that include a poly-A head and poly-T tail which when made single-stranded would provide orientation can be utilized.", "This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNAseH.", "b) Primers that include unique restriction cleavage sites can be utilized.", "Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required.", "c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules.", "4.16.5.1.11 The Recovery of the Re-assorted Sequences The recovery of the re-assorted sequences relies on the identification of cloning vectors with a reduced RI.", "The re-assorted encoding sequences can then be recovered by amplification.", "The products are re-cloned and expressed.", "The recovery of cloning vectors with reduced RI can be effected by: 1) The use of vectors only stably maintained when the construct is reduced in complexity.", "2) The physical recovery of shortened vectors by physical procedures.", "In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on either an agarose gel, or column with a low molecular weight cut off utilizing standard procedures.", "3) The recovery of vectors containing interrupted genes which can be selected when insert size decreases.", "4) The use of direct selection techniques with an expression vector and the appropriate selection.", "Encoding sequences (for example, genes) from related organisms may demonstrate a high degree of homology and encode quite diverse protein products.", "These types of sequences are particularly useful in the present invention as quasi-repeats.", "However, while the examples illustrated below demonstrate the reassortment of nearly identical original encoding sequences (quasi-repeats), this process is not limited to such nearly identical repeats.", "The following example demonstrates the method of the invention.", "Encoding nucleic acid sequences (quasi-repeats) derived from three (3) unique species are depicted.", "Each sequence encodes a protein with a distinct set of properties.", "Each of the sequences differs by a single or a few base pairs at a unique position in the sequence which are designated “A”, “B” and “C”.", "The quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules.", "The number of quasi-repeat units can be controlled by the assembly conditions.", "The average number of quasi-repeated units in a construct is defined as the repetitive index (RI).", "Once formed, the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell.", "The cells are then propagated and “reductive reassortment” is effected.", "The rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired.", "Whether the reduction in RI is mediated by deletion formation between repeated sequences by an “intra-molecular” mechanism, or mediated by recombination-like events through “inter-molecular” mechanisms is immaterial.", "The end result is a reassortment of the molecules into all possible combinations.", "Optionally, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact (e.g., such as catalytic antibodies) with a predetermined macromolecule, such as for example a proteinaceous receptor, peptide oligosaccharide, viron, or other predetermined compound or structure.", "The displayed polypeptides, antibodies, peptidomimetic antibodies, and variable region sequences that are identified from such libraries can be used for therapeutic, diagnostic, research and related purposes (e.g., catalysts, solutes for increasing osmolarity of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or affinity selection.", "The method can be modified such that the step of selecting for a phenotypic characteristic can be other than of binding affinity for a predetermined molecule (e.g., for catalytic activity, stability oxidation resistance, drug resistance, or detectable phenotype conferred upon a host cell).", "4.16.5.1.12 Providing Antibodies Suitable for Affinity Interactions Screening The present invention provides a method for generating libraries of displayed antibodies suitable for affinity interactions screening.", "The method comprises (1) obtaining first a plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotide encoding for said displayed antibody and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and (2) introducing said polynucleotides into a suitable host cell and growing the cells under conditions which promote recombination and reductive reassortment resulting in shuffled polynucleotides.", "CDR combinations comprised by the shuffled pool are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed antibodies comprising CDR permutations and suitable for affinity interaction screening.", "Optionally, the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope (antigen) and thereby selecting a plurality of selected shuffled library members.", "Further, the plurality of selectively shuffled library members can be shuffled and screened iteratively, from 1 to about 1000 cycles or as desired until library members having a desired binding affinity are obtained.", "4.16.5.1.13 Introduction of Mutations into the Original Polynucleotides In another aspect of the invention, it is envisioned that prior to or during recombination or reassortment, polynucleotides generated by the method of the present invention can be subjected to agents or processes which promote the introduction of mutations into the original polynucleotides.", "The introduction of such mutations would increase the diversity of resulting hybrid polynucleotides and polypeptides encoded therefrom.", "The agents or processes which promote mutagenesis can include, but are not limited to: (+)-CC-1065, or a synthetic analog such as (+)-CC-1065-N-3-Adenine), (see.", "Biochem.", "31, 2822-2829 (1992)); a N-acelylated or deacetylated 4′-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis (see, for example, Carcinogenesis vol.", "13, No.", "5, 751-758 (1992); or a N-acetylated or deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis (see also, Id.", "751-758); trivalent chromium, a trivalent chromium salt, a polycyclic aromatic hydrocarbon (“PAH”) DNA adduct capable of inhibiting DNA replication, such as 7-bromomethyl-benz[a]anthracene (“BMA”), tris(2,3-dibromopropyl)phosphate (“Tris-BP”), 1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA), benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II) halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline (“N-hydroxy-IQ”), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine (“N-hydroxy-PhIP”).", "Especially preferred “means for slowing or halting PCR amplification consist of UV light (+)-CC-1065 and (+)-CC-1065-(N-3-Adenine).", "Particularly encompassed means are DNA adducts or polynucleotides comprising the DNA adducts from the polynucleotides or polynucleotides pool, which can be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing.", "4.16.5.1.14 Production Of Hybrid Or Re-Assorted Polynucleotides In another aspect the present invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions according to the present invention which provide for the production of hybrid or re-assorted polynucleotides.", "4.16.5.1.15 Shuffling a Population of Viral Genes of Viral Genomes The invention also provides the use of polynucleotide shuffling to shuffle a population of viral genes (e.g., capsid proteins, spike glycoproteins, polymerases, and proteases) or viral genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses, reoviruses and rhinoviruses).", "In an embodiment, the invention provides a method for shuffling sequences encoding all or portions of immunogenic viral proteins to generate novel combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes which are likely to arise in the natural environment as a consequence of viral evolution; (e.g., such as recombination of influenza virus strains).", "4.16.5.1.16 Generation of Gene Therapy Vectors and Replication-Defective Gene Therapy Constructs The invention also provides a method suitable for shuffling polynucleotide sequences for generating gene therapy vectors and replication-defective gene therapy constructs, such as may be used for human gene therapy, including but not limited to vaccination vectors for DNA-based vaccination, as well as anti-neoplastic gene therapy and other general therapy formats.", "4.16.5.2 Definitions The term “DNA shuffling” is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/10× and/or flp/fit systems and the like.", "The term “amplification” means that the number of copies of a polynucleotide is increased.", "The term “identical” or “identity” means that two nucleic acid sequences have the same sequence or a complementary sequence.", "Thus, “areas of identity” means that regions or areas of a polynucleotide or the overall polynucleotide are identical or complementary to areas of another polynucleotide or the polynucleotide.", "The term “corresponds to” is used herein to mean that a polynucleotide sequence is homologous (ie., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.", "In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.", "For illustration, the nucleotide sequence “TATAC” corresponds to a reference “TATAC” and is complementary to a reference sequence “GTATA.” Genetic instability, as used herein, refers to the natural tendency of highly repetitive sequences to be lost through a process of reductive events generally involving sequence simplification through the loss of repeated sequences.", "Deletions tend to involve the loss of one copy of a repeat and everything between the repeats.", "Quasi-repeated units, as used herein, refers to the repeats to be re-assorted and are by definition not identical.", "Indeed the method is proposed not only for practically identical encoding units produced by mutagenesis of the identical starting sequence, but also the reassortment of similar or related sequences which may diverge significantly in some regions.", "Nevertheless, if the sequences contain sufficient homologies to be reasserted by this approach, they can be referred to as “quasi-repeated” units.", "Reductive reassortment, as used herein, refers to the increase in molecular diversity that is accrued through deletion (and/or insertion) events that are mediated by repeated sequences.", "Repetitive Index (RI), as used herein, is the average number of copies of the quasi-repeated units contained in the cloning vector.", "The term “related polynucleotides” means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.", "The term “population” as used herein means a collection of components such as polynucleotides, portions or polynucleotides or proteins.", "A “mixed population: means a collection of components which belong to the same family of nucleic acids or proteins (i.e., are related) but which differ in their sequence (ie., are not identical) and hence in their biological activity.", "The term “specific polynucleotide” means a polynucleotide having certain end points and having a certain nucleic acid sequence.", "Two polynucleotides wherein one polynucleotide has the identical sequence as a portion of the second polynucleotide but different ends comprises two different specific polynucleotides.", "The following terms are used to describe the sequence relationships between two or more polynucleotides: “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence.", "Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.", "Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.", "A “comparison window,” as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (ie., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.", "Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv.", "Appl.", "Math.", "2: 482 by the homology alignment algorithm of Needlemen and Wuncsch J. Mol.", "Biol.", "48: 443 (1970), by the search of similarity method of Pearson and Lipman Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.", "The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.", "The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or 1) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.", "This “substantial identity”, as used herein, denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 80 percent sequence identity, preferably at least 85 percent identity, often 90 to 95 percent sequence identity, and most commonly at least 99 percent sequence identity as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.", "“Conservative amino acid substitutions” refer to the interchangeability of residues having similar side chains.", "For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.", "Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.", "The term “homologous” or “homeologous” means that one single-stranded nucleic acid nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence.", "The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentrations as discussed later.", "Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.", "The term “heterologous” means that one single-stranded nucleic acid sequence is unable to hybridize to another single-stranded nucleic acid sequence or its complement.", "Thus areas of heterology means that areas of polynucleotides or polynucleotides have areas or regions within their sequence which are unable to hybridize to another nucleic acid or polynucleotide.", "Such regions or areas are, for example areas of mutations.", "The term “cognate” as used herein refers to a gene sequence that is evolutionarily and functionally related between species.", "For example but not limitation, in the human genome the human CD4 gene is the cognate gene to the mouse 3d4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.", "The term “wild-type” means that the polynucleotide does not comprise any mutations.", "A “wild type” protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.", "The term “mutations” means changes in the sequence of a wild-type nucleic acid sequence or changes in the sequence of a peptide.", "Such mutations may be point mutations such as transitions or transversions.", "The mutations may be deletions, insertions or duplications.", "In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.", "Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5′ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5′ direction.", "The direction of 5′ to 3′ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5′ to the 5′ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA and which are 3′ to the 3′ end of the coding RNA transcript are referred to as “downstream sequences”.", "The term “naturally-occurring” as used herein as applied to the object refers to the fact that an object can be found in nature.", "For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.", "Generally, the term naturally occurring refers to an object as present in a non-pathological (un-diseased) individual, such as would be typical for the species.", "The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds (e.g., a VLSIPS peptide array, polynucleotide array, and/or combinatorial small molecule array), biological macromolecule, a bacteriophage peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made form biological materials such as bacteria, plants, fungi, or animal (particular mammalian) cells or tissues.", "Agents are evaluated for potential activity as anti-neoplastics, anti-inflammatories or apoptosis modulators by inclusion in screening assays described hereinbelow.", "Agents are evaluated for potential activity as specific protein interaction inhibitors (i.e., an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which doe snot substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.", "As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.", "Generally, a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition.", "Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.", "Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.", "As used herein the term “physiological conditions” refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell.", "For example, the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions.", "Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions.", "In general, in vitro physiological conditions comprise 50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45 C and 0.001-10 mM divalent cation (e.g., Mg++, Ca++); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein (e.g., BSA).", "A non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).", "Particular aqueous conditions may be selected by the practitioner according to conventional methods.", "For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris HCl, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents and/or membrane fractions and/or anti-foam agents and/or scintillants.", "“Specific hybridization” is defined herein as the formation of hybrids between a first polynucleotide and a second polynucleotide (e.g., a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.", "As used herein, the term “single-chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally liked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]x), and which may comprise additional amino acid sequences at the amino- and/or carboxy-termini.", "For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.", "As an example, a scFv is a single-chain antibody.", "Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and THE.", "Rabbits, eds., (1989) Academic press: San Diego, Calif., pp.", "361-368, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human heavy chain or light chain gene sequence.", "A functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).", "As used herein, the term “complementarity-determining region” and “CDR” refer to the art-recognized term as exemplified by the Kabat and Chothia CDR definitions also generally known as supervariable regions or hypervariable loops (Chothia and Leks (1987) J. Mol.", "Biol.", "196; 901; Clothia et al.", "(1989) Nature 342; 877; E. A. Kabat et al., Sequences of Proteins of Immunological Interest (national Institutes of Health, Bethesda, Md.)", "(1987); and Tramontano et al.", "(1990) J. Mol.", "Biolog.", "215; 175).", "Variable region domains typically comprise the amino-terminal approximately 105-115 amino acids of a naturally-occurring immunoglobulin chain (e.g., amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.", "An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, also called CDR's.", "The extent of the framework region and CDR's have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., 4th Ed., U.S. Department of Health and human services, Bethesda, Md.", "(1987)).", "The sequences of the framework regions of different light or heavy chains are relatively conserved within a specie.", "As used herein, a “human framework region” is a framework region that is substantially identical (about 85 or more, usually 90-95 or more) to the framework region of a naturally occurring human immunoglobulin the framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's.", "The CDR's are primarily responsible for binding to an epitope of an antigen.", "As used herein, the term “variable segment” refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence.", "A variable segment” refers to a portion of a nascent peptide which comprises a random pseudorandom, or defined kernal sequence.", "A variable segment can comprise both variant and invariant residue positions, and the degree of residue variation at a variant residue position may be limited: both options are selected at the discretion of the practitioner.", "Typically, variable segments are about 5 to 20 amino acid residues in length (e.g., 8 to 10), although variable segments may be longer and may comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.", "As used herein, “random peptide sequence” refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a stochastic or random process.", "A random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.", "As used herein “random peptide library” refers to a set of polynucleotide sequences that encodes a set of random peptides, and to the set of random peptides encoded by those polynucleotide sequences, as well as the fusion proteins contain those random peptides.", "As used herein, the term “pseudorandom” refers to a set of sequences that have limited variability, such that, for example, the degree of residue variability at another position, but any pseudorandom position is allowed some degree of residue variation, however circumscribed.", "As used herein, the term “defined sequence framework” refers to a set of defined sequences that are selected on a non-random basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a β-sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations.", "A “defined sequence kernal” is a set of sequences which encompass a limited scope of variability.", "Whereas (1) a completely random 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences, and (2) a pseudorandom 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences but will exhibit a bias for certain residues at certain positions and/or overall, (3) a defined sequence kernal is a subset of sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids).", "A defined sequence kernal generally comprises variant and invariant residue positions and/or comprises variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence.", "Defined sequence kernels can refer to either amino acid sequences or polynucleotide sequences.", "Of illustration and not limitation, the sequences (NNK)10 and (NNM)10, wherein N represents A, T, G, or C; K represents G or T; and M represents A or C, are defined sequence kernels.", "As used herein “epitope” refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding body of an antibody.", "Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.", "As used herein, “receptor” refers to a molecule that has an affinity for a given ligand.", "Receptors can be naturally occurring or synthetic molecules.", "Receptors can be employed in an unaltered state or as aggregates with other species.", "Receptors can be attached, covalently or non-covalently, to a binding member, either directly or via a specific binding substance.", "Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.", "As used herein “ligand” refers to a molecule, such as a random peptide or variable segment sequence, that is recognized by a particular receptor.", "As one of skill in the art will recognize, a molecule (or macromolecular complex) can be both a receptor and a ligand.", "In general, the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor.", "As used herein, “linker” or “spacer” refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.", "4.16.5.3 Methodology Nucleic acid shuffling is a method for in vitro or in vivo homologous recombination of pools of shorter or smaller polynucleotides to produce a polynucleotide or polynucleotides.", "Mixtures of related nucleic acid sequences or polynucleotides are subjected to sexual PCR to provide random polynucleotides, and reassembled to yield a library or mixed population of recombinant hybrid nucleic acid molecules or polynucleotides.", "In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other than primers).", "4.16.5.3.1 Advantage of the Mutagenic Shuffling The advantage of the mutagenic shuffling of this invention over error-prone PCR alone for repeated selection can best be explained with an example from antibody engineering.", "4.16.5.3.2 Inverse Chain Reaction This method differs from error-prone PCR, in that it is an inverse chain reaction.", "In error-prone PCR, the number of polymerase start sites and the number of molecules grows exponentially.", "However, the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same.", "In contrast, in nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time.", "For polynucleotides derived from whole plasmids the theoretical endpoint is a single, large concatemeric molecule.", "Since cross-overs occur at regions of homology, recombination will primarily occur between members of the same sequence family.", "This discourages combinations of CDRs that are grossly incompatible (e.g., directed against different epitopes of the same antigen).", "It is contemplated that multiple families of sequences can be shuffled in the same reaction.", "Further, shuffling generally conserves the relative order, such that, for example, CDR1 will not be found in the position of CDR2.Rare shufflants will contain a large number of the best (eg.", "highest affinity) CDRs and these rare shufflants may be selected based on their superior affinity.", "CDRs from a pool of 100 different selected antibody sequences can be permutated in up to 1006 different ways.", "This large number of permutations cannot be represented in a single library of DNA sequences.", "Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired.", "Error-prone PCR, in contrast, keeps all the selected CDRs in the same relative sequence, generating a much smaller mutant cloud.", "4.16.5.3.3 The Template Polynucleotide The template polynucleotide which may be used in the methods of this invention may be DNA or RNA.", "It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.", "Preferably, the template polynucleotide is from 50 bp to 50 kb.", "It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention, and in fact have been successfully used.", "The template polynucleotide may be obtained by amplification using the PCR reaction (U.S. Pat.", "Nos.", "4,683,202 and 4,683,195) or other amplification or cloning methods.", "However, the removal of free primers from the PCR products before subjecting them to pooling of the PCR products and sexual PCR may provide more efficient results.", "Failure to adequately remove the primers from the original pool before sexual PCR can lead to a low frequency of crossover clones.", "The template polynucleotide often should be double-stranded.", "A double-stranded nucleic acid molecule is recommended to ensure that regions of the resulting single-stranded polynucleotides are complementary to each other and thus can hybridize to form a double-stranded molecule.", "It is contemplated that single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and regions of heterology to the template polynucleotide may be added to the template polynucleotide, at this step.", "It is also contemplated that two different but related polynucleotide templates can be mixed at this step.", "The double-stranded polynucleotide template and any added double- or single-stranded polynucleotides are subjected to sexual PCR which includes slowing or halting to provide a mixture of from about 5 bp to 5 kb or more.", "Preferably the size of the random polynucleotides is from about 10 bp to 1000 bp, more preferably the size of the polynucleotides is from about 20 bp to 500 bp.", "4.16.5.3.4 Use of Double-Stranded Nucleic Acid Having Multiple Nicks Alternatively, it is also contemplated that double-stranded nucleic acid having multiple nicks may be used in the methods of this invention.", "A nick is a break in one strand of the double-stranded nucleic acid.", "The distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.", "This can provide areas of self-priming to produce shorter or smaller polynucleotides to be included with the polynucleotides resulting from random primers, for example.", "The concentration of any one specific polynucleotide will not be greater than 1% by weight of the total polynucleotides, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.", "The number of different specific polynucletides in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.4.16.5.3.5 Increasing the Heterogeneity of the Mixture of Polynucleotides At this step single-stranded or double-stranded polynucleotides, either synthetic or natural, may be added to the random double-stranded shorter or smaller polynucleotides in order to increase the heterogeneity of the mixture of polynucleotides.", "It is also contemplated that populations of double-stranded randomly broken polynucleotides may be mixed or combined at this step with the polynucleotides from the sexual PCR process and optionally subjected to one or more additional sexual PCR cycles.", "Where insertion of mutations into the template polynucleotide is desired, single-stranded or double-stranded polynucleotides having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded polynucleotides may be added in a 10 fold excess by weight as compared to the total nucleic acid.", "Where a mixture of different but related template polynucleotides is desired, populations of polynucleotides from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1: 40.For example, a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations (e.g., mutations yielding an insubstantial alteration in the phenotypic property being selected for).", "In such an example, the ratio of randomly provided wild-type polynucleotides which may be added to the randomly provided sexual PCR cycle hybrid polynucleotides is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.4.16.5.3.5.1 Denaturing and Re-annealing The mixed population of random polynucleotides are denatured to form single-stranded polynucleotides and then re-annealed.", "Only those single-stranded polynucleotides having regions of homology with other single-stranded polynucleotides will re-anneal.", "The random polynucleotides may be denatured by heating.", "One skilled in the art could determine the conditions necessary to completely denature the double-stranded nucleic acid.", "Preferably the temperature is from 80 C to 100 C, more preferably the temperature is from 90 C to 96 C. other methods which may be used to denature the polynucleotides include pressure (36) and pH.", "The polynucleotides may be re-annealed by cooling.", "Preferably the temperature is from 20 C to 75 C, more preferably the temperature is from 40 C to 65 C. If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low annealing temperature, although the process becomes more difficult.", "The degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded polynucleotides.", "Renaturation can be accelerated by the addition of polyethylene glycol (“PEG”) or salt.", "The salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mm.", "The salt may be KCl or NaCl.", "The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to 10%.", "4.16.5.3.5.2 Incubation The annealed polynucleotides are next incubated in the presence of a nucleic acid polymerase and dNTP's (i.e.", "dATP, dCTP, DGTP and dTTP).", "The nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.", "The approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers.", "If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65 C. If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30 C. One skilled in the art could vary the temperature of annealing to increase the number of cross-overs achieved.", "The polymerase may be added to the random polynucleotides prior to annealing, simultaneously with annealing or after annealing.", "The cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid.", "This cycle is repeated for a desired number of times.", "Preferably the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.", "4.16.5.3.6 The Resulting Nucleic Acid The resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to 50 kb.", "This larger polynucleotides may contain a number of copies of a polynucleotide having the same size as the template polynucleotide in tandem.", "This concatemeric polynucleotide is then denatured into single copies of the template polynucleotide.", "The result will be a population of polynucleotides of approximately the same size as the template polynucleotide.", "The population will be a mixed population where single or double-stranded polynucleotides having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.", "These polynucleotides are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.", "It is contemplated that the single polynucleotides may be obtained from the larger concatemeric polynucleotide by amplification of the single polynucleotide prior to cloning by a variety of methods including PCR (U.S. Pat.", "Nos.", "4,683,195 and 4,683,202), rather than by digestion of the concatemer.", "4.16.5.3.7 Vectors Used for Cloning The vector used for cloning is not critical provided that it will accept a polynucleotide of the desired size.", "If expression of the particular polynucleotide is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the polynucleotide to allow expression of the polynucleotide in the host cell.", "Preferred vectors include the pUC series and the pBR series of plasmids.", "4.16.5.3.8 The Resulting Bacterial Population The resulting bacterial population will include a number of recombinant polynucleotides having random mutations.", "This mixed population may be tested to identify the desired recombinant polynucleotides.", "The method of selection will depend on the polynucleotide desired.", "For example, if a polynucleotide which encodes a protein with increased binding efficiency to a ligand is desired, the proteins expressed by each of the portions of the polynucleotides in the population or library may be tested for their ability to bind to the ligand by methods known in the art (i.e.", "panning, affinity chromatography).", "If a polynucleotide which encodes for a protein with increased drug resistance is desired, the proteins expressed by each of the polynucleotides in the population or library may be tested for their ability to confer drug resistance to the host organism.", "One skilled in the art, given knowledge of the desired protein, could readily test the population to identify polynucleotides which confer the desired properties onto the protein.", "It is contemplated that one skilled in the art could use a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee Wis.).", "The recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is encoded by the recombinant DNA molecule.", "The phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell.", "The leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle.", "Thus the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.", "4.16.5.3.9 Cycles of Nucleic Acid Shuffling It is further contemplated that a number of cycles of nucleic acid shuffling may be conducted with polynucleotides from a sub-population of the first population, which sub-population contains DNA encoding the desired recombinant protein.", "In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.", "It is also contemplated that a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type polynucleotides and a sub-population of nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to remove any silent mutations from the sub-population.", "4.16.5.3.10 The Starting Nucleic Acid Any source of nucleic acid, in purified form can be utilized as the starting nucleic acid.", "Thus the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded.", "In addition, a DNA-RNA hybrid which contains one strand of each may be utilized.", "The nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated.", "Preferably the specific nucleic acid sequence is from 50 to 50000 base pairs.", "It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of this invention.", "The nucleic acid may be obtained from any source, for example, from plasmids such a pBR322, from cloned DNA or RNA or from natural DNA or RNA from any source including bacteria, yeast, viruses and higher organisms such as plants or animals.", "DNA or RNA may be extracted from blood or tissue material.", "The template polynucleotide may be obtained by amplification using the polynucleotide chain reaction (PCR) (U.S. Pat.", "Nos.", "4,683,202 and 4,683,195).", "Alternatively, the polynucleotide may be present in a vector present in a cell and sufficient nucleic acid may be obtained by culturing the cell and extracting the nucleic acid from the cell by methods known in the art.", "Any specific nucleic acid sequence can be used to produce the population of hybrids by the present process.", "It is only necessary that a small population of hybrid sequences of the specific nucleic acid sequence exist or be created prior to the present process.", "4.16.5.3.11 Creation of the Initial Population of Sequences The initial small population of the specific nucleic acid sequences having mutations may be created by a number of different methods.", "Mutations may be created by error-prone PCR.", "Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence.", "Alternatively, mutations can be introduced into the template polynucleotide by oligonucleotide-directed mutagenesis.", "In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence.", "The polynucleotide sequence can also be altered by chemical mutagenesis.", "Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.", "other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.", "Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence.", "Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.", "Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.", "Generally, plasmid polynucleotides so mutagenized are introduced into E. coli and propagated as a pool or library of hybrid plasmids.", "Alternatively the small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species (i.e., cognate genes).", "Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.", "Once the mixed population of the specific nucleic acid sequences is generated, the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in the art.", "4.16.5.3.11.1 The Choice of Vector The choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention.", "The templates of this invention may be plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof (e.g., coat protein, spike glycoprotein, capsid protein).", "For example, cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large polynucleotides.", "4.16.5.3.11.2 Clonal Amplification If the mixed population of the specific nucleic acid sequence is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector.", "This is referred to as clonal amplification because while the absolute number of nucleic acid sequences increases, the number of hybrids does not increase.", "Utility can be readily determined by screening expressed polypeptides.", "4.16.5.3.12 Incorporation of Any Sequence Mixture at Any Specific Position The DNA shuffling method of this invention can be performed blindly on a pool of unknown sequences.", "By adding to the reassembly mixture oligonucleotides (with ends that are homologous to the sequences being reassembled) any sequence mixture can be incorporated at any specific position into another sequence mixture.", "Thus, it is contemplated that mixtures of synthetic oligonucleotides, PCR polynucleotides or even whole genes can be mixed into another sequence library at defined positions.", "The insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template.", "Thus, the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.", "This approach of mixing two genes may be useful for the humanization of antibodies sequences into genes may be useful for any therapeutically used protein, for example, interleukin 1, antibodies, tPA and growth hormone.", "The approach may also be useful in any nucleic acid for example, promoters or introns or 3′ untranslated region or 5′ untranslated regions of genes to increase expression or alter specificity of expression of proteins.", "The approach may also be used to mutate ribozymes or aptamers.", "4.16.5.3.13 Creation of Scaffold-Like Proteins Shuffling requires the presence of homologous regions separating regions of diversity.", "Scaffold-like protein structures may be particularly suitable for shuffling.", "The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding.", "Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle which are well-known in the art.", "This shuffling can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.", "4.16.5.4 In vitro Shuffling The equivalents of some standard genetic matings may also be performed by shuffling in vitro.", "For example, a “molecular backcross” can be performed by repeatedly mixing the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.", "As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice.", "It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e.", "immunogenicity).", "Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not, an advantage which cannot be achieved by error-prone mutagenesis or cassette mutagenesis methods.", "Large, functional genes can be assembled correctly from a mixture of small random polynucleotides.", "This reaction may be of use for the reassembly of genes from the highly fragmented DNA of fossils.", "In addition random nucleic acid fragments from fossils may be combined with polynucleotides from similar genes from related species.", "4.16.5.4.1 In Vitro Amplification of a Genome It is also contemplated that the method of this invention can be used for the in vitro amplification of a whole genome from a single cell as is needed for a variety of research and diagnostic applications.", "DNA amplification by PCR is in practice limited to a length of about 40 kb.", "Amplification of a whole genome such as that of E. coli (5,000 kb) by PCR would require about 250 primers yielding 125 forty kb polynucleotides.", "This approach is not practical due to the unavailability of sufficient sequence data.", "On the other hand, random production of polynucleotides of the genome with sexual PCR cycles, followed by gel purification of small polynucleotides will provide a multitude of possible primers.", "Use of this mix of random small polynucleotides as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatemer containing many copies of the genome.", "100 fold amplification in the copy number and an average polynucleotide size of greater than 50 kb may be obtained when only random polynucleotides are used.", "It is thought that the larger concatemer is generated by overlap of many smaller polynucleotides.", "The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA.", "It is expected that this approach will be useful for the mapping of genomes.", "The polynucleotide to be shuffled can be produced as random or non-random polynucleotides, at the discretion of the practitioner.", "4.16.5.5 In Vivo Shuffling In an embodiment of in vivo shuffling, the mixed population of the specific nucleic acid sequence is introduced into bacterial or eukaryotic cells under conditions such that at least two different nucleic acid sequences are present in each host cell.", "The polynucleotides can be introduced into the host cells by a variety of different methods.", "The host cells can be transformed with the smaller polynucleotides using methods known in the art, for example treatment with calcium chloride.", "If the polynucleotides are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences.", "Alternatively, the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.", "In general, in this embodiment, the specific nucleic acids sequences will be present in vectors which are capable of stably replicating the sequence in the host cell.", "In addition, it is contemplated that the vectors will encode a marker gene such that host cells having the vector can be selected.", "This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.", "However, it is contemplated that the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence.", "Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the polynucleotides into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein.", "It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.", "4.16.5.5.1 Homologous Recombination It has been found that when two polynucleotides which have regions of identity are inserted into the host cells homologous recombination occurs between the two polynucleotides.", "Such recombination between the two mutated specific nucleic acid sequences will result in the production of double or triple hybrids in some situations.", "4.16.5.5.2 Increase in the Frequency of Recombination It has also been found that the frequency of recombination is increased if some of the mutated specific nucleic acid sequences are present on linear nucleic acid molecules.", "Therefore, in a preferred embodiment, some of the specific nucleic acid sequences are present on linear polynucleotides.", "4.16.5.5.3 Identification of Host Cell Transformants Containing Desired Sequences After transformation, the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired.", "For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected.", "If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand.", "Alternatively, the protein can be expressed in another system to ensure proper processing.", "Once a subset of the first recombined specific nucleic acid sequences (daughter sequences) having the desired characteristics are identified, they are then subject to a second round of recombination.", "4.16.5.5.4 The Second Cycle of Recombination In the second cycle of recombination, the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above.", "In this way a set of second recombined specific nucleic acids sequences can be identified which have enhanced characteristics or encode for proteins having enhanced properties.", "This cycle can be repeated a number of times as desired.", "It is also contemplated that in the second or subsequent recombination cycle, a backcross can be performed.", "A molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type sequence, such that at least one wild-type nucleic acid sequence and a mutated nucleic acid sequence are present in the same host cell after transformation.", "Recombination with the wild-type specific nucleic acid sequence will eliminate those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.", "4.16.5.5.5 Generation of a Subset of the Specific Nucleic Acid Sequences In another embodiment of this invention, it is contemplated that during the first round a subset of the specific nucleic acid sequences can be generated as smaller polynucleotides by slowing or halting their PCR amplification prior to introduction into the host cell.", "The size of the polynucleotides must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences.", "The size of the polynucleotides will range from 0.03 kb to 100 kb more preferably from 0.2 kb to 10 kb.", "It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be utilized to generate PCR polynucleotides prior to introduction into the host cells.", "The shorter polynucleotide sequences can be single-stranded or double-stranded.", "If the sequences were originally single-stranded and have become double-stranded they can be denatured with heat, chemicals or enzymes prior to insertion into the host cell.", "The reaction conditions suitable for separating the strands of nucleic acid are well known in the art.", "The steps of this process can be repeated indefinitely, being limited only by the number of possible hybrids which can be achieved.", "After a certain number of cycles, all possible hybrids will have been achieved and further cycles are redundant.", "In an embodiment the same mutated template nucleic acid is repeatedly recombined and the resulting recombinants selected for the desired characteristic.", "4.16.5.5.6 Cloning into a Vector Capable of Replicating in a Bacteria Therefore, the initial pool or population of mutated template nucleic acid is cloned into a vector capable of replicating in a bacteria such as E. colil.", "The particular vector is not essential, so long as it is capable of autonomous replication in E. coli.", "In a preferred embodiment, the vector is designed to allow the expression and production of any protein encoded by the mutated specific nucleic acid linked to the vector.", "It is also preferred that the vector contain a gene encoding for a selectable marker.", "The population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells.", "The vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage.", "The concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell.", "Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors.", "This results in the generation of hybrids (daughters) having a combination of mutations which differ from the original parent mutated sequences.", "The host cells are then clonally replicated and selected for the marker gene present on the vector.", "Only those cells having a plasmid will grow under the selection.", "4.16.5.5.7 Testing for the Presence of Favorable Mutations The host cells which contain a vector are then tested for the presence of favorable mutations.", "Such testing may consist of placing the cells under selective pressure, for example, if the gene to be selected is an improved drug resistance gene.", "If the vector allows expression of the protein encoded by the mutated nucleic acid sequence, then such selection may include allowing expression of the protein so encoded, isolation of the protein and testing of the protein to determine whether, for example, it binds with increased efficiency to the ligand of interest.", "4.16.5.5.8 Isolation of the Desired Nucleic Acid Sequence Once a particular daughter mutated nucleic acid sequence has been identified which confers the desired characteristics, the nucleic acid is isolated either already linked to the vector or separated from the vector.", "This nucleic acid is then mixed with the first or parent population of nucleic acids and the cycle is repeated.", "It has been shown that by this method nucleic acid sequences having enhanced desired properties can be selected.", "4.16.5.5.9 Addition of Parental Mutated Sequences to the Cells Containing the First Generation of Hybrids In an alternate embodiment, the first generation of hybrids are retained in the cells and the parental mutated sequences are added again to the cells.", "Accordingly, the first cycle of Embodiment I is conducted as described above.", "However, after the daughter nucleic acid sequences are identified, the host cells containing these sequences are retained.", "The parent mutated specific nucleic acid population, either as polynucleotides or cloned into the same vector is introduced into the host cells already containing the daughter nucleic acids.", "Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above.", "This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained.", "It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the preferred hybrids may come from the parental hybrids or any subsequent generation.", "4.16.5.5.10 “Molecular” Backcross to Eliminate Any Neutral Mutations In an alternative embodiment, the invention provides a method of conducting a “molecular” backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations.", "Neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties.", "Such mutations may however confer on the nucleic acid or peptide undesirable characteristics.", "Accordingly, it is desirable to eliminate such neutral mutations.", "The method of this invention provide a means of doing so.", "In this embodiment, after the hybrid nucleic acid, having the desired characteristics, is obtained by the methods of the embodiments, the nucleic acid, the vector having the nucleic acid or the host cell containing the vector and nucleic acid is isolated.", "The nucleic acid or vector is then introduced into the host cell with a large excess of the wild-type nucleic acid.", "The nucleic acid of the hybrid and the nucleic acid of the wild-type sequence are allowed to recombine.", "The resulting recombinants are placed under the same selection as the hybrid nucleic acid.", "Only those recombinants which retained the desired characteristics will be selected.", "Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA.", "This cycle can be repeated a number of times until all of the silent mutations are eliminated.", "Thus the methods of this invention can be used in a molecular backcross to eliminate unnecessary or silent mutations.", "4.16.5.6 Utility The in vivo recombination method of this invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence.", "However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide.", "The approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin 1, antibodies, tPA and growth hormone.", "This approach may be used to generate proteins having altered specificity or activity.", "The approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes.", "Thus this approach may be used to generate genes having increased rates of expression.", "This approach may also be useful in the study of repetitive DNA sequences.", "Finally, this approach may be useful to mutate ribozymes or aptamers.", "Scaffold-like regions separating regions of diversity in proteins may be particularly suitable for the methods of this invention.", "The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding.", "Examples of such scaffolds are the immunoglobulin beta barrel, and the four-helix bundle.", "The methods of this invention can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.", "The equivalents of some standard genetic matings may also be performed by the methods of this invention.", "For example, a “molecular” backcross can be performed by repeated mixing of the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest.", "As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice.", "It is useful, for example, for the removal of neutral mutations that affect unselected characteristics (i.e.", "immunogenicity).", "Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not.", "4.16.5.7 Peptide Display Methods The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype (e.g., for affinity for a predetermined receptor (ligand).", "An increasingly important aspect of bio-pharmaceutical drug development and molecular biology is the identification of peptide structures, including the primary amino acid sequences, of peptides or peptidomimetics that interact with biological macromolecules.", "one method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor), involves the screening of a large library or peptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the peptide.", "In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods also have been reported.", "One type involves the display of a peptide sequence, antibody, or other protein on the surface of a bacteriophage particle or cell.", "Generally, in these methods each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences.", "Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.", "A well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein.", "The bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule.", "The bacteriophage particles (i.e., library members) which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage sub-population for a subsequent round of affinity enrichment and phage replication.", "After several rounds of affinity enrichment and phage replication, the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor).", "Such methods are further described in PCT patent publication Nos.", "91/17271, 91/18980, and 91/19818 and 93/08278.The latter PCT publication describes a recombinant DNA method for the display of peptide ligands that involves the production of a library of fusion proteins with each fusion protein composed of a first polypeptide portion, typically comprising a variable sequence, that is available for potential binding to a predetermined macromolecule, and a second polypeptide portion that binds to DNA, such as the DNA vector encoding the individual fusion protein.", "When transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the DNA vector encoding it.", "Upon lysis of the host cell, the fusion protein/vector DNA complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).", "4.16.5.7.1 Hybrid Methods for Generating Libraries of Peptides and Like Polymers Other systems for generating libraries of peptides and like polymers have aspects of both the recombinant and in vitro chemical synthesis methods.", "In these hybrid methods, cell-free enzymatic machinery is employed to accomplish the in vitro synthesis of the library members (i.e., peptides or polynucleotides).", "In one type of method, RNA molecules with the ability to bind a predetermined protein or a predetermined dye molecule were selected by alternate rounds of selection and PCR amplification (Tuerk and Gold (1990) Science 249: 505; Ellington and Szostak (1990) Nature 346: 818).", "A similar technique was used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach (1990) Nucleic Acids Res.", "18: 3203; Beaudry and Joyce (1992) Science 257; 635; PCT patent publication Nos.", "92/05258 and 92/14843).", "In a similar fashion, the technique of in vitro translation has been used to synthesize proteins of interest and has been proposed as a method for generating large libraries of peptides.", "These methods which rely upon in vitro translation, generally comprising stabilized polysome complexes, are described further in PCT patent publication Nos.", "88/08453, 90/05785, 90/07003, 91/02076, 91/05058, and 92/02536.Applicants have described methods in which library members comprise a fusion protein having a first polypeptide portion with DNA binding activity and a second polypeptide portion having the library member unique peptide sequence; such methods are suitable for use in cell-free in vitro selection formats, among others.", "4.16.5.7.2 The Displayed Peptide Sequences The displayed peptide sequences can be of varying lengths, typically from 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and often from about 8-15 amino acids long.", "A library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence.", "Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kernal, fixed, or the like.", "The present display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scfv displayed on phage, which enable large-scale screening of scfv libraries having broad diversity of variable region sequences and binding specificities.", "4.16.5.7.3 Sequence Framework Peptide Libraries The present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds (e.g., peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion.", "The random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized.", "The mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or incorporation in a cell.", "4.16.5.7.4 Selecting for the Desired Peptide Using Affinity Enrichment A method of affinity enrichment allows a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected.", "The polynucleotide can then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just VHI, VLI or CDR portions thereof).", "Using these methods, one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of shuffling to converge rapidly to a desired high-affinity peptide or scfv.", "The peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic or diagnostic agent).", "A significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest.", "The peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.", "4.16.5.7.5 Shuffling Sequences Selected by Affinity Screening The present invention also provides a method for shuffling a pool of polynucleotide sequences selected by affinity screening a library of polysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor (e.g., a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like) or epitope (e.g., an immobilized protein, glycoprotein, oligosaccharide, and the like).", "Polynucleotide sequences selected in a first selection round (typically by affinity selection for binding to a receptor (e.g., a ligand)) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.", "The recombined selected polynucleotide sequences are subjected to at least one subsequent selection round.", "The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection.", "Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by back-crossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic.", "Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).", "Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized.", "In one embodiment, selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced.", "Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member.", "The sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence.", "The mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.", "4.16.5.7.6 Peptide Libraries Comprising a Plurality of Individual Library Members The invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein (1) each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and (2) each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is distinct from the variable peptide segment sequences or single-chain antibody sequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).", "4.16.5.7.7 Product-by-Process The invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, (2) shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and (3) screening the shuffled library against the predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the predetermined receptor or epitope to produce a pool of selected shuffled library members.", "4.16.5.8 Antibody Display and Screening Methods The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype (e.g., for affinity for binding a predetermined antigen (ligand).", "Various molecular genetic approaches have been devised to capture the vast immunological repertoire represented by the extremely large number of distinct variable regions which can be present in immunoglobulin chains.", "The naturally-occurring germ line immunoglobulin heavy chain locus is composed of separate tandem arrays of variable segment genes located upstream of a tandem array of diversity segment genes, which are themselves located upstream of a tandem array of joining (i) region genes, which are located upstream of the constant region genes.", "During B lymphocyte development, V-D-J rearrangement occurs wherein a heavy chain variable region gene (VH) is formed by rearrangement to form a fused D segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (VH) of, a heavy chain.", "Similarly, light chain loci rearrange one of several V segments with one of several J segments to form a gene encoding the variable region (VL) of a light chain.", "4.16.5.8.1 Sequence Diversity The vast repertoire of variable regions possible in immunoglobulins derives in part from the numerous combinatorial possibilities of joining V and i segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development.", "Additional sequence diversity in the heavy chain variable regions arises from non-uniform rearrangements of the D segments during V-D-J joining and from N region addition.", "Further, antigen-selection of specific B cell clones selects for higher affinity variants having non-germline mutations in one or both of the heavy and light chain variable regions; a phenomenon referred to as “affinity maturation” or “affinity sharpening”.", "Typically, these “affinity sharpening” mutations cluster in specific areas of the variable region, most commonly in the complementarity-determining regions (CDRs).", "4.16.5.8.2 Prokaryotic Epression Systems In order to overcome many of the limitations in producing and identifying high-affinity immunoglobulins through antigen-stimulated B cell development (i.e., immunization), various prokaryotic expression systems have been developed that can be manipulated to produce combinatorial antibody libraries which may be screened for high-affinity antibodies to specific antigens.", "Recent advances in the expression of antibodies in Escherichia coli and bacteriophage systems (see, “Alternative Peptide Display Methods”, infra) have raised the possibility that virtually any specificity can be obtained by either cloning antibody genes from characterized hybridomas or by de novo selection using antibody gene libraries (e.g., from Ig cDNA).", "Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al.", "(1989) Science 246: 1275; Caton and Koprowski (1990) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 87: 6450; Mullinax et al (1990) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 87: 8095; Persson et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 88: 2432).", "Various embodiments of bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 88: 4363; Clackson et al.", "(1991) Nature 352: 624; McCafferty et al.", "(1990) Nature 348: 552; Burton et al.", "(1991) Proc.", "Natl Acad.", "Sci.", "(U.S.A.) 88: 10134; Hoogenboom et al.", "(1991) Nucleic Acids Res.", "19: 4133; Chang et al.", "(1991) J. Immunol.", "147: 3610; Breitling et al.", "(1991) Gene 104: 147; Marks et al.", "(1991) J. Mol.", "Biol.", "222%: 581; Barbas et al.", "(1992) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 89: 4457; Hawkins and Winter (1992) J. Immunol.", "22: 867; Marks et al.", "(1992) Biotechnology 10: 779; Marks et al.", "(1992) J. Biol.", "Chem.", "267: 16007; Lowman et al (1991) Biochemistry 30: 10832; Lerner et al.", "(1992) Science.", "258: 1313, incorporated herein by reference).", "Typically, a bacteriophage antibody display library is screened with a receptor (e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled (e.g., to screen plaque or colony lifts).", "4.16.5.8.3 Single-Chain Fragment Variable Libraries One particularly advantageous approach has been the use of so-called single-chain fragment variable (scfv) libraries (Marks et al.", "(1992) Biotechnology 10: 779; Winter G and Milstein C (1991) Nature 349: 293; Clackson et al.", "(1991) op.", "cit.", "; Marks et al.", "(1991) J. Mol.", "Biol.", "222: 581; Chaudhary et al.", "(1990) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 87: 1066; Chiswell et al.", "(1992) TIBTECH 10: 80; McCafferty et al.", "(1990) op.cit.", "; and Huston et al-(1988) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 85: 5879).", "Various embodiments of scfv libraries displayed on bacteriophage coat proteins have been described.", "Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods.", "The first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.", "The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s).", "The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH′ In principle, the scfv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.", "Thus, scfv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide.", "After the scfv genes are assembled, they are cloned into a phagemid and expressed at the tip of the M13 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage Pill (gene 3) coat protein.", "Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scfv for binding to a predetermined epitope (e.g., target antigen, receptor).", "The linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence.", "The displayed peptide (s) or single-chain antibody (e.g., scfv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system.", "often polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, and the like.", "Often polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like.", "Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.", "Various other uses of such antibodies are to diagnose and/or stage disease (e.g., neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.", "4.16.5.8.4 Increasing the Combinatorial Diversity of a SCFV Library Various methods have been reported for increasing the combinatorial diversity of a scfv library to broaden the repertoire of binding species (idiotype spectrum) The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunized cells, affording combinatorial diversity in the assortment of VH and VL cassettes which can be combined.", "Furthermore, the VH and VL cassettes can themselves be diversified, such as by random, pseudorandom, or directed mutagenesis.", "Typically, VH and VL cassettes are diversified in or near the complementarity-determining regions (CDRS), often the third CDR, CDR3.Enzymatic inverse PCR mutagenesis has been shown to be a simple and reliable method for constructing relatively large libraries of scfv site-directed hybrids (Stemmer et al.", "(1993) Biotechniques 14: 256), as has error-prone PCR and chemical mutagenesis (Deng et al.", "(1994) J. Biol.", "Chem.", "269: 953 3).", "Riechmann et al.", "(1993) Biochemistry 32: 8848 showed semi-rational design of an antibody scfv fragment using site-directed randomization by degenerate oligonucleotide PCR and subsequent phage display of the resultant scfv hybrids.", "Barbas et al.", "(1992) on.cit.", "attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.", "CDR randomization has the potential to create approximately 1×1020 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants.", "Taken individually or together, the combination possibilities of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding.", "Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems.", "For example, Barbas et al.", "(1992) op.cit.", "only generated 5×107 transformants, which represents only a tiny fraction of the potential diversity of a library of thoroughly randomized CDRS.", "Despite these substantial limitations, bacteriophage.", "display of scfv have already yielded a variety of useful antibodies and antibody fusion proteins.", "A bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al.", "(1994) J. Immunol.", "152: 5368).", "Intracellular expression of an anti-Rev scfv has been shown to inhibit HIV-1 virus replication in vitro (Duan et al.", "(1994) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 91: 5075), and intracellular expression of an anti-p21rar, scfv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al.", "(1993) Biochem.", "Bioshys.", "Res.", "Commun.", "197: 422.Recombinant scfv which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility of scfv (Lilley et al.", "(1994) J. Immunol.", "Meth.", "171: 211).", "Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al.", "(1992) Eur.", "J. Biochess.", "210: 945; Nicholls et al.", "(1993) J. Biol.", "Chem.", "268: 5302).", "4.16.5.8.5 Use of in vitro and in vivo Shuffling Methods to Recombine CDRs If it were possible to generate scfv libraries having broader antibody diversity and overcoming many of the limitations of conventional CDR mutagenesis and randomization methods which can cover only a very tiny fraction of the potential sequence combinations, the number and quality of scfv antibodies suitable for therapeutic and diagnostic use could be vastly improved.", "To address this, the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies.", "Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s).", "In a variation, the CDRs are initially obtained from MRNA (or CDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO92/03918, WO93/12227, and WO94/25585), including hybridomas derived therefrom.", "Polynucleotide sequences selected in a first selection round (typically by affinity selection for displayed antibody binding to an antigen (e.g., a ligand) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRS) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.", "The recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round.", "The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained.", "Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by back-crossing with a human variable region framework to produce human-like sequence antibodies.", "Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined antigen.", "4.16.5.8.6 Controlling the Average Binding Affinity of Selected SCFV Library Members Alternatively, or in combination with the noted variations, the valency of the target epitope may be varied to control the average binding affinity of selected scfv library members.", "The target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art.", "A high density (valency) of predetermined epitope can be used to enrich for scfv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scfv library members.", "4.16.5.8.7 Generating Diverse Variable Segments For generating diverse variable segments, a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kernal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR).", "Similarly, the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like.", "The resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.", "Displayed peptide/polynucleotide complexes (library members) which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique.", "This is accomplished by means of a immobilized macromolecule or epitope specific for the peptide sequence of interest, such as a receptor, other macromolecule, or other epitope species.", "Repeating the affinity selection procedure provides an enrichment of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity enrichment.", "The library members without the desired specificity are removed by washing.", "The degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope.", "A certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing.", "The temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule.", "Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route.", "This may be done either by continued incubation in the presence of a saturating amount of free predetermined macromolecule, or by increasing the volume, number, and length of the washes.", "In each case, the rebinding of dissociated nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.", "Additional modifications of the binding and washing procedures may be applied to find peptides with special characteristics.", "The affinities of some peptides are dependent on ionic strength or cation concentration.", "This is a useful characteristic for peptides that will be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptides are required.", "One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scfv library can be simultaneously screened for a multiplicity of scfv which have different binding specificities.", "Given that the size of a scfv library often limits the diversity of potential scfv sequences, it is typically desirable to us scfv libraries of as large a size as possible.", "The time and economic considerations of generating a number of very large polysome scFv-display libraries can become prohibitive.", "To avoid this substantial problem, multiple predetermined epitope species (receptor species) can be concomitantly screened in a single library, or sequential screening against a number of epitope species can be used.", "In one variation, multiple target epitope species, each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scfv library under suitable binding conditions.", "The collection of beads, comprising multiple epitope species, can then be used to isolate, by affinity selection, scfv library members.", "Generally, subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species.", "This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.", "4.16.5.8.8 Techniques Used to Diversify a Peptide Library or Single-Chain Antibody Library A variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope.", "In one approach, the positive selected peptide/polynucleotide complexes (those identified in an early round of affinity enrichment) are sequenced to determine the identity of the active peptides.", "Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences.", "This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations.", "This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled.", "It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.", "In a variation, mutagenic PCR amplification of positive selected peptide/polynucleotide complexes (especially of the variable region sequences, the amplification products of which are shuffled in vitro and/or in vivo and one or more additional rounds of screening is done prior to sequencing.", "The same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling.", "If desired, shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included.", "Thus, mixtures of synthetic oligonucleotides and PCR produced polynucleotides (synthesized by error-prone or high-fidelity methods) can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).", "4.16.5.8.9 Generation of a Library of CDR-Varient Single-Chain Antibodies The present invention of shuffling enables the generation of a vast library of CDR-variant single-chain antibodies.", "One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling.", "The sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences.", "For example, a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat et al.", "(1991) op cit.", "; the pool (s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence.", "Alternatively, a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are incorporated into the synthetic CDRs at the corresponding position(s).", "Typically, several (e.g., 3 to about 50) known CDR sequences are compared and observed natural sequence variations between the known CDRs are tabulated, and a collection of oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized.", "For example but not for limitation, if a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids.", "In some embodiments, residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences.", "Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences.", "Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.", "The collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence.", "It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions.", "A collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1×10 6 unique CDR sequences are included in the collection, although occasionally 1×107 to 1×108 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare (i.e., less than 0.1 percent) in that position in naturally-occurring human CDRS.", "In general, the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of 10.Such single-chain antibodies generally bind of about at least 1×10 m-, preferably with an affinity of about at least 5×10 (superscript 7) M-1, more preferably with an affinity of at least 1×10 (superscript 8) M-1 to 1×10 (superscript 9) M-1 or more, sometimes up to 1×10 (superscript 10) M-1 or more.", "Frequently, the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, Lselectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.", "4.16.5.8.10 Expression Systems High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems.", "For example, scfv have been produced in plants (Firek et al.", "(1993) Plant Mol.", "Biol.", "23: 861) and can be readily made in prokaryotic systems (Owens R J and Young R J (1994) J. Immunol.", "Meth.", "168: 149; Johnson S and Bird R E (1991) Methods Enzymol 203: 88).", "Furthermore, the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al.", "(1994) Eur.", "J. Immunol.", "24: 952).", "The variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized.", "The polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.", "The DNA expression constructs will typically include an expression control DNA sequence operably linked to the coding sequences, including naturally-associated or heterologous promoter regions.", "Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.", "Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the mutant “engineered” antibodies.", "As stated previously, the DNA sequences will be expressed in hosts after the sequences have been operably linked to an expression control sequence (Le., positioned to ensure the transcription and translation of the structural gene).", "These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.", "Commonly, expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat.", "No.", "4,704,362, which is incorporated herein by reference).", "4.16.5.8.11 Mammalian Tissue Cell Culture In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell culture may also be used to produce the polypeptides of the present invention (see, Winnacker, “From Genes to Clones,” VCH Publishers, N., N.Y. (1987), which is incorporated herein by reference).", "Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, and myeloma cell lines, but preferably transformed Bcells or hybridomas.", "Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al.", "(1986) Immunol.", "Rev.", "89: 49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.", "Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.", "Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the vector.", "Enhancers are cis-acting sequences of between 10 to 300 bp that increase transcription by a promoter.", "Enhancers can effectively increase transcription when either 51 or 31 to the transcription unit.", "They are also effective if located within an intron or within the coding sequence itself.", "Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers.", "Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.", "Mammalian expression vector systems will also typically include a selectable marker gene.", "Examples of suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), or prokaryotic genes conferring drug resistance.", "The first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium.", "Transformed cells can then be identified by their ability to grow on non-supplemented media.", "Examples of prokaryotic drug resistance genes useful as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.", "The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.", "For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment.", "lipofection, or electroporation may be used for other cellular hosts.", "Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation, and micro-injection (see, generally, Sambrook et al., supra).", "Once expressed, the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982)).", "once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Immunological Methods, Vols.", "I and II, Eds.", "Lefkovits and Pemis, Academic Press, New York, N.Y. (1979 and 1981)).", "The antibodies generated by the method of the present invention can be used for diagnosis and therapy.", "By way of illustration and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections.", "For treatment of cancer, the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.", "4.16.5.9 Yeast Two Hybrid Screening Assays Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence.", "The selected library members are pooled and shuffled by in vitro and/or in vivo recombination.", "The shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence (e.g., and SH2 domain) or which bind an alternate predetermined polypeptide sequence (e.g., an SH2 domain from another protein species).", "An approach to identifying polypeptide sequences which bind to a predetermined polypeptide sequence has been to use a so-called “two-hybrid” system wherein the predetermined polypeptide sequence is present in a fusion protein (Chien et al.", "(1991) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 88: 9578).", "This approach identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator (Fields S and Song 0 (1989) Nature 340: 245), the yeast Gal4 transcription protein.", "Typically, the method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation.", "Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a polypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell.", "Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene (e.g., lacz, HIS3) which is operably linked to a Gal4 binding site.", "Typically, the two-hybrid method is used to identify novel polypeptide sequences which interact with a known protein (Silver S C and Hunt S W (1993) Mol.", "Biol.", "Rep. 17: 155; Durfee et al.", "(1993) Genes Devel.", "7: 555; Yang et al.", "(1992) Science 257: 680; Luban et al.", "(1993) Cell 73: 1067; Hardy et al (1992) Genes Devel.", "6; 801; Bartel et al.", "(1993) Biotechniques 14: 920; and Vojtek et al.", "(1993) Cell 74: 205).", "However, variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li B and Fields S (1993) FASEB J.", "7: 957; Lalo et al.", "(1993) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 90: 5524; Jackson et al.", "(1993) Mol.", "Cell.", "Biol.", "13; 2899; and Madura et al.", "(1993) J. Biol-Chem.", "268: 12046).", "Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al.", "(1993) med.", "Microbial.", "8: 1177; Chakrabarty et al.", "(1992) J. Biol.", "Chem.", "267: 17498; Staudinger et al.", "(1993) J. Biol.", "Chem.", "268: 4608; and Milne G T. and Weaver D T (1993) Genes Devel.", "7; 1755) or domains responsible for oligomerization of a single protein (Iwabuchi et al.", "(1993) Oncogene 8; 1693; Bogerd et al.", "(1993) J. Virol.", "67: 5030).", "Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al.", "(1992) Proc.", "Natl.", "Acad.", "Sci.", "(USA) 89: 4159).", "Alternatively, an E. coli/BCCP interactive screening system (Germino et al.", "(1993) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 90: 933; Guarente L (1993) Proc.", "Natl.", "Acad.", "Sci.", "(U.S.A.) 90: 1639) can be used to identify interacting protein sequences (i.e., protein sequences which heterodimerize or form higher order heteromultimers).", "Sequences selected by a two-hybrid system can be pooled and shuffled and introduced into a two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence.", "The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kernals.", "In general, standard techniques of recombination DNA technology are described in various publications, e.g.", "Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel et al., 1987, Current Protocols in Molecular Biology, vols.", "1 and 2 and supplements, and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif., each of which is incorporated herein in their entirety by reference.", "Polynucleotide modifying enzymes were used according to the manufacturers recommendations.", "Oligonucleotides were synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizer using ABI chemicals.", "If desired, PCR amplimers for amplifying a predetermined DNA sequence may be selected at the discretion of the practitioner.", "4.16.5.9.1 Formation of Dimers One microgram samples of template DNA are obtained and treated with U.V.", "light to cause the formation of dimers, including TT dimers, particularly purine dimers.", "U.V.", "exposure is limited so that only a few photoproducts are generated per gene on the template DNA sample.", "Multiple samples are treated with U.V.", "light for varying periods of time to obtain template DNA samples with varying numbers of dimers from U.V.", "exposure.", "4.16.5.9.2 Random Priming Kit A random priming kit which utilizes a non-proofreading polymease (for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems) is utilized to generate different size polynucleotides by priming at random sites on templates which are prepared by U.V.", "light (as described above) and extending along the templates.", "The priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers.", "The dimers formed by U.V.", "exposure serve as a roadblock for the extension by the non-proofreading polymerase.", "Thus, a pool of random size polynucleotides is present after extension with the random primers is finished.", "8.16.5.9.3 Generation of a Selected Mutant Polynucleotide Sequence The present invention is further directed to a method for generating a selected mutant polynucleotide sequence (or a population of selected polynucleotide sequences) typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequences(s) possess at least one desired phenotypic characteristic (e.g., encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like) which can be selected for.", "One method for identifying hybrid polypeptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule (e.g., a receptor), involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.", "4.16.5.9.4 Generating Libraries Suitable for Affinity Interaction Screening or Phenotypic Screening In one embodiment, the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.", "The method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequences, optimally introducing mutations into said polynucleotides or copies, (2) pooling the polynucleotides or copies, (3) producing smaller or shorter polynucleotides by interrupting a random or particularized priming and synthesis process or an amplification process, and (4) performing amplification, preferably PCR amplification, and optionally mutagenesis to homologously recombine the newly synthesized polynucleotides.", "4.16.5.9.5 Producing Hybrid Polynucleotides which Express a Useful Hybrid Polypeptide It is a particularly preferred object of the invention to provide a process for producing hybrid polynucleotides which express a useful hybrid polypeptide by a series of steps comprising: (a) producing polynucleotides by interrupting a polynucleotide amplification or synthesis process with a means for blocking or interrupting the amplification or synthesis process and thus providing a plurality of smaller or shorter polynucleotides due to the replication of the polynucleotide being in various stages of completion; (b) adding to the resultant population of single- or double-stranded polynucleotides one or more single- or double-stranded oligonucleotides, wherein said added oligonucleotides comprise an area of identity in an area of heterology to one or more of the single- or double-stranded polynucleotides of the population; (c) denaturing the resulting single- or double-stranded oligonucleotides to produce a mixture of single-stranded polynucleotides, optionally separating the shorter or smaller polynucleotides into pools of polynucleotides having various lengths and further optionally subjecting said polynucleotides to a PCR procedure to amplify one or more oligonucleotides comprised by at least one of said polynucleotide pools; (d) incubating a plurality of said polynucleotides or at least one pool of said polynucleotides with a polymerase under conditions which result in annealing of said single-stranded polynucleotides at regions of identity between the single-stranded polynucleotides and thus forming of a mutagenized double-stranded polynucleotide chain; (e) optionally repeating steps (c) and (d); (f) expressing at least one hybrid polypeptide from said polynucleotide chain, or chains; and (g) screening said at least one hybrid polypeptide for a useful activity.", "In a preferred aspect of the invention, the means for blocking or interrupting the amplification or synthesis process is by utilization of uv light, DNA adducts, DNA binding proteins.", "In one embodiment of the invention, the DNA adducts, or polynucleotides comprising the DNA adducts, are removed from the polynucleotides or polynucleotide pool, such as by a process including heating the solution comprising the DNA fragments prior to further processing.", "Having thus disclosed exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention.", "Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.", "Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.", "The following examples are to be considered illustrative and thus are not limiting of the remainder of the disclosure in any way whatsoever.", "5.ENGINEERING GOALS 5.1.General Overview: Successive Cycles of Recombination and Screening/Selection The invention provides methods for artificially evolving cells to acquire a new or improved property by recursive sequence recombination.", "Briefly, recursive sequence recombination entails successive cycles of recombination to generate molecular diversity and screening/selection to take advantage of that molecular diversity.", "That is, a family of nucleic acid molecules is created showing substantial sequence and/or structural identity but differing as to the presence of mutations.", "These sequences are then recombined in any of the described formats so as to optimize the diversity of mutant combinations represented in the resulting recombined library.", "Typically, any resulting recombinant nucleic acids or genomes are recursively recombined for one or more cycles of recombination to increase the diversity of resulting products.", "After this recursive recombination procedure, the final resulting products are screened and/or selected for a desired trait or property.", "Alternatively, each recombination cycle can be followed by at least one cycle of screening or selection for molecules having a desired characteristic.", "In this embodiment, the molecule(s) selected in one round form the starting materials for generating diversity in the next round.", "The cells to be evolved can be bacteria, archaebacteria, or eukaryotic cells and can constitute a homogeneous cell line or mixed culture.", "Suitable cells for evolution include the bacterial and eukaryotic, cell lines commonly used in genetic engineering, protein expression, or the industrial production or conversion of proteins, enzymes, primary metabolites, secondary metabolites, fine, specialty or commodity chemicals.", "Suitable mammalian cells include those from, e.g., mouse, rat, hamster, primate, and human, both cell lines and primary cultures.", "Such cells include stem cells, including embryonic stem cells and hemopoietic stem cells, zygotes, fibroblasts, lymphocytes, Chinese hamster ovary (CHO), mouse fibroblasts (NIM), kidney, liver, muscle, and skin cells.", "Other eukaryotic cells of interest include plant cells, such as maize, rice, wheat, cotton, soybean, sugarcane, tobacco, and arabidopsis; fish, algae, fungi (penicillium, aspergillus, podospora, neurospora, saccharomyces), insect (e.g., baculo lepidoptera), yeast (picchia and saccharomyces, Schizosaccharomyces pombe).", "Also of interest are many bacterial cell types, both gram-negative and gram-positive, such as Bacillus subtilis, B. licehniformis, B. cereus, Escherichia coli, Streptomyces, Pseudomonas, Salmonella, Actinomycetes, Lactobacillius, Acelonitcbacter, Deinococcus, and Erwinia.", "The complete genome sequences of E. coli and Bacillus subtilis are described by Blattner et al., Science 277, 1454-1462 (1997); Kunst et al., Nature 390, 249-256 (1997).", "5.2 Identification and Development of New and/or Improved Drugs The genomics revolution, by determining the DNA sequences of great numbers of genes from many different organisms, has considerably broadened the possibilities for drug discovery by identifying, large numbers of molecules that are potential targets of drug action.", "One area of drug development focusing upon generating new antimicrobial drugs.", "New antimicrobial drugs are needed to treat infections by drug resistant organisms, and new methods are urgently needed to facilitate making such discoveries.", "Technical advances in molecular biology, automated methods for high throughput screening and chem ical syntheses have led to an increase in the number of target based screens utilized for antimicrobial drug discovery and in the number of compounds being analyzed.", "The invention relates to procedures that can be applied to identifying compounds that bind to and modulate the function of target components of a cell whose function is known or unknown, and cell components that are not amenable to other screening methods.", "The invention relates to generating and/or identifying a compound that binds to and modulates (inhibits or enhances) the function of a component of a cell, thereby producing a phenotypic effect in the cell.", "Within these procedures are methods for identifying a biomolecule that 1) binds to, in vitro, a component of a cell that has been isolated from other constituents of the cell and that 2) causes, in vivo, as seen in an assay upon intracellular expression of the biomolecule, a phenotypic effect in the cell which is the usual producer and host of the target cell component.", "In an assay demonstrating characteristic 2) above, intracellular production of the biomolecule can be in cells grown in culture or in cells introduced into an animal.", "Further methods within these procedures are those methods comprising an assay for a phenotypic effect in the cell upon intracellular production of the biomolecule, either in cells in culture or in cells that have been introduced into one or more animals, and an assay to identify one or more compounds that behave as competitors of the biomolecule in an assay of binding to the target cell component.", "5.2.1.Procedure for Identifying and/or Designing Compounds with Antimicrobial Activity Against a Pathogen The invention further relates to methods particularly well suited to a procedure for identifying and/or designing compounds with antimicrobial activity against a pathogen whose target cell component is the subject of studies to identify such compounds.", "A common mechanism of action of an antimicrobial agent is binding to a component of the cells of the pathogen treated with the antimicrobial.", "The procedure includes methods for identifying biomolecules that bind to a chosen target in vitro, methods for identifying biomolecules that also bind to the chosen target and modulate its function during infection of a host mammal in vivo, and methods for identifying compounds that compete with the biomolecules for sites on the target in competitive binding assays.", "Compounds identified by this procedure are candidates for drugs with antimicrobial activity against the pathogen.", "5.3 Producing Proteins with Improved Affinities Polynucleotide sequences selected in a first selection round (typically by affinity selection for binding to a receptor (e.g., a ligand)) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.", "The recombined selected polynucleotide sequences are subjected to at least one subsequent selection round.", "The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection.", "Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral sequences (i.e., having insubstantial functional effect on binding), such as for example by back-crossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic.", "Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).", "Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized.", "In one embodiment, selected library members are cloned in a prokaryotic vector (e.g., plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced.", "Individual selected library members can then be manipulated (e.g., by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member.", "The sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation (i.e., comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence.", "The mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.", "The invention also provides a product-by-process, wherein selected polynucleotide sequences having (or encoding a peptide having) a predetermined binding specificity are formed by the process of: (1) screening a displayed peptide or displayed single-chain antibody library against a predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, (2) shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and (3) screening the shuffled library against the predetermined receptor (e.g., ligand) or epitope (e.g., antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the predetermined receptor or epitope to produce a pool of selected shuffled library members.", "In one embodiment, the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.", "The method comprises (1) obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed polypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequences, optimally introducing mutations into said polynucleotides or copies, (2) pooling the polynucleotides or copies, (3) producing smaller or shorter polynucleotides by interrupting a random or particularized priming and synthesis process or an amplification process, and (4) performing amplification, preferably PCR amplification, and optionally mutagenesis to homologously recombine the newly synthesized polynucleotides.", "It is a particularly preferred object of the invention to provide a process for producing hybrid polynucleotides which express a useful hybrid polypeptide by a series of steps comprising: (a) producing polynucleotides by interrupting a polynucleotide amplification or synthesis process with a means for blocking or interrupting the amplification or synthesis process and thus providing a plurality of smaller or shorter polynucleotides due to the replication of the polynucleotide being in various stages of completion; (b) adding to the resultant population of single- or double-stranded polynucleotides one or more single- or double-stranded oligonucleotides, wherein said added oligonucleotides comprise an area of identity in an area of heterology to one or more of the single- or double-stranded polynucleotides of the population; (c) denaturing the resulting single- or double-stranded oligonucleotides to produce a mixture of single-stranded polynucleotides, optionally separating the shorter or smaller polynucleotides into pools of polynucleotides having various lengths and further optionally subjecting said polynucleotides to a PCR procedure to amplify one or more oligonucleotides comprised by at least one of said polynucleotide pools; (d) incubating a plurality of said polynucleotides or at least one pool of said polynucleotides with a polymerase under conditions which result in annealing of said single-stranded polynucleotides at regions of identity between the single-stranded polynucleotides and thus forming of a mutagenized double-stranded polynucleotide chain; (e) optionally repeating steps (c) and (d); (f) expressing at least one hybrid polypeptide from said polynucleotide chain, or chains; and (g) screening said at least one hybrid polypeptide for a useful activity.", "In a preferred aspect of the invention, the means for blocking or interrupting the amplification or synthesis process is by utilization of UV light, DNA adducts, DNA binding proteins.", "In one embodiment of the invention, the DNA adducts, or polynucleotides comprising the DNA adducts, are removed from the polynucleotides or polynucleotide pool, such as by a process including heating the solution comprising the DNA fragments prior to further processing.", "Having thus disclosed exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention.", "Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.", "5.3.1.Antibody Production High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems.", "For example, scfv have been produced in plants (Firek et al, 1993) and can be readily made in prokaryotic systems (Owens and Young, 1994; Johnson and Bird, 1991).", "Furthermore, the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al, 1994).", "The variable region encoding sequence may be isolated (e.g., by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized.", "The polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell (e.g., from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.", "The antibodies generated by the method of the present invention can be used for diagnosis and therapy.", "By way of illustration and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections.", "For treatment of cancer, the antibodies will typically bind to an antigen expressed preferentially on cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.", "5.3.1.1.Modified Variable Regions Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods.", "The first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.", "The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s).", "The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH In principle, the scfv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.", "Thus, scfv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide.", "After the scfv genes are assembled, they are cloned into a phagemid and expressed at the tip of the M13 phage (or similar filamentous bacteriophage) as fusion proteins with the bacteriophage PIII (gene 3) coat protein.", "Enriching for phage expressing an antibody of interest is accomplished by panning the recombinant phage displaying a population scfv for binding to a predetermined epitope (e.g., target antigen, receptor).", "The linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence.", "The displayed peptide (s) or single-chain antibody (e.g., scfv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system.", "Often polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies (e.g., chimeric or fully-human), antibody fragments, and the like.", "Often polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully-human), antibody fragments, and the like.", "Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography.", "Various other uses of such antibodies are to diagnose and/or stage disease (e.g., neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.", "If it were possible to generate scfv libraries having broader antibody diversity and overcoming many of the limitations of conventional CDR mutagenesis and randomization methods which can cover only a very tiny fraction of the potential sequence combinations, the number and quality of scfv antibodies suitable for therapeutic and diagnostic use could be vastly improved.", "To address this, the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies.", "Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s).", "In a variation, the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells (e.g., plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/122227, and WO 94/25585), including hybridomas derived therefrom.", "Polynucleotide sequences selected in a first selection round (typically by affinity selection for displayed antibody binding to an antigen (e.g., a ligand) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences.", "The recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round.", "The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection until an antibody of the desired binding affinity is obtained.", "Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral antibody framework sequences (i.e., having insubstantial functional effect on antigen binding), such as for example by back-crossing with a human variable region framework to produce human-like sequence antibodies.", "Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined antigen.", "Alternatively, or in combination with the noted variations, the valency of the target epitope may be varied to control the average binding affinity of selected scfv library members.", "The target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art.", "A high density (valency) of predetermined epitope can be used to enrich for scfv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scfv library members.", "For generating diverse variable segments, a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence kernal set of peptide sequences can be inserted by ligation into a predetermined site (e.g., a CDR).", "Similarly, the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like.", "The resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly (i.e., may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.", "A variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope.", "In one approach, the positive selected peptide/polynucleotide complexes (those identified in an early round of affinity enrichment) are sequenced to determine the identity of the active peptides.", "Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences.", "This mixture of (slightly) degenerate oligonucleotides is then cloned into the variable segment sequences at the appropriate locations.", "This method produces systematic, controlled variations of the starting peptide sequences, which can then be shuffled.", "It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.", "In a variation, mutagenic PCR amplification of positive selected peptide/polynucleotide complexes (especially of the variable region sequences, the amplification products of which are shuffled in vitro and/or in vivo and one or more additional rounds of screening is done prior to sequencing.", "The same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling.", "If desired, shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included.", "Thus, mixtures of synthetic oligonucleotides and PCR produced polynucleotides (synthesized by error-prone or high-fidelity methods) can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).", "5.3.1.2.Modified CDR Regions The present invention of shuffling enables the generation of a vast library of CDR-variant single-chain antibodies.", "One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling.", "The sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences.", "For example, a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat (Kabat et al, 1991); the pool (s) of synthetic CDR sequences are calculated to encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence.", "Alternatively, a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently (i.e., in at least 5 percent of known CDR sequences) are incorporated into the synthetic CDRs at the corresponding position(s).", "Typically, several (e.g., 3 to about 50) known CDR sequences are compared and observed natural sequence variations between the known CDRs are tabulated, and a collection of oligonucleotides encoding CDR peptide sequences encompassing all or most permutations of the observed natural sequence variations is synthesized.", "For example but not for limitation, if a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids.", "In some embodiments, residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences.", "Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences.", "Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.", "The collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence.", "It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions.", "A collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually not more than about 1×10 6 unique CDR sequences are included in the collection, although occasionally 1×107 to 1×108 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare (i.e., less than 0.1 percent) in that position in naturally-occurring human CDRS.", "In general, the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of 10.Such single-chain antibodies generally bind of about at least 1×10 m-, preferably with an affinity of about at least 5×1 M-1, more preferably with an affinity of at least 1×108 M-1 to 1×109 M-1 or more, sometimes up to 1×10 M-1 or more.", "Frequently, the predetermined antigen is a human protein, such as for example a human cell surface antigen (e.g., CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule (e.g., thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, Lselectin), or nonhuman disease associated macromolecule (e.g., bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.", "5.4 Increased Expression in a Recombinant Host In one embodiment of this invention, it provides for increasing expression of a gene or trait of interest in a recombinant host of interest.", "The hosts can include but are not limited to bacteria, fungi, protozoans, viruses, animals, insects, and plants.", "5.5 Metabolite Shifting In one embodiment of this invention, it provides for metabolite shifting.", "5.6 Creating a Modified Plant with Desired Traits One aspect of the present invention relates to the use of trait DNA molecules which are heterologous to the plant—e.g., DNA molecules that confer disease resistance to plants transformed with the DNA construct.", "The present invention is useful in plants for imparting resistance to a wide variety of pathogens including viruses, bacteria, fungi, viroids, phytoplasmas, nematodes, and insects.", "The present invention may also be used in mammals to impart genetic traits.", "Resistance, inter alia, to the following viruses can be achieved by the method of the present invention: tomato spotted wilt virus, impatiens necrotic spot virus, groundnut ringspot virus, potato virus Y, potato virus X, tobacco mosaic virus, turnip mosaic virus, tobacco etch virus, papaya ringspot virus, tomato mottle virus, tomato yellow leaf curl virus, or -combinations thereof.", "Resistance, inter alia, to the following bacteria can also be imparted to plants in accordance with present invention: Pseudomonas solancearum, Pseudomonas syringae pv.", "tabaci, Xanthamonas campestris pv.", "pelargonii, and Agrobacterium tumefaciens.", "Plants can be made resistant, inter alia, to the following fungi by use of the method of the present invention: Fusarium oxysporum and Phytophthora infestans.", "Suitable DNA molecules include a DNA molecule encoding a coat protein, a replicase, a DNA molecule not encoding protein, a DNA molecule encoding a viral gene product, or combinations thereof.", "The present invention is also used to confer traits other than disease resistance on plants.", "For example, DNA molecules which impart a plant genetic trait can be used as the DNA trait molecule of the present invention.", "In this aspect of the present invention, suitable trait DNA molecules encode for desired color, enzyme production, or combinations thereof.", "In another embodiment of this invention, it provides for engineering plants with desired traits, including output (e.g., producing increased amounts of a desired vitamin, mineral, or genetically engineered and introduced molecule such as an antibody) and input (e.g., drought and/or salinity resistance) traits.", "5.7 Plant Gene Expression This invention is related to the genetic engineering of plants and to a means and method (use of DNA construct) for conferring a plurality of traits, including resistance to viruses, to a plant using a vector encoding a plurality of genes, such as coat protein genes, protease genes, or replicase genes.", "The field of the invention is plant genetics, including genetic mapping and restriction fragment length polymorphism technology.", "The present invention also relates to: (i) the production of mature proteins in plant cells, including the production of proteins in mature secreted form.", "(i) the development of techniques for the commercial production of transgenic plants.", "5.7.1 General Considerations The present invention provides a chimeric recombinant DNA molecule comprising: a plurality of DNA sequences, each of which comprises a plant-functional promoter linked to a coding region, which encodes a virus-associated coat protein, wherein said DNA sequences are preferably linked in-tandem so that they are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule to impart resistance to said viruses; as well as methods for transforming plants with the chimeric constructs and for selecting plants which express at least one of said DNA sequences imparting viral resistance.", "Methods of making a genetically modified plant comprising regenerating a whole plant from a plant cell that has been transfected with DNA sequences comprising a first gene whose expression results in an altered plant phenotype linked to a transiently active promoter, the gene and promoter being separated by a blocking sequence flanked on either side by specific excision sequences, a second gene that encodes a recombinase specific for the specific excision sequences linked to a repressible promoter, and a third gene that encodes the repressor specific for the repressible promoter.", "Also a method for making a genetically modified hybrid plant by hybridizing a first plant regenerated from a plant cell that has been transfected with DNA sequences comprising a first gene whose expression results in an altered plant phenotype linked to a transiently active promoter, the gene and promoter being separated by a blocking sequence flanked on either side by specific excision sequences to a second plant regenerated from a second plant cell that has been transfected with DNA sequences comprising a second gene that encodes a recombinase specific for the specific excision sequences linked to a promoter that is active during seed germination, and growing a hybrid plant from the hybrid seed.", "Plant cells, plant tissues, plant seed and whole plants containing the above DNA sequences are also claimed.", "The present invention is also directed to a DNA construct formed from a fusion gene which includes a trait DNA molecule and a silencer DNA molecule.", "The trait DNA molecule has a length that is insufficient to impart a desired trait to plants transformed with the trait DNA molecule.", "The silencer DNA molecule is operatively coupled to the trait DNA molecule with the trait and silencer DNA molecules collectively having sufficient length to impart the trait to plants transformed with the DNA construct.", "Expression systems, host cells, plants, and plant seeds containing the DNA construct are disclosed.", "The present invention is also directed to imparting multiple traits to a plant.", "The present invention is also directed to methods of introgressing one or more desired quantitative traits into a plant comprising screening one or more restriction fragment length polymorphisms (RFLP) for association with desired quantitative traits (QT), selecting one or more RFLP's showing association with the desired QT's, developing a mathematical model based on the magnitude of the association of RFLP(s) to predict the degree of expression of the desired QT's, and using the thus-selected RFLP(s) and the mathematical model in a plant breeding program to predict the degree of introgression and expression of the desired QT's in plant progeny.", "A method for producing one of the following proteins in transgenic monocot plant cells is disclosed: (i) mature, glycosylated α1-antitrypsin (AAT) having the same N-terminal amino acid sequence as mature AAT produced in humans and a glycosylation pattern which increases serum halflife substantially over that of mature non-glycosylated AAT; (ii) mature, glycosylated antithrombin m (ATIII) having the same N-terminal amino acid sequence as mature ATIII produced in humans; (iii) mature human serum albumin (HSA) having the same N— temninal amino acid sequence as mature HSA produced in humans and having the folding pattern of native mature HSA as evidenced by its bilirubin-binding characteristics; and (iv) mature, active subtilisin BPN′ (BPN′) having the same N-terminal amino acid sequence as BPN′ produced in Bacillus.", "Monocot plants cells are transformed with a chimeric gene which includes a DNA coding sequence encoding a fusion protein having an (i) N-terminal moiety corresponding to a rice α-amylase signal sequence peptide and, (iii) immediately adjacent the C-terminal amino acid of said peptide, a protein moiety corresponding to the mature protein to be produced.", "A process for commercially propagating plants by tissue culture in such a way as both to conserve desired plant morphology and to transform the plant with respect to one or more desired genes.", "The method includes the steps of (a) creating an Agrobacterium vector containing the gene sequence desired to be transferred to the propagated plant, preferably together with a marker gene; (b) taking one or more petiole explants from a mother plant and inoculating them with the Agrobacterium vector; (c) conducting callus formation in the petiole sections in culture, in the dark; and (d) culturing the resulting callus in growth medium containing a benzylamino growth regulator such as benzylaminopurine or, most preferably, benzylaminopurine-riboside.", "Additional optional growth regulators including auxins and cytokinins (indole butyric acid, benzylamine, benzyladenine, benzylaminopurine, alpha naphthylacetic acid and others known in the art) may also be present.", "Preferably, the petiole tissue is taken from Pelargonium x domesticum and the Agrobacterium vector contains an antisense gene for ACC synthase or ACC oxidase to prevent ACC synthase or ACC oxidase expression and, in turn, the ethylene formation for which these enzymes are precursors.", "5.7.2.Production of Virus Resistant Plants 5.7.2.1 Production of Virus Resistant Plants Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques.", "Several different types of host resistance to viruses are recognized.", "The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.", "One potential application would be to engineer a plant that is resistant to potyviruses.", "Potyviruses are a distinct group of plant viruses which are pathogenic to various crops, and which demonstrate cross-infectivity between plant members of different families.", "One example is that expression of the coat protein genes from tobacco mosaic virus, alfalfa mosaic virus, cucumber mosaic virus, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus.", "Some evidence of heterologous protection has also been reported.", "For example, see references Namba et al., Phytopathology, 82, 940 (1992) Stark et al., Biotechnology, 1, 1257 (1989).", "5.7.2.2 Using “Pathogen Driven Resistance” (PDR) for Developing Virus Resistant Transgenic Plants Control of plant virus diseases took a major step forward in the last decade when it was shown in 1986 that the tobacco mosaic virus (“TMV”) coat protein gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel, P., et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science, 232: 738-43 (1986)).", "The concept of pathogen-derived resistance (“PDR”), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford, J. C., et al.", "“The Concept of Parasite-Derived Resistance—Deriving Resistance Genes from the Parasite's Own Genome,” J. Theor.", "Biol., 113: 395-405 (1985)) was introduced at about the same time.", "Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann.", "Rev.", "Photopathol., 33: 323-43 (1995)).", "Only eight years after the report by Beachy and colleagues (Powell-Abel, P., et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science, 232: 738-43 (1986)), Grumet, R., “Development of Virus Resistant Plants via Genetic Engineering,” Plant Breedinq Reviews, 12: 47-49 (1994) reviewed the PDR literature and listed the successful development of virus resistant transgenic plants to at least 11 different groups of plant viruses.", "5.7.2.2.1 Utilizing The Coat Protein Genes The vast majority of reports have utilized the coat protein genes of the viruses that are targeted for control (e.g., Grumet, R., “Development of Virus Resistant Plants via Genetic Engineering,” Plant Breeding Reviews, 12: 47-49 (1994)).", "Additional examples are included in the following references: Fuchs, M., et al., Bio/Technology 13: 1466-73 (1995); Tricoli, D. M., et al, Bio/Technology, 13: 1458-65 (1995); Fitch, M. M. M., et al., Bio/Technology, 1466-72 (1992); Tennant, P. F., et al., Phytopathology, 84: 1359-66 (1994).", "5.7.2.2.2 Other Effective Viral Genes Interestingly, remarkable progress has been made in developing virus resistant transgenic plants despite a poor understanding of the mechanisms involved in the various forms of pathogen-derived resistance (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann.-Rev.", "Photopathol., 33: 323-43 (1995)).", "Various reports have utilized proteins other than the coat protein genes to confer resistance (Golemboski, D. B., et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 87: 6311-15 (1990); Beck, D. L., et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 91: 10310-14 (1994); Maiti, I.", "B., et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 90: 6110-14 (1993).", "Furthermore, the viral genes can be effective in the translatable and nontranslatable sense forms, and less frequently antisense forms (e.g., Baulcombe, D. C., “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell, 8: 1833-44 (1996); Dougherty, W. G., et al., “Transgenes and Gene Suppression: Telling us Something New?,” Current Opinion in Cell Biology, 7: 399-05 (1995); Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann.", "Rev.", "Photopathol.", "33: 323-43 (1995)).", "5.7.2.2.3.RNA-Mediated Resistance 5.7.2.2.3.1 Description (A Form of PDR) RNA-mediated resistance is the form of PDR where there is clear evidence that viral proteins do not play a role in conferring resistance to the transgenic plant.", "The first clear cases for RNA-mediated resistance were reported in 1992 for tobacco etch (“TEV”) potyvirus (Lindbo, et al., “Pathogen-Derived Resistance to a Potyvirus Immune and Resistance Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,” Mol.", "Plant Microbe Interact., 5: 144-53 (1992)), for potato virus Y (“PVY”) potyvirus by Van Der Vlugt, R. A.", "A., et al., “Evidence for Sense RNA-Mediated Protection to PVY in Tobacco Plants Transformed with the Viral Oat Protein Cistron,” Plant Mol.", "Biol., 20: 631-39 (1992), and for tomato spotted wilt (“TSWV”) tospovirus by de Haan, P., et al., “Characterization of RNA-Mediated Resistance to Tomato Spotted Wilt Virus in Transgenic Tobacco Plants,” Bio/Technology 10: 1133-37 (1992).", "others confirmed the occurrence of RNA-mediated resistance with potyviruses (Smith, H. A., et al., “Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,” Plant Cell, 6: 1441-53 (1994)), potexviruses (Mueller, E., et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal, 7: 1001-13 (1995)), and TSWV and other topsoviruses (Pang, S. Z., et al., “Resistance of Transgenic Nicotiana Benthamiana Plants to Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence of Involvement of the N Protein and N Gene RNA in Resistance,” Phytopathology, 84: 243-49 (1994); Pang, S.-Z., et al., “Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt Virus and Impatiens Necrotic Spot Tospoviruses,” Bio/Technology 11: 819-24 (1993)).", "More recent work has shown that RNA-mediated resistance also occurs with the comovirus cowpea mosaic virus (Sijen, T., et al., “RNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,” Plant Cell, 8: 2227-94 (1996)) and squash mosaic virus (Jan, F.-J., et al., “Genetic and Molecular Analysis of Squash Plants Transformed with Coat Protein Genes of Squash Mosaic Virus,” Phytopathology, 86:S16-17 (1996)).", "5.7.3 Creating Transgenic Plants with Controllable Genes This invention also relates to certain transgenic plants and involves a method of creating transgenic plants with controllable genes.", "More particularly, the invention relates to transgenic plants that have been modified such that expression of a desired introduced gene can be limited to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location, or a combination of these situations.", "5.7.3.1 Inducible Gene Promoter: “Gene Switch” Various gene expression control elements that are operable in one or more species of organisms are known.", "Examples are mentioned in PCT Application WO 90/08826 and PCT application WO 94/03619.A Tetracycline-controlled plant-active repressor-operator system can be utilized as described in various references: Gatz and Quail (1988) and Gatz, et al.", "(1992), (Hoppe-Seyler), 372: 659-660 (1991); Gatz and Quail, 1988; and (Gatz, et al., 1992).", "5.7.4 Recombinant Production of Proteins A major commercial focus of biotechnology is the recombinant production of proteins, including both industrial enzymes and proteins that have important therapeutic uses.", "5.7.4.1.Alternative Protein Expression System to Overcome Problems of Microbial and Mammalian Systems It would therefore be desirable to produce selected therapeutic and industrial proteins in a protein expression system that largely overcomes problems associated with microbial and mammalian-cell systems.", "In particular, production of the proteins should allow large volume production at low cost, and yield properly processed and glycosylated proteins.", "The production system should also have a relatively stable genotype from generation to generation.", "These aims are achieved, in the present invention, for the therapeutic proteins AAT, HSA, and antithrombin m (ATM), and the industrial enzyme subtilisin BPN′.", "5.7.4.2.Uses Various proteins of interest could be produced such as but not limited to: 1) Human α1-antitrypsin (AAT; Carrell, P., et al., Nature (1992) 298: 329; involved in cirrhosis and liver failure: e.g., Wu, Y., et al., BioEssays 13(4): 163 (1991), 2) Human Antithrombin III (ATIII) (potentially useful in the prevention of thrombosis and pulmonary embolism), 3) Human Serum Albumin (Geisow, M. J. et al.", "(1977) Biochem.", "J.", "163: 477-484; HSA is used to expand blood volume and raise low blood protein levels in cases of shock, trauma, and post-surgical recovery.", "HSA is often administered in emergency situations to stabilize blood pressure).", "4) Subtilisin BPN′ is an important industrial enzyme, particularly for use as a detergent enzyme.", "5.7.5 Practical Method for the Commercial Production of Transgenic Plants Translating genetic engineering theory into practice, however, and then furthermore into a commercially practical reality, requires ingenuity.", "Gene transplantation in plants has already been accomplished at this writing—and examples are cited below—but heretofore no practical method for the commercial production of transgenic plants has been perfected.", "Genetic engineering of plants may involve any and/or all of the following steps: tissue culture propagation, gene transplantation (eg., with Agrobacterium and T-DNA), the binary system (using binary vectors).", "A general reference is Buchanan, B.", "B. et al., Biochemistry and Molecular Biology of Plants, ASPP Publications, 2001.Exemplary publications and patents which disclose transgenic plants and various techniques therefor are summarized below.", "Pellegrineschi, A., et al., Bio/Technology, Vol.", "12 (January, 1994) discloses transformation of root cultures by inoculating stem and leaf fragments with Agrobacterium rhizoaenes.", "An important plasmid in this species of Agrobacterium is the root-inducing plasmid which can be used to transfer to the plant genome the genes necessary for improved root growth in culture.", "The use of sterilized petioles as the source of explant material for plant transformation and culture is disclosed.", "U.S. Pat.", "No.", "5,276,268 to Strauch et al., entitled 11 Phosphinothricin-Resistance Gene, and Its Use,” is directed to the transfer of phosphinothricin-resistance gene into plants using Agrobacterium species.", "A modification of the binary vector method is discussed, and the phosphinothricin-resistance gene nucleic acid sequences are provided.", "U.S. Pat.", "No.", "5,283,184; U.S. Pat.", "No.", "5,286,635.5.7.6 Method of Identifying and Characterizing the Role of Individual Plant Genes in Quantitative Trait Expression One area in which biotechnology may have a significant impact on plant improvement is in the development of new methods to identify and characterize the role of individual plant genes in quantitative trait expression.", "Following the development of a new class of plant molecular markers based on restriction fragment length polymorphisms, termed “RFLPs”, (Helentjaris et al., Plant Mol.", "Bio.", "5: 109-118 (1985)) (“Helentjaris et al.", "I”), the processes to identify such loci and discriminate gene effects have been invented and are described and claimed herein.", "This and all other publications noted herein are hereby incorporated by reference.", "This will undoubtedly benefit plant improvement, not only within the context of conventional breeding approaches, but also by providing a means for identifying appropriate loci for future cloning and direct gene transfer efforts.", "5.7.7.Fusion Genes The present invention is directed to a DNA construct formed from a fusion gene which includes a trait DNA molecule and a silencer DNA molecule.", "In an alternative embodiment of the present invention, the DNA construct can be a fusion gene comprising a plurality of trait DNA molecules at least some of which having a length that is insufficient to impart that trait to plants transformed with that trait DNA molecule.", "However, the plurality of trait DNA molecules collectively have a length sufficient to impart their traits to plants transformed with the DNA construct and to effect post-transcriptional silencing of the fusion gene.", "Expression systems, host cells, plants, and plant seeds containing this embodiment of the DNA construct are disclosed.", "The present invention also provides a recombinant chimeric DNA molecule comprising a plurality of DNA sequences each of which comprises a promoter operably linked to a DNA sequence which encodes a virus-associated protein, such as a coat protein (cp), a protease, or a replicase, wherein said DNA sequences are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule to impart resistance to infection by each of said viruses.", "Preferably, the DNA sequences are linked in tandem, i.e., exist in head to tail orientation relative to one another.", "Also, preferably substantially equal levels of resistance to infection by each of said viruses occurs in plant cells transformed with said plurality of DNA sequences.", "Preferably, each DNA sequence is also linked to a 3′ non-translated DNA sequence which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed mRNA sequences.", "Preferably, the virus is a plant-associated virus, such as a potyvirus.", "Thus, the present DNA molecule can be employed as a chimeric recombinant “expression construct,” or “expression cassette” to prepare transgenic plants that exhibit increased resistance to infection by at least two plant viruses, such as potyviruses.", "The present cassettes also preferably comprise at least one selectable marker gene or reporter gene which is stably integrated into the genome of the transformed plant cells in association with the viral genes.", "The selectable marker and/or reporter genes facilitate identification of transformed plant cells and plants.", "Preferably, the virus gene array is flanked by two or more selectable marker genes, reporter genes or a combination thereof.", "Another aspect of the present invention is a method of preparing a virus-resistant plant, such as a dicot, comprising: (a) transforming plant cells with a chimeric recombinant DNA molecule comprising a plurality of DNA sequences, each comprising a promoter functional in said plant cells, operably linked to a DNA sequence, which encodes a protein associated with a virus which is capable of infecting said plant; (b) regenerating said plant cells to provide a differentiated plant; and (c) identifying a transformed plant which expresses the DNA sequences so as to render the plant resistant to infection by said viruses, preferably at substantially equal levels of resistance to infection by each virus.", "Yet another object of the present invention is to provide a method for providing resistance to infection by viruses in a susceptible Cucurbitaceae plant which comprises: (a) transforming Cucurbitaceae plant cells with a DNA molecule encoding a plurality of proteins from viruses which are capable of infecting said Cucurbitaceae plant; (b) regenerating said plant cells to provide a differentiated plant; and (c) selecting a transformed Cucurbitaceae which expresses the virus proteins at levels sufficient to render the plant resistant to infection by said viruses.", "It is a further object of the present invention to provide multi-virus resistant transformed plant which contains stably-integrated DNA sequences encoding virus proteins.", "5.7.8 Controlling Gene Expression with External Stimulus The present invention involves, in one embodiment, the creation of a transgenic plant that contains a gene whose expression can be controlled by application of an external stimulus.", "This system achieves a positive control of gene expression by an external stimulus, without the need for continued application of the external stimulus to maintain gene expression.", "The present invention also involves, in a second embodiment, the creation of transgenic parental plants that are hybridized to produce a progeny plant expressing a gene not expressed in either parent.", "By controlling the expression of genes that affect the plant phenotype, it is possible to grow plants under one set of conditions or in one environment where one phenotype is advantageous, then either move the plant or plant its seed under another set of conditions or in another environment where a different phenotype is advantageous.", "This technique has particular utility in agricultural and horticultural applications.", "In accordance with one embodiment of the invention, a series of sequences is introduced into a plant that includes a transiently-active promoter linked to a structural gene, the promoter and structural gene being separated by a blocking sequence that is in turn bounded on either side by specific excision sequences, a repressible promoter operably linked to a gene encoding a site-specific recombinase capable of recognizing the specific excision sequences, and a gene encoding a repressor specific for the repressible promoter whose function is sensitive to an external stimulus.", "Without application of the external stimulus, the structural gene is not expressed.", "Upon application of the stimulus, repressor function is inhibited, the recombinase is expressed and effects the removal of the blocking sequence at the specific excision sequences, thereby directly linking the structural gene and the transiently-active promoter.", "In a modification of this embodiment, the sequences encoding the recombinase can be introduced separately into the plant via a viral vector.", "In an alternative embodiment, no repressor gene or repressible promotor is used.", "Instead, the recombinase gene is linked to a germination-specific promotor and introduced into a separate plant from the other sequences.", "The plant containing the transiently-active promotor, blocking sequence, and structural gene is then hybridized with the plant containing the recombinase gene, producing progeny that contain all of the sequences.", "When the second transiently-active promotor becomes active, the recombinase removes the blocking sequence in the progeny, allowing expression of the structural gene in the progeny, whereas it was not expressed in either parent.", "In still another embodiment, the recombinase gene is simply linked to an inducible promoter.", "Exposure of the plant to the induce specific for the inducible promoter leads to the expression of the recombinase gene and the excision of the blocking sequence.", "In all of these embodiments, the structural gene is expressed when the transiently-active promoter becomes active in the normal course of growth and development, and will continue to be expressed so long as the transiently-active promoter is active, without the necessity of continuous external stimulation.", "This system is particularly useful for developing seed, where a particular trait is only desired during the first generation of plants grown from that seed, or a trait is desired only in subsequent generations.", "5.7.9.Preparing Plants which are Resistant to Multiple Viruses It is still a further object of the present invention to provide virus resistant transformed plant cells which contain a plurality of viral genes, i.e., 2-7 or more genes, which are expressed as virus proteins, such as coat proteins, proteases and/or replicases, from the same virus strain, from different virus strains as from different members of the virus group, such as the potyvirus group.", "Representative viruses from which these DNA sequences can be isolated include, but are not limited to, potato virus X (PVX), potyviruses such as potato virus Y (PVY), cucomovirus (CMV), tobacco vein mottling virus, watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), bean common mosaic virus, bean yellow mosaic virus, soybean mosaic virus, peanut mottle virus, beet mosaic virus, wheat streak mosaic virus, maize dwarf mosaic virus, sorghum mosaic virus, sugarcane mosaic virus, johnsongrass mosaic virus, plum pox virus, tobacco etch virus, sweet potato feathery mottle virus, yam mosaic virus, and papaya ringspot virus (PRV), cucomoviruses, including CMA and comovirus.", "5.7.3.4.1 Using Short Fragments of Viral Genes to Impart Resistance Rather than attempting to incorporate full length viral genes in a plant, the present invention uses short fragments of such genes to impart resistance to the plant against a plurality of viral pathogens.", "These short fragments, which each by themselves have insufficient length to impart such resistance, are more easily and cost effectively produced than full length genes.", "There is no need to include in the plant separate promoters for each of the fragments; only a single promoter is required.", "Moreover, such viral gene fragments can preferably be incorporated in a single expression system to produce transgenic plants with a single transformation event.", "5.7.10 Imparting Other Traits to Plants In addition to conferring on plants resistance to multiple viral diseases, the present invention can be utilized to impart other traits to plants.", "It is often desirable to incorporate a number of traits to a transgenic plant besides disease resistance.", "For example, color, enzyme production, etc.", "may be desirable traits to confer on a plant.", "However, transforming plants with a plurality of such traits encounter the same difficulties discussed above with respect to disease resistance.", "The present invention may be likewise useful in alleviating these problems with respect to traits other than disease resistance.", "Thus, the present invention provides a genetic engineering methodology by which multiple traits can be manipulated and tracked as a single gene insert, i.e., as a construct which acts as a single gene which segregates as a single Mendelian locus.", "Although the invention is exemplified via virus resistance genes, in practice, any combination of genes could be linked.", "Therefore one could track a block of genes that provide traits such as disease resistance, plus enhanced herbicide resistance, plus extended shelf life, and the like, by simply tracking the linked selectable marker or reporter gene which has been incorporated into the transformation vector.", "It was also discovered that when multiple tandem genes are inserted, they preferably all exhibit substantially the same degrees of efficacy, and more preferably substantially equal degrees of efficacy, wherein the term “substantial” as it relates to viral resistance is defined with reference to the assays described in the examples hereinbelow.", "For example, if one examines numerous transgenic lines containing an intact ZYMV and WMV-2 coat protein insert, one finds that if a line is immune to infection by ZYMV it is also immune to infection by WMV-2.Similarly, if a line exhibits a delay in symptom development to ZYMV it will also exhibit a delay in symptom development to WMV2.Finally, if a line is susceptible to ZYMV it win be susceptible to WMV-2.This phenomenon is unexpected.", "If there were not a correlation between the efficacy of each gene in these multiple gene constructs this approach as a tool in plant breeding would probably be prohibitively difficult to use.", "Even with single gene constructs, one must test numerous transgenic plant lines to find one that displays the appropriate level of efficacy.", "The probability of finding a line with useful levels of expression can range from 10-50% (depending on the species involved).", "If the efficacy of individual genes in a Ti plasmid containing multiple genes were independent, the probability of finding a transgenic line that was resistant to each targeted virus would decrease dramatically.", "For example, in a species in which there is a 10% probability of identifying a line with resistance using a single gene insert, is transformed with a triple-gene construct CZW and each gene display an independent levels of efficacy, the probability of finding a line with resistance to CMV, ZYMV and WMV-2 would be 0.1×0.1×0.1=0.001 or 0.1%.", "However, since the efficacy of multivalent genes is not independent of each other the probability of finding a line with resistance to CMV, ZYMV and WMV-2 is still 10% rather than 0.1%.", "Obviously this advantage becomes more pronounced as constructs containing four or more genes are used.", "5.7.0.1.Production of a Mature Heterologous Protein in Transformed Monocot Plant Cells In one aspect, the invention includes a method of producing, in monocot plant cells, a mature heterologous protein of interest.", "The method includes obtaining monocot cells transformed with a chimeric gene having (i) a monocot transcriptional regulatory region, inducible by addition or removal of a small molecule, or during seed maturation, (ii) a first DNA sequence encoding the heterologous protein, and (iii) a second DNA sequence encoding a signal peptide.", "The second DNA sequence is operably linked to the transcriptional regulatory region and to the first DNA sequence.", "The first DNA sequence is in translation-frame with the second DNA sequence, and the two sequences encode a fusion protein.", "5.7.10.2 Inducing the Transcriptional Regulatory Region In other embodiments of the method, the transcriptional regulatory region may be a promoter derived from a rice or barley α-amylase gene, including RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, RAmy3E, pM/C, gKAmyl41, gKAmyl55, Amy32b, or HV18.The chimeric gene may further include, between the transcriptional regulatory region and the fusion protein coding sequence, the 5′ untranslated region (5′ UTR) of an inducible monocot gene such as one of the rice or barley α-amylase genes described above.", "One preferred 5′ UTR is that from the RAmy1A gene, which is effective to enhance the stability of the gene transcript.", "The chimeric gene may further include, downstream of the coding sequence, the 3′ untranslated region (3′ UTR) from an inducible monocot gene, such as one of the rice or barley α-amylase genes mentionedabove.", "One preferred 3′UTR is from the RAmylA gene.", "Where the method is employed in protein production in a monocot cell culture, preferred promoters are the RAmy3D and RAmy3E gene promoters, which are upregulated by sugar depletion in cell culture.", "Where the gene is employed in protein production in germinating seeds, a preferred promoter is the RAmylA gene promoter, which is upregulated by gibberellic acid during seed germination.", "Where gene is upregulated during seed maturation, a preferred promoter is the barley endosperm-specific B1-hordein promoter.", "5.7.11 Indentifying such Loci and Discrinating Gene Effects of Restriction Fragment Length Polymorphisms The development of new methods to identify and characterize the role of individual plant genes in quantitative trait expression has significant impact on plant improvement.", "Following the development of a new class of plant molecular markers based on restriction fragment length polymorphisms, termed “RFLPs”, (Helentjaris et al., Plant Mol.", "Bio.", "5: 109-118 (1985)) (“Helentjaris et al.", "I”), the processes to identify such loci and discriminate gene effects have been invented and are described and claimed herein.", "Additional RFLP reference is (Roberts, Nuc.", "Acids Res.", "10: 117-144 (1982)).", "The utility of isozyme markers or morphological markers in studies is frequently limited by a lack of informativeness in lines of interest or by an insufficient availability or chromosomal distribution of the loci.", "Over 300 RFLPs covering all ten maize chromosomes have been characterized (Helentjaris et al., Trends in Genetics.", "3: 217-221 (1987)).", "Various plant genetic linkage maps based on RFLP markers have been constructed (Helentjaris et al., Theor.", "Appl.", "Genet.", "72: 761-769 (1986); Brassica Figdore et al., Theor.", "Appl.", "Genetics.", "75: 833-840 (1988); Slocum et al., In “Genetic Maps” (S. J. O'Brien, ed.", "), 5th Edition, Cold Spring Harbor Press, N.Y. (1990); Wright et al., MNL 61: 89-90 (1987); Helentjaris et al., Weber and Helentjaris, Genetics 121: 583-590 (1989).", "5.7.12 Isozyme Variation in Plant Breeding The use of isozyme variation in plant breeding is like RFLP technology, one of indirect selection.", "(Tanskley and Orton, Isozymes in Plant Genetics and Breeding 1B (Elsevier, N.Y. 1983; Vallejos and Tanksley, Theor.", "Appl.", "Genet.", "66: 241-247 (1983); Stuber et al., Crop.", "Sci.", "22: 737-740 (1982)).", "Maize is perhaps the best characterized plant system in terms of isozymes and yet only about two dozen isozyme loci have been located and it is rare for more than a dozen.", "Particular Definitions The terms below have the following meaning, unless indicated otherwise in the specification.", "As used in this specification, a “transiently-active promoter” is any promoter that is active either during a particular phase of plant development or under particular environmental conditions, and is essentially inactive at other times.", "A “plant active promoter” is any promoter that is active in cells of a plant of interest.", "Plant-active promoters can be of viral, bacterial, fungal, animal or plant origin.", "A gene that results in an altered plant phenotype is any gene whose expression leads to the plant exhibiting a trait or traits that would distinguish it from a plant of the same species not expressing the gene.", "Examples of such altered phenotypes include a different growth habit, altered flower or fruit color or quality, premature or late flowering, increased or decreased yield, sterility, mortality, disease susceptibility, altered production of secondary metabolites, or an altered crop quality such as taste or appearance.", "A gene and a promoter are to be considered to be operably linked if they are on the same strand of DNA, in the same orientation, and are located relative to one another such that the promoter directs transcription of the gene (i.e.", "in cis).", "The presence of intervening DNA sequences between the promoter and the gene does not preclude an operable relationship.", "A “blocking sequence” is a DNA sequence of any length that blocks a promoter from effecting expression of a targeted gene.", "A “specific excision sequence” is a DNA sequence that is recognized by a site-specific recombinase.", "A “recombinase” is an enzyme that recognizes a specific excision sequence or set of specific excision sequences and effects the removal of, or otherwise alters, DNA between specific excision sequences.", "A “repressor element” is a gene product that acts to prevent expression of an otherwise expressible gene.", "A repressor element can comprise protein, RNA or DNA.", "A “repressible promoter” is a promoter that is affected by a repressor element, such that transcription of the gene linked to the repressible promoter is prevented.", "“Expression” means transcription or transcription followed by translation of a particular DNA molecule.", "As used herein, with respect to a DNA sequence or “gene”, the term “isolated” is defined to mean that the sequence is either extracted from its context in the viral genome by chemical means and purified and/or modified to the extent that it can be introduced into the present vectors in the appropriate orientation, i.e., sense or antisense.", "“Cell culture” refers to cells and cell clusters, typically callus cells, growing on or suspended in a suitable growth medium.", "“Germination” refers to the breaking of dormancy in a seed and the resumption of metabolic activity in the seed, including the production of enzymes effective to break down starches in the seed endosperm.", "“Inducible” means a promoter that is upregulated by the presence or absence of a small molecules.", "It includes both indirect and direct inducement.", "“Inducible during germination” refers to promoters which are substantially silent but not totally silent prior to germination but are turned on substantially (greater than 25%) during germination and development in the seed.", "Examples of promoters that are inducible during germination are presented below.", "“Small molecules”, in the context of promoter induction, are typically small organic or bioorganic molecules less than about 1 kDal.", "Examples of such small molecules include sugars, sugar-derivatives (including phosphate derivatives), and plant hormones (such as, gibberellic or absissic acid).", "“Specifically regulatable” refers to the ability of a small molecule to preferentially affect transcription from one promoter or group of promoters (e.g., the a-amylase gene family), as opposed to non-specific effects, such as, enhancement or reduction of global transcription within a cell by a small molecule.", "“Seed maturation” or “grain development” refers to the period starting with fertilization in which metabolizable reserves, e.g., sugars, oligosaccharides, starch, phenolics, amino acids, and proteins, are deposited, with and without vacuole targeting, to various tissues in the seed (grain), e.g., endosperm, testa, aleurone layer, and scutellar epithelium, leading to grain enlargement, grain filling, and ending with grain desiccation.", "“Inducible during seed maturation” refers to promoters which are turned on substantially (greater than 25%) during seed maturation.", "“Heterologous” is defined to mean not identical, e.g.", "different in nucleotide and/or amino acid sequence, phenotype or an independent isolate.", "“Heterologous DNA” or “foreign DNA” refers to DNA which has been introduced into plant cells from another source, or which is from a plant source, including the same plant source, but which is under the control of a promoter or terminator that does not normally regulate expression of the heterologous DNA.", "“Heterologous protein” is a protein, including a polypeptide, encoded by a heterologous DNA.", "A “transcription regulatory region” or “promoter” refers to nucleic acid sequences that influence and/or promote initiation of transcription.", "Promoters are typically considered to include regulatory regions, such as enhancer or inducer elements.", "“Chimeric” is defined to mean the linkage of two or more DNA sequences which are derived from different sources, strains or species, i.e., from bacteria and plants, or that two or more DNA sequences from the same species are linked in a way that does not occur in the native genome.", "Thus, the DNA sequences useful in the present invention may be naturally occurring, semi-synthetic or entirely synthetic.", "The DNA sequence may be linear or circular, Le, may be located on an intact or linearized plasmid, such as the binary plasmids described below.", "A “chimeric gene,” in the context of the present invention, typically comprises a promoter sequence operably linked to DNA sequence that encodes a heterologous gene product, e.g., a selectable market gene or a fusion protein gene.", "A chimeric gene may also contain further transcription regulatory elements, such as transcription termination signals, as well as translation regulatory signals, such as, termination codons.", "“Operably linked” refers to components of a chimeric gene or an expression cassette that function as a unit to express a heterologous protein.", "For example, a promoter operably linked to a heterologous DNA, which encodes a protein, promotes the production of functional mRNA corresponding to the heterologous DNA.", "A “product” encoded by a DNA molecule includes, for example, RNA molecules and polypeptides.", "“Removal” in the context of a metabolite includes both physical removal as by washing and the depletion of the metabolite through the absorption and metabolizing of the metabolite by the cells.", "“Substantially isolated” is used in several contexts and typically refers to the at least partial purification of a protein or polypeptide away from unrelated or contaminating components.", "Methods and procedures for the isolation or purification of proteins or polypeptides are known in the art.", "“Stably transformed” as used herein refers to a cereal cell or plant that has foreign nucleic acid stably integrated into its genome which is transmitted through multiple generations.", "“α1-antitrypsin or “AAT” refers to the protease inhibitor which has an amino acid sequence substantially identical or homologous to AAT protein.", "“Antithrombin III” or “ATIII” refers to the heparin-activated inhibitor of thrombin and factor Xa, and which has an amino acid sequence substantially identical or homologous to ATIII protein.", "Human serum albumin” or “HSA” refers to a protein which has an amino acid sequena substantially identical or homologous to the mature HSA protein.", "“Subtilisin” or “subtilisin BPN′” or “BPN′” refers to the protease enzyme produced naturally by B. amyloliquefaciens.", "“proBPN” refers to a form of BPN′ having an approximately 78 amino-acid “pro” moiety that functions as a chaperon polypeptide to assist in folding and activation of the BPN'.", "“Codon optimization” refers to changes in the coding sequence of a gene to replace native codons with those corresponding to optimal codons in the host plant.", "A DNA sequence is “derived from” a gene, such as a rice or barley α-amylase gene, if it corresponds in sequence to a segment or region of that gene.", "Segments of genes which may be derived from a gene include the promoter region, the 5′ untranslated region, and the 3′ untranslated region of the gene.", "5.7.13 General Approach Generally, the nomenclature and laboratory procedures with respect to standard recombinant DNA technology can be found in Sambrook, et al., MOLECULAR-CLONING—A LABORATORY MANUAL, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989 and in S. B. Gelvin and R. A. Schilperoot, PLANT MOLECULAR BIOLOGY, 1988.Other general references are provided throughout this document.", "The procedures therein are known in the art and are provided for the convenience of the reader.", "Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail in, or example, European Patent Application Publication Number 223,452, published Nov. 29, 1986, which is incorporated herein by reference.", "Enzymes are obtained from commercial sources and are used according to the vendor's recommendations or other variations known in the art.", "General references containing such standard techniques include the following: R. Wu, ed.", "(1979) Methods Emzymology, Vol.", "68; J. H. Miller (1972) Experiments in Molecular Genetics; J. Sambrook et al.", "(1989) Molecular Cloning: A Laboratory Manual 2nd Ed.", "; D. M. Glover, ed.", "(1985) DNA Cloning Vol.", "II; H. G. Polites and K. R. Marotti (1987) “A step-wise protocol for cDNA synthesis, “Biotechniques 4; 514-520; S. B. Gelvin and R. A. Schilperoort, eds.", "Introduction, Expression, and Analysis of Gene Products in Plants, all of which are incorporated by reference.", "The recombinase/excision sequence system can be any one that selectively removes DNA in a plant genome.", "The excision sequences are preferably unique in the plant, so that unintended cleavage of the plant genome does not occur.", "Several examples of such systems are discussed in Sauer, U.S. Pat.", "No.", "4,959,317 and in Sadowski (1993).", "A preferred system is the bacteriophage CRE/LOX system, wherein the CRE protein performs site-specific recombination of DNA at LOX sites.", "Other systems include the resolvases (Hall, 1993), FLP (Pan, et al., 1993), SSVI encoded integrase (Muskhekishvili, et al., 1993), and the maize Ac/Ds transposon system (Shen and Hohn, 1992).", "5.7.13.1 Control of Plant Gene Expression 5.7.13.1.1 Using a Transiently-Active Promoter This invention relates to a method of creating transgenic plants wherein the expression of certain plant traits is ultimately under external control.", "In one embodiment the control is achieved through application of an external stimulus; in another embodiment it is achieved through hybridization, in still another embodiment it is achieved by direct introduction of a recombinase or recombinase gene into a plant.", "The transgenic plants of the present invention are prepared by introducing into their genome a series of functionally interrelated DNA sequences, containing the following basic elements: a plant-active promoter that is active at a particular stage in plant development or under particular environmental conditions (“transiently-active promoter”), a gene whose expression results in an altered plant phenotype which is linked to the transiently-active promoter through a blocking sequence separating the transiently-active promoter and the gene, unique specific excision sequences flanking the blocking sequence, wherein the specific excision sequences are recognizable by a site-specific recombinase, a gene encoding the site-specific recombinase, an alternative repressible promoter linked to the recombinase gene, and an alternative gene that encodes the repressor specific for the repressible promoter, the action of the repressor being responsive to an applied or exogenous stimulus.", "While these elements may be arranged in any order that achieves the interactions described below, in one embodiment they are advantageously arranged as follows: a first DNA sequence contains the transiently-active promotor, a first specific excision sequence, the blocking sequence, a second specific excision sequence, and the gene whose expression results in an altered plant phenotype; a second DNA sequence contains the repressible promoter operably linked to the recombinase gene, and optionally an enhancer; and a third DNA sequence containing the gene encoding the repressor specific for the repressible promoter, itself linked to a promoter functional and constitutive in plants.", "The third DNA sequence can conveniently act as the blocking sequence located in the first DNA sequence, but can also occur separately without altering the function of the system.", "This embodiment can be modified such that the recombinase sequence is introduced separately via a viral vector.", "In an alternative embodiment, an advantageous arrangement is as follows: a first plant containing a DNA sequence comprising the transiently-active promotor, a first specific excision signal sequence, the blocking sequence, a second specific excision signal sequence, and the gene whose expression results in an altered plant phenotype; a second plant containing a DNA sequence comprising a constitutive plant-active promotor operably linked to the recombinase gene (the two plants being hybridized to produce progeny that contain all of the above sequences).", "When a plant contains the basic elements of either embodiment, the gene whose expression results in an altered plant phenotype is not active, as it is separated from its promoter by the blocking sequence.", "In the first embodiment, absent the external stimulus, the repressor is active and represses the promoter that controls expression of the recombinase; in the alternative embodiment the recombinase is not present in the same plant as the first DNA sequence.", "Such a plant will not display the altered phenotype, and will produce seed that would give rise to plants that also do not display the altered phenotype.", "When the 5 stimulus to which the repressor is sensitive is applied to this seed or this plant, the repressor no longer functions, permitting the expression of the site-specific recombinase, or alternatively, when the recombinase is introduced via hybridization it is expressed during germination of the seed, either of which effects the removal of the blocking sequence between the specific excision signal sequences.", "Upon removal of the blocking sequence, the transiently-active promoter becomes directly linked to the gene whose expression results in an altered plant phenotype.", "A plant grown from either treated or hybrid seed, or a treated plant, will still not exhibit the altered phenotype, until the transiently-active promoter becomes active during the plant's development, after which the gene to which it is linked is expressed, and the plant will exhibit an altered phenotype.", "5.7.13.2 Transgenic Plants that Produce Seeds that Cannot Germinate In a preferred embodiment, the present invention involves a transgenic plant or seed which, upon treatment with an external stimulus produces plants that produce seed that cannot germinate (but that is unaltered in other respects).", "If the transiently-active promoter is one that is active only in late embryogenesis, the gene to which it is linked will be expressed only in the last stages of seed development or maturation.", "If the gene linked to this promoter is a lethal gene, it will render the seed produced by the plants incapable of germination.", "In the initially-transformed plant cells, this lethal gene is not expressed, not only because the promoter is intrinsically inactive, but because there is a blocking sequence separating the lethal gene from its promoter.", "Also within the genome of these cells are the genes for the recombinase, linked to a repressible promoter, and the gene coding for the repressor.", "The repressor is expressed constitutively and represses the expression of the recombinase.", "These plant cells can be regenerated into a whole plant and allowed to produce seed.", "The mature seed is exposed to a stimulus, such as a chemical agent, that inhibits the function of the repressor.", "Upon inhibition of the repressor, the promotor driving the recombinase gene is depressed and the recombinase gene is expressed.", "The resulting recombinase recognizes the specific excision sequences flanking the blocking sequence, and effects the removal of the blocking sequence.", "The late embryogenesis promoter and the lethal gene are then directly linked.", "The lethal gene is not expressed, however, because the promoter is not active at this time in the plant's life cycle.", "This seed can be planted, and grown to produce a desired crop of plants.", "As the crop matures and produces a second generation of seed, the late embryogenesis promoter becomes active, the lethal gene is expressed in the maturing second generation seed, which is rendered incapable of germination.", "In this way, accidental reseeding, escape of the crop plant to areas outside the area of cultivation, or germination of stored seed can be avoided.", "5.7.13.1.3 Trangenic Plants Hybridized to Display Phenotype Not Seen in Either Parent In an alternative preferred embodiment, the present invention involves a pair of transgenic plants that are hybridized to produce progeny that display a phenotype not seen in either parent.", "In this alternative embodiment a transiently-active promotor that is active only in late embryogenesis can be linked to a lethal gene, with an intervening blocking sequence bounded by the specific excision sequences.", "These genetic sequences can be introduced into plant cells to produce one transgenic parent plant.", "The recombinase gene is linked to a germination-specific promotor and introduced into separate plant cells to produce a second transgenic parent plant.", "Both of these plants can produce viable seed if pollinated.", "If the first and second transgenic parent plants are hybridized, the progeny will contain both the blocked lethal gene and the recombinase gene.", "The recombinase is expressed upon germination of the seed and effects the removal of the blocking sequence, as in the first embodiment, thereby directly linking the lethal gene and the transiently-active promotor.", "As in the first embodiment, this promotor becomes active during maturation of the second generation seed, resulting in seed that is incapable of germination.", "Ideally, the first parent employs a male-sterility gene as the blocking sequence, and includes an herbicide resistance gene.", "In this way, self-pollination of the first transgenic parent plant is avoided, and self-pollinated second transgenic parent plants can be eliminated by application of the herbicide.", "In the hybrid progeny, the male-sterility gene is removed by the recombinase, resulting in hybrid progeny capable of self-pollination.", "5.7.13.1.4.Linking the Recombinase Gene to an Inducible Promoter In another embodiment, the recombinase gene is linked to an inducible promoter.", "Examples of such promoters include the copper, controllable gene expression system (Mett et al., 1993) and the steroid-inducible gene system (Schena et al., 1991).", "Exposure of the transgenic plant to the inducer specific for the inducible promoter leads to expression of the recombinase gene and the excision of the blocking sequence.", "The gene that results in an altered plant phenotype is then expressed when the transiently active promoter becomes active.", "The gene or genes linked to the plant development promoter can be any gene or genes whose expression results in a desired detectable phenotype.", "In one method, Gene(s) can be Linked to the Plant Development Promoter to control expression developmentally.", "In those embodiments employing a repressible promoter system, the gene encoding the repressor is responsive to an outside stimulus, or encodes a repressor element that is itself responsive to an outside stimulus, so that repressor function can be controlled by the outside stimulus.", "The stimulus is preferably one to which the plant is not normally exposed, such as a particular chemical, temperature shock, or osmotic shock.", "A preferred system is the Tn10 tet repressor system, which is responsive to tetracycline.", "Gatz and Quail (1988); Gatz, et al.", "(1992).", "Examples of other repressible promoter systems are described by Lanzer and Bujard (1988) and Ptashne, et al.", "5.7.14.Conferring Viral Resistance to the Plant To practice the present invention, a viral gene must be isolated from the viral genome and inserted into a vector containing the genetic regulatory sequences necessary to express the inserted gene.", "Accordingly, a vector must be constructed to provide the regulatory sequences such that they will be functional upon inserting a desired gene.", "When the expression vector/insert construct is assembled, it is used to transform plant cells which are then used to regenerate plants.", "These transgenic plants carry the viral gene in the expression vector/insert construct.", "The gene is expressed in the plant and increased resistance to viral infection is conferred thereby.", "5.7.14.1.Table of Selected Literature References to Methods of Isolating, Cloning and Expressing Viral Genes The nucleotide sequences encoding the coat protein genes and nuclear inclusion genes of a number of viruses have been determined and the genes have been inserted into expression vectors.", "The expression vectors contain the necessary genetic regulatory sequences for expression of an inserted gene.", "The coat protein gene is inserted such that those regulatory sequences are functional and the genes can be expressed when incorporated into a plant genome.", "Selected literature references to methods of isolating, cloning and expressing viral genes are listed on Table 3, below.", "TABLE 3 Cloned Genes From RNA Viruses Viral Gene Reference Papaya ringspot cp M. M. Fitch et al., Bio/Technology, 10, 1466(1992) Potato virus X cp K. Ling et al., Bio/Technology, 2, 752 (1991); A. Hoekema et al., Bio/Technology, 7, 273 (1989) Watermelon Mosaic Virus H. Quemada et al., J. Gen.", "Virol., 71, 1451 II cp (1990); S. Namba et al., Phytopathology, 82, 940 (1992) Zucchini yellow Mosaic S. Namba et al., Phytopathology, 82, 940 Virus cp (1992) Tobacco Mosaic Virus cp R. S. Nelson et al., Bio/Technology, 6, 403 (1988); P. Powell Abel et al., Science, 232, 738 (1986) Alfalfa Mosaic Virus cp Loesch-Fries et al., EMBO J., 6, 1845 (1987); N. E. Turner et al., EMBO J., 6, 1181 (1987) Soybean Mosaic Virus cp D. M. Stark et al., Biotechnology, 7, 1257 (1989) Cucumber Mosaic Virus H. Q. Quemada et al., Molec.", "Plant Pathol., strain C cp 81, 794 (1991) Cucumber Mosaic Virus UpJohn Co. (PCT W090/02185) strain WL cp Tobacco etch virus cp Allison et al., Virology, 147, 309 (1985) Tobacco etch virus nu- J. C. Carrington et al., J.", "Virol., 61, 2540 clear inclusion protein (1987) Pepper Mottle Virus cp W. G. Dougherty et al., Virology, 146, 282 (1985) Potato virus Y cp D. D. Shukla et al., Virology, 152, 118 (1986) Potato virus Y nuclear European Patent Application 578, 627 inclusion protein Potato virus X cp C. Lawson et al., Biotechnology, 8, 127 (1990) Tobacco streak virus C. M. Van Dun et al., Virology, 164, 383 (TSV) cp (1988) 5.7.15 Disease Resistance One aspect of the present invention relates to the use of trait DNA molecules which are heterologous to the plant—e.g., DNA molecules that confer disease resistance to plants transformed with the DNA construct.", "5.7.15.1 Sense/Antisense Orientation The DNA molecule conferring disease resistance can be positioned within the DNA construct in sense orientation.", "Alternatively, it can have an antisense orientation.", "Antisense RNA technology involves the production of an RNA molecule that is complementary to the messenger RNA molecule of a target gene; the antisense RNA can potentially block all expression of the targeted gene.", "In the anti-virus context, plants are made to express an antisense RNA molecule corresponding to a viral RNA (that is, the antisense RNA is an RNA molecule which is complementary to a plus sense RNA species encoded by an infecting virus).", "Such plants may show a slightly decreased susceptibility to infection by that virus.", "Such a complementary RNA molecule is termed antisense RNA.", "5.7.16 Other Traits The present invention is also used to confer traits other than disease resistance on plants.", "For example, DNA molecules which impart a plant genetic trait can be used as the DNA trait molecule of the present invention.", "In this aspect of the present invention, suitable trait DNA molecules encode for desired color, enzyme production, or combinations thereof.", "5.7.16.1 Gene Silencing The silencer DNA molecule of the present invention can be selected from virtually any nucleic acid which effects gene silencing.", "This involves the cellular mechanism to degrade mRNA homologous to the transgene mRNA.", "The silencer DNA molecule can be heterologous to the plant, need not interact with the trait DNA molecule in the plant, and can be positioned 3′ to the trait DNA molecule.", "For example, the silencer DNA molecule can be a viral cDNA molecule, a jellyfish green fluorescence protein encoding DNA molecule, a plant DNA molecule, or combinations thereof.", "While not wishing to be bound by theory, by use of the construct of the present invention, it is believed that post-transcriptional gene silencing is achieved.", "More particularly, the silencer DNA molecule is believed to boost the level of heterologous RNA within the cell above a threshold level.", "This activates the degradation mechanism by which viral resistance is achieved.", "5.7.16.1.1 Trait & Silencer DNA Molecules Encoding RNA Molecules—Translatable It is possible for the DNA construct of the present invention to be configured so that the trait and silencer DNA molecules encode RNA molecules which are translatable.", "As a result, that RNA molecule will be translated at the ribosomes to produce the protein encoded by the DNA construct.", "Production of proteins in this manner can be increased by joining the cloned gene encoding the DNA construct of interest with synthetic double-stranded oligonucleotides which represent a viral regulatory sequence (i.e., a S′ untranslated sequence).", "See U.S. Pat.", "No.", "4,820,639 to Gehrke and U.S. Pat.", "No.", "5,849,527 to Wilson which are hereby incorporated by reference.", "5.7.16.1.2 Trait & Silencer DNA Molecules Encoding RNA Molecules—Not Translatable Alternatively, the DNA construct of the present invention can be configured so that the trait and silencer DNA molecules encode mRNA which is not translatable.", "This is achieved by introducing into the DNA molecule one or more premature stop codons, adding one or more bases (except multiples of 3 bases) to displace the reading frame, removing the translation initiation codon, etc.", "See U.S. Pat.", "No.", "5,583,021 to Dougherty et al., which is hereby incorporated by reference.", "5.7.17 Recombinant DNA Technology The subject DNA construct can be incorporated in cells using conventional recombinant DNA technology.", "Generally, this involves inserting the DNA construct into an expression system to which the DNA construct is heterologous (i.e.", "not normally present).", "The heterologous DNA construct may be inserted into the expression system or vector in proper sense orientation and correct reading frame.", "The vector contains the necessary elements for the transcription of the inserted sequences.", "5.7.18 Incorporation into a Host Cell Once the DNA construct has been cloned into an expression system, it is ready to be incorporated into a host cell.", "Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system.", "Suitable host cells include, but are not limited to, bacteria, virus, plant, is and the like cells.", "5.7.18.1 Plant Transformation—Production of Mature Proteins in Plants Expression vectors for use in the present invention comprise a chimeric gene (or expression cassette), designed for operation in plants, with companion sequences upstream and downstream from the expression cassette.", "The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from bacteria to the desired plant host.", "For transformation of plants, the chimeric gene is placed in a suitable expression vector designed for operation in plants.", "The vector includes suitable elements of plasmid or viral origin that provide necessary characteristics to the vector to permit the vectors to move DNA from bacteria to the desired plant host.", "Suitable components of the expression vector, including an inducible promoter, coding sequence for a signal peptide, coding sequence for a mature heterologous protein, and suitable termination sequences, are discussed below.", "One exemplary vector is the p3Dvl.O (p3D(AAT)v1.0) vector described herein.", "5.7.18.1.2 Transformation Vector Vectors containing a chimeric gene of the present invention may also include selectable markers for use in plant cells (such as the nptII kanamycin resistance gene, for selection in kanamycin-containing or the phosphinothricin acetyltransferase gene, for selection in medium containing phosphinothricin (PPT).", "The vectors may also include sequences that allow their selection and propagation in a secondary host, such as sequences containing an origin of replication and a selectable marker such as antibiotic or herbicide resistance genes, e.g., HPH (Hagio et al., Plant Cell Reports 14: 329 (1995); van der Elzer, Plant Mol.", "Biol.", "5: 299-302 (1985)).", "Typical secondary hosts include bacteria and yeast.", "In one embodiment, the secondary host is Escherichia coli, the origin of replication is a colEl-type, and the selectable marker is a gene encoding ampicillin resistance.", "Such sequences are well known in the art and are commercially available as well (e.g., Clontech, Palo Alto, Calif.; Stratagene, La Jolla, Calif.).", "The vectors of the present invention may also be modified to intermediate plant transformation plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T-DNA border region from Agrobacterium tumefaciens, and chimeric genes or expression cassettes (described above).", "Further, the vectors of the invention may comprise a disarmed plant tumor inducing plasmid of Agrobacterium tumefaciens.", "5.7.19 Plant Expression Vector Production of Mature Proteins in Plants 5.7.19.1 Suitable Vectors Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pER322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see F. W. Studier et.", "al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technolog vol.", "185 (1990), which is hereby incorporated by reference), and any derivatives thereof.", "Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.", "The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference.", "5.7.19.2 Host-Vector Systems A variety of host-vector systems may be utilized to carry out the present invention.", "Primarily, the vector system must be compatible with the host cell used.", "Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.", "); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.", "The expression elements of these vectors vary in their strength and specificities.", "Depending upon the host-vector system utilized, any one of a number of suitable transcription and, perhaps, translation elements can be used.", "5.7.19.3 Signal Sequences for Production of Mature Proteins In addition to encoding the protein of interest, the chimeric gene encodes a signal sequence (or signal peptide) that allows processing and translocation of the protein, as appropriate.", "Suitable signal sequences are described in above-referenced PCT application WO 95/14099.The plant signal sequence is placed in frame with a heterologous nucleic acid encoding a mature protein, forming a construct which encodes a fusion protein having an N-terminal region corresponding to the signal peptide and, immediately adjacent to the C-terminal amino acid of the signal peptide, the N-terminal amino acid of the mature heterologous protein.", "The expressed fusion protein is subsequently secreted and processed by signal peptidase cleavage precisely at the junction of the signal peptide and the mature protein, to yield the mature heterologous protein.", "In another embodiment of the invention, the coding sequence in the fusion protein gene, in at least the coding region for the signal sequence, may be codon-optimized for optimal expression in plant cells, e.g., rice cells, as described below.", "5.7.20 Promotors Transcription Dependent upon the Presence of a Promotor Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.", "The DNA sequences of eucaryotic promoters differ from those of procaryotic promotors.", "Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.", "The segment of DNA referred to as the promoter is responsible for the regulation of the transcription of DNA into mRNA.", "A number of promoters which function in plant cells are known in the art and may be employed in the practice of the present invention.", "These promoters may be obtained from a variety of sources such as plants or plant viruses, and may include but are not limited to promoters isolated from the caulimovirus group such as the cauliflower mosaic virus 35S promoter (CaMV35S), the enhanced cauliflower mosaic virus 35S promoter (enh CaMV35S), the figwort mosaic virus full-length transcript promoter (FMV35S), and the promoter isolated from the chlorophyll alb binding protein.", "Other useful promoters include promoters which are capable of expressing the potyvirus proteins in an inducible manner or in a tissue-specific manner in certain cell types in which the infection is known to occur.", "For example, the inducible promoters from phenylalanine ammonia lyase, chalcone synthase, hydroxyproline rich glycoprotein, extensin, pathogenesis-related proteins (e.g.", "PR—I a), and wound-inducible protease inhibitor from potato may be useful.", "Preferred promoters for use in the present viral gene expression cassettes include the constitutive promoters from CaMV, the Ti genes nopaline synthase (Bevan et al., Nucleic Acids Res.", "11, 369-385 (1983)) and octopine synthase (Depicker et al., J. Mol.", "Appl.", "Genet., 1, 561-564 (1982)), and the bean storage protein gene phaseolin.", "The poly(A) addition signals from these genes are also suitable for use in the present cassettes.", "The particular promoter selected is preferably capable of causing sufficient expression of the DNA coding sequences to which it is operably linked, to result in the production of amounts of the proteins or the RNAs effective to provide viral resistance, but not so much as to be detrimental to the cell in which they are expressed.", "The promoters selected should be capable of functioning in tissues including but not limited to epidermal, vascular, and mesophyll tissues.", "The actual choice of the promoter is not critical, as long as it has sufficient transcriptional activity to accomplish the expression of the preselected proteins or antisense RNA, and subsequent conferral of viral resistance to the plants.", "Promotors vary in their “strength” (i.e.", "their ability to promote transcription).", "For the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene.", "Depending upon the host cell system utilized, any one of a number of suitable promotors may be used.", "For instance, when cloning in E. coli, its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, Ipp, and the like, may be used to direct high levels of transcription of adjacent DNA segments.", "Additionally, a hybrid trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.", "5.7.20.1 Promoters that Transcribe the cereal A-Amylase Genes and Sucrose Synthase Genes The transcription regulatory or promoter region is chosen to be regulated in a manner allowing for induction under selected cultivation conditions, e.g., sugar depletion in culture or water uptake followed by gibberellic acid production in germinating seeds.", "Suitable promoters, and their method of selection are detailed in above-cited PCT application WO 95/14099.Examples of such promoters include those that transcribe the cereal a-amylase genes and sucrose synthase genes, and are repressed or induced by small molecules, like sugars, sugar depletion or phytohormones such as gibberellic acid or absissic acid.", "Representative promoters include the promoters from the rice a-amylase RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, and RAmy3E genes, and from the pM/C, gKAmyl41, gKAmyl55, Amy32b, and HV18 barley (x-amylase genes.", "These promoters are described, for example, in ADVANCES IN PLANT BIOTECHNOLOGY Ryu, D. D. Y., et al, Eds., Elsevier, Amsterdam, 1994, p. 37, and references cited therein.", "Other suitable promoters include the sucrose synthase and sucrose-6-phosphate-synthetase (SPS) promoters from rice and barley.", "Other suitable promoters include promoters which are regulated in a manner allowing for induction under seed-maturation conditions.", "Examples of such promoters include those associated with the following monocot storage proteins: rice glutelins, oryzins, and prolamines, barley hordeins, wheat gliadins and glutelins, maize zeins and glutelins, oat glutelins, and sorghum kafirins, millet pennisetins, and rye secalins.", "A preferred promoter for expression in germinating seeds is the rice a-amylase RAmy1A promoter, which is upregulated by gibberellic acid.", "Preferred promoters for expression in cell culture are the rice a-amylase RAmy3D and RAmy3E promoters which are strongly upregulated by sugar depletion in the culture.", "These promoters are also active during seed germination.", "A preferred promoter for expression in maturing seeds is the barley endosperm-specific BI-hordein promoter (Brandt, A., et al., (1985) Carlsberg Res.", "Commun.", "50: 333-345).", "The chimeric gene may further include, between the promoter and coding sequences, the 5′ untranslated region (5′ UTR) of an inducible monocot gene, such as the 5′ UTR derived from one of the rice or barley a-amylase genes mentioned above.", "One preferred 5′ UTR is that derived from the RAmy1A gene, which is effective to enhance the stability of the gene transcript.", "5.7.20.2 Use of Inducers Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced.", "In certain operations, the addition of specific inducers is necessary for efficient transcription of the inserted DNA.", "For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).", "A variety of other operons, such as trp, pro, etc., are under different controls.", "5.7.20.3 Transcription Initiation Signals Specific initiation signals are also required for efficient gene transcription in procaryotic cells.", "These transcription initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.", "The DNA expression vector, which contains a promotor, may also contain any combination of various “strong” transcription initiation signals.", "5.7.20.4 Translation Dependent on Shine-Dalgarno Sequence (in Procaryotes) Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes.", "Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgamo (“SD”) sequence on the mRNA.", "This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein.", "The SD sequences are complementary to the 31-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome.", "For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzmmology, 68: 473 (1979), which is hereby incorporated by reference.", "The non-translated leader sequence can be derived from any suitable source and can be specifically modified to increase the translation of the mRNA.", "The 5′ non-translated region can be obtained from the promoter selected to express the gene, an unrelated promoter, the native leader sequence of the gene or coding region to be expressed, viral RNAs, suitable eucaryotic genes, or a synthetic gene sequence.", "The present invention is not limited to the constructs presented in the following examples.", "5.7.20.5 A4.Codon-Optimized Coding Sequences In accordance with one aspect of the invention, it has been discovered that a severalfold enhancement of expression level can be achieved in plant cell culture by modifying the native coding sequence of a heterologous gene by contain predominantly or exclusively, highest-frequency codons found in the plant cell host.", "The method will be illustrated for expression of a heterologous gene in rice plant cells, it being recognized that the method is generally applicable to any monocot.", "As a first step, a representative set of known coding gene sequence from rice is assembled.", "The sequences are then analyzed for codon frequency for each amino acid, and the most frequent codon is selected for each amino acid.", "This approach differs from earlier reported codon matching methods, in which more than one frequent codon is selected for at least some of the amino acids.", "The optimal codons selected in this manner for rice and barley are shown in Table 4.TABLE 4 Amino Acid Rice Preferred Codon Barley Preferred Codon Ala A GCC Arg R CGC Asn N AAC Asp D GAC Cys C UGC Gln Q CAG Glu E GAG Gly G GGC His H CAC Ile I AUC Leu L CUC Lys K AAG Phe F UUC Pro P CCG CCC Ser S AGC UCC Thr T ACC Tyr Y UAC Val V GUC GUG stop UAA UGA As indicated above, the fusion protein coding sequence in the chimeric gene is constructed such that the final (C-terminal) codon in the signal sequence is immediately followed by the codon for the N-terminal amino acid in the mature form of the heterologous protein.", "TABLE 5 Location (Asn) (in N-Glycosylation Sites mature protein) Amino Acid Substitution Asn Asn Ser 61 Thr Asn Ser Asn Asn Ser 76 Thr Asn Ser Asn Met Ser 123 Thr Met Ser Asn Gly Thr 218 Ser Gly Thr′ Asn Trp Thr 240 Thr Trp Thr ′improved thermostability; Bryan, et al., Proteins: Structure, Function, and Genetics 1: 326 (1986).", "5.7.20.6 Termination Sequence Coupled To The Fusion End The present invention can also utilize a termination sequence operatively coupled to the fusion gene to end transcription.", "Suitable transcription termination sequences include the termination region of a 3′ non-translated region.", "This will cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed mRNA sequence.", "The termination region or 31 non-translated region will be additionally one of convenience.", "The termination region may be native with the promoter region or may be derived from another source, and preferably includes a terminator and a sequence coding for polyadenylation.", "Suitable 3′non-translated regions include but are not limited to: (1) the 3′ transcribed, non-translated regions containing the polyadenylated signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene or the 35S promoter terminator gene, and (2) plant genes like the soybean 7S storage protein genes and the pea small subunit of the ribulose 1,5-bisphosphate carboxylase-oxygenase (ssRUBISCO) E9 gene.", "The termination region or 3′ non-translated region which is employed is one which will cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3′ end of the transcribed mRNA sequence.", "The termination region may be native with the promoter region, native with the structural gene, or may be derived from another source, and preferably include a terminator and a sequence coding for polyadenylation.", "Suitable 3′ non-translated regions of the chimeric plant gene include but are not limited to: (1) the 3′ transcribed, non-translated regions containing the polyadenylation signal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopaline synthase (NOS) gene, and (2) plant genes like the soybean 7S storage protein genes.", "5.7.20.7 Transcription and Translation Terminators for Production of Mature Proteins The chimeric gene may also include, downstream of the coding sequence, the 3′ untranslated region (Y UTR) from an inducible monocot gene, such as one of the rice or barley a-amylase genes mentioned above.", "One preferred 3′ UTR is that derived from the RAmy1A gene.", "This sequence includes non-coding sequence 5′ to the polyadenylation site, the polyadenylation site, and the transcription termination sequence.", "The transcriptional termination region may be selected, particularly for stability of the mRNA to enhance expression.", "Polyadenylation tails (Alber and Kawasaki, 1982, Mol.", "and Appl.", "Genet.", "1: 419-434) are also commonly added to the expression cassette to optimize high levels of transcription and proper transcription termination, respectively.", "Polyadenylation sequences include but are not limited to the Agrobacterium octopine synthetase signal (Gielen, et al., EMBO J.", "3: 835-846 (1984) or the nopaline synthase of the same species (Depicker, et al., Mol.", "Appl.", "Genet.", "1: 561-573 (1982).", "Since the ultimate expression of the heterologous protein will be in a eukaryotic cell (in this case, a member of the grass family), it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicing machinery.", "If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code (Reed and Maniatis, Cell 41: 95-105 (1985).", "5.7.20.9 Selectable Marker Gene Selectable marker genes may be incorporated into the present expression cassettes and used to select for those cells or plants which have become transformed.", "The marker gene employed may express resistance to an antibiotic, such as kanamycin, gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracyline, chloramphenicol, and the like.", "Other markers could be employed in addition to or in the alternative, such as, for example, a gene coding for herbicide tolerance such as tolerance to glphosate, sulfonylurea, phosphinothricin, or bromoxynil.", "Additional means of selection could include resistance to methotrexate, heavy metals, complementation providing prototrophy to an auxotrophic host, and the like.", "For example, see Table 1 of PCT WO/91/10725, cited above.", "The present invention also envisions replacing all of the virus-associated genes with an array of selectable marker genes.", "The particular marker employed will be one which will allow for the selection of transformed cells as opposed to those cells which were not transformed.", "Depending on the number of different host species one or more markers may be employed, where different conditions of selection would be useful to select the different host, and would be known to those of skill in the art.", "A screenable marker or “reporter gene” such as the 0-glucuronidase gene or luciferase gene may be used in place of, or with, a selectable marker.", "Cells transformed with this gene may be identified by the production of a blue product on treatment with 5-bromo-4-chloro-3-indoyl-β-D-glucuronide (X-Gluc).", "In developing the present expression construct, the various components of the expression construct such as the DNA sequences, linkers, or fragments thereof will normally be insetted into a convenient cloning vector, such as a plasmid or phage, which is capable of replication in a bacterial host, such as E. coli.", "Numerous cloning vectors exist that have been described in the literature.", "After each cloning, the cloning vector may be isolated and subjected to further manipulation, such as restriction, insertion of new fragments, ligation, deletion, resection, insertion, in vitro mutagenesis, addition of polylinker fragments, and the like, in order to provide a vector which will meet a particular need.", "5.7.20.10 Transferring Recombinant DNA into Plant Cell 5.7.20.10.1 Use of Micropipettes or Polyethylene Glycol In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA.", "Crossway, Mol.", "Gen. Genetics, 202: 179-85 (1985), which is hereby incorporated by reference.", "The genetic material may also be transferred into the plant cell using polyethylene glycol.", "Krens, et al., Nature, 296: 72-74 (1982), which is hereby incorporated by reference.", "5.7.4.12.2 Particle Bombardment (Biolistic Transformation) Another approach to transforming plant cells with the DNA construct is particle bombardment (also known as biolistic transformation) of the host cell.", "This can be accomplished in one of several ways.", "The first involves propelling inert or biologically active particles at cells.", "This technique is disclosed in U.S. Pat.", "Nos.", "4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which are hereby incorporated by reference.", "5.7.4.12.3 Fusion of Protoplasts with Other Entities Yet another method of introduction is fusion of protoplasts with other entities,—either minicells, cells, lysosomes or other fusible lipid-surfaced bodies.", "Fraley, et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 79: 1859-63 (1982), which is hereby incorporated by reference.", "5.7.4.12.4 Electroporation The DNA molecule may also be introduced into the plant cells by electroporation.", "Fromm et al., Proc.", "Natl.", "Acad.", "Sci.", "USA, 82: 5824 (1985), which is hereby incorporated by reference.", "In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette.", "Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids.", "Electroporated plant protoplasts reform the cell wall, divide, and regenerate.", "5.7.4.12.5 Infection with Agrobacterium tumefaciens or A. rhizogenes Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene.", "Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants.", "Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C. Methods are explained in various references such as: J. Schell, Science, 237: 1176-83 (1987); (U.S. Pat.", "No.", "5,258,300); Herrera-Estrella, Nature, 303, 209 (1983), Biotechnica (published PCT application PCT WO/91/10725), and U.S. Pat.", "No.", "4,940,838.5.7.21 Method for Making Genetically Recombinant Plants in Commercially Feasible Numbers In one preferred embodiment, the invention provides for a process for propagating plants by tissue culture in such a way as both to conserve desired plant morphology and to transform the plant with respect to one or more desired genes.", "The method includes the steps of (a) creating an Agrobacterium vector containing the gene sequence desired to be transferred to the propagated plant, preferably together with a marker gene; (b) taking one or more petiole explants from a mother plant and inoculating them with the Agrobacterium vector; (c) conducting callus formation in the petiole sections in culture, in the dark; and (d) culturing the resulting callus in growth medium having a benzylamino growth regulator such as benzylaminopurine or, most preferably, benzylaminopurineriboside.", "Additional optional growth regulators including auxins and cytokinins (indole butyric acid, benzylamine, benzyladenine, benzylaminopurine, alpha naphthylacetic acid and others known in the art) may also be present.", "Preferably, the petiole tissue is taken from Pelarcronium x domesticum and the Acrobacterium vector contains an antisense gene for ACC synthase or ACC oxidase to prevent ACC synthase or ACC oxidase expression and, in turn, preventing ethylene formation.", "Pelargoniums propagated in culture using the present technique are resistant to wilting and petal shatter, and are morphologically conserved due to the use of petiole explants specifically and the particular culture media disclosed.", "Using a probe for the transposon, the mutated gene can be isolated.", "Then, using the DNA adjacent to the transposon in the isolated, mutated gene as a probe, the normal wild-type allele of the target gene can be isolated.", "Such techniques are taught, for example, in McLaughlin and Walbot, Genetics, Vol.", "117 pp.", "771-776 (1987), as well as numerous other references.", "5.7.21.1 Reporter Gene In addition to the functional gene and the selectable marker gene, the DNA sequences may also contain a reporter gene which facilitates screening of the transformed shoots and plant material for the presence and expression of endogenous DNA sequences.", "Exemplary reporter genes include β-glucuronidase and luciferase.", "5.7.21.2.Transfer Regions of a Suitable Plasmid As described above, the exogenous DNA sequences are introduced into the area of the explants by incubation with Agrobacterium cells which carry the sequences to be transferred within a transfer DNA (T-DNA) region found on a suitable plasmid, typically the Ti plasmid.", "Ti plasmids contain two regions essential for the transformation of plant cells.", "One of these, the T-DNA region, is transferred to the plant nuclei and induces tumor formation.", "The other, referred to as the virulence (vir) region, is essential for the transfer of the T-DNA but is not itself transferred.", "By inserting the DNA sequence to be transferred into the T-DNA region, introduction of the DNA sequences to the plant genome can be effected.", "Usually, the Ti plasmid will be modified to delete or to inactivate the tumor-causing genes so that they are suitable for use as a vector for the transfer of the gene constructs of the present invention.", "Other plasmids may be utilized in conjunction with Agrobacterium for transferring the DNA sequences of the present invention to the plant cells.", "The construction of recombinant Ti plasmids may be accomplished using conventional recombinant DNA techniques, such as those described by Sambrook et al.", "(1989).", "Frequently, the plasmids will include additional selective marker genes which permit manipulation and construction of the plasmids in suitable hosts, typically bacterial hosts other than Agrobacterium, such as E. coli.", "In addition to the above-described kanamycin resistance marker gene, other exemplary genes are the tetracycline resistance gene and the ampicillin resistance gene, among others.", "5.7.21.3 Confirming Transformation After green transformed shoots are approximately ½″ tall, they can then be transplanted to soil within a greenhouse or elsewhere in a conventional manner for tissue culture plantlets.", "Transformation of the resulting plantlets can be confirmed by assaying activity for the selection marker, or by assaying the plant material for any of the phenotypes which have been introduced by the exogenous DNA.", "Suitable assay techniques include polymerase chain reaction (PCR), restriction enzyme digestion, Southern blot hybridization and Northern blot hybridization.", "5.7.21.4.Commercial Production The present invention represents a breakthrough in the commercial production and genetically transformed plants.", "Because the method uses petiole tissue from a grower's mother plant (a stock plant), the starting petiole explants have a commercially desirable morphology to begin with—by definition.", "However, if the mother plant could be improved by genetic transformation of some type, for example to deactivate a gene which expresses an enzyme in the ethylene synthesis pathway, the progeny of the mother plant may thus be improved in this one way over their parent stock.", "The petiole tissue from the stock plant, plus the genetic transformation from the Agrobacterium, yield both an improved genetic makeup of the commercially produced plants—although with preserved desired morphology from the mother plant—and at the same time the high yields possible only with the generation of many plantlets in a single generation's growth in tissue culture.", "In summary, with the present method a single genetically transformed mother plant can yield literally thousands of offspring plants.", "No one in the prior art has attempted to combine these two previously disparate technologies to achieve a unique method in which the result is no less than a commercially viable technique for making genetically recombinant plants in commercially feasible numbers.", "5.7.22 Transformation of Plant Cells Using Alternative Methods A variety of techniques are available for the introduction of the genetic material into or transformation of the plant cell host.", "However, the particular manner of introduction of the plant vector into the host is not critical to the practice of the present invention, and any method which provides for efficient transformation may be employed.", "In addition to transformation using plant transformation vectors derived from the tumor-inducing (Ti) or root-inducing (RI) plasmids of Agrobacterium, alternative methods could be used to insert the DNA constructs of the present invention into plant cells.", "Such methods may include, for example, the use of liposomes, transformation using viruses or pollen, chemicals that increase the direct uptake of DNA (Paszkowski et al., EMBO J., 3, 2717 (1984)), microinjection (Crossway et al., Mol.", "Gen.", "Genet., 202, 179 (1985)), electroporation (Fromm et al., Proc.", "Natl.", "Acad.", "Sci.", "US, 82, 824 (1985)), or high-velocity microprojectiles (Klein et al., Nature, 327, 70 (1987)).", "5.7.22.1 Plant Tissue Source or Cultured Plant Cell The choice of plant tissue source or cultured plant cells for transformation will depend on the nature of the host plant and the—transformation protocol.", "Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like.", "The tissue source is regenerable, in that it will retain the ability to regenerate whole, fertile plants following transformation.", "5.7.22.2 Conditions During Transformation The transformation is carried out under conditions directed to the plant tissue of choice.", "The plant cells or tissue are exposed to the DNA carrying the present multi-gene expression cassette for an effective period of time.", "This may range from a less-than-one-second pulse of electricity for electroporation, to a two-to-three day co-cultivation in the presence of plasmid-beazing Agrobacterium cells.", "Buffers and media used will also vary with the plant tissue source and transformation protocol.", "Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet Corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.", "Following treatment with DNA, the plant cells or tissue may be cultivated for varying lengths of time prior to selection, or may be immediately exposed to a selective agent such as those described hereinabove.", "5.7.22.3 Inhibitory Agent Protocols involving exposure to Agrobacterium will also include an agent inhibitory to the growth of the Agrobacterium cells.", "Commonly used compounds are antibiotics such as cefotaxime and carbenicillin.", "The media used in the selection may be formulated to maintain transformed callus or suspension culture cells in an undifferentiated state, or to allow production of shoots from callus, leaf or stem segments, tuber disks, and the like.", "5.7.23 Method for Transformation of The Target Plant The methods used for the actual transformation of the target plant are not critical to this invention.", "The transformation of the plant is preferably permanent, e.g.", "by integration of introduced sequences into the plant genome, so that the introduced sequences are passed onto successive plant generations.", "There are many plant transformation techniques well-known to workers in the art, and new techniques are continually becoming known.", "Any technique that is suitable for the target plant can be employed with this invention.", "For example, the sequences can be introduced in a variety of forms, such as a strand of DNA, in a plasmid, or in an artificial chromosome, to name a few.", "The introduction of the sequences into the target plant cells can be accomplished by a variety of techniques, as well, such as calcium phosphate-DNA co-precipitation, electroporation, microinjection, Agrobacterium infection, liposomes or microprojectile transformation.", "Those of ordinary skill in the art can refer to the literature for details, and select suitable techniques without undue experimentation.", "5.7.23.1 Introduction of Sequences into Target Plant Cells It is possible to introduce the recombinase gene, in particular, into the transgenic plant in a number of ways.", "The gene can be introduced along with all of the other basic sequences, as in the first preferred embodiment described above.", "The repressible promoter/recombinase construct can be also introduced directly via a viral vector into a transgenic plant that contains the other sequence components of the system.", "Still another method of introducing all the necessary sequences into a single plant is the second preferred embodiment described above, involving a first transgenic plant containing the transiently-active promoter/structural gene sequences and the blocking sequence, and a second transgenic plant containing the recombinase gene linked to a germination-specific plant-active promotor, the two plants being hybridized by conventional to produce hybrid progeny containing all the necessary sequences.", "It is also possible to introduce the recombinase itself directly into a transgenic plant as a conjugate with a compound such as biotin, that is transported into the cell.", "See Horn, et al.", "(1990).", "5.7.4.15.2 Direct or Vectored Transformation Various methods for direct or vectored transformation of plant cells, e.g., plant protoplast cells, have been described, e.g., in above-cited PCT application WO 95/14099.As noted in that reference, promoters directing expression of selectable markers used for plant transformation (e.g., nptII) should operate effectively in plant hosts.", "One such promoter is the nos promoter from native Ti plasmids (Heffera-Estrella, et al., Nature 303: 209-213 (1983).", "Others include the 35S and 19S promoters of cauliflower mosaic virus (Odell, et al., Nature 313: 810-812 (1985) and the 2′ promoter (Velten, et al., EMBO J.", "3: 2723-2730 (1984).", "In one preferred embodiment, the embryo and endosperm.", "of mature seeds are removed to exposed scutulum tissue cells.", "The cells may be transformed by DNA bombardment or injection, or by vectored transformation, e.g., by Agrobacteriwn infection after bombarding the scuteller cells with microparticles to make them susceptible to Agrobacteriwn infection (Bidney et al., Plant Mol.", "Biol.", "18: 301-313, 1992).", "One preferred transformation follows the methods detailed generally in Sivamani, E. et al., Plant Cell Reports 15: 465 (1996); Zhang, S., et al., Plant Cell Reports 15: 465 (1996); and Li, L., et al., Plant Cell Reports 12: 250 (1993).", "5.7.24 Subculturing Cells or Callus Growing in Normally Inhibitory Concentrations of the Selective Agents Cells or callus observed to be growing in the presence of normally inhibitory concentrations of the selective agents are presumed to be transformed and may be subcultured several additional times on the same medium to remove non-resistant sections.", "The cells or calli can then be assayed for the presence of the viral gene cassette, or may be subjected to known plant regeneration protocols.", "In protocols involving the direct production of shoots, those shoots appearing on the selective media are presumed to be transformed and may be excised and rooted, either on selective medium suitable for the production of roots, or by simply dipping the excised shoot in a root-inducing compound and directly planting it in vermiculite.", "5.7.25 Selecting for Multi-Viral Resistance In order to produce transgenic plants exhibiting multi-viral resistance, the viral genes must be taken up into the plant cell and stably integrated within the plant genome.", "Plant cells and tissues selected for their resistance to an inhibitory agent are presumed to have acquired the selectable marker gene encoding this resistance during the transformation treatment.", "Since the marker gene is commonly linked to the viral genes, it can be assumed that the viral genes have similarly been acquired.", "Southern blot hybridization analysis using a probe specific to the viral genes can then be used to confirm that the foreign genes have been taken up and integrated into the genome of the plant cell.", "This technique may also give some indication of the number of copies of the gene that have been incorporated.", "Successful transcription of the foreign gene into mRNA can likewise be assayed using Northern blot hybridization analysis of total cellular RNA and/or cellular RNA that has been enriched in a polyadenylated region.", "mRNA molecules encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the same polarity to that of the viral genomic RNA such that they are capable of base pairing with viral specific RNA of the opposite polarity to that of viral genomic RNA under conditions described in Chapter 7 of Sambrook et al.", "(1989).", "mRNA molecules also encompassed within the scope of the invention are those which contain viral specific sequences derived from the viral genes present in the transformed vector which are of the opposite polarity to that of the viral genomic RNA such that they are capable of base pairing with viral genomic RNA under conditions described in Chapter 7 of Sambrook et al.", "(1989).", "The presence of a viral gene can also be detected by immunological assays, such as the double-antibody sandwich assays described by Namba, et al., Gene, 107, 181 (1991) as modified by Clark et al., J. Gen.", "Virol., 34, 475 (1979).", "See also, Namba et al., Phytopathology, 82, 940 (1992).", "Virus resistance can be assayed via infectivity studies as generally disclosed by Namba et al., ibid., wherein plants are scored as symptomatic when any inoculated leaf shows veinclearing, mosaic or necrotic symptoms.", "It is understood that the invention is operable when either sense or anti-sense viral specific RNA is transcribed from the expression cassettes described above.", "That is, there is no specific molecular mechanism attributed to the desired phenotype and/or genotype exhibited by the transgenic plants.", "Thus, protection against viral challenge can occur by any one or any number of mechanisms.", "It is also understood that virus resistance can occur by the expression of any virally encoded gene.", "Thus, transgenic plants expressing a coat protein gene or a non-coat protein gene can be resistant to challenge with a homologous or heterologous virus.", "5.7.26 Cell Culture Production of Mature Heterologous Protein Transgenic cells, typically callus cells, are cultured under conditions that favor plant cell growth, until the cells reach a desired cell density, then under conditions that favor expression of the mature protein under the control of the given promoter.", "Preferred culture conditions are described herein.", "Purification of the mature protein secreted into the medium is by standard techniques known by those of skill in the art.", "In one embodiment of the invention, in which BPN′ is secreted as the proBPN′ form of the enzyme, the chaperon “pro” moiety of the enzyme facilitates enzyme folding and is cleaved from the enzyme, leaving the active mature form of BPN'.", "In another embodiment, the mature enzyme is co-expressed and co-secreted with the “pro” chaperon moiety, with conversion of the enzyme to active form occurring in presence of the free chaperon (Eder et al., Biochem.", "(1993) L2: 18-26; Eder et al, (1993) J. Mol.", "Biol.", "223: 293-304).", "In yet another embodiment of the invention, the BPN′ is secreted in inactive form at a pH that may be in the 6-8 range, with subsequent activation of the inactive form, e.g., after enzyme isolation, by exposure to the “pro” chaperon moiety, e.g., immobilized to a solid support.", "In both of these embodiments, the culture medium is maintained at a pH of between 5 and 6, preferably about 5.5 during the period of active expression and secretion of BPN', to keep the BPN', which is normally active at alkaline pH, at a pH below optimal activity.", "5.7.26.1 Production of Mature Heterologous Protein in Germinatin Seeds In this embodiment, monocot cells transformed as above are used to regenerate plants, seeds from the plants are harvested and then germinated, and the mature protein is isolated from the germinated seeds.", "Plant regeneration from cultured protoplasts or callus tissue is carried by standard methods, e.g., as described in Evans et al., HANDBOOK OF PLANT CELL CULTURE Vol.", "1: (MacMillan Publishing Co. New York, 1983); and Vasil I. R.", "(ed.", "), CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS, Acad.", "Press, Orlando, Vol.", "1, 1984, and Vol.", "111, 1986, and as described in the above-cited PCT application.", "To achieve maximum production of recombinant protein from malting, the malting procedure may be modified to accommodate de-hulled and de-embryonated seeds, as described in above-cited PCT application WO 95/14099.In the absence of sugars from the endosperm, there is expected to be a 5 to 10 fold increase in RAmy3D promoter activity and thus expression of heterologous protein.", "Alternatively when embryoless half-seeds are incubated in 10 mM CaCl2 and 5 μM gibberellic acid, there is a 50 fold increase in RAmy1A promoter activity.", "5.7.27 Regeneration of the Transformed Plant Cells After transformation, the transformed plant cells must be regenerated.", "The methods used to regenerate transformed cells into whole plants are not critical to this invention, and any method suitable for the target plant can be employed.", "The literature describes numerous techniques for regenerating specific plant types, (e.g., via somatic embryogenesis, Umbeck, et al., 1987) and more are continually becoming known.", "Those of ordinary skill in the art can refer to the literature for details and select suitable techniques without undue experimentation.", "Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol.", "1: (MacMillan Publishing Co., New York, 1983); and Vasil T. R.", "(ed.", "), Cell Culture and Somatic Cell Genetics of Plants, Acad.", "Press, Orlando, Vol.", "1, 1984, and Vol.-III (1986), which are hereby incorporated is by reference.", "It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.", "Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided.", "Callus tissue is formed and shoots may be induced from callus and subsequently rooted.", "Alternatively, embryo formation can be induced in the callus tissue.", "These embryos germinate as natural embryos to form plants.", "The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins.", "It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa.", "Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture.", "If these three variables are controlled, then regeneration is usually reproducible and repeatable.", "5.7.28 Breeding Techniques After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing.", "Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.", "Seed from plants regenerated from tissue culture is grown in the field and self-pollinated to generate true breeding plants.", "The progeny from these plants become true breeding lines which are evaluated for viral resistance in the field under a range of environmental conditions.", "The commercial value of viral-resistant plants is greatest if many different hybrid combinations with resistance are available for sale.", "The farmer typically grows more than one kind of hybrid based on such differences as maturity, disease and insect resistance, color or other agronomic traits.", "Additionally, hybrids adapted to one part of a country are not adapted to another part because of differences in such traits as maturity, disease and insect tolerance, or public demand for specific varieties in given geographic locations.", "Because of this, it is necessary to breed viral resistance into a large number of parental lines so that many hybrid combinations can be produced.", "Adding viral resistance to agronomically elite lines is most efficiently accomplished when the genetic control of viral resistance is understood.", "This requires crossing resistant and sensitive plants and studying the pattern of inheritance in segregating generations to ascertain whether the trait is expressed as dominant or recessive, the number of genes involved, and any possible interaction between genes if more than one are required for expression.", "With respect to transgenic plants of the type disclosed herein, the transgenes exhibit dominant, single gene Mendelian behavior.", "This genetic analysis can be part of the initial efforts to covert agronomically elite, yet sensitive lines to resistant lines.", "A conversion process (backcrossing) is carried out by crossing the original resistant line with a sensitive elite line and crossing the progeny back to the sensitive parent.", "The progeny from this cross will segregate such that some plants carry the resistance gene(s) whereas some do not.", "Plants carrying the resistance gene(s) will be crossed again to the sensitive parent resulting in progeny which segregate for resistance and sensitivity once more.", "This is repeated until the original sensitive parent has been converted to a resistant line, yet possesses all of the other important attributes originally found in the sensitive parent.", "A separate backcrossing program is implemented for every sensitive elite line that is to be converted to a virus resistant line.", "Subsequent to the backcrossing, the new resistant lines and the appropriate combinations of lines which make good commercial hybrids are evaluated for viral resistance, as well as for a battery of important agronomic traits.", "Resistant lines and hybrids are produced which are true to type of the original sensitive lines and hybrids.", "This requires evaluation under a range of environmental conditions under which the lines or hybrids will be grown commercially.", "Parental lines of hybrids that perform satisfactorily are increased and utilized for hybrid production using standard hybrid production practices.", "5.7.28.1 Use of Conventional Cultivation Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants.", "Alternatively, transgenic seeds are recovered from the transgenic plants.", "These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.", "5.7.28.2 Plant Varieties The present invention can be used to make a variety of transgenic plants.", "The method is particularly suited for use with plants that are planted as a yearly crop from seed.", "These include, but are not limited to, fiber crops such as cotton and flax; dicotyledonous seed crops such as soybean, sunflower and peanut; annual ornamental flowers; monocotyledonous grain crops such as maize, wheat and sorghum; leaf crops such as tobacco; vegetable crops such as lettuce, carrot, broccoli, cabbage and cauliflower; and fruit crops such as tomato, zucchini, watermelon, cantaloupe and pumpkin.", "The present invention can be utilized in conjunction with a wide variety of plants or their seeds.", "Suitable plants include dicots and monocots.", "More particularly, useful crop plants can include: alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, papaya, and sugarcane.", "Examples of suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.", "The plants used in the process of the present invention are derived from monocots, particularly the members of the taxonomic family known as the Gramineae.", "This family includes all members of the grass family of which the edible varieties are known as cereals.", "The cereals include a wide variety of species such as wheat (Triticwn sps.", "), rice (Oryza sps.)", "barley (Hordewn sps.)", "oats, (Avena sps.)", "rye (Secale sps.", "), corn (Zea sps.)", "and millet (Pennisettum sps.).", "In the present invention, preferred family members are rice and barley.", "5.7.29 Identification and Localization and Introgression into Plants of Desired Multigenic Traits with RFLP Technology The invention typically involves genetic linkage maps constructed with RFLP technology and the use of RFLP probes to correlate those probes with Quantitative Trait Loci (QTL) and the degree of inheritance of particular multigenic traits (For references see: PTC numbers WO 96/21031; WO 97/17429; WO 98/37223; WO 98/36085; U.S. Pat.", "Nos.", "5,925,808 and 5,385,835)." ] ]

[ [ "Electrode and electron emission applications for n-type doped nanocrystalline materials", "An electrode having a surface of an electrically conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1 is disclosed as is a method of remediating toxic materials with the electrode.", "An electron emission device incorporating an electrically conducting ultrananocrystalline diamond having not less that 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1 is disclosed." ], [ "1.An electrode having a surface of an electrically conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1.2.The electrode of claim 1, wherein the ultrananocrystalline diamond is a film.", "3.The electrode of claim 1, wherein the ultrananocrystalline diamond has grain boundaries that are about 0.2 to about 2.0 nm wide and the conductivity at ambient temperature is not less than about 1 (Ωcm)−1.4.The electrode of claim 1, wherein the conductivity at ambient temperature is not less than about 10 (Ωcm)−1.5.The electrode of claim 1, wherein the film has a thickness less than about 2000 Å and is substantially pin-hole free.", "6.The electrode of claim 1, wherein the source of carbon is one or more of CH4 or a precursor thereof and C2H2 or a precursor thereof and a C60 compound.", "7.The electrode of claim 6, wherein the nitrogen is present in the source gas in an amount of less than about 20% by volume.", "8.The electrode of claim 7, wherein the atomic percent of carbon in the source gas is about 1% and the nitrogen is present in an amount less than about 25% by volume, the balance being a nobel gas.", "9.An electrode having a surface of an electrically conducting ultrananocrystalline diamond having an average grain size of about 15 nm or less and nitrogen present in an amount of not less than about 1019 atoms/cm3 made by the process of providing a source of carbon and a source of nitrogen and subjecting the sources of carbon and nitrogen in vapor form to an energy source in an noble-gas atmosphere to create a plasma to form an ultrananocrystalline material, wherein carbon is present in an amount less than about 2 atom percent of the source gas.", "10.The electrode of claim 9, wherein the ultrananocrystalline diamond is a film having a thickness less than about 2000 Å.", "11.A method of remediating aqueous solutions having toxic material therein, comprising subjecting the aqueous solution having toxic material therein to an electrical potential between two electrodes, at least one of which has a surface of an electrically conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1.12.A method of stimulating nerves comprising establishing an electrical potential across the nerve using an electrode conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1.13.An electron emission device incorporating an electrically conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1.14.The electron emission device of claim 13, wherein the device is a cold cathode used in one or more of a flat panel display, a traveling wave tube, and electrical instrument such as mass spectrometers or electron microscopes, ion beam accelerators, an x-ray machine, in a thruster or a sensor in a semiconductor-based sensor or actuation device.", "15.An electrochemical cell having an anode and a cathode and an aqueous based electrolyte wherein at least one of said anode or cathode has a surface of an electrically conducting ultrananocrystalline diamond having not less than 1019 atoms/cm3 nitrogen with an electrical conductivity at ambient temperature of not less than about 0.1 (Ω·cm)−1." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The use of diamond as an electronic material has remained elusive for many years.", "The problem lies in the difficulty of finding a way to dope diamond so that it's ambient temperature conductivity and carrier mobility are sufficiently high to make diamond-based devices work at room or ambient temperature.", "Traditional doping with nitrogen does not work, since nitrogen forms a deep donor level 1.7 eV below the conduction band, and thus is not thermally activated at room temperature.", "This is due to the fact that nitrogen is very reluctant to insert into the diamond lattice, and all efforts to dope microcrystalline diamond with electrically active nitrogen have to date met with very limited success.", "The inventors and others at Argonne National Laboratory have worked for several years developing the use of microwave plasma enhanced chemical vapor deposition (MPCVD) to produce ultrananocrystalline diamond (UNCD) thin films.", "These films are grown using argon-rich plasmas rather than the traditional hydrogen-rich plasmas, which are routinely used to grow microcrystalline diamond films, as disclosed in U.S. Pat.", "No.", "5,462,776, the disclosure of which is incorporated by reference.", "The UNCD films have grain boundaries are almost atomically abrupt (−0.5 nm).", "and have been measured on the average of 0.3 to 0.4 nm.", "These UNCD films exhibit exceptional mechanical, and tribological properties, the latter particularly applicable to the development of a new microelectromechanical system (MEMS) technology based on UNCD.", "For purposes of this application, UNCD shall be defined as films grown from C 2 dimers, as set forth in the '776 patent." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>This invention relates to n-type doping of UNCD films, that is films with average grain size of less than about 15 nm, as opposed to films with larger grain sizes, such as microcrystalline or nanocrystalline diamond.", "When nitrogen gas was added to gas mixtures used to grow UNCD, the conductivity of the films unexpectedly increased by more than five orders of magnitude, while the grain boundaries and the grain size become larger.", "Accordingly, it is an object of the present invention to provide an electrically conducting ultrananocrystalline diamond film having about 10 19 atoms/cm 3 nitrogen with an electrical conductivity of not less than about 0.1 Ω −1 cm −1 having a voltammetric response in the presence of Fe(CN) 6 −3/−4 , Ru(NH 3 ) 6 +2/+3 , methyl viologen and 4-tert-butylcatechol similar to high quality microcrystalline diamond, indicating that the nanocrystalline films are active without any conventional pretreatment, and posses semimetallic electronic properties over a potential range from 0.5 to −1.5 V (vs. SCE).", "Another object of the present invention is to provide an electrically conducting ultrananocrystalline diamond film of the type set forth useful as diamond film electrodes with continuous pin-hole free surface at thickness in the order of about 750 Å to about 2000 Å in electrochemical cells operating at over voltages of about 2.5 volts to degrade or destroy organic contaminants.", "Another object of the invention is to provide field emission devices using the nitrogen doped UNCD films disclosed herein as flat panel displays, cold cathode devices in traveling wave tubes, satellite thrusters, x-ray machines and devices.", "The invention consists of certain novel features and a combination of parts hereinafter fully described, illustrated in the accompanying drawings, and particularly pointed out in the appended claims, it being understood that various changes in the details may be made without departing from the spirit, or sacrificing any of the advantages of the present invention." ], [ "RELATED APPLICATIONS This application, pursuant to 37 C.F.R.", "1.78(c), claims priority based on provisional application Ser.", "No.", "60/239,173 filed on Oct. 9, 2000 and provisional application Ser.", "No.", "60/314,142 filed on Aug. 22, 2001.CONTRACTUAL ORIGIN OF THE INVENTION The United States Government has rights in this invention pursuant to Contract No.", "W-31-109-ENG-38 between the U.S. Department of Energy (DOE) and The University of Chicago representing Argonne National Laboratory.", "BACKGROUND OF THE INVENTION The use of diamond as an electronic material has remained elusive for many years.", "The problem lies in the difficulty of finding a way to dope diamond so that it's ambient temperature conductivity and carrier mobility are sufficiently high to make diamond-based devices work at room or ambient temperature.", "Traditional doping with nitrogen does not work, since nitrogen forms a deep donor level 1.7 eV below the conduction band, and thus is not thermally activated at room temperature.", "This is due to the fact that nitrogen is very reluctant to insert into the diamond lattice, and all efforts to dope microcrystalline diamond with electrically active nitrogen have to date met with very limited success.", "The inventors and others at Argonne National Laboratory have worked for several years developing the use of microwave plasma enhanced chemical vapor deposition (MPCVD) to produce ultrananocrystalline diamond (UNCD) thin films.", "These films are grown using argon-rich plasmas rather than the traditional hydrogen-rich plasmas, which are routinely used to grow microcrystalline diamond films, as disclosed in U.S. Pat.", "No.", "5,462,776, the disclosure of which is incorporated by reference.", "The UNCD films have grain boundaries are almost atomically abrupt (−0.5 nm).", "and have been measured on the average of 0.3 to 0.4 nm.", "These UNCD films exhibit exceptional mechanical, and tribological properties, the latter particularly applicable to the development of a new microelectromechanical system (MEMS) technology based on UNCD.", "For purposes of this application, UNCD shall be defined as films grown from C2 dimers, as set forth in the '776 patent.", "SUMMARY OF THE INVENTION This invention relates to n-type doping of UNCD films, that is films with average grain size of less than about 15 nm, as opposed to films with larger grain sizes, such as microcrystalline or nanocrystalline diamond.", "When nitrogen gas was added to gas mixtures used to grow UNCD, the conductivity of the films unexpectedly increased by more than five orders of magnitude, while the grain boundaries and the grain size become larger.", "Accordingly, it is an object of the present invention to provide an electrically conducting ultrananocrystalline diamond film having about 1019 atoms/cm3 nitrogen with an electrical conductivity of not less than about 0.1 Ω−1 cm−1 having a voltammetric response in the presence of Fe(CN)6−3/−4, Ru(NH3)6+2/+3, methyl viologen and 4-tert-butylcatechol similar to high quality microcrystalline diamond, indicating that the nanocrystalline films are active without any conventional pretreatment, and posses semimetallic electronic properties over a potential range from 0.5 to −1.5 V (vs. SCE).", "Another object of the present invention is to provide an electrically conducting ultrananocrystalline diamond film of the type set forth useful as diamond film electrodes with continuous pin-hole free surface at thickness in the order of about 750 Å to about 2000 Å in electrochemical cells operating at over voltages of about 2.5 volts to degrade or destroy organic contaminants.", "Another object of the invention is to provide field emission devices using the nitrogen doped UNCD films disclosed herein as flat panel displays, cold cathode devices in traveling wave tubes, satellite thrusters, x-ray machines and devices.", "The invention consists of certain novel features and a combination of parts hereinafter fully described, illustrated in the accompanying drawings, and particularly pointed out in the appended claims, it being understood that various changes in the details may be made without departing from the spirit, or sacrificing any of the advantages of the present invention.", "BRIEF DESCRIPTION OF THE DRAWINGS For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawings a preferred embodiment thereof, from an inspection of which, when considered in connection with the following description, the invention, its construction and operation, and many of its advantages should be readily understood and appreciated.", "FIG.", "1(a) is a graphical representation of the relationship of the concentration of CN radicals as a function of nitrogen in the plasma; FIG.", "1(b) is a graphical representation of the relationship of the concentration of C2 radicals as a function of nitrogen in the plasma; FIG.", "2(a) is a graphical representation of the relationships of total nitrogen content (left axis) and room-temperature conductivity (right axis) in a UNCD film as a function of nitrogen in the plasma; FIG.", "2(b) is an Arrhenius plot of conductivity data obtained in the temperature range 300-4.2 K for a series of UNCD films synthesized using different nitrogen concentrations in the plasma as shown; FIG.", "3 is a graphical representation of relationship of the concentration of nitrogen incorporated in the UNCD films versus the percent nitrogen in the feed gas of the plasma; FIGS.", "4(a)-(d) are UV Raman spectra of UNCD films: a) without nitrogen in the gas chemistry, and with b) 2%, c) 10% and d)20% nitrogen, showing that all the nitrogen-added films have approximately the same sp2;sp3 ratio, which is increased 25-30% over the non-nitrogen film; FIG.", "5 is EELS spectra of a UNCD film with 2% nitrogen and without nitrogen in the plasma, showing a distinct shoulder in the nitrogen film indicating sp2 bonded carbon; FIGS.", "6(a)-6(d) Low and high resolution TEM micrographs of a.)", "0% N2 b.)", "5% N2 UNCD, c.) 10% N2 UNCD, and d.) 20% N2 UNCD films.", "Low resolution micrographs are on the left, high resolution on the right.", "The figures are scaled so that the low resolution micrographs are 350 nm by 350 nm and the high resolution ones are 35 nm by 35 nm; FIG.", "7 is a graphical representation of the relationship of the onset field emission as a function of the percent nitrogen in the plasma; FIGS.", "8(A)-(B) are graphical representations of the Visible Raman spectra of (A) nanocrystalline diamond films deposited from 0, 2, 4 and 10% N2 in the source gas mixture and (B) is a microcrystalline, boron-doped diamond film; FIG.", "9 is a graphical representation of a UV Raman spectra of nanocrystalline diamond films deposited from 1, 5 and 10% N2 in the source gas mixture; FIGS.", "10(A)-(B) are graphical representations of SIMS data for nanocrystalline diamond films.", "(A) Plot of the N/C atomic concentration ratio versus the percentage of N2 added to the source gas mixture, and (B) depth profiles for the atomic carbon and nitrogen concentrations in a film deposited from 1% CH4/5% N2/95% Ar.", "FIG.", "11 are cyclic voltammetric i-E curves for nanocrystalline diamond thin films deposited from 1% CH4/1% N2/98% Ar, 1% CH4/2% N2/97% AR, 1% CH4/4% N2/95% Ar and 1% CH4/5% N2/94% Ar.", "Electrolyte: 1M KCl.", "Scan rate: 0.1 V/s.", "FIGS.", "12(A)-(B) are cyclic voltammetric i-E curves for nanocrystalline diamond thin films deposited from (A) 1% CH4/99% Ar, (B) 1% CH4/2% N2/97% Ar, (c) 1% CH4/4% N2/95% Ar and (D) 1% CH4/5% N2/94% Ar.", "Electrolyte: 0.1 M HClO4.Scan rate: 0.1 V/s; FIGS.", "13(A)-(B) are graphical representation of cyclic voltammetric data for nanocrystalline diamond thin films.", "(A) Plots of the oxidation peak current versus the scan rate1/2.Analyte concentration: 1 mM.", "Electrolyte: 1M KCl.", "(B) Plots of the oxidation peak current versus the analyte concentration.", "Electrolyte: 1M KCl.", "Scan rate: 0.1 V/s; FIG.", "14 show oxidation-reduction reactions for a variety of compounds; and FIG.", "15 is a schematic diagram of a field-emission cold cathode using the n-doped UNCD.", "DESCRIPTION OF THE PREFERRED EMBODIMENT This invention relates to the incorporation of dopants into UNCD thin films, in particular, the incorporation of nitrogen via the addition of N2 gas to the carbon containing noble gas plasma.", "When we use CH4 it is a short hand for the sources of carbon set forth above, and when we use argon it is a short hand for any noble gas.", "We have shown that nitrogen-containing UNCD thin films can be used as electrochemical electrodes over a 4 eV potential range and exhibit semimetal-like electronic properties.", "A possible explanation is that nitrogen may introduce changes in morphology and electronic structure within the GBs that may lead to enhanced electronic transport, since simulation indicate that introduction of nitrogen into high-angle twist diamond GBs is energetically favored by 3-5 eV compared to substation into the grains.", "The inventive films were grown on a variety of metals and non-metals using microwave plasma chemical vapor deposition with gas mixtures of Ar/CH4(1%-2%)/N2(1-20%) at total pressures of 100 Torr and 800 W of microwave power, while the substrates were maintained at temperatures from about 350 to 800° C. Essentially all the grains of UNCD films have the stated grain sizes, and by essentially all we mean greater than about 90% and preferably greater than about 95%.", "Moreover, UNCD films may be produced using up to about 2% by volume of CH4 or a precursor thereof or C2H2 or a precursor thereof or a C60 compound.", "UNCD films exhibit a number of interesting materials properties, including enhanced field emission, and electrochemical, as well as mechanical, tribological, and conformal coating properties suitable for microelectromechanical system devices.", "The number densities of the C2 and CN radicals formed in the plasma increase proportionally with nitrogen content in the plasma up to 5%, as measured by absorption spectroscopy.", "Secondary ion mass spectroscopy (SIMS) data show that the content of nitrogen in the film saturates at about 1×1019 atoms/cm3 (−0.2% total nitrogen content in the film) when the nitrogen concentration in the plasma is 5%.", "The conductivity at room temperature increases dramatically with nitrogen concentration, from 0.016 (1% N2) to 143 Ω−1 cm−1 (20% N2).", "This is to be compared with the best values reported previously: 10−6 Ω−1 cm−1 for nitrogen-doped microcrystalline diamond and 0.33 Ω−1 cm−1 for phosphorous-doped microcrystalline diamond films.", "Grain boundaries (GBs) in UNCD are believed to be high-energy, high-angle GBs.", "Molecular dynamics simulations of diamond (100) twist GBs have revealed that they have a large fraction os sp2-bonded atoms.", "Tight-binding calculations for 13 and Σ29 GBs revealed that electronic states are introduced into the band gap of the UNCD films, due to dangling bonds and π-bonded carbon atoms in the GBs.", "Temperature dependent conductivity and Hall measurements are both indicative of multiple, thermally activated conduction mechanisms with effective activation energies of <0.1 eV.", "This behavior is very similar to highly-boron-doped microcrystalline diamond.", "However, the inventors do not believe that nitrogen is acting in the manner boron does.", "It is believed that conduction occurs via the grain boundaries and not the grains.", "Tight-binding molecular dynamic simulations have shown that nitrogen incorporation into the high-angle grain boundaries is favored by 3-5 eV over substitution into the bulk.", "Nitrogen increases the amount of three-fold coordinated carbon atoms in the grain boundaries (GB) and leads to additional electronic states near the Fermi level.", "The inventors believe that GB conduction involving carbon πEr-states in the GB is responsible for the high conductivities.", "It has been shown that many of these states near the Fermi-level are delocalized over several carbon nearest neighbors.", "Some of the inventive films were grown either on Si(100) or insulating silica (SiO2) substrates (for transport measurements) at 800° C., using a CH4(1%)/Ar/N2 gas mixture at a total gas pressure of 100 Torr and 800 W microwave power.", "However, other substrates, such as various metals and non-metals may also be used.", "The average C2 and CN radical densities in the plasma were determined in situ using absorption spectroscopy.", "These results are shown in FIGS.", "1(a) and (b).", "Equivalent widths of rotational lines within the d3 Pa3P(0,0) Swan band of C2 and the B2S+-X2+(0,0) violet band of CN were integrated and converted into column densities using published values of the band oscillator strengths weighted by the appropriate Hönl-London and Boltzmann factors using a gas temperature of 1600 K, which had been determined previously by rotational analysis.", "As shown in FIGS.", "1(a) and (b) the densities of both the C2 and CN radicals increase substantially as N2 gas is added to the plasma, while their ratio changes as well.", "For small additions of N2 (1%-5%), the effect is to increase the density of C2 dimers by one order of magnitude.", "As the N2 content approaches 8%, the relative density of C2 to CN decreases by a factor of 5.This trend in the data is also reflected by accompanying changes in film morphology, total nitrogen content, and conductivity, as discussed below.", "High-resolution transmission electron micrographs (HRTEM) from UNCD films synthesized using either 1% or 20% N2 in the plasma show substantial microstructural changes, as shown in FIG.", "6(a)-6(d).", "For low-nitrogen partial pressures (<5%) the morphology of the films remains largely unchanged, with the average grain size and average GB widths increasing only slightly.", "However, in films made using 10% or more N2, both the average grain size and average GB widths increase significantly, to 12 and 1.5 nm, respectively.", "Films made using 20% N2 have average grain sizes about 15 nm and average GB width of 2 nm.", "The contrast in the HRTEM images between the GBs and the diamond grains suggests that the GBs are less dense than the grains.", "We believe this is evidence of an increase in sp2 bonding in these regions of the films.", "The inventive films have a substantially different microstructure than prior art films.", "For instance, Zhou et al.", "in J. Appl.", "Phys.", "82(9), 1 Nov. 1997 report a nanocrystalline thin film grown from N2/CH4 microwave plasma.", "The Zhou et al.", "films were grown in an entirely different plasma than the inventive materials described herein.", "The Zhou et al.", "plasma contained no nobel gas, whereas the predominant portion of the plasma used to grow the inventive material is a nobel gas.", "With N2 present in the 20-25 volume percent range and carbon present in the 2 atom percent range, the nobel gas would be present in an amount of at least 73 volume percent for the inventive process and materials produced thereby.", "At stated, the Zhou et al.", "material does not have the same microstructure as the inventive films.", "The inventive materials have a clear grain+GB morphology, whereas the films studies by Zhou et al.", "do not, as shown in FIG.", "3 of that paper.", "Furthermore, the average grain size of the Zhou et al.", "material is believed to be substantially larger (about 30-50 nm, based again on FIG.", "3 of their paper) than the average grain size of the inventive material, which is between about 2 nm or less to about 15 nm.", "Four-point-probe conductivity measurements in the temperature range 300-4.2 K were performed using both linear and van der Pauws geometries.", "These results are shown in FIGS.", "2(a) and (b).", "In addition, FIG.", "2(a) shows secondary ion mass spectroscopy (SIMS) data for the total nitrogen content in the films as a function of the percentage of N2 gas added to the plasma.", "Along with these data is a plot of the room-temperature conductivities for the same films.", "The SIMS data indicate that the nitrogen content in the films initially increases but then saturates at −2×1020 atoms/cm−3 for 5% N2 in the plasma, which corresponds to about 0.2% total nitrogen content in the film.", "The increase in room-temperature conductivity is both dramatic and unexpected, increasing from 0.016 Ω−1cm−1 (for 1% N2) to 143 Ω−1 cm−1 (for 20% N2), which represents an increase by roughly five orders of magnitude over undoped UNCD films.", "The latter value is much higher than any other previously reported for n-type diamond and is comparable to heavily boron-doped p-type diamond.", "Materials made with source gases having up to about 23-25% N2 show substantially conductivity, but at 25% N2 it is believed the conductivity begins to decrease.", "Temperature-dependent conductivity data in the range of 300-4.2 K are shown in the Arrhenius plot in FIG.", "2(b).", "These data are remarkable for several reasons.", "First, it is clear that these films exhibit finite conduction for temperatures even as low as 4.2 K. This behavior is also seen in heavily boron-doped diamond thin films.", "Also, these curves are clearly not simple straight lines in the Arrhenius plot, which is indicative of multiple, thermally activated conduction mechanism with different activation energies.", "These curves can be modeled by a summation of exponential functions as has been done in other studies where impurity conduction due to boron doping is dominant over the normal band conduction in doped single-crystal and polycrystalline diamond.", "We do not, however, expect that the present case is an example of degenerate doping of UNCD with nitrogen.", "Hall measurements (mobility, carrier concentration, Hall coefficient) have been made on two of the UNCD films grown with 10% and 20% nitrogen in the plasma.", "The carrier concentrations for the 10% and 20% N2 samples, were found to be 2.0×1019 and 1.5×1020 cm−3, respectively.", "The latter concentration is two orders of magnitude larger than any previous result for n-type diamond, and comparable to the carrier density in heavily boron-doped diamond.", "We also find reasonable high room-temperature carrier mobilities of 5 and 10 cm2/Vs for the 10% and 20% films, respectively.", "The negative value of the Hall coefficients indicates that electrons are the majority carriers in each of these films.", "It is seen therefore that the electrical conductivity of a nitrogen doped UNCD material can be systematically and reproducibly adjusted, permitting a material or film to be made with a predetermined electrical conductivity.", "For instance, adding 5% nitrogen results in a material having a conductivity of about 0.1 (Ω cm)−1 while adding 10% nitrogen results in a material having a conductivity of about 30 (Ω cm)−1, see FIGS.", "2 (a)(b).", "The ability to predetermine and vary the conductivity of UNCD materials is entirely new and unexpected.", "Previously materials were made and then their conductivities were measured, but there was no method of making materials having a specifically desired conductivity, until this invention.", "We believe that conduction occurs via the grain boundaries based on the above data and the following considerations.", "Nitrogen in microcrystalline diamond thin films usually forms a deep donor level with an activation energy of 1.7 eV.", "Therefore, it is unlikely that the enhanced conductivity in UNCD is due to nitrogen doping of the grains as previously believed, but rather at the grain boundaries.", "With the theoretical calculations indicating that nitrogen is favored by 3-5 eV for GB doping, we believe that the nitrogen in these films is present predominantly in the GBs and not within the grains.", "Using ultrananocrystalline diamond rather than microcrystalline or even nanocrystalline diamond because the smaller the grains, the larger number of grain boundaries and it is at the grain boundaries that the effective nitrogen doping occurs.", "Our tight-binding calculations assuming nitrogen substitution in the GBs shows that new electronic states associated with carbon Tr bonds and dangling bonds are introduced into the fundamental gap, and that there are unoccupied states available near the Fermi level.", "When nitrogen is introduced into the GBs, the associated carbon dangling-bond state is above the Fermi level and donates electron-to-carbon defect states near the Fermi level, causing it to shift upward (i.e., toward the conduction band).", "Thus, it is not unreasonable to believe that nearest-neighbor hopping or other thermally activated conduction mechanisms could occur in the GBs and result in greatly enhanced electron transport.", "The conduction may occur via the new carbon states in the band gap.", "Other films produced according to this invention were prepared by mechanically polishing n-type silicon wafers (resistivity 0.001-1.0 Ω-cm) with 0.1 micron diamond powder for approximately 10 minutes.", "The Si substrates were then placed in the PECVD chamber.", "The films were grown at 800° C., 100 Torr total pressure, 100 sccm total gas flow rate, and 800 W microwave power.", "These conditions are by way of example only and are not meant to limit the invention.", "It is now within the skill of the art to produce ultrananocrystalline diamond using a variety of conditions and techniques.", "The content of the source gas mixture was changed by successively adding N2 to replace argon in 1% CH4/99% Ar plasmas.", "Films with 1% CH4 and 0% N2/99% Ar to 20% N2/79% Ar were grown and were approximately one micron in thickness.", "The films were then characterized using secondary ion mass spectrometry (SIMS), transmission electron microscopy (TEM), UV Raman spectroscopy, and scanning electron microscopy (SEM).", "SIMS analysis was performed using a high-mass resolution SIMS.", "It is necessary to examine the CN ion because the hydrocarbon masses interfere with the positive nitrogen secondary ions, and there are no stable nitrogen negative secondary ions.", "High mass resolution is required to analyze CN (26.003 amu) to distinguish it from C2H2 (26.015 amu).", "FIG.", "3 displays the secondary ion mass spectroscopy results as nitrogen concentration in the film versus the percent nitrogen in the plasma during film growth.", "Since the base pressure of the PECVD system is approximately 1 mTorr, about 8×1018 atoms/cm3 of nitrogen, slightly less than 0.01 atomic percent, is present in the UNCD film due to atmospheric nitrogen contamination.", "With the addition of 1% N2 to the plasma, the concentration of nitrogen in the film increases an order of magnitude to 2.5×1020 atoms/cm3, and continues to rise until about 5% nitrogen is added to the plasma.", "No further increase in nitrogen in the film is observed even when 20% N2 is added to the plasma.", "The concentration of nitrogen incorporated in the film therefore saturates at about 8×1020 atoms/cm3 TEM electron diffraction patterns for a film without added nitrogen and one with 2% nitrogen can be completely indexed on the diamond lattice, no other crystalline phase was found.", "The grain size distribution of such films is on the order of 3-15 nm.", "FIGS.", "4(a)-(d) show the UV Raman spectra of UNCD films with varying degrees of nitrogen content.", "The introduction of nitrogen results in an increase in the peak at 1580 cm−1 relative to the peak at 1332 cm−1, which is the phonon peak for diamond.", "The relative ratio of sp2 to sp3, however, remains roughly independent of the nitrogen concentration.", "By integrating the areas under the Raman curves in FIGS.", "4(a)-(b), the present increase in the sp2:sp3 ratio for the nitrogen films is calculated as 25-30%.", "FIG.", "5 shows the electron energy loss spectra (EELS) for UNCD films without nitrogen and with 2% nitrogen to the plasma, respectively.", "The EELS of the nitrogen-grown diamond film reveals the K-edge δ* peak at 291 and a distinct π* peak originating form the sp2 carbon K edge at 286 eV.", "The film grown without nitrogen shows only the δ* peak by EELS measurements.", "The field emission measurements were carried out on an apparatus previously described in an article published by D. Zhou et al.", "in J. Electrochem soc.", "The field emission measurements were carried out on an apparatus previously described in an article published by D. Zhou et al.", "in J. Electrochem Soc.", "144(1997) L224, the disclosure of which is hereby incorporated by references.", "Briefly, a negative potential is applied to the sample, and the emission current is measured using a Keithley electrometer.", "A CCD camera is used to estimate the initial starting gap, typically 50 to 100 microns.", "The distance has been calibrated with a foil with a thickness of 500 microns.", "The anode is a 2.0 mm diameter tungsten probe with the edges slightly rounded to avoid edge effects.", "An ambient pressure of 10−8 Torr is maintained in the test chamber by a turbo molecular pump and an ion pump.", "The applied voltage is varied, and the collected current as a function of applied voltage is recorded on a computer system.", "The applied voltage divided by the gap distance yields the applied field.", "The results of the field emission measurements are shown in FIG.", "7.The film without added nitrogen had an average onset field of 23 V/μm, with a best onset value of 10 V/μm.", "The figure shows that for nitrogen containing films the average onset field required for emission immediately drops to below 5 V/μm for 1% nitrogen added to the plasma, and remains relatively constant for up to 20% N2 in the plasma.", "Some film areas had onset fields as low as 2 V/μm.", "The measurements represent the average values of at least 12 different areas on one or two samples with the given percent nitrogen.", "Some of the films had a few areas that did not emit below fields of 40 V/μm.", "These spots were not included in the averages.", "In general, it was observed that several films without added nitrogen showed distinctly higher onset fields for all examined areas than the nitrogen-added films.", "Table 1 shows a summary of the data for all the films.", "Field emitters have a wide variety of applications to multiple devices involving electron emission from fabricated field emitter sharp tips or edge structures that are localized at the center of a hole (see FIG.", "15).", "The application of a potential between the gate electrode and the field emitter tip produces a very high field on the tip, which results in field-induced emission of electrons.", "Current materials used for producing the field emitter have relatively high threshold fields for emission (about 10 V/μm) and/or they exhibit unstable emission current.", "Undoped or n-type or p-type doped UNCD exhibit both very low threshold field (4 V/μm) and stable emission current, the two main requirements for operation of cold cathodes based on field emission.", "UNCD based cold cathodes can be used in multiple applications, some of which are: 1.Field emission flat panel displays, where the electrons emitted from a high-density array of tips (FIG.", "15) impact on a phosphor screen on a glass substrate located in front of the tip array.", "The emission from the tip array is controlled by an electronic microcircuit that provides the processing signal for image production.", "2.Cold cathode for traveling wave tubes used for high power, high frequency devices included in many systems such as radar, communication devices and others.", "3.Micrometer size field emission sources for fabrication of miniaturized sensors integrated in semiconductor-based sensor/actuation devices.", "4.Cold cathodes for instruments based on electron emission such as mass spectrometers and field emission electron microscopes.", "5.Cold cathodes for large systems such as ion beam accelerators, where the electrons are used to generate plasmas needed to produce the ion beams; X-ray sources, where the electrons are used to generate X-rays via electron impact on solid anodes.", "6.Field emission sources to provide electrons for creating a plasma in a confined chamber for use as a spacecraft thruster.", "The impulse is provided by momentum transfer to the thruster chamber by ion expelled from the plasma.", "N-type doped UNCD materials, as disclosed herein have applications to all the devices described above.", "Although FIG.", "15 shows one emitter, in use as is well known in the art, an array of high density emitters may be employed, as for instance in flat panel displays.", "TABLE 1 Nitrogen Average Field Percent Surface Concentration Emission Onset Nitrogen Roughness (×1020 at/cc) (V/μm) 0 36-44 0.08 23 1 24 2.5 3 2 25 4.0 5 5 27 7.5 10 10 29 7.0 6 20 27 8.0 4.6 The volume fraction of grain boundaries can be determined using the following equation given by Palumbo et al., Aust, Scripta Metall-Matev.", "24(1990), 1347, 2347 Vgb=[3 Δ(d−Δ)2]/d3 (Eq.", "1) Where Δ is the grain boundary thickness and d is the average diameter of the grains.", "If one chooses 10 nm for the average grain size and a grain boundary width of 0.32 nm, as known, the volume fraction of grain boundaries in 0.09, or 9%, of the film.", "If 40% of the grain boundaries are sp2 bonded, then 3.6% of the film is sp2, assuming all sp2 bonded carbon atoms are in the grain boundaries.", "A 30% increase in sp2 character as given by the UV Raman equates to 4.7% of the film being sp2 (0.036×1.3).", "If 9% of the carbon atoms in the film are in the grain boundaries, this means that (0.0468/0.09)=0.52 or 52% of the grain boundaries would have to be sp2 bonded to rationalize the UV Raman data.", "These calculations based on experimental values demonstrate the inventors' belief that nitrogen preferentially enters the grain boundary and that the neighboring bonds change from a four-coordinated (sp3) to a three-coordinated (sp2) configuration.", "There are several possible reasons for nitrogen incorporation to improve the field emission characteristics.", "The inventors' experiments show an increase in the number of sp2 bonds with nitrogen incorporation.", "The increase in the number sp2 bonds may lower the activation energy for conduction by, for example, increasing the density of states within the band gap of diamond.", "The transport of electrons to the vacuum interface may be enhanced by increased connectivity within the grain boundaries through an increase in sp2 bonding.", "Both phenomena acting in concert may reduce a barrier for the emission of electrons.", "These experiments show that nitrogen incorporated into UNCD their films lower the onset fields from diamond thin films to 5 V/μm or less, making it a potential candidate for field emission displays.", "Recent density-functional based tight-binding (DFTB) calculations have been performed, which may explain the increase in the sp2:sp3 in the films with nitrogen and show that nitrogen substitution into the grain boundaries rather than into the diamond lattice, is energetically favorable by 2.6 to 5.6 eV, depending on the specific grain boundary site.", "The calculations suggest that three-fold coordinated sites are the lowest energy sites for nitrogen and that these promote sp2 bonding in the neighboring carbon.", "The theoretical calculations are thus in agreement with the experimental results, which show a 25-30% relative increase in sp2 bonding.", "In summary, nitrogen-doped UNCD thin films have been synthesized using a microwave plasma CVD technique with a CH4/Ar/N2 gas mixture.", "Other carbon containing gases also are applicable, as well as other deposition methods and other noble gases, as previously stated.", "The morphology and transport properties of the films are both greatly affected by the presence and amount of CN in the plasma, which varies as N2 gas is added.", "The HRTEM data indicated that the grain size and GB width of the UNCD films increase with the addition of N2 in the plasma.", "Our transport measurements indicate that these films have the highest n-type electrical conductivity reported thus far in phase-pure diamond thin films.", "The electrochemical characterization reveals that these films have a wide working potential window in aqueous media (˜4V), a very low background voltammetric signal, and excellent activity for several aqueous-based redox analytes without any pretreatment.", "Cyclic voltammetric ΔEp-values of 60 to 90 mV (0.1 V/s) for Fe(CN)6−3/−4, Ru(NH3)6+3/+2 and methyl viologen are observed.", "Evidence for the important role of the sp2-bonded carbon atoms in the grain boundaries, thus removing the Tr bonding.", "The electrochemical response for the redox analytes is almost totally inhibited after 60 minutes of treatment due to excessive film resistance caused by the loss of the π bonding.", "FIG.", "8A shows a series of visible Raman spectra for films deposited with and without N2 in the source gas mixture.", "FIG.", "8B shows the spectrum for a microcrystalline diamond film, for comparison.", "Three bands are observed for all four nanocrystalline films (FIG.", "8A): 1125, 1339 and 1560 cm−1.The broad band at 1339 cm−1 is assigned to the first-order phonon mode for diamond, reflective of the sp3-bonded microstructure.", "The peak is shifted to higher wavenumbers from the expected 1332 cm−1 position.", "Normally, microcrystalline diamond films have a sharp peak at 1332±2 cm−1 with a linewidth of 5 to 8 cm−1 (FIG.", "8B).", "The significantly broadened and shifted diamond line for the nanocrystalline films results from the decreasing grain size to the nanometer scale.", "To a first approximation, the linewidth is a measure of the phonon lifetime.", "The more defects there are (i.e., grain boundaries), the shorter the phonon lifetime and the broader the linewidth.", "The amorphous sp2-bonded carbon peak at 1560 cm−1 results from the π-bonded carbon atoms in the grain boundaries.", "The position and intensity of this broad peak depends on the deposition conditions used, the wavelength of the excitation photon and how microstructurally ordered the nondiamond carbon phase is.", "The spectra also contain a feature centered at 1125 cm−1.Broadening and shifting of the diamond band as the grain size decreases to the nanometer level, with the concomitant development of scattering intensity in the 1400-1600 cm−1 region is observed.", "The latter is due to an increasing fraction of π-bonded carbon atoms in the grain boundaries being probed.", "The band at 1125 cm−1 is a characteristic feature of nanocrystalline diamond but an unequivocal assignment has not been made.", "Semiquantitative analysis of the Raman data was performed and the results are presented in Table 2.TABLE 2 Visible Raman spectroscopic peak intensity ratios for nanocrystalline diamond films.", "Film I1339/I1560 I1339/I1125 I1560/I1125 1% CH4/99% Ar 1.45 2.27 1.59 1% CH4/2% N2/99% Ar 1.29 2.33 1.89 1% CH4/4% N2/99% Ar 1.30 2.44 1.89 1% CH4/5% N2/99% Ar 1.30 2.65 2.10 The relative intensity ratios of the three peaks in the nanocrystalline diamond spectra are shown as a function of the N2 percentage in the source gas mixture.", "It can be seen that the ratio of the diamond to the nondiamond band intensities, I1339/I1560, is largest for the film deposited without N2 and decreases for the films deposited with the gas.", "Interestingly, the ratio is independent of the N2 level.", "An assumption is often made that the relative band intensities reflect the volume fractions of diamond and nondiamond carbon present.", "In making this assumption, one must consider that the optical probing depth (i.e., sampled volume) can vary with the microstructure of the nondiamond phase.", "Also, the scattering cross sections for the different types of nondiamond carbon phases possible (mixtures of sp2- and sp3-bonded carbon) are unknown.", "Therefore, these data should be used in a relative not an obsolute sense.", "The decrease in the I1339/I1560 intensity ratio when N2 is added results from the increased fraction of π-bonded carbon atoms in the grain boundaries.", "The trends in the I1339/I1125 and I1560/I1125 band intensity ratios and the N2 added are interesting and more difficult to interpret.", "The data reveal that both the diamond and nondiamond band intensities relative to the 1125 cm−1 band intensity with increasing levels of N2.In other words, both the diamond and nondiamond peaks grow in intensity, relative to the 1125 cm−1 peak, with decreasing grain or cluster size.", "If the 1125 cm−1 intensity were directly related to defect-induced states brought about by the nanocrystalline morphology, then one would expect this intensity to increase with the decreasing grain or cluster size.", "Therefore, we suppose that this peak results from a film property other than defect-induced states.", "The effect of film thickness, grain and cluster size, temperature and the wavelength of the excitation photon will need to be studied systematically to better understand the origins of this band.", "FIG.", "9 shows the UV Raman spectra for films deposited from CH4/Ar with N2 added.", "The use of visible excitation often gives rise to an intense background luminescence that can mask the Raman line in nanocrystalline diamond, even in films with low sp2 carbon content.", "Also, the Raman signal for sp2-bonded carbon (amorphous or graphitic) is approximately 50 times more sensitive than diamond using visible excitation (514.5 nm).", "The signal for diamond is expected to increase relative to that for amorphous or graphitic carbon as the excitation wavelength is shifted toward the UV.", "For example, the spectrum for a 1% CH4/1% N/98% Ar film shows a moderately intense diamond line at 1332 cm−1 with a linewidth of 25 cm−1.There is no band present at 1125 cm−1 (this region of the spectrum is not shown), but there is a broad band centered near 1550 cm−1, due to the sp2-bonded carbon in the grain boundaries.", "The band intensities for the diamond and nondiamond carbon are roughly the same, but the peak area for the latter is significantly larger.", "The sp3/sp2 band intensity ratios are 1.0, 0.56, and 0.25 for the 1%, 5%, and 10% N2 levels, respectively, indicating that the fraction of Tr-bonded grain boundaries increase with N2 added.", "FIGS.", "10A and B show dynamic SIMS data for the nitrogen and carbon atomic concentrations in the nanocrystalline films.", "The actual nitrogen and carbon atomic concentrations, as well as the N/C atomic ratios, are listed in Table 3.TABLE 3 Secondary ion mass spectrometry data for nanocrystalline diamond films.", "Film N(atoms/cm3) C(atoms/cm3) N/C 1% CH4/99% Ar 7.88 × 1018 1.32 × 1022 5.97 × 10−4 1% CH4/1% N2/97% Ar 1.89 × 1020 1.29 × 1022 1.46 × 10−2 1% CH4/2% N2/97% Ar 2.30 × 1020 1.29 × 1022 1.96 × 10−2 1% CH4/5% N2/97% Ar 5.30 × 1020 1.20 × 1022 4.42 × 10−2 1% CH4/10% N2/97% Ar 4.17 × 1020 1.22 × 1022 3.42 × 10−2 FIG.", "10A shows a plot of the N/C atomic ratio versus the percentage of N2 in the source gas mixture.", "There is a near linear increase in the ratio with N2 added up to the 5% level.", "Above 5%, the amount incorporated levels off.", "The N/C for 0% N2 in the source gas mixture is not zero but rather 5.97×10-4; about two orders of magnitude lower than the ratio in the films deposited from gas mixtures containing N2.FIG.", "10B shows profiles of the carbon and nitrogen concentrations as a function of depth for a film approximately 1 μm thick.", "The concentration of nitrogen is as high as approximately 5×1020 atoms/cm3 with uniform distribution through the film.", "FIG.", "11 shows a series of cyclic voltammetric i-E curves in 1 M KCl for nanocrystalline diamond films containing different levels of incorporated nitrogen.", "It is clear that the responses between −500 and 1000 mV are very similar irrespective of the level of nitrogen incorporated.", "The background currents are low and featureless within this potential range.", "Each is also unchanging with cycle number indicating that the surface structure is stable.", "The magnitude of the anodic current at 250 mV is approximately 0.4 μA or 2.0 μA/cm2 (geom.)", "for all of the nanocrystalline films.", "This is slightly lower than the 2.7 μA/cm2 reported previously for nanocrystalline diamond films deposited from C60/A6.For comparison, the background current for polished glassy carbon at this potential and scan rate is near 20 μA/cm2.In summary, very minor differences are seen in the background voltammograms between −500 and 1000 mV with varying levels of incorporated nitrogen.", "This indicates that the excess surface charge density int his potential region is not affected significantly by the nitrogen concentration, and the introduction of nitrogen in the grain boundaries does not introduce detectable levels of electroactive carbon sites.", "FIGS.", "12(A)-(D) show cyclic voltammetric i-E curves in 0.1 M HClO4 for nanocrystalline diamond films containing different levels of incorporated nitrogen.", "The voltammograms cover a wider potential range than those in FIG.", "11, allowing determination of the full working potential window.", "All the films have an anodic limit of approximately 2400 mV (100 μA or 500 μA/cm2).", "The current at this potential is mainly due to oxygen evolution and, to a much lesser extent, the oxidation of carbon atoms on the surface.", "The surface oxidation processes may involve both the diamond and grain boundary carbon, and are evidenced indirectly by the anodic charge passed between 1400 and 2200 mV just prior to the exponentially increasing current for oxygen evolution.", "Previously, we have reported a well defined anodic peak near 1.6 V for C60/Ar nanocrystalline films, and have attributed this peak to the oxidation of sp2-bonded grain boundary carbon atoms.", "However, the currents for this proposed redox process in the present films are much lower than what was reported previously.", "The main difference between the nitrogen-containing films is the apparent overpotential for hydrogen evolution.", "There is a progressive decrease in the overpotential for hydrogen evolution (−100 μA or −500 μA/cm2) with increasing levels of incorporated nitrogen.", "The films deposited with 0, 2, 4 and 5%.", "N2 added to the source gas mixture have working potential windows of 4.27, 4.05, 3.85 and 3.78 V, respectively.", "The electrochemical activity of the nitrogen-containing films was probed using Fe(CN)6−3/−4, Ru(NH3)6+3+2, methyl viologen and 4-methylcatechol.", "The voltammetric response of these and other aqueous-based analytes at clean, boron-doped microcrystalline diamond has been discussed in detail previously.", "TABLE 4 Cyclic voltammetric ΔEp values for nanocrystalline diamond film.", "Fe Ru Film (CN)6−3/−4 (NH3)6+2/+3 MV+2/+1 4-MC R(Ω) 1% CH4/99% Ar 103 219 100 201 1535 ± 30 1% CH4/2% N2/ 93 73 54 382 177 ± 3 97% Ar 1% CH4/4% N2/ 91 69 50 312 106 ± 7 97% Ar 1% CH4/5% N2/ 88 61 51 387 81 ± 4 97% Ar Table 4 shows the cyclic voltammetric ΔEp values at 0.1 V/s with iR correction.", "It can be seen that the largest uncompensated resistance, most of which is the bulk resistance of the electrode, is observed for the 1% CH4/99% Ar film.", "The uncompensated resistance is significantly lower for the films containing nitrogen with a trend of decreasing resistance with increasing nitrogen incorporation.", "The iR corrected data reveal that the ΔEp values for methyl viologen, Ru(NH3)6+3/+2 and Fe(CN)6−2/−3 all decrease with increasing nitrogen incorporation.", "These relatively low ΔEp's were obtained even though the films received no pretreatment prior to use.", "This reflects the material's chemical inertness and resistance to fouling by adsorbed impurities.", "The rate of electron transfer for Fe(CN)6−3/−4 at metal and sp2 carbon electrodes is strongly affected several factors.", "First, the rate is strongly influenced by the fraction of edge plane exposed on sp2 carbon electrodes (i.e., electronic properties), but relatively insensitive to the surface-oxygen functionalities terminating the edge plane carbon atoms as long as a thick oxide film is not present.", "The rate of electron transfer increases proportionally with the fraction of exposed edge plane, as detected by Raman spectroscopy.", "Second, surface cleanliness is important as is the electrolyte type and concentration.", "For example, the involvement of specifically adsorbed cations (e.g., K+) through a possible surface-bridging interaction has been proposed.", "The rate of electron transfer increases with electrolyte composition in the order of LiCl NaCl KCl.", "At the 1 m electrolyte concentration, the rate is about a factor of 10 higher in KCl than in LiCl at both gold and glassy carbon electrodes.", "Third, adsorbed monolayers on sp2 carbon electrodes can decrease the rate of electron transfer.", "It has been observed the Δ(ΔEp) increase from 5 to 140 mV after modification of the polished grassy carbon surface with adsorbed monolayers.", "The level of increase depends on the type and coverage of the adsorbate.", "The rate of electron transfer is also influenced by the physiochemical properties of boron-doped diamond.", "ΔEp is very sensitive to the surface termination with the smallest ΔEp observed at the clean, hydrogen-terminated surface.", "After oxygen termination, Δ(ΔEp) increases by over 125 mV but it is reversibly reduced to the original value after removal of the oxygen functionalities by hydrogen plasma treatment (42).", "The sensitivity of the kinetics to surface oxygen is in sharp contrast to the minor effects these functionalities have on the response at sp2 carbon electrodes.", "The rate of electron transfer is also sensitive to the electrolyte composition and ionic strength with the largest rates observed in Kcl and the smallest in LiCl.", "However, the difference at the 1 M concentration level is only a factor of 2 to 3 rather than 10, as is the case for metal and glassy carbon electrodes.", "All the evidence at sp2 carbon and diamond electrodes suggest the involvement of some non-oxide surface site.", "Typical ΔEp values of 85 to 95 mV for the nitrogen-incorporated nanocrystalline diamond indicate that these films possess the requisite surface structure, chemical composition and electronic properties to support rapid electron transfer for this particular mechanistically-complicated redox system.", "The rate of electron transfer for Ru(NH3)6+3/+2, in contrast with Fe(CN)6−3/−4, is relatively insensitive to the surface microstructure, surface oxides and adsorbed monolayers on sp2 carbon electrodes.", "The rate of electron transfer is insensitive to surface modification with the strong implication that electron transfer does not depend on an interaction with a surface site or functional group.", "The most important factor affecting the rate of electron transfer is the electronic properties of the electrode, specifically the density of electronic states near the formal potential of the redox system.", "Of course with metal and glassy carbon electrodes, a low density of electronic states is never an issue.", "However, with the semiconducting/semimetallic properties of diamond, the potential-dependent electronic density of states is an influential factor.", "This is why ΔEp values of 60 to 75 mV are good in agreement with the 70 to 80 mV values often observed for boron-doped microcrystalline diamond films at 0.1 V/s.", "The formal potential (i.e., cyclic voltammetric Ep/2 value) for this couple is −218 mV vs. SCE.", "The valence band position of boron-doped microcrystalline diamond has been estimated to be approximately 550 mV vs. SCE.", "Given the 5.5 eV bandgap and the assumption that the interfacial energetics of nanocrystalline diamond are similar, this means that the formal potential falls within the bandgap (i.e., between the valence and conduction band positions).", "Therefore, this redox system is not expected to exchange charge directly with either the valence or the conduction band.", "The nearly reversible response indicates that there must be a high density of electronic states present within the bandgap at this potential.", "These electronic states arise from the nitrogen incorporated and or/the nitrogen-related defects introduced.", "Theoretical work will be discussed below which indicates the bandgap density of electronic states arises from the Tr-bonded carbon in the grain boundaries.", "Methyl viologen also involves simple outer sphere electron transfer at diamond and most other electrodes.", "The rate of electron transfer at diamond is relatively insensitive to surface oxides, grain boundaries and defect density, and the presence of nondiamond carbon impurities.", "Like Ru(NH3)6+3/+2, the most important factor influencing the rate of electron transfer is the density of electronic states at the formal potentials for the two redox reactions.", "Nearly reversible voltammetric behavior (ΔEp's from 60 to 90 mV at 0.2 V/s) is typically observed for both the MV+2/MV+ and MV+/MV0 redox couples having a formal potentials of −725 and −1050 mV vs. SCE, respectively.", "MV can form surface phases depending on the experimental conditions and these deposits complicate the process of directly relating the ΔEp to the rate of electron transfer.", "The formal potentials are well into the bandgap region, even more negative than the formal potential for Ru(NH3)6+3/+2.The relatively low ΔEp of 50 to 60 mV for nitrogen-incorporated nanocrystalline diamond indicates these electrodes contain a high density of bandgap electronic states, even these negative potentials.", "4-methylcatechol exhibits more electrochemical irreversibility as evidenced by the ΔEp of 200 to 400 mV.", "Also, there is a trend of increasing ΔEp with increasing nitrogen-incorporation.", "The more irreversible behavior is also characteristic of all the catechols and catecholamines investigated so far at microcrystalline diamond.", "Typical ΔEp values of 450 to 700 mV at 0.1 V/s are observed.", "For comparison, ΔEp at polished glassy carbon under identical conditions is in the range of 125 to 175 mV (36).", "The formal potential is positive of that for Fe(CN)6−3/−4 so a low density of electronic states is not the reason for the large ΔEp.", "The ΔEp at microcrystalline diamond is largely unaffected by chaning the surface termination from hydrogen to oxygen leading to the conclusion that surface carbon-oxygen functionalities are not influential.", "We believe a lack of adsorption on diamond is one of the reasons for the relatively slow electrode kinetics.", "Recent voltammetric and coulometric studies of several catechols and catecholamines revealed no evidence for adsorption even at solution concentrations as low as 2 μM.", "Indirect support for this belief also comes from the knowledge that other polar analytes, such as 2,6-anthraquinone-disulfonate, adsorb weakly on diamond at very low coverages.", "A detailed study has been presented showing that low ΔEp values correlate with catechol and catecholamine adsorption on glassy carbon, and surface treatments that decreased adsorption also increased ΔEp.", "FIGS.", "13(A) and (B) show plots of the voltammetric oxidation peak current as a function of the square root of the scan rate and the solution concentration for all four redox analytes.", "In all cases, the peak current varies linearly with the square root of the scan rate (r20.995) and all plots intercept the y-axis near the origin.", "This indicates the reactions are limited by semi-infinite linear diffusion of the reactant to the electrode.", "The voltammetric peak current also varies linearly with the concentrations (r2) 0.992) for all analytes from the 0.1 to 1 mM level.", "All plots intercept the y-axis near the origin, as expected.", "Electrochemically, these films are excellent electrodes.", "They are characterized by a wide working potential window (˜4 V), low background current and a very active response for Fe(CN)6−3/−4, Ru(NH3)6+/+2 and MV+2/+ without any conventional pretreatment.", "ΔEp's in the range of 60 to 90 mV (0.1 V/s) are observed for these three redox systems depending on the nitrogen incorporation.", "ΔEp for 4-MC was significantly larger at 200 to 400 mV (0.1 V/s) indicative of slower electrode reaction kinetics compared to the other three redox systems.", "Ultrananocrystalline diamond films doped with nitrogen to render them electrically conducting can be used as electrochemical electrodes which span a potential range of over 4 eV in aqueous solutions such as 0.1M HClO4.FIGS.", "12(A)-(D) shows cyclic voltammetric i-E curves for films deposited from source gas mixtures of methane and vapor containing different amount of nitrogen.", "The n-UNCD electrodes are useful for a wide range of oxidation reduction reactions as illustrated in FIG.", "14 for Fe(CN)6−3/−4, Ru(NH3)6+3/+2, IrCl6−2/−3, and methyl violagen with a high degree of electrochemical activity.", "More sluggish electrode kinetics are observed for 4-methylcatechol.", "Apparent heterogeneous electron transfer rate constants of 10−2 to 10−1cm/5 are observed for the highly active reactions without any pretreatment.", "The fact that continuous pinhole free n-type UNCD films can be grown at thicknesses at least an order of magnitude lower than 4 p-type microcrystalline diamond make the UNCD electrode extremely useful for industrial applications.", "While particular embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects.", "Therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.", "The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation.", "The actual scope of the invention is intended to be defined in the following claims when viewed in their proper perspective based on the prior art." ] ]

[ [ "Apparatus and method for detecting transmitting rate of turbo decoder", "The present invention relates to an apparatus and method for detecting a data rate in a turbo decoder for a mobile communication system.", "When a rate selector selects one data rate among a plurality of data rates, a turbo decoder repeatedly decodes an input data frame within a predetermined repetition limit number using the selected data rate and outputs the decoded data.", "A CRC detector performs CRC check on the decoded data and outputs the CRC check result, and a decoding state measurer measures decoding quality depending on the decoded data and outputs decoding state information.", "A controller then sets the repetition limit number to a predetermined minimum value, controls the repetition limit number according to the decoding state information, controls the rate selector and determines a data rate of the input data depending on the CRC check result." ], [ "1.A method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the coded data in the frame at a selected data rate of the plurality of the data rates by the turbo decoder and calculating a current decoding state value indicating an average of absolute values of log likelihood ratio (llr) values being identical to decoded values of the coded data, output from the turbo decoder; (b) calculating a current under-decoding state value defined as a difference between the current decoding state value and a previous decoding state value; (c) performing CRC (Cyclic Redundancy Check) checking on the decoded data, if the current decoding state value is larger than a first threshold or the current under-decoding state value is larger than a second threshold; and (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good.", "2.The method as claimed in claim further comprising the step of (e) repeating the steps (a) to (d) within an iteration limit number until the selected data rate is determined as a data rate of the coded data, if the CRC checkingy result is not good.", "3.The method as claimed in claim 2, further comprising the step of repeating the steps (a) to (e) until another selected data rate of the plurality of the data rates is determined as a data rate of the coded data, if a data rate of the coded data is not detected from the selected data rate.", "4.The method as claimed in claim 3, wherein the iteration limit number is changed based on the current decoding state value and the current under-decoding state value during every decoding.", "5.The method as claimed in claim 4, wherein the step (a) comprises the step of setting the iteration limit number to a predetermined, minimum value.", "6.The method as claimed in claim 5, wherein the iteration limit number is set to a predetermined maximum value, if the current decoding state value is larger than the first threshold or the current under-decoding state value is larger than the second threshold.", "7.The method as claimed in claim 5, wherein the iteration limit number is set to a predetermined maximum value, if the current decoding state value is larger than the first threshold and the current under-decoding state value is less than or equal to the second threshold.", "8.The method as claimed in claim 7, wherein the iteration limit number is an intermediate value between the minimum value and the maximum value, if the current decoding state value is less than or equal to the first threshold and the current under-decoding state value is larger than the second threshold.", "9.The method as claimed in claim 6 or 8, further comprising the step of returning to the step (a) without performing CRC checking on the decoded data, if the current decoding state value is less than or equal to the first threshold and the current under-decoding state value is less than or equal to the second threshold.", "10.The method as claimed in any of claims 1 to 3, wherein the decoding state value is calculated by m =  llr ⁡ ( a )  +  llr ⁡ ( a + 1 )  +  llr ⁡ ( a + 2 )  ⁢ ⋯ ⁢  llr ⁡ ( b )  b - ( a - 1 ) where m represents the decoding state value, ‘a’ and ‘b’ represent constants, wherein 0≦a<b<FL where FL represents a frame length associated with the selected data rate, and llr(n) represents a soft output value of an nth bit from the turbo decoder for the FL.", "11.A method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the data coded at a selected data rate of the plurality of the data rates and outputting decoded data; (b) controlling an iteration limit number depending on decoding state information measured for the decoded data, if a number of decoding performed at the selected data rate is less than the iteration limit number; (c) performing CRC checking on the decoded data, if the number of decoding is larger than or equal to the iteration limit number; (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good; (e) repeating the steps (a) to (d) within the iteration limit number until the selected data rate is determined as the data of the coded data, if the CRC checking result is not good; and (f) repeating the steps (a) to (e) until another selected data rate of the plurality of the data rates is determined as the data rate of the coded data, if the selected data rate is not determined as the data rate of the coded data.", "12.The method as claimed in claim 11, wherein the decoding state information includes a current decoding state value indicating an average of absolute values of log likelihood ratio (llr) values being identical to soft output values of the decoded data, and also includes a current under-decoding state value defined as a difference between a current decoding state value and a previous decoding state value.", "13.The method as claimed in claim 12, wherein the decoding state value is calculated by m =  llr ⁡ ( a )  +  llr ⁡ ( a + 1 )  +  llr ⁡ ( a + 2 )  ⁢ ⋯ ⁢  llr ⁡ ( b )  b - ( a - 1 ) where m represents the decoding state value, ‘a’ and ‘b’ represent constants, wherein 0≦a<b<FL where FL represents a frame length associated with the selected data rate, and llr(n) represents a soft output value of an nth bit from the turbo decoder for the FL.", "14.The method as claimed in claim 12, wherein the step (a) comprises the step of setting the iteration limit number to a predetermined minimum value.", "15.The method as claimed in claim 14, wherein in the step (b), the iteration limit number is set to a predetermined maximum value, if the current decoding state value is larger than a first threshold or the current under-decoding state value is larger than a second threshold.", "16.The method as claimed in claim 14, wherein in the step (b), the iteration limit number is set to a predetermined maximum value, if the current decoding state value is larger than a first threshold and the current under-decoding state value is less than or equal to a second threshold.", "17.The method as claimed in claim 16, wherein in the step (b), the iteration limit number is set to an intermediate vague between the minimum value and the maximum value, if the current decoding state value is less than or equal to the first threshold and the current under-decoding state value is larger than the second threshold.", "18.The method as claimed in claim 15 or 17, further comprising the step of returning to the step (a) without performing CRC checking on the decoded data, if the current decoding state value is less than or equal to a first threshold and the current under-decoding state value is less than or equal to a second threshold.", "19.An apparatus for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, the apparatus comprising: a data rate determiner for selecting a data rate from a plurality of data rates; a turbo decoder for iteratively decoding an input data frame within an iteration limit number using the selected data rate, and outputting decoded data; a CRC detector for performing CRC checking on the decoded data and outputting a CRC checking result; a decoding state measurer for measuring decoding quality using the decoded data and outputting decoding state information; and a controller for first determining the iteration limit number as a minimum value, controlling the iteration limit number based on the decoding state information, controlling the data rate determiner, and determining a data rate of the input data based on the CRC checking result.", "20.The apparatus as claimed in claim 19, wherein the decoding state information includes a current decoding state value indicating an average of absolute values of log likelihood ratio (llr) values being identical to soft output values of the decoded data, and also includes a current under-decoding state value defined as a difference between a current decoding state value and a previous decoding state value.", "21.The apparatus as claimed in claim 20, wherein the decoding state value is calculated by m =  llr ⁡ ( a )  +  llr ⁡ ( a + 1 )  +  llr ⁡ ( a + 2 )  ⁢ ⋯ ⁢  llr ⁡ ( b )  b - ( a - 1 ) where in represents the decoding state value, ‘a’ and ‘b’ represent constants, wherein 0≦a<b<FL where FL represents a frame length associated with the selected data rate, and llr(n) represents a soft output value of an nth bit from the turbo decoder for the FL.", "22.The apparatus as claimed in claim 20, wherein the controller sets the iteration limit number to a predetermined maximum value, if the current decoding state value is larger than a first threshold or the current under-decoding state value is larger than a second threshold.", "23.The apparatus as claimed in claim 20, wherein the controller sets the iteration limit number to a predetermined maximum value if the current decoding state value is larger than a first threshold and the current under-decoding state value is less than or equal to a second threshold.", "24.The apparatus as claimed in, claim 23, wherein the controller sets the iteration limit member to an intermediate value between the minimum value and the maximum value, if the current decoding state value is less than or equal to the first threshold and the current under-decoding state value is larger than the second threshold." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>1.Field of the Invention The present invention relates generally to an apparatus and method for detecting a data rate, and in particular, to an apparatus and method for detecting a data rate of a turbo decoder in a mobile communication system.", "2.Description of the Related Art In general, an encoder and a decoder are used in a digital mobile communication system to correct an error of a forward channel.", "The digital mobile communication system transmits and receives data in a radio environment, so it employs a coding technique to prevent generation of noises in a transmission channel.", "The mobile communication system typically uses a turbo coding technique for the coding technique.", "3GPP (3 rd Generation Partnership Projection) or 3GPP2 (3 rd Generation Partnership Projection 2), an ongoing standardization work on the mobile communication system, specifies that transmission data can be transmitted at different data rates.", "Here, the data rate depends upon a length (or size) of a frame decoded by a turbo decoder.", "For example, a data rate of 2.4 Kbps corresponds to a decoded frame length of 24 bits/frame, and a data rate of 4.8 Kbps corresponds to a decoded frame length of 48 bits/frame.", "When transmitting data at various data rates, a transmitter generally transmits the data to a receiver along with information on a data rate of the currently transmitted data.", "However, transmitting the data along with the data rate of the transmission data causes a waste of transmission power, especially when the transmission data has a low data rate.", "Therefore, there is a demand for a method for transmitting data without information on its data rate.", "There is a known blind rate detection (BRD) technique, in which a transmitter transmits data without information on its data rate and a receiver detects the data rate based on only the received data.", "When data is transmitted in the BRD mode after being subject to turbo encoding the results of CRC (Cyclic Redundancy Code) checking performed on an output of a turbo decoder are typically used to detect a data rate.", "That is, the transmitter adds CRC information to transmission data before transmission.", "Then, a channel decoder of the receiver decodes the received data at all of its available data rates and determines whether the decoded data includes noises, through CRC checking.", "If it is determined through the CRC checking performed at a specific data rate that the frame is not damaged, the receiver detects the corresponding data rate.", "A procedure for detecting a data rate by the receiver using the CRC checking will be described in detail with reference to FIG.", "1 .", "FIG.", "1 illustrates a procedure for detecting a data rate by a receiver through CRC checking of the BRD technique in a mobile communication system according to the prior art.", "A detailed description of the conventional procedure for detecting a data rate will now be made with reference to FIG.", "1 .", "In step 10 , the receiver performing the turbo decoding receives frame data over a radio channel.", "In this procedure, a description of a radio processing process and a channel decoding process will not be provided.", "After receiving the frame data, the receiver sets count values i and j to ‘0’, in step 12 .", "Here, i represents a value for counting a decoding iteration number (or decoding frequency) to perform turbo decoding on one frame at a selected data rate.", "Further, j represents a value for counting the number of types of the data rates.", "That is, if the turbo decoding is iteratively performed 6 times, the i value represents a decoding iteration count value for limiting the turbo decoding to be performed up to 6 times, and if the number of the types of the data rates is 7, the j value represents a data rate type count value for performing the decoding at up to 7 data rates.", "After the step 12 , the receiver sets an iteration limit number to its maximum value in step 14 .", "Here, the “iteration limit number” refers to the maximum number of iterative decoding at a specific data rate, i.e., a value for setting a threshold for the iteration count value ‘i’ of the turbo decoder.", "In step 16 , the receiver performs turbo decoding at a data rate set to the j value.", "For example, if decoding on voice data is performed at 2 Kbps, 4 Kbps and 8 Kbps, then the receiver performs the decoding on the data with a frame length FL(j) associated with 2 Kbps or 8 Kbps, whichever determined first.", "That is, the receiver performs the decoding at one initial data rate.", "After the decoding, the receiver analyzes the result of CRC checking performed on the decoded data in step 18 .", "As the result of the analysis, if it is determined that the CRC is ‘good’, the receiver performs a data rate detection process for the current j value in step 20 .", "Otherwise, the receiver determines whether the decoding iteration count value i is less than the iteration limit number, in order to iterate the decoding up to the iteration limit number.", "As the result of the determination, if the decoding iteration count value i is less than the iteration limit number, the receiver proceeds to step 24 , and otherwise, proceeds to step 26 .", "In the step 24 , the receiver increases the decoding iteration count value i by 1, and then returns to the step 16 .", "The reason for iteratively performing the decoding at one data rate will be described with reference to FIGS.", "2 and 3 .", "FIG.", "2 illustrates distribution of a decoding state value m(i) indicating the quality of a data frame when data with a frame size 60 transmitted over a radio channel is subject to iterative turbo decoding by a turbo decoder with a frame size 60 .", "FIG.", "3 illustrates distribution of an m(i) value when data with a frame size 40 transmitted over a radio channel is subject to iterative turbo decoding by a turbo decoder with a frame size 60 .", "In FIG.", "2 , a part represented by a solid line represents a part having an m(i) value of correctly decoded frame, while a part represented by dots represents an incorrectly decoded part.", "When performing the decoding for second time, the receiver iteratively performs the decoding for restoration, using the incorrectly decoded frames represented by the dots.", "Then, the incorrectly decoded frames are divided again into restored frames and non-restored frames, as shown in a second graph of FIG.", "2 .", "If the process is iterated several times, the frame data will be more correctly decoded, thus increasing a probability that the CRC will be detected in a ‘good’ state.", "However, in the case where the data is decoded with a frame length associated with another data rate, even though the decoding is performed several times, the decoding results will continuously show an error state as shown in FIG.", "3 .", "Turning back to FIG.", "1 , the iterative limit number is typically set to 6.If the CRC is not ‘good’ after iterating the decoding six times at one data rate, the receiver determines in step 26 whether the data rate type count value j is larger than the number N of the data rates.", "As the result of the determination, if the data rate type count value j is not less than the number N, e.g., 3, of the data rates, the receiver proceeds to step 28 to perform a data rate detection failure process, considering that the decoding and the CRC checking have been completed for all of the available data rates.", "However, if the count value j indicates that the decoding has not yet performed at all of the data rates, i.e., if the j value is less than N, the receiver increases the count value j by 1 and sets the decoding iteration count value i to 0 in step 30 , and then returns to the step 16 to repeat the steps 16 to 30 .", "However, the turbo decoding technique for detecting a data rate through the above process causes a waste of time in detecting the data rate, especially when there is a great difference between a data rate at which the data frame has been transmitted and the initial data rate at which the turbo decoding is performed.", "That is, for example, if it is assumed that the number of the data rates to be detected is N and the iteration number is 8, the decoding is performed (N−1)*8 times at the worst.", "In addition, an increase in the number of the data rates for the transmission data causes an increase in the detection time and also causes an increase in power consumption for detecting the data rate." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>It is, therefore, an object of the present invention to provide an apparatus and method for controlling an iteration limit number for turbo decoding when detecting a data rate of a turbo-coded data in a BRD mode.", "It is another object of the present invention to provide an apparatus and method for reducing a delay time and power consumption when detecting a data rate of turbo-coded data in a BRD mode.", "According to one aspect of the present invention, A method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the coded data in the frame at a selected data rate of the plurality of the data rates by the turbo decoder and calculating a current decoding state value indicating an average of absolute values of log likelihood ratio (llr) values being identical to decoded values of the coded data, output from the turbo decoder; (b) calculating a current under-decoding state value defined as a difference between the current decoding state value and a previous decoding state value; (c) performing CRC (Cyclic Redundancy Check) checking on the decoded data, if the current decoding state value it larger than a first threshold or the current under-decoding state value is larger than a second threshold; and (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good.", "According to a second aspect of the present invention, a method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the data coded at a selected data rate of the plurality of the data rates and outputting decoded data; (b) controlling an iteration limit number depending on decoding state information measured for the decoded data, if a number of decoding performed at the selected data rate is less than the iteration limit number; (c) performing CRC checking on the decoded data, if the number of decoding is larger than or equal to the iteration limit number; (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good; (e) repeating the steps (a) to (d) within the iteration limit number until the selected data rate is determined as the data of the coded data, if the CRC checking result is not good; and (f) repeating the steps (a) to (e) until another selected data rate of the plurality of the data rates is determined as the data rate of the coded data, if the selected data rate is not determined as the data rate of the coded data.", "According to a third aspect of the present invention, an apparatus for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted., and detecting a data rate of the coded data, the apparatus comprising: a data rate determiner for selecting a data rate from a plurality of data rates; a turbo decoder for iteratively decoding an input data frame within an iteration limit number using the selected data rate, and outputting decoded data; a CRC detector for performing CRC checking on the decoded data and outputting a CRC checking result; a decoding state measurer for measuring decoding quality using the decoded data and outputting decoding state information; and a controller for first determining the iteration limit number as a minimum value, controlling the iteration limit number based on the decoding state information, controlling the data rate determiner, and determining a data rate of the input data based on the CRC checking result." ], [ "BACKGROUND OF THE INVENTION 1.Field of the Invention The present invention relates generally to an apparatus and method for detecting a data rate, and in particular, to an apparatus and method for detecting a data rate of a turbo decoder in a mobile communication system.", "2.Description of the Related Art In general, an encoder and a decoder are used in a digital mobile communication system to correct an error of a forward channel.", "The digital mobile communication system transmits and receives data in a radio environment, so it employs a coding technique to prevent generation of noises in a transmission channel.", "The mobile communication system typically uses a turbo coding technique for the coding technique.", "3GPP (3rd Generation Partnership Projection) or 3GPP2 (3rd Generation Partnership Projection 2), an ongoing standardization work on the mobile communication system, specifies that transmission data can be transmitted at different data rates.", "Here, the data rate depends upon a length (or size) of a frame decoded by a turbo decoder.", "For example, a data rate of 2.4 Kbps corresponds to a decoded frame length of 24 bits/frame, and a data rate of 4.8 Kbps corresponds to a decoded frame length of 48 bits/frame.", "When transmitting data at various data rates, a transmitter generally transmits the data to a receiver along with information on a data rate of the currently transmitted data.", "However, transmitting the data along with the data rate of the transmission data causes a waste of transmission power, especially when the transmission data has a low data rate.", "Therefore, there is a demand for a method for transmitting data without information on its data rate.", "There is a known blind rate detection (BRD) technique, in which a transmitter transmits data without information on its data rate and a receiver detects the data rate based on only the received data.", "When data is transmitted in the BRD mode after being subject to turbo encoding the results of CRC (Cyclic Redundancy Code) checking performed on an output of a turbo decoder are typically used to detect a data rate.", "That is, the transmitter adds CRC information to transmission data before transmission.", "Then, a channel decoder of the receiver decodes the received data at all of its available data rates and determines whether the decoded data includes noises, through CRC checking.", "If it is determined through the CRC checking performed at a specific data rate that the frame is not damaged, the receiver detects the corresponding data rate.", "A procedure for detecting a data rate by the receiver using the CRC checking will be described in detail with reference to FIG.", "1.FIG.", "1 illustrates a procedure for detecting a data rate by a receiver through CRC checking of the BRD technique in a mobile communication system according to the prior art.", "A detailed description of the conventional procedure for detecting a data rate will now be made with reference to FIG.", "1.In step 10, the receiver performing the turbo decoding receives frame data over a radio channel.", "In this procedure, a description of a radio processing process and a channel decoding process will not be provided.", "After receiving the frame data, the receiver sets count values i and j to ‘0’, in step 12.Here, i represents a value for counting a decoding iteration number (or decoding frequency) to perform turbo decoding on one frame at a selected data rate.", "Further, j represents a value for counting the number of types of the data rates.", "That is, if the turbo decoding is iteratively performed 6 times, the i value represents a decoding iteration count value for limiting the turbo decoding to be performed up to 6 times, and if the number of the types of the data rates is 7, the j value represents a data rate type count value for performing the decoding at up to 7 data rates.", "After the step 12, the receiver sets an iteration limit number to its maximum value in step 14.Here, the “iteration limit number” refers to the maximum number of iterative decoding at a specific data rate, i.e., a value for setting a threshold for the iteration count value ‘i’ of the turbo decoder.", "In step 16, the receiver performs turbo decoding at a data rate set to the j value.", "For example, if decoding on voice data is performed at 2 Kbps, 4 Kbps and 8 Kbps, then the receiver performs the decoding on the data with a frame length FL(j) associated with 2 Kbps or 8 Kbps, whichever determined first.", "That is, the receiver performs the decoding at one initial data rate.", "After the decoding, the receiver analyzes the result of CRC checking performed on the decoded data in step 18.As the result of the analysis, if it is determined that the CRC is ‘good’, the receiver performs a data rate detection process for the current j value in step 20.Otherwise, the receiver determines whether the decoding iteration count value i is less than the iteration limit number, in order to iterate the decoding up to the iteration limit number.", "As the result of the determination, if the decoding iteration count value i is less than the iteration limit number, the receiver proceeds to step 24, and otherwise, proceeds to step 26.In the step 24, the receiver increases the decoding iteration count value i by 1, and then returns to the step 16.The reason for iteratively performing the decoding at one data rate will be described with reference to FIGS.", "2 and 3.FIG.", "2 illustrates distribution of a decoding state value m(i) indicating the quality of a data frame when data with a frame size 60 transmitted over a radio channel is subject to iterative turbo decoding by a turbo decoder with a frame size 60.FIG.", "3 illustrates distribution of an m(i) value when data with a frame size 40 transmitted over a radio channel is subject to iterative turbo decoding by a turbo decoder with a frame size 60.In FIG.", "2, a part represented by a solid line represents a part having an m(i) value of correctly decoded frame, while a part represented by dots represents an incorrectly decoded part.", "When performing the decoding for second time, the receiver iteratively performs the decoding for restoration, using the incorrectly decoded frames represented by the dots.", "Then, the incorrectly decoded frames are divided again into restored frames and non-restored frames, as shown in a second graph of FIG.", "2.If the process is iterated several times, the frame data will be more correctly decoded, thus increasing a probability that the CRC will be detected in a ‘good’ state.", "However, in the case where the data is decoded with a frame length associated with another data rate, even though the decoding is performed several times, the decoding results will continuously show an error state as shown in FIG.", "3.Turning back to FIG.", "1, the iterative limit number is typically set to 6.If the CRC is not ‘good’ after iterating the decoding six times at one data rate, the receiver determines in step 26 whether the data rate type count value j is larger than the number N of the data rates.", "As the result of the determination, if the data rate type count value j is not less than the number N, e.g., 3, of the data rates, the receiver proceeds to step 28 to perform a data rate detection failure process, considering that the decoding and the CRC checking have been completed for all of the available data rates.", "However, if the count value j indicates that the decoding has not yet performed at all of the data rates, i.e., if the j value is less than N, the receiver increases the count value j by 1 and sets the decoding iteration count value i to 0 in step 30, and then returns to the step 16 to repeat the steps 16 to 30.However, the turbo decoding technique for detecting a data rate through the above process causes a waste of time in detecting the data rate, especially when there is a great difference between a data rate at which the data frame has been transmitted and the initial data rate at which the turbo decoding is performed.", "That is, for example, if it is assumed that the number of the data rates to be detected is N and the iteration number is 8, the decoding is performed (N−1)*8 times at the worst.", "In addition, an increase in the number of the data rates for the transmission data causes an increase in the detection time and also causes an increase in power consumption for detecting the data rate.", "SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide an apparatus and method for controlling an iteration limit number for turbo decoding when detecting a data rate of a turbo-coded data in a BRD mode.", "It is another object of the present invention to provide an apparatus and method for reducing a delay time and power consumption when detecting a data rate of turbo-coded data in a BRD mode.", "According to one aspect of the present invention, A method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the coded data in the frame at a selected data rate of the plurality of the data rates by the turbo decoder and calculating a current decoding state value indicating an average of absolute values of log likelihood ratio (llr) values being identical to decoded values of the coded data, output from the turbo decoder; (b) calculating a current under-decoding state value defined as a difference between the current decoding state value and a previous decoding state value; (c) performing CRC (Cyclic Redundancy Check) checking on the decoded data, if the current decoding state value it larger than a first threshold or the current under-decoding state value is larger than a second threshold; and (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good.", "According to a second aspect of the present invention, a method for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted, and detecting a data rate of the coded data, comprising the steps of: (a) decoding the data coded at a selected data rate of the plurality of the data rates and outputting decoded data; (b) controlling an iteration limit number depending on decoding state information measured for the decoded data, if a number of decoding performed at the selected data rate is less than the iteration limit number; (c) performing CRC checking on the decoded data, if the number of decoding is larger than or equal to the iteration limit number; (d) determining the selected data rate as a data rate of the coded data transmitted from the transmitter, if the CRC checking result is good; (e) repeating the steps (a) to (d) within the iteration limit number until the selected data rate is determined as the data of the coded data, if the CRC checking result is not good; and (f) repeating the steps (a) to (e) until another selected data rate of the plurality of the data rates is determined as the data rate of the coded data, if the selected data rate is not determined as the data rate of the coded data.", "According to a third aspect of the present invention, an apparatus for decoding coded data in one frame transmitted from a transmitter at one data rate of a plurality of data rates by a turbo decoder of a receiver having no information on a data rate at which the coded data is transmitted., and detecting a data rate of the coded data, the apparatus comprising: a data rate determiner for selecting a data rate from a plurality of data rates; a turbo decoder for iteratively decoding an input data frame within an iteration limit number using the selected data rate, and outputting decoded data; a CRC detector for performing CRC checking on the decoded data and outputting a CRC checking result; a decoding state measurer for measuring decoding quality using the decoded data and outputting decoding state information; and a controller for first determining the iteration limit number as a minimum value, controlling the iteration limit number based on the decoding state information, controlling the data rate determiner, and determining a data rate of the input data based on the CRC checking result.", "BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which: FIG.", "1 illustrates a procedure for detecting a data rate by a receiver through CRC checking of a BRD technique in a mobile communication system according to the prior art; FIG.", "2 illustrates a decoding state of data when data with a frame size 60 transmitted over a radio channel is subject to turbo decoding by a turbo decoder with a frame size 60; FIG.", "3 illustrates a decoding state of data when data with a frame size 40 transmitted over a radio channel is subject to turbo decoding by a turbo decoder with a frame size 60; FIG.", "4 illustrates a block diagram of a radio receiver for performing turbo decoding according to a preferred embodiment of the present invention; FIG.", "5 illustrates a procedure for detecting a data rate in a BRD mode by a receiver performing turbo decoding according to a first embodiment of the present invention; FIG.", "6 illustrates a change in Δ(i) when rates are identical to each other; FIG.", "7 illustrates a change in Δ(i) when rates are not identical to each other; and FIG.", "8 illustrates a procedure for detecting a data rate in the BRD mode by a receiver performing turbo decoding according to a second embodiment of the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A preferred embodiment of the present invention will be described in detail herein below with reference to the accompanying drawings.", "FIG.", "4 illustrates a block diagram of a radio receiver for performing turbo decoding according to a preferred embodiment of the present invention.", "A structure and operation of the radio receiver for performing the turbo decoding according to the present invention will be described in detail with reference to FIG.", "4.A radio signal transmitted from a transmitter is received at an antenna ANT of the receiver through a radio environment.", "A demodulator 101 then demodulates the radio signal.", "A signal output from the demodulator 101 is descrambled and despread by a descrambler and despreader 103.The output signal is provided to a buffer 105.The buffer 105 sequentially buffers the received signal according to the order of decoding and provides its output to a decoder 107.The decoder 107, a turbo decoder according to the present invention, performs decoding according to a data rate (i.e., frame length) determined by a data rate determiner 115.The frame length of actual data transmitted and received in the mobile communication system becomes 39, 42, 49, 55, 58, 61, 65, 75 and 81 bits.", "For example, when the data rate determiner 115 desires to perform the decoding with a frame length of 39 bits, the turbo decoder 107 reads a data frame with a length of 39 bits from the buffer 105 and decodes the read data frame.", "The decoded data is provided to a CRC detector 109 and a decoding state measurer 111.The CRC detector 109 performs hard decision and CRC checking on the output of the turbo decoder 107, and provides the CRC checking result to a controller 113.The decoding state measurer 111 measures decoding quality for the output of the turbo decoder 107, and provides the measured result to the controller 113.Based on the result values, the controller 113 manages a decoding iteration count value i, an iteration limit number, a data rate type count value j and the number N of the data rates, and detects a data rate transmitted from the transmitter.", "In addition, the controller 113 creates a signal to be provided to the data rate determiner 115 by receiving a value calculated by the decoding state measurer 111.The data rate determiner 115 determines the frame length associated with q data rate for the decoding based on a signal provided from the controller 113, and provides the determined frame length to the turbo decoder 107.A control operation of the radio receiver having the above structure will be described with reference to FIGS.", "5 to 8.FIG.", "5 illustrates a procedure for detecting a data rate in a BRD mode by a receiver performing turbo decoding according to a first embodiment of the, present invention.", "A detailed description of a method for detecting a data rate using CRC checking of the BPD technique by the receiver will be described with reference to FIGS.", "4 and 5.In step 200, the controller 113 sets a decoding iteration count value i and a data rate type count value j to 0.For example, if the data frame with a length of 39, 42, 49, 55, 58, 61, 65, 75 and 81 bits can be transmitted through a radio channel, then the number N of the types of the data rates is 9 and the j value will be counted from 0 to 8.A data rate for decoding, i.e., the frame length is determined based on the j value.", "The frame length FL(j) associated with the data rate type count value j determined by the controller 113 is provided to the decoder 107 from the data rate determiner 115.In step 202, the controller 111 sets an iteration limit number to a predetermined minimum value.", "This is to perform the iterative decoding as minimum times as possible each time the data rate FL(j) is changed.", "In step 204, the turbo decoder 107 decodes a data stream read from the buffer 105 using the FL(j) provided from the data rate determiner 115, and provides the decoded data to the CRC detector 109 and the decoding state measurer 111.The output of the turbo decoder 107 is a non-binary soft output value.", "The CRC detector 109 performs hard decision and CRC checking on the decoded data, and the decoding state measurer 111 measures a state (or quality) of the decoded data.", "The controller 113 determines in step 206 whether the decoding iteration result of the determination, if the decoding iteration count value i is less than the iteration limit number, the controller 113 proceeds to step 208, and otherwise proceeds to step 216.In the step 208, the controller 113 calculates a decoding state value m(i) indicating quality of the currently decoded frame and an under-decoding state value Δ(i) defined as a difference between a decoding state value during current decoding and a decoding state value during previous decoding.", "The m(i) value is calculated by m ⁡ ( i ) = ∑ n = a b ⁢ ⁢  llr i ⁡ ( n )  b - ( a - 1 ) =  llr i ⁡ ( a )  +  llr i ⁡ ( a + 1 )  +  llr i ⁡ ( a + 2 )  ⁢ ⋯ ⁢  llr i ⁡ ( b )  b - ( a - 1 ) ( 1 ) In Equation (1), |•| represents an absolute value, and ‘a’ and ‘b’ represent given constants, where 0≦a<b<FL(j).", "Further, llr (Log Likelihood Ratio) becomes a non-binary soft output value decoded by the turbo decoder 107.That is, llri(n) is an nth soft output value among FL(j) soft output values from the decoder 107 during ith iterative decoding for FL(j), i.e., a value required when it is converted to a binary value by hard decision.", "For example, if the llr value is a negative number, the hard decision result is ‘1’, and if the llr value is a positive number, the hard decision result is ‘0’.", "The llr value is used for measuring decoding quality, because as its absolute value is higher, there is a high probability that the hard decision will be properly performed.", "That is, it can be considered that as the absolute value of the llr value is higher, the decoding quality is also higher.", "Here, the m(i) value can be an average (average of abs(llr)) of the absolute values of the FL(j) soft output values from the decoder 107.However, during an actual decoding operation, it is possible to obtain the similar result, even by using an average of the absolute values of some of the output values, i.e., ath to bth output values, among the FL(j) soft output values.", "In addition, the under-decoding state value Δ(i) is calculated by Δ(i)=m(i)−m(i−1) (2) In the formula, m(i) represents a decoding state value during the current decoding and m(i−1) represents a decoding state value during the previous decoding.", "As a result, it is noted that Δ(i) indicates how much the decoding quality is improved, by the iterative decoding at one data rate.", "Here, the Δ(i) is ‘0’ during initial decoding.", "Based on Equation (2), FIG.", "6 illustrates a change in the Δ(i) when an input frame length (an actual data rate to be used during transmission) is identical to a decoder frame length (a data rate used during the current decoding).", "It is noted herein that iteration of the decoding causes a change in the Δ(i).", "Further, FIG.", "7 illustrates a change in the Δ(i) when the input frame length is not identical to the decoder frame length.", "It is noted herein that the Δ(i) is not changed even though the decoding is iterated.", "As illustrated, a change in the Δ(i) becomes a basis for determining whether a data rate is detected.", "After calculating the m(i) value and the Δ(i) value based on Equations (1) and (2) in the step 208, the controller 113 determines in step 210 whether the m(i) value is larger than a first threshold.", "As the result of the determination, if the m(i) value is larger than the first threshold, the controller 113 proceeds to step 214, and otherwise proceeds to step 212.Here, the first threshold can be set to the minimum value among the values having a high probability that the m(i) will be normally decoded.", "The controller 113 determines in step 212 whether the Δ(i) value is larger than a second threshold.", "Likewise, the second threshold can be set to the minimum value among the values having a high probability that the Δ(i) will be normally decoded.", "As the result of the determination, if the Δ(i) is larger than the second threshold, the controller 113 proceeds to step 214, and otherwise proceeds to step 216.In the step 214, the controller 113 changes the iteration limit number to its maximum value, considering that it is necessary to iterate the decoding as many times as possible, and then proceeds to step 216.In the step 216, the controller 113 determines whether the CRC checking result received from the CRC detector 109 is ‘good’.", "If the CRC checking result is ‘good’, the controller 113 performs a data rate detection success process in step 218.That is, the controller 113 determines the data rate used during the decoding process of the step 204 as an actual data rate transmitted from the transmitter.", "However, if the CRC checking result is not ‘good’, the controller 113 determines in step 220 whether the decoding iteration count value i is less than the iteration limit number.", "As the result of the determination, if the i value is less than the iteration limit number, the controller 113 increase the i value by 1 in step 222, and then returns to the step 202.However, if the i value is not less than the iteration limit number, i.e., if the turbo decoding has already been performed as D many times as the set iteration limit number, the controller 113 determines in step 224 whether the data rate type count value j is less than the number N of the types of the frames.", "As the result of the determination, if the j value is less than the number of the types of the frames, the controller 113 increases the data rate type count value j by 1 and sets the decoding iteration count value i to 0 in step 228, and then returns to the step 202.However, if the j value is not less than the number N of the data rates, the controller 113 performs a data rate detection failure process in step 226.Summarizing the above processes, when the count values i and j and the decoder frame size are determined, the decoder 107 performs decoding by first setting the iteration limit number to its minimum value.", "If one of the m(i) value and the Δ(i) value is larger than its associated threshold, the controller 113 increases the iteration limit number to its maximum value, considering that there is a high probability that the CRC checking result will be ‘good’ for the current frame size, and then attempts to detect a data rate by performing CRC checking on every decoding result after increasing the iteration limit number to its maximum value.", "If the CRC checking result is not ‘good’ even though the decoding is iterated as many times as the maximum iteration limit number, the controller 113 considers that the data rate used for the current decoding is not identical to a data rate used during the transmission.", "In the case where the data rate is selected in error, the probability that the m(i) value or the Δ(i) value will be larger than their associated thresholds, is almost close to 0.Thus, the decoding at the current data rate will be iterated as many times as the minimum iteration limit number.", "If the m(i) value or the Δ(i) value are larger than their associated thresholds, it can be considered that there is a very high probability that the data rate will be detected even though the initial CRC checking result is not ‘good’.", "Therefore, the decoding at the current data rate is iterated as many times as possible.", "As described above, the receiver according to the present invention reduces time and power required for detecting a data rate by iteratively decoding the data rate with the lower probability as many times as the minimum iteration limit number and iteratively decoding the data rate with the higher probability as many times as the maximum iteration limit number.", "FIG.", "8 illustrates a procedure for detecting a data rate in the BRD mode by a receiver performing turbo decoding according to a second embodiment of the present invention.", "A detailed description of the procedure for detecting a data rate according to the second embodiment of the present invention will be made with reference to FIGS.", "4 and 8.Steps 300 to 304 of FIG.", "8 are equal in operation to the steps 200 to 204 of FIG.", "5, so the description will be omitted for simplicity.", "The controller 113 determines in step 306 whether the decoding iteration count value i is less than the iteration limit number.", "As the result of the determination, if the decoding iteration count value i is less than the iteration limit number, the decoding state measurer 111 calculates the m(i) and the Δ(i) using the data decoded by the decoder 107 in step 308.The m(i) and the Δ(i) are calculated based on Equations (1) and (2), respectively.", "After the calculation, the controller 113 determines in step 310 whether the decoding iteration count value i is equal to 0.If the decoding iteration count value i is equal to 0, the m(i) and the Δ(i) are not required, since the decoding is initial decoding.", "Therefore, in this case, the controller 113 proceeds to step 320.However, if the decoding iteration count value i is not 0, the controller 113 determines in step 312 whether the m(i) value is larger than a first threshold.", "In step 314, the controller 113 sets the iteration limit number to its maximum value and then proceeds to step 320.However, if the m(i) value is not larger than the first threshold, the controller 113 determines in step 316 whether the Δ(i) value larger than a second threshold.", "As the result of the determination, if the Δ(i) is larger than the second threshold, the controller 113 proceeds to step 31S, and otherwise proceeds to step 324.In the step 318, the controller 113 sets the iteration limit number to a predetermined intermediate value.", "Herein, for example, the minimum value can be set to 2 or 3, the intermediate value can be set to D, and the maximum value can be set to 8.After setting the iteration limit number, the controller 113 determines whether a CRC state is ‘good’, by using CRC checking results received from the CRC detector 109.As the result of the determination, if the CRC state is ‘good’, the controller 113 performs a data rate detection success process in step 322.That is, the controller 113 determines the data rate used during the decoding of the step 304 as a data rate transmitted from the transmitter.", "However, if the CRC is not ‘good’, the controller 113 determines in step 324 whether the decoding iteration count value i is less than the iteration limit number.", "As the result of the determination, if the decoding iteration count value i is less than the iteration limit number, the controller 113 increases the decoding iteration count value i by 1 in step 326, and then returns to the step 304.However, if the decoding iteration count value i is not less than the iteration limit number, the controller 113 determines in step 328 whether the data rate type count value j is less than the number N of the data rates.", "As the result of the determination, if the data rate type count value j is not less than the number N of the data rates, the controller 113 performs a data rate detection failure process in step 330.However, if the data rate type count value j is less than the number N of the data rates, the controller 113 increases the j value by 1 and sets the decoding iteration count value i to 0 in step 332, and then returns to the step 302.In FIG.", "8, unlike in FIG.", "5, if the m(i) value is larger than the first threshold, the iteration limit number is set to its maximum value, and if the m(i) value is not larger than the first threshold and the Δ(i) value is larger than the second threshold, the iteration limit number is set to its intermediate value.", "That is, if there is a high probability that the data rate can be detected, the iteration limit number is set to its maximum value, and otherwise, the iteration limit number is set to its intermediate value, thereby making it possible to detect the data rate more rapidly.", "In addition, in FIG.", "8, unlike in FIG.", "5, if the m(i) value is not larger than the first threshold and the Δ(i) value is not larger than the second threshold, the CRC checking is not performed.", "This is because it can be considered that there is a very low probability that the data rate will be detected, when both of the m(i) value and the Δ(i) value are not larger than their associated thresholds.", "Another reason is because the CRC checking result may become ‘good’ even though the input frame length is not identical to the decoder frame length due to serious damage of a received data frame.", "In this case, an incorrect data rate may be detected.", "Shown in Table 1 are the iteration limit numbers based on the m(i) and the Δ(i) for the first embodiment of FIG.", "5 and the second embodiment of FIG.", "8.TABLE 1 m(i) > First Threshold Yes Yes No No Δ(i) > Second Yes No Yes No Threshold Maximum Maximum Maximum Minimum Value Value Value Value Maximum Maximum Intermediate Minimum Value Value Value Value (No CRC) While the invention has been shown and described with reference to a certain preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.", "As described above, it is possible to rapidly detect a data rate by using the method for detecting the data rate in the BRD mode according to the present invention.", "In addition, the increase in detection speed of the data rate contributes to a reduction in power consumption, and a reduction in delay time enables rapid connection." ] ]

[ [ "Methods and composition for visualizing and interfering with chromosomal tethering of extrachromosomal molecules", "The invention provides methods and compositions for visualizing and interfering with chromosomal tethering and segregation of extrachromosomal molecules." ], [ "1.A preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag.", "2.The preselected molecule of claim 1, wherein the extrachromosomal molecule is a double minute chromosome.", "3.The preselected molecule of claim 1, wherein the extrachromosomal molecule is a viral nucleic acid sequence.", "4.The vector of claim 3, wherein the viral genome is maintained as an episome within a cell.", "5.The preselected molecule of claim 3, wherein the viral nucleic acid sequence is from an RNA virus.", "6.The preselected molecule of claim 3, wherein the viral nucleic acid sequence is from a DNA virus.", "7.The preselected molecule of claim 3, wherein the viral nucleic acid sequence is from Flaviviridae, Retroviridae, Hepadnaviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, a Hepatitis C virus, a Papillomavirus, an Epstein-Barr virus, an Influenza virus or a Polyomavirus.", "8.The preselected molecule of claim 1, wherein the extrachromosomal molecule comprises an oncogene.", "9.The preselected molecule of claim 8, wherein the oncogene is selected from sis, erbB, fins, sea, kit, ros, mpl, eyk, erbA, H-ras, K-ras, crk, src, abl, fps, fes, fgr, yes, mos, raf, mil, akt, jun, fos, myc, myb, ets, rel, mat, ski or qin.", "10.The preselected molecule of claim 1, wherein the tag is a reporter gene.", "11.The preselected molecule of claim 10, wherein the reporter gene is green fluorescent protein, cyan fluorescent protein, red fluorescent or yellow fluorescent protein.", "12.The preselected molecule of claim 1, wherein the tag is a selection marker.", "13.The preselected molecule of claim 12, wherein the selection marker is resistance to chloramphenicol, rifampicin, ampicillin or blasticidin.", "14.The preselected molecule of claim 13, wherein the selection marker is a blasticidin resistance gene (bsr) driven by an SRα promoter.", "15.The preselected molecule of claim 1, wherein the tag is a binding site for a detectable trans-acting element that binds to the tag.", "16.The preselected molecule of claim 1, comprising a vector that integrates into an extrachromosomal molecule.", "17.The preselected molecule of claim 16, wherein the vector comprises Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) vector sequences.", "18.The preselected molecule of claim 16, comprising EBNA-1 sequences and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites.", "19.The preselected molecule of claim 18, wherein the EBNA-1 binding sites are located at two distinct regions within the vector.", "20.The preselected molecule of claim 18, wherein the EBNA-1 binding sites have dyad symmetry within the vector.", "21.The preselected molecule of claim 13, comprising an FR element from oriP.", "22.The preselected molecule of claim 18, comprising a plurality of tandem repeats of a lac operator (lacO).", "23.The preselected molecule of claim 13, further comprising a nucleotide sequence that interferes with chromosomal tethering.", "24.The preselected molecule of claim 13, further comprising a polynucleotide that encodes a lac repressor (lacR)-GFP fusion protein that binds to the lac operator.", "25.A preselected molecule comprising a reporter gene fused to a lac repressor-nuclear localization signal.", "26.The preselected molecule of claim 25, wherein the reporter gene is green fluorescent protein, cyan fluorescent protein, red fluorescent or yellow fluorescent protein.", "27.The preselected molecule of claim 26, further comprising retroviral sequences.", "28.The preselected molecule of claim 26, further comprising plasmid sequences.", "29.A vector comprising a histone H2B gene fused to a reporter gene.", "30.The vector of claim 29, wherein the reporter gene is green fluorescent protein, cyan fluorescent protein, red fluorescent or yellow fluorescent protein.", "31.The vector of claim 29, further comprising retroviral sequences.", "32.The vector of claim 29, further comprising plasmid sequences.", "33.A recombinant chromosomal tethering polypeptide.", "34.The polypeptide of claim 33, operably linked to a cellular chromatid and an extrachromosomal molecule.", "35.The polypeptide of claim 34, wherein the extrachromosomal molecule is a double minute chromosome.", "36.The polypeptide of claim 33, operably linked to a cellular chromatid and to an oriP-containing vector.", "37.The polypeptide of claim 33, wherein the polypeptide comprises a cellular polypeptide.", "38.The polypeptide of claim 33, wherein the polypeptide comprises a viral polypeptide.", "39.A cell comprising the preselected molecule of claim 1, the vector of claim 29 or the polypeptide of claim 33.40.The cell of claim 39, wherein the cell is a COLO320DM cell.", "41.A method of visualizing chromosomal tethering of an extrachromosomal molecule comprising contacting the vector of claims 13, 25, or 29, with a cell suspected of containing an extrachromosomal molecule.", "42.The method of claim 41, wherein the extrachromosomal molecule is a double minute chromosome.", "43.The method of claim 41, wherein the extrachromosomal molecule is a virus.", "44.A method of interfering with chromosomal tethering of extrachromosomal molecule by contacting the vector of claims 13, 25, or 29, with a cell suspected of containing an extrachromosomal molecule.", "45.The method of claim 44, wherein the extrachromosomal molecule is a double minute chromosome.", "46.The method of claim 44, wherein the extrachromosomal molecule is a virus.", "47.A method to identify at least one agent that modulates chromosomal tethering of an extrachromosomal molecule comprising: (a) contacting a cell that contains an extrachromosomal molecule with a test agent; and (b) determining if the agent causes an increase or decrease in segregation of the extrachromosomal molecule when the cell divides.", "48.The method of claim 47, wherein the extrachromosomal molecule is operably linked to a tag.", "49.The method of claim 48, wherein the tag is a reporter gene.", "50.The method of claim 49, wherein the reporter gene is green fluorescent protein, cyan fluorescent protein, red fluorescent or yellow fluorescent protein.", "51.The method of claim 47, wherein the extrachromosomal molecule is operably linked to a selection marker.", "52.The method of claim 51, wherein the selection marker is resistance to chloramphenicol, rifampicin, ampicillin or blasticidin.", "53.The method of claim 47, wherein the cell is the cell of claim 39.54.The method of claim 47, wherein the cell comprises an extrachromosomal molecule into which a vector of claim 13 has integrated.", "55.The method of claim 47, wherein the extrachromosomal molecule is a double minute chromosome or a virus.", "56.A method of treating cancer comprising administering a pharmaceutical composition comprising a compound that inhibits tethering of an extrachromosomal molecule to a chromosome.", "57.The method of claim 56, wherein the extrachromosomal molecule is a virus or a double minute chromosome.", "58.A method of treating a viral infection comprising administering a pharmaceutical composition comprising a compound that inhibits tethering of a viral extrachromosomal molecule to a cellular chromatid.", "59.A method for identifying an antiviral agent, comprising: (a) contacting a cell comprising a viral acentric extrachromosomal molecule with a test compound; and (b) identifying a compound that inhibits association of the viral acentric extrachromosomal molecule with a cellular chromatid.", "60.The method according to claim 59, further comprising (c) determining if the compound inhibits the association of a tethering polypeptide with the cellular chromatid or with the viral acentric extrachromosomal molecule.", "61.The method of claim 60, wherein the tethering polypeptide is Epstein-Barr nuclear antigen or herpesvirus latent nuclear antigen.", "62.A method for identifying an anticancer agent, comprising: (a) contacting a cell comprising an extrachromosomal molecule with a test compound; and (b) identifying a compound that inhibits association of the extrachromosomal molecule with a cellular chromatid.", "63.The method according to claim 62, further comprising (c) determining if the compound inhibits association of a tethering polypeptide with the cellular chromatid or with the extrachromosomal molecule.", "64.The method according to claim 63, wherein the polypeptide is Epstein-Barr nuclear antigen or herpesvirus latent nuclear antigen.", "65.A chromosomally integrating vector that specifically labels double-minute chromosomes (DMs).", "66.The vector of claim 65, wherein the vector is an Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) vector sequences.", "67.The vector of claim 65, wherein the vector comprises an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions.", "68.The vector of claim 65, wherein the vector comprises a plurality of tandem repeats of a lac operator (lacO).", "69.The vector of claim 65, wherein the vector comprises a reporter gene.", "70.The vector of claim 69, wherein the reporter gene is GFP or YFP.", "71.The vector of claim 65, wherein the vector comprises a selection marker.", "72.The vector of claim 71, wherein the selection marker is a blasticidin resistance gene (bsr) driven by SR promoter.", "73.The vector of claim 65, wherein the vector further comprises a nucleotide sequence that interferes with chromosomal tethering.", "74.The vector of claim 65, wherein the vector further encodes a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO.", "75.The vector of claim 65, wherein the vector is a modified virus.", "76.The vector of claim 65, wherein the vector is a modified animal virus.", "77.The vector of claim 65, wherein the vector is a DNA virus.", "78.The vector of claim 65, wherein the vector is a member of the Herpesviridae, Papovaviridae or Adenoviridae.", "79.A plasmid vector comprising retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal.", "80.A plasmid vector comprising retroviral vector, a gene encoding YFP fused to lac repressor-nuclear localization signal.", "81.A plasmid vector comprising a histone H2B gene and a CFP gene." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>The replication and subsequent faithful segregation of duplicated chromosomes are crucial for the proper transmission of the cellular genome to daughter cells.", "In higher eukaryotes, the nuclear membrane breaks down at the beginning of mitosis, and subsequently spindle microtubules attach to centromeric kinetochores to assure the even distribution of sister chromatids.", "At the end of mitosis, the nuclear membrane reassembles around each group of chromosomes to form two daughter nuclei.", "Therefore, it would be reasonable to assume that acentric DNA molecules should not be maintained stably in nuclei as they do not attach to microtubules, and that acentric DNA molecules should be dispersed throughout the cytoplasm subsequent to nuclear membrane breakdown.", "In many cases, however, acentric DNA molecules, lacking functional centromeres, exhibit a surprisingly high stability in dividing cells.", "Examples of such stably-transmitted acentric DNA molecules in human cells include cellular acentric chromosomes called double minute chromosomes (DMs) and extrachromosomally replicating viral DNAs.", "DMs are cancer specific genomic anomalies known to harbor amplified oncogenes and drug resistance genes (Alitalo and Schwab, 1986; Hahn, 1993; Wahl, 1989).", "They are autonomously replicating, acentric, atelomeric, circular chromatin bodies, and usually 1-2 megabase pairs in size.", "Although they apparently lack functional centromeres (Barker and Hsu, 1978; Levan and Levan, 1978), their segregation efficiency is much higher than expected (Kimmel et al., 1992; Pauletti et al., 1990).", "Clues to the mechanisms underlying the efficient segregation came from light and electron microscopic observations showing that DMs frequently associated with mitotic chromosomes (Barker and Hsu, 1978; Hamkalo et al., 1985; Jack et al., 1987; Levan and Levan, 1978).", "These observations were extended using a fusion protein of human histone H2B and Aequorea victoria green fluorescent protein (H2B-GFP) to reveal DM clusters tethered to segregating daughter chromosomes in living cancer cells (Kanda et al., 1998).", "Time-lapse microscopy demonstrated that DMs could ‘hitchhike’ on segregating chromosomes from anaphase to telophase, indicating how chromosome tethering could contribute to increased segregation efficiency.", "There is a continuing need for improved visualization of viral and cellular acentric extrachromosomal molecules that tether to chromosomes.", "There is a further need to identify compounds that effectively interfere with the chromosomal tethering of these viral and cellular acentric extrachromosomal molecules.", "Abbreviations: Epstein-Barr virus (EBV); lac operator (lacO); Epstein-Barr nuclear antigen-1 (EBNA-1); green fluorescent protein (GFP); yellow fluorescent protein (YFP); red fluorescent protein (RFP); cyan fluorescent protein (CFP); Chinese hamster ovary cells (CHO); double minute chromosome (DM); lac repressor (lacR); fluorescent in situ hybridization (FISH); ampicillin (Amp); family of repeats (FR)." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>The invention provides a preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag.", "The extrachromosomal molecule can be any molecule that segregates with cellular chromosomes during cell division.", "For example, the extrachromosomal molecule may be a double minute chromosome or a viral nucleic acid sequence from a DNA or RNA virus.", "The viral genome may be maintained as an episome within a cell.", "Examples of sources of the viral nucleic acid include, but are not limited to Flaviviridae, Retroviridae, Hepadnaviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, a Hepatitis C virus, a Papillomavirus, an Epstein-Barr virus, an Influenza virus or a Polyomavirus.", "Alternatively, the extrachromosomal molecule may be an oncogene, such as sis, erbB, fins, sea, kit, ros, mpl, eyk, erbA, H-ras, K-ras, crk, src, abl, fps, fes, fgr, yes, mos, raf, mil, akt, jun, fos, myc, myb, ets, rel, maf, ski or qin.", "The tag includes any nucleic acid sequence that encodes a selection marker (such as a blasticidin resistance gene (bsr), ampicillin, rifampicin, chloramphenicol or other art recognized drug resistance selection marker gene).", "Such selection markers may be driven by any operably linked promoter, such as the SRα promoter.", "The tag may be a reporter gene (such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein or cyan fluorescent protein) that allows the extrachromosomal molecule to be detected.", "The tag may also be a binding site for a detectable trans-acting element that binds to the tag and thereby allows detection of the extrachromosomal molecule.", "The tag can be operably linked to the extrachromosomal molecule through integration of an integrating vector, into the extrachromosomal molecule.", "The preselected nucleic acid may further contain an integrating vector that specifically labels double-minute chromosomes (DMs).", "The vector may contain Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) sequences.", "The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions.", "The vector may contain a plurality of tandem repeats of a lac operator (lacO).", "The vector may contain a nucleotide sequence that interferes with chromosomal tethering such as an antisense message to a tethering protein, such as the Epstein-barr nuclear antigen.", "The vector may encode a fusion protein that binds to an extrachromosomal molecule such as a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO that may be integrated into the extrachromosomal molecule.", "Examples of the present invention include, but are not limited to, a plasmid vector containing a retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal, or a vector containing a gene encoding YFP fused to lac repressor-nuclear localization signal, or a histone H2B gene fused to a CFP gene.", "The present invention further provides a chromosomal tethering polypeptide.", "The polypeptide can operably link a cellular chromatid and an extracellular molecule.", "Such an extracellular molecule may be an oriP-containing vector.", "This polypeptide may be of cellular or viral origin.", "The tethering protein may also be a peptidomimetic or a fusion polypeptide having a chromatid binding domain and a domain that binds to an extrachromosomal molecule.", "The present invention further provides a method of visualizing chromosomal tethering of extrachromosomal molecules, such as viral acentric extrachromosomal molecules (DAE) or double-minute chromosomes (DM), by contacting a vector of the invention with a cell suspected of containing a DM or a DAE.", "The extrachromosomal molecule, such as the DM or DAE, may associate with a cellular chromatid through the action of a tethering polypeptide.", "The present invention provides a method of interfering with chromosomal tethering of extrachromosomal molecules, such as viral or cellular acentric extrachromosomal molecules, by administering a vector that specifically labels the extrachromosomal molecule, such as a double-minute chromosome (DM).", "Additionally, the vector may produce a product that inhibits the expression or function of a tethering polypeptide, such as an antisense message to the Epstein-Barr nuclear antigen or the herpesvirus latent nuclear antigen.", "The present invention also provides a method to identify an agent that modulates segregation of extrachromosomal molecules into daughter cells following division of a parent cell.", "Such methods can be used to identify agents that are useful for treating cancer, viral infections or other afflictions that involve extrachromosomal molecules.", "The present invention also provides a method of treating cancer or viral infections by administering a pharmaceutical composition containing a compound that inhibits the tethering of viral or cellular acentric extrachromosomal molecules to a chromosome.", "The present invention further comprises a chromosomally integrating vector that specifically labels DMs.", "The vector may be an Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) vector sequences.", "The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions.", "Further, the vector may contain a plurality of tandem repeats of a lac operator (lacO).", "The vector may contain a reporter gene, such as GFP or YFP, and/or may contain a selection marker, such as a blasticidin resistance gene (bsr) driven by SR promoter.", "The vector may further contain a nucleotide sequence that interferes with chromosomal tethering.", "The vector may contain a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO.", "The vector may be a modified virus, such as a modified animal virus.", "It may be a DNA virus, such as a member of the Herpesviridae, Papovaviridae or Adenoviridae.", "The present invention provides a plasmid vector comprising retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal.", "It also provides a plasmid vector comprising retroviral vector, a gene encoding YFP fused to lac repressor-nuclear localization signal.", "Moreover, the present invention provides a plasmid vector comprising a histone H2B gene and a CFP gene." ], [ "BACKGROUND OF THE INVENTION The replication and subsequent faithful segregation of duplicated chromosomes are crucial for the proper transmission of the cellular genome to daughter cells.", "In higher eukaryotes, the nuclear membrane breaks down at the beginning of mitosis, and subsequently spindle microtubules attach to centromeric kinetochores to assure the even distribution of sister chromatids.", "At the end of mitosis, the nuclear membrane reassembles around each group of chromosomes to form two daughter nuclei.", "Therefore, it would be reasonable to assume that acentric DNA molecules should not be maintained stably in nuclei as they do not attach to microtubules, and that acentric DNA molecules should be dispersed throughout the cytoplasm subsequent to nuclear membrane breakdown.", "In many cases, however, acentric DNA molecules, lacking functional centromeres, exhibit a surprisingly high stability in dividing cells.", "Examples of such stably-transmitted acentric DNA molecules in human cells include cellular acentric chromosomes called double minute chromosomes (DMs) and extrachromosomally replicating viral DNAs.", "DMs are cancer specific genomic anomalies known to harbor amplified oncogenes and drug resistance genes (Alitalo and Schwab, 1986; Hahn, 1993; Wahl, 1989).", "They are autonomously replicating, acentric, atelomeric, circular chromatin bodies, and usually 1-2 megabase pairs in size.", "Although they apparently lack functional centromeres (Barker and Hsu, 1978; Levan and Levan, 1978), their segregation efficiency is much higher than expected (Kimmel et al., 1992; Pauletti et al., 1990).", "Clues to the mechanisms underlying the efficient segregation came from light and electron microscopic observations showing that DMs frequently associated with mitotic chromosomes (Barker and Hsu, 1978; Hamkalo et al., 1985; Jack et al., 1987; Levan and Levan, 1978).", "These observations were extended using a fusion protein of human histone H2B and Aequorea victoria green fluorescent protein (H2B-GFP) to reveal DM clusters tethered to segregating daughter chromosomes in living cancer cells (Kanda et al., 1998).", "Time-lapse microscopy demonstrated that DMs could ‘hitchhike’ on segregating chromosomes from anaphase to telophase, indicating how chromosome tethering could contribute to increased segregation efficiency.", "There is a continuing need for improved visualization of viral and cellular acentric extrachromosomal molecules that tether to chromosomes.", "There is a further need to identify compounds that effectively interfere with the chromosomal tethering of these viral and cellular acentric extrachromosomal molecules.", "Abbreviations: Epstein-Barr virus (EBV); lac operator (lacO); Epstein-Barr nuclear antigen-1 (EBNA-1); green fluorescent protein (GFP); yellow fluorescent protein (YFP); red fluorescent protein (RFP); cyan fluorescent protein (CFP); Chinese hamster ovary cells (CHO); double minute chromosome (DM); lac repressor (lacR); fluorescent in situ hybridization (FISH); ampicillin (Amp); family of repeats (FR).", "SUMMARY OF THE INVENTION The invention provides a preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag.", "The extrachromosomal molecule can be any molecule that segregates with cellular chromosomes during cell division.", "For example, the extrachromosomal molecule may be a double minute chromosome or a viral nucleic acid sequence from a DNA or RNA virus.", "The viral genome may be maintained as an episome within a cell.", "Examples of sources of the viral nucleic acid include, but are not limited to Flaviviridae, Retroviridae, Hepadnaviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, a Hepatitis C virus, a Papillomavirus, an Epstein-Barr virus, an Influenza virus or a Polyomavirus.", "Alternatively, the extrachromosomal molecule may be an oncogene, such as sis, erbB, fins, sea, kit, ros, mpl, eyk, erbA, H-ras, K-ras, crk, src, abl, fps, fes, fgr, yes, mos, raf, mil, akt, jun, fos, myc, myb, ets, rel, maf, ski or qin.", "The tag includes any nucleic acid sequence that encodes a selection marker (such as a blasticidin resistance gene (bsr), ampicillin, rifampicin, chloramphenicol or other art recognized drug resistance selection marker gene).", "Such selection markers may be driven by any operably linked promoter, such as the SRα promoter.", "The tag may be a reporter gene (such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein or cyan fluorescent protein) that allows the extrachromosomal molecule to be detected.", "The tag may also be a binding site for a detectable trans-acting element that binds to the tag and thereby allows detection of the extrachromosomal molecule.", "The tag can be operably linked to the extrachromosomal molecule through integration of an integrating vector, into the extrachromosomal molecule.", "The preselected nucleic acid may further contain an integrating vector that specifically labels double-minute chromosomes (DMs).", "The vector may contain Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) sequences.", "The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions.", "The vector may contain a plurality of tandem repeats of a lac operator (lacO).", "The vector may contain a nucleotide sequence that interferes with chromosomal tethering such as an antisense message to a tethering protein, such as the Epstein-barr nuclear antigen.", "The vector may encode a fusion protein that binds to an extrachromosomal molecule such as a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO that may be integrated into the extrachromosomal molecule.", "Examples of the present invention include, but are not limited to, a plasmid vector containing a retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal, or a vector containing a gene encoding YFP fused to lac repressor-nuclear localization signal, or a histone H2B gene fused to a CFP gene.", "The present invention further provides a chromosomal tethering polypeptide.", "The polypeptide can operably link a cellular chromatid and an extracellular molecule.", "Such an extracellular molecule may be an oriP-containing vector.", "This polypeptide may be of cellular or viral origin.", "The tethering protein may also be a peptidomimetic or a fusion polypeptide having a chromatid binding domain and a domain that binds to an extrachromosomal molecule.", "The present invention further provides a method of visualizing chromosomal tethering of extrachromosomal molecules, such as viral acentric extrachromosomal molecules (DAE) or double-minute chromosomes (DM), by contacting a vector of the invention with a cell suspected of containing a DM or a DAE.", "The extrachromosomal molecule, such as the DM or DAE, may associate with a cellular chromatid through the action of a tethering polypeptide.", "The present invention provides a method of interfering with chromosomal tethering of extrachromosomal molecules, such as viral or cellular acentric extrachromosomal molecules, by administering a vector that specifically labels the extrachromosomal molecule, such as a double-minute chromosome (DM).", "Additionally, the vector may produce a product that inhibits the expression or function of a tethering polypeptide, such as an antisense message to the Epstein-Barr nuclear antigen or the herpesvirus latent nuclear antigen.", "The present invention also provides a method to identify an agent that modulates segregation of extrachromosomal molecules into daughter cells following division of a parent cell.", "Such methods can be used to identify agents that are useful for treating cancer, viral infections or other afflictions that involve extrachromosomal molecules.", "The present invention also provides a method of treating cancer or viral infections by administering a pharmaceutical composition containing a compound that inhibits the tethering of viral or cellular acentric extrachromosomal molecules to a chromosome.", "The present invention further comprises a chromosomally integrating vector that specifically labels DMs.", "The vector may be an Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) vector sequences.", "The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions.", "Further, the vector may contain a plurality of tandem repeats of a lac operator (lacO).", "The vector may contain a reporter gene, such as GFP or YFP, and/or may contain a selection marker, such as a blasticidin resistance gene (bsr) driven by SR promoter.", "The vector may further contain a nucleotide sequence that interferes with chromosomal tethering.", "The vector may contain a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO.", "The vector may be a modified virus, such as a modified animal virus.", "It may be a DNA virus, such as a member of the Herpesviridae, Papovaviridae or Adenoviridae.", "The present invention provides a plasmid vector comprising retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal.", "It also provides a plasmid vector comprising retroviral vector, a gene encoding YFP fused to lac repressor-nuclear localization signal.", "Moreover, the present invention provides a plasmid vector comprising a histone H2B gene and a CFP gene.", "BRIEF DESCRIPTION OF THE DRAWINGS FIG.", "1.EBV vector used for specific labeling of DMs.", "The EBV-lacO vector contains the EBNA-1 gene and oriP sequence, which has total of 24 EBNA-1 binding sites in two distinct regions (family of repeats and dyad symmetry) (Reisman et al., 1985).", "The vector has 256 tandem repeats of the lac operator (lacO), to which lac repressor (lacR)-GFP fusion protein binds with high affinity.", "The vector also has a blasticidin resistance gene (bsr) driven by SRα promoter as a drug selection marker.", "FIG.", "2.DMs are preferred targets for EBV vector integration.", "FIG.", "2A: FISH analyses demonstrating the co-localization of lacO repeats with DMs.", "DMs are observed as small scattered dots in mechanically spread chromosomes of colcemid-treated cells.", "In two color photography, signals detected using a c-myc cosmid probe were visualized as green, and lacO repeat probe were visualized as red.", "Chromosomes were counter-stained with DAPI and were visualized as blue.", "Intrachromosomal c-myc loci, lacking any overlapping red signals, are shown by arrowheads in the merged image.", "The scale bar is 10 μm.", "FIG.", "2B: Frequency of co-localization of DMs and lacO repeats.", "Paired black dots represent DMs with overlapping lacO signals.", "Fifty metaphase spreads were examined for co-localization, and they were divided into three categories; complete overlap (left), partial overlap (middle), and no overlap (right).", "The numbers of metaphase spreads in each category are shown.", "FIG.", "2C: FISH analyses using chromatin fibers prepared from untransfected COLO320DM cells (top) and cells transfected with the EBV-lacO vector (bottom).", "In a colored photograph, the lacO signals were visualized as red, and c-myc signals were visualized as green on the same chromatin fibers.", "DAPI was visualized as blue in the colored photograph.", "The scale bar is 10 μm.", "FIG.", "2D: Genomic DNAs (10 μg) of parental COLO320DM cells (lanes 1 through 3, ten copies and 1 copy equivalent of EBV-lacO plasmids added in lanes 1 and 2, respectively) and lacO integrated cells (lane 4) were digested with HindIII, which clipped out an 11 kb fragment containing the lacO repeats from the EBV-lacO plasmid.", "Digested DNA samples were analyzed by Southern blotting using 32P-labelled lacO repeat (SalI-XhoI fragment of pSV2-dhfr 8.32) (Robinett et al., 1996) as a probe.", "An arrow indicates the expected size of the fragment containing the lacO repeats.", "FIG.", "3.Localization of DM-integrated and extrachromosomal EBV vectors.", "FIGS.", "3A, 3C: Prometaphase chromosome rosettes of COLO320DM cells, having either EBV-lacO vectors integrated into DMs after stable transfection (FIG.", "3A) or transiently-transfected EBV-lacO vectors (FIG.", "3C), are shown.", "Green fluorescent dots representing the lacR-GFP protein recruited to the lacO repeats in the vectors were visualized in the colored photograph.", "Chromosomes were counterstained with DAPI (blue) in the colored photographs.", "FIGS.", "3B, 3D: In a color photograph, the localization of EBNA-1 protein in the same cells as FIGS.", "3A and 3C was visualized by indirect immunofluorescence as red.", "FIG.", "3E: Distribution of native (unlabelled) DMs in an anaphase cell of the parental COLO320DM cell line.", "Note that DMs make clusters and attach to mitotic chromosomes when cells are not treated with colcemid.", "FIG.", "3F: Distribution of lacO/lacR-GFP labelled DMs in an anaphase cell of the established cell line.", "The scale bar is 10 μm.", "FIG.", "4.Mitotic behavior of DMs, centromeres, and microtubules In a colored photograph, cells containing labelled DMs visualized as green were processed for immunofluorescence analyses while preserving the fluorescence of lacR-GFP.", "Centromeres (visualized as red in A, C, E, G, and I) and microtubules (visualized as red in B, D F, H, and J) in the same cells were detected by indirect immunofluorescence.", "In a color photograph, chromosomes were counterstained with DAPI (blue).", "DMs associating with distal chromosomal arms, but not with telomeres, are shown by arrows (C, D).", "DMs being incorporated into micronuclei are shown by arrows (I, J).", "The scale bar is 10 μm.", "FIG.", "5.Real time observation of DM behavior using different fluorescent proteins.", "(A) CFP and YFP can be used for dual color labeling.", "Entire chromosomes, including DMs, were stained with H2B-CFP (left), while lacO-tagged DMs are specifically stained with lacR-YFP (middle).", "Merged image is shown in right.", "The scale bar is 10 μm.", "(B) Time lapse images of DM behavior at metaphase-anaphase transition during mitosis.", "Dual color images were collected at the indicated time points (min).", "The scale bar is 10 μm.", "FIG.", "6.Microtubule inhibitors disrupt the peripheral localization of Dms Cells with lacO/lacR-GFP labelled DMs, treated with either taxol (A) or vinblastine (B), were processed for immunofluorescence analyses to detect microtubules (red) in a colored photograph while preserving the fluorescence of lacR-GFP (green) in a color photograph.", "Chromosomes were counterstained with DAPI (blue) in a color photograph.", "Note that DMs are no longer at the periphery but still associating with chromosomes.", "The scale bar is 10 μm.", "FIG.", "7.Model to explain the difference of the dynamics of various acentric DNA molecules.", "For simplicity, only one pair of mono-oriented prometaphase sister chromatids is drawn in each panel.", "The four panels show: (A) the behavior of normal mono-oriented sister chromatids; (B) a chromosome arm severed by laser microsurgery (Rieder et al., 1986); (C) a pair of DMs attached to the tip of chromosomes (large white circles); and (D) EBV vectors randomly associating with chromosomes (small white circles).", "A spindle pole is shown as a gray circle, and microtubules are shown as solid lines.", "Microtubule-mediated forces are shown as black arrows, while interacting forces between DMs and chromosomes are shown as white arrows (C).", "The size of each arrow represents the relative strength of each force.", "DETAILED DESCRIPTION OF THE INVENTION Recent studies revealed that chromosome tethering may be a common mechanism for enhancing the transmission of extrachromosomally replicating viruses into daughter nuclei (Bastien and McBride, 2000; Ilves et al., 1999; Lehman and Botchan, 1998; Marechal et al., 1999; Skiadopoulos and McBride, 1998).", "One of the best characterized episomal vectors is based on the Epstein-Barr virus (EBV) replicon, which utilizes the cis-acting oriP sequence and the virally-encoded EBNA-1 protein (Mackey and Sugden, 1999).", "OriP is composed of two clusters of EBNA-1 binding sites referred to as the family of repeats and the dyad symmetry element (Reisman et al., 1985).", "It has been shown that EBNA-1 both enables autonomous replication of oriP-containing plasmids in human cells (Yates et al., 1985) and mediates the nuclear retention of the plasmids (Krysan et al., 1989; Middleton and Sugden, 1994).", "Since EBNA-1 protein localizes on mitotic chromosomes (Grogan et al., 1983; Marechal et al., 1999; Petti et al., 1990), it is reasonable to infer that EBNA-1 could recruit oriP plasmids to mitotic chromosomes.", "Consistent with this, fluorescence in situ hybridization (FISH) previously demonstrated that EBV vectors did associate with mitotic chromosomes (Simpson et al., 1996; Westphal et al., 1998).", "Such chromosome tethering should facilitate the efficient segregation of EBV vectors into daughter nuclei when the nuclear membrane reforms at the end of mitosis.", "The observation of viral association with host chromosomes, “hitchhiking,” raised the intriguing question of whether DMs achieve efficient segregation by a similar mechanism.", "This possibility was explored by introducing EBV vectors into DM-harboring cells.", "Unexpectedly, EBV vectors frequently integrated into DMs subsequent to DNA transfection and selection.", "This observation enabled a method to preferentially integrate exogenous DNA into DMs.", "Cell lines with DM-EBV chimeras were derived in which lac operator (lacO) repeats were introduced as part of the EBV vector.", "These lacO-tagged DMs were readily detected using a fusion protein between the lac repressor (lacR) and green fluorescent protein (GFP), as previously demonstrated for visualizing homogeneously stained regions in CHO cells (Robinett et al., 1996).", "This provided a powerful tool for analyzing the mitotic behavior of DMs.", "Different distributions of free EBV vectors and DM-EBV chimeras were found, although they both hitchhiked onto mitotic chromosomes.", "Possible molecular mechanisms governing the behavior of these acentric molecules are discussed and described herein.", "DEFINITIONS A “cis-acting” element or sequence refer to DNA or RNA sequences whose function require them to be on the same molecule.", "An example of a cis-acting element is an origin of replication.", "An “extrachromosomal molecule” of the invention includes nucleic acid molecules which segregate during cell division through interaction with cellular chromatin.", "Examples of such extrachromosomal molecules include, but are not limited to, viral acentric extrachromosomal molecules, viral genomes and double-minute chromosomes.", "Extrachromosomal molecules also include nucleic acid constructs containing a nucleic acid sequence that allows interaction of the nucleic acid construct with cellular chromatin and provides for segregation of the nucleic acid construct with the cellular chromatin during cell division.", "Such nucleic acid sequences include origins of replication, such as oriP and the like, as well as fragments of such origins such as the FR element of oriP.", "It is well within the skill in the art to identify such sequences and link such sequences into nucleic acid constructs of the invention.", "Such constructs can also include retroviral vectors, plasmids, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes and the like.", "The term “modulate” or “modulates” means an increase or decrease in the occurrence of an event.", "For example, an agent that modulates the segregation of an extrachromosomal molecule in a cell will either increase or decrease the efficiency of extrachromosomal molecule segregation to sister cells following division of a cell treated with the agent.", "“Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other.", "For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter).", "Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.", "A “polypeptide” of the invention includes proteins found in nature as well as poly-amino acids that are not found in nature.", "Such polypeptides include fusion proteins as well as poly-amino acids that contain non-natural amino acids.", "Persons of skill in the art realize that many amino acid derivatives exist that can be linked into a chain of amino acids in a desired manner and that fusion proteins can be designed through recombinant techniques.", "The terms “polypeptide,” “protein” and “peptide” are used synonymously herein.", "A “reporter gene” is a nucleic acid that expresses a detectable polypeptide.", "Examples of such reporter genes include those that encode for green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, or other polypeptides that may be detected.", "A “selection marker” is a nucleic acid sequence that confers resistance to a chemical, such as a drug, to a cell.", "Examples of such chemicals include chloramphenicol, ampicillin, rifampicin, blasticidin or the like.", "A “tag” of the invention includes a marker that is associated with an extrachromosomal molecule and that allows detection of an extrachromosomal molecule.", "Examples of a tag include, but are not limited to, a reporter gene that expresses a polypeptide that can be detected, such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, or other detectable polypeptides.", "A tag can also be a nucleic acid sequence that encodes a selection marker such as chloramphenicol, ampicillin, rifampicin, blasticidin resistance or the like.", "Such nucleic acid sequences are well known in the art.", "A tag can also be a cis-acting element that is recognized by a trans-acting element.", "An example of a cis-acting element is a nucleic acid sequence that encodes a repressor binging site, such as the lac-repressor binding site found within the lac operator (lacO).", "This cis-acting element can be integrated into an extrachromosomal molecule and bound by a detectable trans-acting element that allows detection of an extrachromosomal molecule.", "An example of such a trans-acting element is a fusion polypeptide of a repressor polypeptide, such as the lac repressor (lacR), and a detectable polypeptide, such as green fluorescent protein.", "Such trans-acting factors may also include a nuclear localization signal that will cause the fusion polypeptide to localize to the nucleus.", "It will be recognized that there are many combinations of such cis-acting and trans-acting elements that are within the scope of the invention.", "A “tethering polypeptide” is a polypeptide that provides an association of an extrachromosomal molecule with a cellular chromatid.", "A tethering polypeptide may comprise a cellular protein or a viral protein.", "Examples of such a tethering polypeptide are the Epstein-Barr Nuclear Antigen-1 protein (EBNA-1) and the Herpesvirus late nuclear antigen (LANA).", "A “recombinant tethering polypeptide” is a tethering polypeptide produced through recombinant methods.", "An example of such a recombinant polypeptide is a protein having the N-terminal cellular chromatid binding domain of EBNA-1 linked to the C-terminal oriP binding domain of EBNA-1 through an recombinantly produced amino acid linkage.", "Alternatively, such a polypeptide can have the N-terminal cellular chromatid binding domain of EBNA-1 fused to a domain that would bind to a recombinantly designed cis-acting element within an extrachromosomal molecule.", "An example of such a recombinant polypeptide is a protein having the N-terminal cellular chromatid binding domain of EBNA-1 linked to the lac repressor binding domain of the lac repressor protein.", "A “trans-acting” element refers to a polypeptide or nucleic acid whose function requires them not to be directly linked to the same molecule.", "An example includes a polypeptide that binds to a cis-acting element that is operably linked to an extrachromosomal molecule.", "I.", "A Preselected Nucleic Acid Molecule Comprising an Extrachromosomal Molecule Operably Linked to a Tag.", "The invention provides a preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag.", "It is contemplated that the extrachromosomal molecule is any molecule that is tethered to a cellular chromatid during cellular division.", "Such tethering allows the extrachromosomal molecule to be segregated into progeny cells upon division of the parent cell.", "The invention provides a method to tag the extrachromosomal molecule which allows the extrachromsomal molecule to be detected during chromosome segregation and cell division.", "The tag may become operably linked to the extrachromosomal molecule through integration of an integrating vector, which contains the tag, into the extrachromosomal molecule.", "Thus, the invention provides integrating vectors that are able to integrate into an extrachromosomal molecule and operably link a tag into the extrachromosomal molecule.", "Extrachromosomal molecules: The extrachromosomal molecule can be any molecule that segregates with cellular chromosomes during cell division.", "Examples of such extrachromosomal molecules include, but are not limited to, double minute chromosomes, viral acentric extrachromosomal molecules, cellular acentric extrachromosomal molecules and recombinantly engineered extrachromosomal molecules.", "Double minute chromosomes, viral acentric extrachromosomal molecules and cellular acentric extrachromosomal molecules are well known in the art.", "Recombinantly engineered extrachromosomal molecules include, but are not limited to, plasmid and viral based vectors into which a nucleic acid sequence has been inserted that allows the extrachromosomal molecule to associate with a cellular chromatid and be segregated into daughter cells during cell division.", "Nucleic acid sequences that may be operably linked into extrachromosomal molecules include, but are not limited to, an origin of replication such as EBV-oriP, or nucleic acid sequences that are able to interact with tethering proteins, such as the FR element of EBV-oriP.", "A recombinantly engineered extrachromosomal molecule can also contain nucleic acid sequences that allow the molecule to be detected within a cell.", "Such nucleic acid sequences include reporter genes that encode fluorescent proteins or selection markers that confer chemical, i.e.", "drug, resistance to a cell.", "It is contemplated that a recombinantly engineered extrachromosomal molecule may be introduced into a cell such that it is segregated into progeny cells during division of the parent cell.", "It is further contemplated that such extrachromosomal molecules may be constructed to associate with recombinantly engineered tethering polypeptides, described below.", "This allows for the production of recombinantly engineered extrachromosomal molecules and tethering polypeptides that specifically associate with each other and allow specific interactions of the recombinant extrachromosomal molecules with chromatids to be assessed.", "Tag: A tag includes any nucleic acid sequence which provides for detection of an operably linked extrachromosomal molecule.", "A tag may confer a detectable trait onto an extrachromosomal molecule.", "Examples of traits that may be conferred include selection markers and reporter genes.", "Selection markers include genes that confer resistance to a chemical or drug such as blasticidin, ampicillin, rifampicin or chloramphenicol.", "Reporter genes encode a detectable polypeptide such as a fluorescent protein as described herein.", "Additionally, a tag may function as a cis-acting binding site for a detectable trans-acting element that binds to the tag when the tag has integrated into an extrachromosomal molecule.", "Binding of the detectable trans-acting element to the cis-acting tag, that is operably linked to the extrachromosomal molecule, allows the extrachromosomal molecule to be detected.", "Binding sites for trans-acting elements include nucleic acid sequences to which a detectable trans-acting element will bind and thereby tag the extrachromosomal molecule containing the binding site.", "There are many combinations of binding sites and trans-acting elements that are well known in the art.", "These combinations are exemplified by the lac repressor binding site (lacO) and the lac repressor (lacR).", "Thus, a cis-acting lac repressor binding site may be operably linked to an extrachromosomal molecule through integration of an integrating vector, which contains the tag, into the extrachromosomal molecule.", "A trans-acting fusion protein having the lac repressor fused to a fluorescent protein can bind to the cis-acting lac repressor binding site and allow the extrachromosomal molecule to be detected.", "Such methods are within the scope of the invention and are well known to those of skill in the art.", "Integrating vector: The invention provides vectors that can integrate into the extrachromosomal molecules discussed above.", "The vectors of the invention may be derived from a plasmid, virus, retrovirus, phagemid or other cloning vehicles and vectors known in the art.", "Such vectors may include a nucleic acid sequence that promotes the insertion of the vector into an extrachromosomal molecule.", "In one embodiment, the vectors of the invention include Epstein-Barr nucleic acid sequences, like that encoding the origin of replication (oriP).", "Optionally, such vectors may encode the ENBA-1 protein, lac operator repeats and lac repressor-fluorescent protein fusion protein individually or in combination.", "One example of a vector of the invention is the EBV-lacO vector (FIG.", "1).", "Chromosomal tethering polypeptide: The invention provides chromosomal tethering polypeptides.", "A tethering polypeptide of the invention includes those that promote the association of an extrachromosomal molecule with a cellular chromatid such that the extracellular molecule is segregated with the cellular chromatid during cell division.", "Tethering polypeptides can be isolated according to the methods of the invention or be created through recombinant DNA technology according to methods known in the art.", "Such recombinant molecules include, but are not limited to, a fusion polypeptide having a chromosome binding domain linked to an extrachromosomal molecule binding domain.", "An example of a tethering polypeptide is a polypeptide having the N-terminal chromosome binding domain of EBNA-1 linked to the C-terminal oriP binding domain of EBNA-1.In another example, a tethering polypeptide can have the N-terminal chromosome binding domain of EBNA-1 linked to any domain that is able to bind to a site within an extrachromosomal molecule.", "This type of tethering polypeptide is exemplified by a fusion protein having the N-terminal chromosome binding domain of EBNA-1 fused to a lac repressor.", "Cells: The invention provides cells that contain an extrachromosomal molecule that has an operably linked tag as described above.", "The cells of the invention include, but are not limited to, mammalian cells, insect cells, bacteria, yeast and any other cell which can contain an extrachromosomal molecule that segregates with a cellular chromatid during cell division.", "Examples of cells which may be used include 3T3, BHK21, MDCK, HeLa, PtK1, L6, PC12, COLO320DM and SP2 cells.", "Additional cells may be obtained from the American Type Culture Collection.", "(Hay et al., eds., American Type Culture Collection Catalogue of Cell Lines and Hybridomas, 6th ed.", "Rockville, Md.", ": American Type Culture Collection, 1988).", "These cells may be grown under any condition that allows them to divide.", "Conditions for cell and tissue culture are well known in the art.", "Ham, Proc.", "Natl.", "Acad.", "Sci.", "USA 53:288 (1965); Loo et al., Science, 236:200 (1987); Sato et al., eds.", "Growth of Cells in Hormonally Defined Media.", "Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1982).", "II.", "A Method to Visualize Chromosomal Tethering of an Extrachromosomal Molecule to a Cellular Chromatid.", "The invention provides a method to visualize tethering of an extrachromosomal molecule to a cellular chromatid.", "The method involves tagging the extrachromosomal molecule and visually detecting the tag in order to determine whether the extrachromosomal molecule is tethered to a cellular chromatid.", "The extrachromosomal molecule may be tagged by contacting the molecule with a vector of the invention such that the vector integrates into the molecule.", "Integration of the vector into the extrachromsomal molecule incorporates a cis-acting element into the extrachromosomal molecule.", "Such cis-acting elements may include, but are not limited to, nucleic acid sequences to which trans-acting elements bind and thereby tag the extrachromosomal molecule.", "In one embodiment, the extrachromosomal molecules are tagged with a fluorescent polypeptide.", "The fluorescent polypeptide may then be visualized through use of a microscope as described herein.", "The present method may be used to screen for agents that modulate chromosomal tethering and segregation of extrachromosomal molecules as described herein.", "Alternatively, the above method may be used as a diagnosis tool to determine if cells contain extrachromosomal molecules that are segregated during cell division.", "During such a diagnosis scheme, cells could be obtained through biopsy of tissue suspected of containing extrachromosomal molecules.", "These cells would then be contacted with a vector of the invention that would integrate into and tag extrachromosomal molecules contained within the suspect cells.", "The cells could then be cultured and the presence of extrachromosomal molecules could then be determined.", "Such a method would be useful because extrachromosomal molecules are known to encode oncogenes and drug resistance genes that cause proliferative disorders.", "III.", "A Method to Interfere with Tethering of Extrachromosomal Molecules to a Cellular Chromatid.", "The present invention also provides a method for treating a test cell that contains a tagged extrachromosomal molecule with a candidate agent and then determining if the candidate agent inhibits tethering of the extrachromosomal molecule to a cellular chromomatid.", "In one embodiment, the method involves use of a cell that contains a fluorescently tagged extrachromosomal molecule.", "Such molecules can be produced according to the methods described herein.", "A control cell is used that has the tagged extrachromosomal molecule but that is not treated with a candidate agent.", "A test cell has a tagged extrachromosomal molecule and is treated with a candidate agent to produce a treated cell.", "The control cell and the treated cell are allowed to begin cell division and the linkage of the extrachromosomal molecule in the test cell and the control cell is then determined and compared.", "If a candidate agent interferes with tethering, the extrachromosomal molecule will not be associated with a cellular chromatid through cell division.", "In one embodiment, the extrachromosomal molecule may be fluorescently tagged according to the methods described herein and the effects of a candidate agent on tethering can be determined through use of fluorescent microscopy as described herein.", "It is understood that any trait that can be integrated into an extrachromosomal molecule and detected in order to determine the tethering of an extrachromosomal molecule to a cellular chromatid is within the scope of the invention.", "IV.", "A Method to Identify an Agent that Modulates Segregation of Extrachromosomal Molecules into Daughter Cells during Division of a Parent Cell.", "The method involves treating a test cell that contains a tagged extrachromosomal molecule with a candidate agent and then determining if the candidate agent increases or decreases segregation of the extrachromosomal molecule during cell division when compared to a control cell.", "In one embodiment, the method involves use of a cell having a fluorescently tagged extrachromosomal molecule.", "Methods to prepare a fluorescently tagged extrachromosomal molecule are disclosed herein.", "Segregation of the fluorescently tagged extrachromosomal molecule can then be followed through use of fluorescence microscopy.", "Alternatively, fluorescent activated cell sorting (FACS) can be used to determine segregation efficiency.", "Through use of FACS, cells containing an extrachromosomal molecule can be sorted from those that do not.", "This method provides an automated procedure to rapidly screen numerous candidate agents for their ability to inhibit or increase segregation of extrachromosomal molecules.", "Alternatively, an extrachromosomal molecule having a selection marker may be used within the method of the invention.", "In this embodiment, a cell having an extrachromosomal molecule that encodes a selective marker is treated with a test agent to produce a test cell.", "The test cell is allowed to undergo cell division and is then subjected to selection according to the selection marker.", "A untreated control cell having an extrachromosomal molecule that encodes the selective marker is allowed to divide in parallel and then is subjected to selection according to the selection marker.", "The number of resistant cells of the test cell and the control cell is then compared to determine if the candidate agent increased or decreased segregation of the extrachromosomal molecule.", "It is understood that any trait that can be integrated into an extrachromosomal molecule and detected in order to determine segregation efficiency of the extrachromosomal molecule is within the scope of the invention.", "V. A method of Treating Cancer or Viral Infection.", "The invention provides a method of treating cancer or viral infection.", "The method involves contacting cells with an agent that interferes with the tethering of extrachromosomal molecules to a cellular chromatid which causes loss of the extrachromosomal molecule upon cell division.", "Such agents can be identified according to the methods disclosed herein and administered to an animal in need thereof according to methods well known in the pharmaceutical arts.", "The agents of the invention may be formulated into a variety of acceptable compositions.", "In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate.", "Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.", "Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.", "Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.", "Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids also are made.", "The compounds may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.", "Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.", "They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.", "For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.", "Such compositions and preparations should contain at least 0.1% of active compound.", "The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.", "The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.", "The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.", "When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol.", "Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form.", "For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.", "A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.", "Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.", "In addition, the active compound may be incorporated into sustained-release preparations and devices.", "The active compound may also be administered intravenously or intraperitoneally by infusion or injection.", "Solutions of the active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant.", "Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils.", "Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.", "The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.", "In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.", "The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.", "The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.", "The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.", "In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.", "Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.", "Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.", "In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.", "For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids.", "However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.", "Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.", "Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.", "Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.", "The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.", "Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.", "Examples of useful dermatological compositions that can be used to deliver the compounds of the present invention to the skin are known to the art; for example, see Jacquet et al.", "(U.S. Pat.", "No.", "4,608,392), Geria (U.S. Pat.", "No.", "4,992,478), Smith et al.", "(U.S. Pat.", "No.", "4,559,157) and Wortzman (U.S. Pat.", "No.", "4,820,508).", "Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models.", "Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat.", "No.", "4,938,949.Generally, the concentration of the compound(s) of the present invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.", "The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.", "The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.", "In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.", "The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.", "Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM.", "This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient.", "Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).", "The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.", "The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.", "EXAMPLE I Materials and Methods Plasmids: EBV oriP and EBNA-1 coding sequences derived from pCEP4 (Invitrogen) and a blasticidin resistance gene (Izumi et al., 1991) derived from pYN3215-bsr (kindly provided by Dr. Fumio Hanaoka, Osaka University) were subcloned into pMBL19 to make pMBL19-EBVbsr.", "pMBL19, which has a bacterial p15A ori, was chosen for its ability to subclone unstable inserts (Nakano et al., 1995).", "Lac operator (lacO) repeats (256 direct repeats) derived from pSV2-dhfr 8.32 (Robinett et al., 1996) were subcloned into the pMBL19-EBVbsr to make EBV-lacO vector using STBL2 competent cells (Life Technologies, Grand Island, N.Y.) (Belmont et al., 1999).", "pCLMFG-lacR-GFP was constructed by subcloning a gene encoding EGFP (Clontech, Palo Alto, Calif.) fused to lac repressor-nuclear localization signal (p3′SSdimerClonEGFP) (Robinett et al., 1996) into a splicing retroviral vector pCLMFG-MCS (kindly provided by Dr. Nikunj Somia, Salk Institute), a derivative of the pMFG vector (Dranoff et al., 1993).", "pCLMFG-lacR-YFP was constructed in the same way using EYFP gene (Clontech).", "A histone H2B-CFP fusion gene was made by swapping the GFP gene of H2B-GFPN1 (Kanda et al., 1998) with the ECFP gene (Clontech).", "The H2B-CFP gene was subcloned into a pCLNRX vector (Naviaux et al., 1996).", "Production of VSV-G pseudotyped retroviruses was performed by co-transfection of each retroviral vector and pMD.G (the plasmid encoding the envelope protein VSV-G) into 293 gp/bsr cells as described (Miyoshi et al., 1997).", "Establishing cell lines with lacO-tagged DMs: COLO320DM cells harboring DMs containing an amplified c-myc gene were grown as described (Kanda et al., 1998).", "Exponentially growing cells (1×107) were transfected with 5 μg of the EBV-lacO vector using electroporation (BioRad, Hercules, Calif.), resuspended in 10 ml of culture medium, and plated into two 10 cm dishes (8 ml, 2 ml for each dish).", "Blasticidin (15 μg/ml, Calbiochem, San Diego, Calif.) was added to the transfected cells 24 hours after transfection, and cells were selected for 14 days.", "Drug resistant cells were further grown under reduced blasticidin concentration (5 μg/ml).", "Blasticidin resistant colonies were isolated 4 weeks after transfection and then re-plated into 48 well dishes.", "Cells were expanded in media containing blasticidin (5 μg/ml) and 12 fast growing clones were selected for infection with the lacR-GFP retrovirus.", "Punctate staining in nuclei was observed in all clones analyzed, and three independent clones which exhibited the brightest fluorescent dots by lacR-GFP staining were chosen for further FISH analyses as these clones were expected to contain the highest number of EBV-lacO vectors.", "This strategy was repeated independently three times to confirm the reproducibility of the experimental data.", "Fluorescence in situ hybridization (FISH): Cells were treated with colcemid (100 μg/ml) for 50 minutes, and chromosome spreads were prepared by conventional fixation.", "For dual color FISH, c-myc cosmid DNA was labelled with biotin, while the lacO repeat (SalI-XhoI fragment of pSV2-dhfr 8.32) (Robinett et al., 1996) was labelled with digoxigenin using random prime labeling.", "Denaturation, hybridization, and washing were performed as previously described (Shimizu et al., 1996).", "Signals were detected using FITC-avidin (10 μg/ml, Vector Laboratories, Burlingame, Calif.) and rhodamine-conjugated sheep anti-digoxigenin antibody (4 μg/ml, Boehringer Mannheim, Indianapolis, Ind.).", "Chromosomes were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, 1 μg/ml) in VectaShield (Vector).", "Chromatin fibers were prepared on slide glasses as previously described for fiber-FISH (Parra and Windle, 1993).", "Signals were detected using 3 sequential steps of a signal amplification protocol as follows: (1) FITC-avidin (5 μg/ml) and anti-digoxigenin monoclonal antibody (1 μg/ml, Boehringer Mannheim); (2) biotinylated goat anti-avidin (1 μg/ml, Vector) and digoxigenin-labelled sheep anti-mouse IgG (2 μg/ml, Boehringer Mannheim); (3) FITC-avidin (5 μg/ml) and rhodamine-conjugated sheep anti-digoxigenin antibody (4 μg/ml).", "Visualizing DM-integrated and free EBV vectors by in vivo lacR-GFP staining: One of the established cell lines, containing DMs tagged with lacO repeat, was infected with lacR-GFP retrovirus.", "The infected cells were subcloned by limited dilution to obtain sublines in which DMs are more uniformly labelled with lacR-GFP.", "For time lapse imaging, the same cell line was infected with H2B-CFP and lacR-YFP viruses simultaneously, and the double-labelled cells were subcloned by limited dilution.", "For visualizing EBV vectors in transiently transfected cells, a subline of COLO320DM cells stably expressing lacR-GFP protein was established by retrovirus infection and subcloning of lacR-GFP positive cells.", "The established cells were transfected with the EBV-lacO vector by electroporation, and the transfected cells were harvested for immunofluorescence analyses at 3 days posttransfec Immunofluorescence staining: Cells were harvested by gentle pipetting, attached to slide glasses by cytospin (500 rpm, 1 minute, Shandon, Pittsburgh, Pa.), and fixed with 3.7% formaldehyde for 10 minutes.", "When indicated, cells were treated with either taxol (10 μM paclitaxel, Sigma Chemical Co., St. Louis, Mo.)", "or vinblastine (10 μg/ml, Sigma) for 3 hours prior to harvesting them in order to disrupt microtubules.", "Slides were washed with PBS three times, and treated with blocking buffer (2.5% BSA, 0.2M glycine, 0.1% TritonX-100) for 30 minutes.", "Primary and secondary antibodies were diluted in the blocking buffer.", "Primary antibodies were rabbit anti EBNA-1 serum K67-3 (1:1000, kindly provided by Dr. Jaap Middeldorp, Free University Hospital, Amsterdam, the Netherlands), human CREST autoantiserum hACA-M (for detecting centromeres, 1:2000 dilution) (Sullivan et al., 1994), and monoclonal anti α-tubulin (for detecting microtubules, 1:2000 dilution, Sigma).", "Following incubation for 60 minutes at room temperature, glass slides were washed three times with PBS.", "Secondary antibodies were Texas-Red conjugated anti-rabbit IgG (1:500, Jackson ImmunoResearch, West Grove, Pa.), Cy5 conjugated goat anti-human IgG (1:1000, Amersham, Piscataway, N.J.), and rhodamine conjugated anti mouse IgG (1:1000, Boehringer Mannheim), respectively.", "Following incubation for 60 minutes at room temperature, slides were washed three times with PBS, and chromosomes were counterstained with DAPI (1 μg/ml).", "Fluorescence of lacR-GFP was preserved well by this protocol.", "Microscopy: All images appearing in the Figures were collected using a DeltaVision microscope system (Applied Precision Inc. Issaquah, Wash.) with either a 63×/NA 1.4 or a 100×/NA 1.35 oil immersion objective.", "For fixed specimens (except for the images of FIG.", "2), three-dimensional data sets were collected to visualize EBV vectors and DMs as they distributed in multiple focal planes.", "Optical sections were collected at 0.2-μm focal intervals; pixel size was 0.111 μm for 63× objective and 0.0669 μm for 100× objective.", "Out-of-focus contamination was removed from each optical section via deconvolution processing and two-dimensional images were created by projecting the three-dimensional data stacks using the software supplied with the DeltaVision system.", "For observation of living specimens, cells were grown on 40 mm cover slips pretreated with fibronectin (25 μg/ml in PBS) and placed in an FCS2 chamber system (Bioptechs, Butler, Pa.) with prewarmed medium (containing 20 mM HEPES pH 7.3).", "Special filter sets required for CFP and YFP detection (Ellenberg et al., 1999) were installed into the DeltaVision microscope system.", "Single slice images were collected every 2 minutes using 100× objective equipped with an objective heater (Bioptechs).", "Pixel size was 0.1338 μm and a binning factor of 2 was used to minimize the total exposure time during observation.", "Pseudo-color images were created using Adobe Photoshop (Adobe Systems, San Jose, Calif.).", "Results DMs are Preferred Targets for EBV Vector Integration FISH analyses revealed that stably transfected EBV vectors frequently co-localized with DMs.", "One explanation of this finding was that EBV vectors integrated into DMs.", "To facilitate the tracking of transfected EBV vectors, two hundred and fifty-six direct repeats of the lac operator (lacO) were added to the EBV vector (EBV-lacO) (FIG.", "1).", "If the EBV-lacO vectors recombined with DMs, the DMs were tagged with lacO repeats and were rapidly detected in cells expressing a fusion protein between the lac repressor and GFP (lacR-GFP) (FIG.", "1) (Robinett et al., 1996).", "The EBV-lacO vector was transfected into COLO320DM cells, which contain DMs encompassing c-myc loci, and then blasticidin resistance encoded by the EBV vector was selected for.", "Drug resistant colonies were obtained at frequencies of approximately 1×10−4˜10−5.It should be noted that both the oriP sequence and the EBNA-1 gene were required for obtaining transformants, as no colonies arose in transfections employing vectors lacking either element (transformation efficiency <1×10−7).", "Three drug resistant colonies were expanded into cell lines and analyzed by dual color FISH analyses using a c-myc cosmid probe (to detect DMs) and a lacO repeat DNA (to visualize EBV-lacO vectors).", "The result revealed that, in all three clones, the signals generated by both probes frequently overlapped in metaphase chromosome spreads (FIG.", "2A).", "Analysis of 50 metaphase spreads of one of the established clones showed that 50% (25/50) of the spreads exhibited complete colocalization of lacO/DM signals, and 90% (45/50) of the metaphase spreads contained at least one pair of DMs with overlapping lacO signals (FIG.", "2B).", "On the average, each chromosome spread contained 18.3 (±19.2) pairs of DMs, and 12.7 (±13.2) DMs had overlapped lacO signals.", "It is noteworthy that lacO signals never overlapped with intrachromosomal c-myc signals (FIG.", "2A), and no evidence of EBV-lacO integration into chromosomes was observed in >50 metaphase spreads in three different clones.", "Thus, the combined data strongly suggest that the majority of transfected EBV vectors preferentially co-localize with DMs.", "Direct evidence was obtained that EBV-lacO vectors integrated into DMs using FISH analysis of stretched chromatin fibers (Parra and Windle, 1993).", "This analysis revealed that arrays of lacO signals were detected on the same DNA fibers exhibiting c-myc signals (FIG.", "2C).", "Semi-quantitative Southern blotting revealed approximately 130 copies of lacO repeats per cell, and several bands in addition to those of the expected-size were detected (FIG.", "2D).", "As there are approximately 13 DMs with integrated lacO repeats per cell (see above), it was concluded that each DM contains approximately 10 copies of EBV-lacO (i.e., 130/13).", "The complex patterns observed by fiber-FISH, together with the extra bands observed in Southern blotting, indicate that the integration events are likely to be complex, or that further rearrangements occurred subsequent to the initial integration.", "EBV-lacO/DM Chimeras and Free EBV Vectors Behave Differently in Prometaphase Cells The established cell lines containing the lacO-integrated DMs were infected with retrovirus expressing the lacR-GFP fusion protein.", "Retroviral infection resulted in readily detectable lacR-GFP protein expression in approximately 80% of the recipient cells, and approximately 20% of which exhibited punctate fluorescent dots representing DMs.", "The lacR-GFP expressing populations were subcloned to yield clones in which 60% of the cells had punctate fluorescent DMs.", "The distribution of EBV-lacO/DM chimeras in prometaphase cells, in which chromosomes aggregate briefly into a single, wheel-shaped ring called a chromosome rosette (Nagele et al., 1995) was first focused on.", "It was found that the fluorescent dots always attached to the periphery of the chromosome rosette (FIG.", "3A).", "Immunofluorescence analyses revealed that EBNA-1 protein colocalized with EBV-integrated DMs (FIG.", "3B), indicating that EBNA-1 was recruited to the oriP sequences that had been integrated into DMs.", "The mitotic distribution of EBV-lacO vectors after transient transfection into COLO320DM cells (see methods) was also examined.", "It was inferred that at 3 days posttransfection, most EBV plasmids were not integrated, and one or a few DMs may have contained integrated EBV plasmids.", "The fluorescent dots representing the transiently transfected EBV-lacO vectors were found to associate randomly with prometaphase chromosomes (FIG.", "3C), corresponding well with previous FISH results indicating no preferential peripheral localization (Simpson et al., 1996; Westphal et al., 1998).", "In this case, EBNA-1 staining was rather diffuse on chromosomes, as observed previously (Grogan et al., 1983; Petti et al., 1990), but still colocalized with the EBV vectors (FIG.", "3D).", "These data are consistent with free EBV-lacO vectors associating randomly with mitotic chromosomes, while DM-integrated EBV vectors localizing at the periphery.", "Importantly, it was found that native DMs and EBV-integrated DMs displayed equivalent mitotic behavior (FIG.", "3E, F), although the latter had co-localizing EBNA-1 protein (FIG.", "3B).", "Therefore, it is likely that the mitotic behavior of the chimeric extrachromosomal molecules faithfully represents that of native DMs.", "These observations justify the use of EBV-lacO/DMs as a tool to analyze DM dynamics.", "Mitotic Behaviors of DMs, Centromeres, and Microtubules The fluorescent labeling strategy for DMs described above enables visualization of DMs, centromeres, and microtubules simultaneously in various phases of mitosis.", "Centromeres and spindle microtubules were detected by indirect immunofluorescence while preserving the fluorescence of lacR-GFP.", "This analysis confirmed that DMs lack centromeric antigens (Barker and Hsu, 1978; Levan and Levan, 1978) (FIG.", "4A, C, E, G, I), and that DMs do not associate with kinetochore microtubules (FIG.", "4D, F, H).", "DMs and centromeres exhibited distinctly different behaviors during the phases of mitosis.", "In interphase cells (FIG.", "4A, B), DMs and centromeres were dispersed independently in nuclei.", "However, in prometaphase cells (FIG.", "4C, D), paired fluorescent DM dots attached to the periphery of the chromosome rosettes (Nagele et al., 1995), as described above, while centromeres localized centrally as they were pulled inward by the attached microtubules.", "In metaphase cells (FIG.", "4E,F), DMs did not strictly align on metaphase plates, but rather associated with the periphery of the aligned chromosomes.", "At the metaphase-anaphase transition (FIG.", "4G,H), centromeres of sister chromatids were pulled apart by the attached spindle microtubules.", "In contrast, some DMs were still clearly observed as double dots in anaphase cells, demonstrating that the connections between sister minute chromosomes are not readily broken at the metaphase-anaphase transition.", "The majority of DMs, still attached to the distal chromosomal arms, lagged behind centromeric regions that were pulled toward opposing poles in anaphase cells (FIG.", "4G,H).", "In telophase cells (FIG.", "4I, J), most DMs were incorporated into daughter nuclei along with the chromosomes, while a minority became entrapped in micronuclei (Shimizu et al., 1996; Tanaka and Shimizu, 2000).", "Real Time Observation of DM Behavior using Dual-Color Fluorescent Protein Labeling The above results with fixed cells could mask movements occurring in living cells, and it does not provide a dynamic view of DM behavior.", "These concerns were addressed by taking advantage of the in vivo expression of distinguishable fluorescent fusion proteins (Ellenberg et al., 1999) in order to analyze the dynamics of DMs and chromosomes in living mitotic cells.", "Cells with lacO-labelled DMs were simultaneously infected with two different retroviruses expressing either H2B-CFP or lacR-YFP.", "H2B-CFP should label chromosomes and DMs, as described previously (Kanda et al., 1998), while lacR-YFP should label only DMs as descried above.", "Chromosomes and DMs in the same cell were observed with minimal spectral overlap (FIG.", "5A).", "Representative time lapse images demonstrating the behavior of DMs at the metaphase-anaphase transition are shown (FIG.", "5B).", "DMs were found at the tips of chromosome arms in late prometaphase cells (time 00).", "Subsequently, paired sister chromatids were observed to align on metaphase plates (time 04).", "When sister chromatids started to separate at the onset of anaphase (time 06), DMs quickly changed their position and lagged behind segregating chromosomes (time 08, 10).", "In early G1 phase, significant numbers of DMs were still observed as paired dots (data can be seen in the attached movie), confirming the previous observation obtained by a premature chromosome condensation protocol (Takayama and Uwaike, 1988).", "These time lapse images correspond very well with the data of fixed cells (FIG.", "4).", "Taken together, the peripheral localization of DMs in prometaphase chromosome rosettes and their lagging behavior in anaphase cells strongly suggest that DMs may be repelled from the spindle poles.", "Microtubule Inhibitors Disrupt DM Peripheral Localization The ability to readily visualize DMs without using FISH protocol has enabled the investigation of the mechanisms that contribute to their behavior during mitosis.", "The possibility that DMs are held away from the spindle poles by the microtubule-mediated antipolar force, to which normal chromosome arms are also known to be subjected (Fuller, 1995; Rieder et al., 1986) was examined.", "DM-labelled cells were treated with either a microtubule stabilizer (taxol) or destabilizer (vinblastine).", "DMs and microtubules were visualized by lacR-GFP and immunofluorescence staining, respectively.", "Microtubules of taxol-treated cells showed multiple aster-like structures (FIG.", "6A), while vinblastine-treated cells exhibited rod-like microtubules (FIG.", "6B).", "In both cases, chromosome organization was completely disrupted and DMs were no longer attached to the periphery of clustered chromosomes.", "Rather, DMs distributed randomly, although they were still associated closely with chromosomes (FIG.", "6).", "These results support the idea that DMs are repelled from the spindle poles via microtubule-mediated antipolar forces and that microtubules do not mediate the attachment of DMs to mitotic chromosomes.", "Discussion The mechanisms underlying the precision of chromosome segregation are being elucidated with increasing detail.", "It now appears that some autonomously replicating DNA viruses achieve high efficiency segregation not merely by their high copy number, but rather by having devised strategies to associate with chromosomes (Bastien and McBride, 2000; Ilves et al., 1999; Lehman and Botchan, 1998; Marechal et al., 1999; Skiadopoulos and McBride, 1998).", "The data herein highlights the role of chromosomes as “cargo ships” on which both viral replicons and cellular DMs are loaded to enable their efficient transmission to daughter nuclei.", "It was determined that EBV vectors integrate into DMs at high frequency.", "This targeted integration of EBV vectors into DMs was totally unexpected since EBV vectors containing oriP and the EBNA-1 gene are usually maintained as extrachromosomal elements without integrating into chromosomes (Yates et al., 1985).", "It was found that EBV vectors randomly associated with mitotic chromosomes as well as DMs after transient transfection (FIG.", "3C).", "This observation corresponds well with the known noncovalent association of EBV vectors with mitotic chromosomes (Harris et al., 1985; Marechal et al., 1999; Simpson et al., 1996; Westphal et al., 1998).", "However, after stable transfection into DM-harboring cells, it was observed that EBV vectors recombined with DMs, and that the chimeric molecules of EBV vectors and DMs were always found at the periphery of mitotic chromosomes (FIG.", "3A).", "Free extrachromosomal EBV vectors randomly associating with mitotic chromosomes after stable transfection were not detected.", "Interestingly, it was found that the same EBV-lacO vector could be maintained extrachromosomally in stably transfected HeLa cells, which do not contain DMs (data not shown).", "Therefore, the EBV vectors appear to remain as independent extrachromosomal molecules in cells without DMs, while there is a high probability of integration of multiple EBV plasmids into DMs.", "The molecular basis of preferential integration into DMs remains a mystery.", "It is possible that DMs and normal chromosomes have different tendencies to undergo recombination.", "Another possibility is that EBV replicons and DMs share the same replication machinery during S phase of the cell cycle, which increases the probability of recombination between replication intermediates.", "This would be consistent with the observed requirements for both oriP and EBNA-1 to recombine with DMs.", "The heterogeneity in the number and fraction of DMs containing integrated EBV-lacO sequences (FIG.", "2B) is most easily explained if it is assumed that a single integration event occurred at an early stage of selection shortly after transfection.", "Replication of the EBV-lacO/DM chimeras, followed by their unequal selection, could have led to eventual emergence of clones with dissimilar numbers of chimeric extrachromosomal molecules.", "EBV vectors are thought to provide a general strategy for tagging DMs derived from different chromosomal loci as EBV-DM chimeras were obtained in the CRL2270 neuroblastoma line containing extrachromosomally amplified N-myc amplicons (data not shown).", "The chimeric extrachromosomal molecules of DMs and EBV-lacO vectors appear to exhibit the same behavior as native (unlabelled) DMs.", "The visualization strategy involving lacR-GFP can be used in combination with immunofluorescence, as it does not require harsh denaturation of DNA, and it preserves chromosomal fine structures far better than FISH (Robinett et al., 1996).", "This sensitive methodology enabled the visualization of DMs together with centromeres and microtubules (FIGS.", "4, 6), and to track the dynamics of DMs and chromosomes in living human cells (FIG.", "5).", "The data confirms and further extends previous analyses using fixed cells (Levan and Levan, 1978) and previous findings obtained using H2B-GFP staining (Kanda et al., 1998).", "A model is proposed in which DMs are subject to two “forces” in mitotic cells (FIG.", "7).", "First, DMs appear to be pushed away from the poles, as they are always found at the periphery of prometaphase chromosome rosettes, and they lag behind segregating chromosomes in anaphase cells.", "It is well known that, while the kinetochore microtubules pull chromosomes poleward, another force appears to repel chromosomal arms (Fuller, 1995) (FIG.", "7A).", "Laser microsurgery experiments demonstrated that severed chromosomal arms immediately moved radially outward to the periphery of the aster (Rieder et al., 1986) (FIG.", "7B), indicating the existence of such an astral exclusion force.", "It has been proposed that plus-oriented kinesin-related microtubule motor proteins, distributed along chromosomal arms, mediate this astral exclusion force (Fuller, 1995).", "Since DMs originate from chromosomes, in this case the distal part of chromosome 8 (8q24; c-myc locus), it is reasonable to assume that DMs are covered with such motor proteins and subject to a microtubule-mediated antipolar force (FIG.", "7C).", "The present observations that peripheral localization of DMs became apparent only after nuclear membrane breakdown and chromosomes attached to microtubules, and that disrupting microtubule organization prevented the peripheral localization of DMs, support the model.", "The antipolar force working on chromosomal fragments is likely to be proportional to their size (Fuller, 1995).", "This can partly explain the difference between the behavior of severed chromosomal arms and that of DMs, since bigger acentric chromosome fragments should be subject to a stronger antipolar force compared to smaller DMs (FIG.", "7B, C).", "Random chromosomal association of free EBV vectors can be explained by the lack of plus-oriented motor proteins and astral exclusion force on free EBV vectors (FIG.", "7D).", "It is unlikely that DMs specifically associate with telomeres, as some DMs associated with chromosomal arms at positions clearly distinct from telomeric regions (FIG.", "4C, D).", "The second “force” acting on DMs keeps them attached to mitotic chromosomes (FIG.", "7C).", "This force is not affected by microtubule disruption.", "Although there is a study demonstrating that DMs associate with chromosomes via nucleolar material (Levan and Levan, 1978), no further experimental data supporting the idea has been presented.", "The present finding that DMs are frequent recombinational targets of EBV vectors leads to another proposed model in which DMs may somehow mimic the behavior of viral vectors.", "It has been suggested that chromosome tethering of EBV vectors is mediated by the cis-acting oriP sequence and trans-acting viral protein EBNA-1 (Krysan et al., 1989; Mackey and Sugden, 1999; Marechal et al., 1999; Middleton and Sugden, 1994; Simpson et al., 1996).", "It was recently found that EBNA-1 appears to serve as a bridge between chromosomes and oriP-containing vectors (Kanda T et al, manuscript in preparation).", "This observation raises the possibility that DMs may also have cis-acting sequences that recruit cellular transacting factors to them to mediate chromosome association.", "This possibility is strengthened by the finding that DMs consist of multiple copies of amplicons, each copy of which contains cellular replication origin(s) that are usually associated with scaffold/matrix attachment regions (S/MAR) (Carroll et al., 1991; Pemov et al., 1998).", "A recent study showed that an episomal vector containing a human S/MAR sequence and SV40 origin is associated with mitotic chromosomes (Baiker et al., 2000).", "Therefore, it is conceivable that DMs containing multiple S/MARs attach to chromosomal scaffolds, which then gives the appearance that they are associating with mitotic chromosomes, even though there is no direct connection 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[ [ "Process for cracking an olefin-rich hydrocarbon feedstock", "A process for cracking an olefin containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst selected from an MFI-type crystalline silicate having a silicontalurniniumn atomic ratio of a least 180 and an MEL-type crystalline silicate having a silicon/aluminium atomic ration of from 150 to 800 which has been subjected to a steaming step, at an inlet temperature of from 500 to 600° C., at an olefin partial pressure of from 0.1 to 2 bars and the feedstock being passed over the catalyst at an LHSV of from 5 to 30 h−1 to produce an effluent with an olefin content of lower molecular weight than that of the feedstock, intermittently removing a first fraction of the catalyst from the moving bed reactor, regenerating the first fraction of the catalyst in a regenerator and intermittently feeding into the moving bed reactor a second fraction of the catalyst which has been regenerated in the regenerator, the catalyst regeneration rate being controlled whereby the propylene purity is maintained constant at a value corresponding to the average value observed in a fixed bed reactor using the same feedstock:, catalyst and cracking conditions, for example at least 94 wt %." ], [ "1.A process for cracking an olefin-containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst selected from an MFI-type crystalline silicate having a silicon/aluminum atomic ratio of at least 180 and an MEL-type crystalline silicate having a silicon/aluminum atomic ratio from 150 to 800 which has been subjected to steaming step, at an inlet temperature of from 500 to 600° C., at an olefin partial pressure of from 0.1 to 2 bars and the feedstock being passed over the catalyst at an LHSV of from 5 to 30 h−1 to produce an effluent containing propylene and with an olefin content of lower molecular weight than the olefin content of the feedstock with concomittant deactivation of said catalyst, removing a first fraction of the deactivated catalyst from the moving bed reactor and transferring said deactivated catalyst to a regenerator, regenerating said deactivated catalyst in said regenerator to produce a second fraction of regenerated catalyst and recycling said regenerated catalyst to the moving bed reactor, continuing the transfer of deactivated catalyst and the recycle of regenerated catalyst while carrying out the cracking of the olefin-containing hydrocarbon feedstock, the catalyst regeneration and recycle rate being controlled to maintain the propylene purity at a relatively constant value corresponding to the average value observed in a fixed bed reactor using the same feedstock, catalyst and cracking conditions.", "2.The process of claim 1 wherein the catalyst regeneration and recycle rate is controlled to provide an ethylene yield in the effluent on an olefin basis which is less than 10 wt %.", "3.The process of claim 1, wherein the effluent has a propylene purity of at least 94 wt % propylene based upon the total C3 content of the effluent.", "4.The process of claim 1, wherein the olefin content of the effluent is within ±15 wt % of the olefin content of the feed stock.", "5.The process of claim 1, wherein said first fraction of the catalyst is intermittently removed from said moving bed reactor.", "6.The process of claim 5, wherein said second fraction of the regenerated catalyst is intermittently supplied from said regenerator to said moving bed reactor.", "7.The process of claim 1, wherein said catalyst is regenerated in said regenerator by supplying an oxidizing gas containing oxygen in amount within the range of 0.2 to 2 volume percent of said oxidizing gas.", "8.The method of claim 1, wherein the regeneration of catalyst in said regenerator involves a supply of an initial oxygen-containing gas to the regenerator and a supply of a second oxygen-containing gas to the regenerator at a point downstream of the introduction of said initial oxygen-containing gas, said second oxygen-containing gas having a higher oxygen content than said initial oxygen-containing gas.", "9.The process of claim 8, wherein said second oxygen-containing gas contains from 5 to 21 volume percent oxygen.", "10.The process of claim 1, wherein said moving bed reactor comprises a first stage reactor and a second stage reactor connected in series with said first stage reactor, wherein the effluent from the first stage reactor is heated and then supplied to the inlet of said second stage reactor.", "11.The process of claim 10, wherein the contact time of the reaction mixture with the catalyst in the second reactor is greater than the contact time of the reaction mixture with the catalyst in the first stage reactor.", "12.A process for cracking an olefin-containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst selected from an MFI-type crystalline silicate having a silicon/aluminum atomic ratio of at least 180 and an MEL-type crystalline silicate having a silicon/aluminum atomic ratio from 150 to 800 which has been subjected to steaming step, at an inlet temperature of from 500 to 600° C., at an olefin partial pressure of from 0.1 to 2 bars and the feedstock being passed over the catalyst at an LHSV of from 5 to 30 h−1 to produce an effluent with an olefin content of lower molecular weight than that of the feedstock with concomittant deactivation of said catalyst, removing a first fraction of the deactivated catalyst from the moving bed reactor and transferring said deactivated catalyst to a regenerator, regenerating said deactivated catalyst in said regenerator to produce a second fraction of regenerated catalyst and recycling said regenerated catalyst to the moving bed reactor, continuing the transfer of deactivated catalyst and the recycle of regenerated catalyst while carrying out the cracking of the olefin-containing hydrocarbon feedstock, the catalyst regeneration and recycle rate being controlled to provide that all of the catalyst in the moving bed reactor is regenerated in a period of from 20 to 240 hours.", "13.The process of claim 12 wherein the propylene purity is maintained at a relative constant value corresponding to the average value obtained in a fixed bed reactor using the same feedstock, catalyst and cracking conditions.", "14.The process of claim 12 where the regeneration and recycle rate is controlled to proceed on ethylene yield on an olefin basis which is less than 10 wt %.", "15.The process of claim 12, wherein the propylene yield of said process is within the range of 30 to 50 wt % propylene with a selectivity to propylene of at least 92 wt % of the total amount of propylene and propane in the effluent.", "16.The process of claim 15, wherein the olefin content of the effluent is within the range of ±10 wt % of the olefin content of the feed stock.", "17.The method of claim 12, wherein the regeneration of catalyst in said regenerator involves a supply of an initial oxygen-containing gas to the regenerator and a supply of a second oxygen-containing gas to the regenerator at a point downstream of the introduction of said initial oxygen-containing gas, said second oxygen-containing gas having a higher oxygen content than said initial oxygen-containing gas.", "18.The process of claim 17, wherein said second oxygen-containing gas contains from 5 to 21 volume percent oxygen.", "19.The process of claim 12, wherein said moving bed reactor comprises a first stage reactor and a second stage reactor connected in series with said first stage reactor, wherein the effluent from the first stage reactor is heated and then supplied to the inlet of said second stage reactor.", "20.The process of claim 17, wherein the contact time of the reaction mixture with the catalyst in the second stage reactor is greater than the contact time of the reaction mixture with the catalyst in the first stage reactor.", "21.A process for cracking an olefin-containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst to produce an effluent with an olefin content of lower molecular weight than that of the feedstock with concomittant deactivation of said catalyst, removing a deactivated catalyst from the moving bed reactor and regenerating said deactivated catalyst to produce a regenerated catalyst which is recycled to said moving bed reactor, continuing the removal of deactivated catalyst and the recycle of regenerated catalyst while carrying out the cracking of the olefin-containing hydrocarbon feedstock, the catalyst regeneration and recycle rate being controlled to provide a propylene content in the effluent that is maintained relatively constant at a value corresponding to the initial value observed in the effluent from a fixed bed reactor during an initial period of an olefin cracking process using the same feedstock, catalyst and cracking conditions.", "22.The process of claim 21, wherein the initial period of the fixed bed reaction is the first 10 to 40 hours of said reaction.", "23.The process according to claim 21, wherein said moving bed reactor comprises a first stage reactor and a second stage reactor connected in series with said first stage reactor, wherein the effluent from the first stage reactor is heated and then supplied to the inlet of said second stage reactor.", "24.The process of claim 23, wherein the contact time of the reaction mixture with the catalyst in the second reactor is greater than the contact time of the reaction mixture with the catalyst in the first stage reactor.", "25.The process of claim 21, wherein fresh makeup catalyst is supplied to said moving bed reactor along with said regenerated catalyst recycled to said moving bed reactor." ], [ "The present invention relates to a process for cracking an olefin-rich hydrocarbon feedstock which is selective towards light olefins in the effluent.", "In particular, olefinic feedstocks from refineries or petrochemical plants can be converted selectively so as to redistribute the olefin content of the feedstock in the resultant effluent.", "It is known in the art to use zeolites to convert long chain paraffins into lighter products, for example in the catalytic de-waxing of petroleum feedstocks.", "While it is not the objective of de-waxing, at least parts of the paraffinic hydrocarbons are converted into olefins.", "It is known in such processes to use crystalline silicates for example of the MFI or MEL type, the three-letter designations “MFI” and “MEL” each representing a particular crystalline silicate structure type as established by the Structure Commission of the International Zeolite Association.", "Examples of a crystalline silicate of the MFI type are the synthetic zeolite ZSM-5 and silicalite and other MFI type crystalline silicates are known in the art.", "An example of a crystalline silicate of the MEL type is the synthetic zeolite ZSM-11.EP-A-0305720 discloses the production of gaseous olefins by catalytic conversion of hydrocarbons.", "EP-B-0347003 discloses a process for the conversion of a hydrocarbonaceous feedstock into light olefins.", "WO-A-90/11338 discloses a process for the conversion of C2-C12 paraffinic hydrocarbons to petrochemical feedstocks, in particular to C2 to C4 olefins.", "U.S. Pat.", "No.", "5,043,522 and EP-A-0395345 disclose the production of olefins from paraffins having four or more carbon atoms.", "EP-A-0511013 discloses the production of olefins from hydrocarbons using a steam activated catalyst containing phosphorous and H-ZSM-5.U.S.", "Pat.", "No.", "4,810,356 discloses a process for the treatment of gas oils by de-waxing over a silicalite catalyst.", "GB-A-2156845 discloses the production of isobutylene from propylene or a mixture of hydrocarbons containing propylene.", "GB-A-2159833 discloses the production of a isobutylene by the catalytic cracking of light distillates.", "It is known in the art that for the crystalline silicates exemplified above, long chain olefins tend to crack at a much higher rate than the corresponding long chain paraffins.", "It is further known that when crystalline silicates are employed as catalysts for the conversion of paraffins into olefins, such conversion is not stable against time.", "The conversion rate decreases as the time on stream increases, which is due to formation of coke (carbon) which is deposited on the catalyst.", "These known processes are employed to crack heavy paraffinic molecules into lighter molecules.", "However, when it is desired to produce propylene, not only are the yields low but also the stability of the crystalline silicate catalyst is low.", "For example, in an FCC unit a typical propylene output is 3.5 wt %.", "The propylene output may be increased to up to about 7-8 wt % propylene from the FCC unit by introducing the known ZSM-5 catalyst into the FCC unit to “squeeze” out more propylene from the incoming hydrocarbon feedstock being cracked.", "Not only is this increase in yield quite small, but also the ZSM-5 catalyst has low stability in the FCC unit.", "There is an increasing demand for propylene in particular for the manufacture of polypropylene.", "The petrochemical industry is presently facing a major squeeze in propylene availability as a result of the growth in propylene derivatives, especially polypropylene.", "Traditional methods to increase propylene production are not entirely satisfactory.", "For example, additional naphtha steam cracking units which produce about twice as much ethylene as propylene are an expensive way to yield propylene since the feedstock is valuable and the capital investment is very high.", "Naphtha is in competition as a feedstock for steam crackers because it is a base for the production of gasoline in the refinery.", "Propane dehydrogenation gives a high yield of propylene but the feedstock (propane) is only cost effective during limited periods of the year, making the process expensive and limiting the production of propylene.", "Propylene is obtained from FCC units but at a relatively low yield and increasing the yield has proven to be expensive and limited.", "Yet another route known as metathesis or disproportionation enables the production of propylene from ethylene and butene.", "Often, combined with a steam cracker, this technology is expensive since it uses ethylene as a feedstock which is at least as valuable as propylene.", "Thus there is a need for a high yield propylene production method which can readily be integrated into a refinery or petrochemical plant, taking advantage of feedstocks that are less valuable for the market place (having few alternatives on the market).", "EP-A-0921179 in the name of Fina Research S.A. discloses the production of olefins by catalytic cracking of an olefin-rich hydrocarbon feedstock which is selective towards light olefins in the effluent.", "While it is disclosed in that document that the catalyst has good stability, i.e.", "high activity over time, and a stable olefin conversion and a stable product distribution over time, nevertheless the catalyst stability still requires improvement, particularly when higher inlet temperature within the broad range disclosed (500 to 600° C.) are employed in conjunction with a single reactor.", "That specification exemplifies the use of a fixed bed reactor, although it is disclosed that a moving bed reactor, of the continuous catalytic reforming type, or a fluidised bed reactor may be employed for the olefin-cracking process.", "During hydrocarbon conversion reactions, a carbonaceous material, i.e., coke, can be formed and deposited on a catalyst thereby causing it to lose activity.", "The deposited carbonaceous material on the catalyst affects the amount of active catalyst centres on the catalyst and thereby influences the extent of the hydrocarbon conversion reaction, and hence the conversion to desired products and by-products.", "The presence of carbonaceous material on the catalyst results in a changing product distribution that affects the downstream fractionation section and the recycle rate of unconverted hydrocarbon feed.", "For most hydrocarbons conversion process the loss of activity can be compensated by increasing the reaction temperature up to a value where undesirable side reactions become important or up to a value which becomes impracticable.", "Thus, it is further known in hydrocarbon conversion processes partially to regenerate a catalyst using a moving bed reactor.", "U.S. Pat.", "No.", "3,838,039 discloses a method of operating a continuous hydrocarbon process employing catalyst particles in which catalyst activity is maintained by continuous regeneration.", "EF-A-0273592 discloses a process for continuous de-waxing of hydrocarbon oils including reactivation of partially spent catalyst.", "U.S. Pat.", "No.", "5,157,181 discloses a moving bed hydrocarbon conversion process incorporating partial regeneration of a co-catalyst.", "U.S. Pat.", "No.", "3,978,150 discloses a continuous paraffin dehydrogenation process incorporating partial catalyst regeneration.", "U.S. Pat.", "No.", "5,336,829 discloses a continuous process for the dehydrogenation of paraffinic to olefinic hydrocarbons incorporating catalyst regeneration.", "U.S. Pat.", "No.", "5,370,786 discloses a method of operating a continuous conversion process employing solid catalyst particles in which the catalyst may be regenerated.", "U.S. Pat.", "No.", "4,973,780 discloses the alkylation of benzene in a moving bed incorporating partial catalyst regeneration.", "U.S. Pat.", "No.", "5,849,976 discloses a moving bed solid catalyst hydrocarbon alkylation process incorporating partial catalyst regeneration.", "U.S. Pat.", "No.", "5,087,783 discloses the transalkylation of benzene in a moving bed, incorporating partial catalyst reactivation.", "EP-A-0385538 discloses a process for the conversion of a straight-run hydrocarbonaceous feedstock containing hydrocarbons having such a boiling range that an amount thereof boils at a temperature of at least 330° C., such as a gas oil, in a moving bed reactor which may incorporate catalyst regeneration of the zeolite catalyst.", "EP-A-0167325 discloses a process for changeover of a moving bed catalytic cracking unit's catalyst inventory from conventional catalyst to ZSM-5 containing catalyst, the feedstock comprising an oil changestock for example a blend of crude oils or a gas oil fraction.", "U.S. Pat.", "No.", "4,927,526 discloses a process for catalytically cracking hydrocarbon feedstock in a cracking unit to a product comprising gasoline with an increased octane number in the presence of a cracking catalyst, under cracking conditions.", "The process may employ moving bed catalytic cracking, with changeover of the catalyst inventory.", "While the use of a moving bed employing partial catalyst regeneration or reactivation has been known in the art for some time, this, to the applicant's knowledge, has not been disclosed heretofore for use in an olefin-cracking process.", "The olefin-cracking process as disclosed in EP-A-0921179 may be carried out at high reaction temperature close to the temperature of thermal cracking of hydrocarbon molecules.", "However, raising the reaction temperature in order to compensate the loss of catalytic activity in the olefin-cracking process is limited, as it will favour undesirable side reactions that are not the result of the presence of the catalyst.", "Moreover, the surface temperatures required to heat up the feed mixture in for instance a fire heater can become so high that thermal cracking of the feed starts.", "When the olefin-cracking process of EP-A-0921179 is applied in a fixed bed reactor, it is observed that at the start of the catalytic cycle small amounts of less desired products like propane are produced.", "This results in a lower propylene purity of the C3 fraction.", "Moreover, the ethylene production rate is higher at the start of the catalytic cycle than after some time.", "The amount of the less desired product, propane, decreases during the operation and also the ethylene product decreases.", "During an important period of time the propylene yield remains fairly constant while those of propane and ethylene progressively decreases.", "These variations during the use of the catalyst in a fixed bed reactor are the result of a changing performance of the catalyst caused by the carbonaceous material laydown.", "It is an object of the present invention to provide a process for using the less valuable olefins present in refinery and petrochemical plants as a feedstock for a process which, in contrast to the prior art processes referred to above, catalytically converts olefins into lighter olefins, and in particular propylene, and which process has improved catalyst stability.", "It is another object of the invention to provide a process for producing olefins having a high propylene yield and purity, most particularly substantially constantly over the whole time of the process.", "The present invention provides a process for cracking an olefin-containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst selected from an MFI-type crystalline silicate having a silicon/aluminium atomic ratio of at least 180 and an MEL-type crystalline silicate having a silicon/aluminium atomic ratio of from 150 to 800 which has been subjected to a steaming step, at an inlet temperature of from 500 to 600° C., at an olefin partial pressure of from 0.1 to 2 bars and the feedstock being passed over the catalyst at an LHSV of from 5 to 30 h−1 to produce an effluent with an olefin content of lower molecular weight than that of the feedstock, intermittently removing a first fraction of the catalyst from the moving bed reactor, regenerating the first fraction of the catalyst in a regenerator and intermittently feeding into the moving bed reactor a second fraction of the catalyst which has been regenerated in the regenerator, the catalyst regeneration rate being controlled whereby the propylene purity is maintained constant at a value corresponding to the average value observed in a fixed bed reactor using the same feedstock, catalyst and cracking conditions, for example at least 94 wt %.", "Preferably, the catalyst regeneration rate is controlled whereby the ethylene yield on an olefin basis is less than 10 wt %.", "The present invention further provides a process for cracking an olefin-containing hydrocarbon feedstock which is selective towards light olefins in the effluent, the process comprising passing a hydrocarbon feedstock containing one or more olefins through a moving bed reactor containing a crystalline silicate catalyst selected from an MFI-type crystalline silicate having a silicon/aluminium atomic ratio of at least 180 and an MEL-type crystalline silicate having a silicon/aluminium atomic ratio of from 150 to 800 which has been subjected to a steaming step, at an inlet temperature of from 500 to 600° C., at an olefin partial pressure of from 0.1 to 2 bars and the feedstock being passed over the catalyst at an LHSV of from 5 to 30 h−1 to produce an effluent with an olefin content of lower molecular weight than that of the feedstock, intermittently removing a first fraction of the catalyst from the moving bed reactor, regenerating the first fraction of the catalyst in a regenerator and intermittently feeding into the moving bed reactor a second fraction of the catalyst which has been regenerated in the regenerator, the catalyst regeneration rate being controlled whereby all of the catalyst in the moving bed reactor is regenerated in a period of from 20 to 240 hours.", "Preferably, the regeneration rate is controlled whereby the propylene purity is maintained constant at a value corresponding to the average value obtained in a fixed bed reactor using the same feedstock, catalyst and cracking conditions, for example at least 94 wt %.", "More preferably, the regeneration rate is controlled whereby the ethylene yield on an olefin basis is less than 10 wt %.", "The present invention still further provides the use of catalyst regeneration of a moving bed reactor for the catalytic cracking of an olefin-containing feedstock which is selective towards lighter olefins, the catalyst regeneration being employed to average out propylene purity to higher values observed in a fixed bed reaction during an initial period, typically from 10 to 40 hours, of the olefin-cracking process.", "Preferably, the catalyst regeneration is also employed to average out the high ethylene yield during the initial period and the low ethylene yield during the final period observed in a fixed bed reactor.", "The feedstock having at least C4+ hydrocarbons may be an effluent from a fluidised bed catalytic cracking (FCC) unit in an oil refinery.", "The present invention provides a solution to the problem of loss of activity of the catalyst by the addition of the steps of removing deactivated catalyst from, and feeding reactivated catalyst into, the catalytic conversion zone which compensates for loss of activity without raising the reaction temperature, in particular, by using a moving bed reactor in which the catalyst circulates between a catalytic conversion zone and a catalyst regeneration zone.", "A moving bed reactor/regeneration combination still provides the possibility to operate the reaction section and regeneration section independently as they are physically isolated by means of lock hoppers and valves between the different sections.", "Each section can thus operate at its own optimal conditions and moreover the regeneration section can be temporarily shut down while the reaction section continues to operate.", "When employing a moving bed reactor in which intermittently catalyst is withdrawn and regenerated and consequently re-injected into the catalytic reaction zone, the catalytic performance of the catalyst in the catalytic reaction zone can be maintained constant.", "This will result in a constant product distribution over time.", "Moreover, the less desired product formation, observed at the start of the catalytic cycle in fixed bed reactors, can thus be moderated because the catalytic performance in a moving bed reactor is an average of the catalytic performance observed in fixed bed reactors.", "The present invention is predicated on the discovery by the inventor that in order to achieve a propylene purity i.e.", "a proportion of propylene in the total C3 content of the effluent, of at least 94 wt %, and preferably also to achieve an ethylene yield on an olefin basis below 10 wt %, then the use of a moving bed reactor with catalyst regeneration enables these average values to be achieved on a continuous basis, more particularly by regulating the catalyst regeneration according to the desired propylene purity, and optionally depending on the ethylene content, which is dependent upon the particular commercial requirements for the proportion of ethylene in the effluent, whereby the entire catalyst content of the moving bed reactor is regenerated in a period of from 20 to 240 hours.", "The particular period within which the entire body of catalyst in the moving bed reactor is regenerated depends on a number of factors, including the nature of the particular catalyst, temperature, LHSV, feedstock content, etc.", "Fundamentally, the catalyst regeneration is carried out so that the average values of propylene purity, and preferably also ethylene yield on an olefin basis, are such as to enable high purity propylene to be produced, with the averaging essentially overcoming the technical problem of low propylene purity and optionally high ethylene yield on an olefin basis during the initial period of a fixed bed reactor, typically up to the first 10 to 40 e.g.", "20 or 30 hours, of the olefin cracking process.", "This overcomes the technical problem present in the prior art, in particular in EP-A-0921179, of low propylene purity, and optionally also high ethylene yield on an olefin basis, reducing the ability of the catalyst to produce acceptable chemical grade purity propylene, and optionally low ethylene content, over acceptable run times.", "The preferred embodiment of the present invention can thus provide a process using a catalyst for the production of a catalytic reactor effluent characterised by a constant composition by utilising a moving bed reactor in which the catalyst circulates between a catalytic conversion zone and a catalyst regeneration zone.", "The preferred embodiments of the present invention can also provide a process using a catalyst whereby the formation of less desired products over fresh catalyst is tempered to an average acceptable level by utilising a moving bed reactor in which the catalyst circulates between a catalytic conversion zone and a catalyst regeneration zone.", "The present invention can thus provide a process wherein olefin-rich hydrocarbon streams (products) from refinery and petrochemical plants are selectively cracked not only into light olefins, but particularly into propylene.", "In one embodiment, the olefin-rich feedstock is passed over an MFI-type crystalline silicate catalyst with a particular Si/Al atomic ratio of either at least 180 attained after a steaming/de-alumination treatment or at least 300 with the catalyst having been prepared by crystallisation using an organic template and having been unsubjected to any subsequent steaming or de-alumination process.", "In another embodiment, the olefin-rich feedstock is passed over an MEL-type crystalline silicate catalyst, with a particular Si/Al atomic ratio and which has been steamed for example at a temperature of at least 300° C. for a period of at least 1 hour with a water partial pressure of at least 10 kPa.", "The feedstock may be passed over the catalyst at a temperature ranging between 500 to 600° C., an olefin partial pressure of from 0.1 to 2 bars and an LHSV of from 5 to 30 h−1.This can yield at least 30 to 50% propylene based on the olefin content in the feedstock, with a selectivity to propylene for the C3 species propylene and propane (i.e.", "a C3−/C3s ratio) of at least 92% by weight.", "In this specification, the term “silicon/aluminium atomic ratio” is intended to mean the Si/Al atomic ratio of the overall material, which may be determined by chemical analysis.", "In particular, for crystalline silicate materials, the stated Si/Al ratios apply not just to the Si/Al framework of the crystalline silicate but rather to the whole material.", "The feedstock may be fed either undiluted or diluted with an inert gas such as nitrogen.", "In the latter case, the absolute pressure of the feedstock constitutes the partial pressure of the hydrocarbon feedstock in the inert gas.", "In accordance with the present invention, cracking of olefins is performed in the sense that olefins in a hydrocarbon stream are cracked into lighter olefins and selectively into propylene.", "The feedstock and effluent preferably have substantially the same olefin content by weight.", "Typically, the olefin content of the effluent is within ±15 wt %, more preferably ±10 wt %, of the olefin content of the feedstock.", "The feedstock may comprise any kind of olefin-containing hydrocarbon stream.", "The feedstock may typically comprise from 10 to 100 wt % olefins and furthermore may be fed undiluted or diluted by a diluent, the diluent optionally including a non-olefinic hydrocarbon.", "In particular, the olefin-containing feedstock may be a hydrocarbon mixture containing normal and branched olefins in the carbon range C4 to C10, more preferably in the carbon range C4 to C6, optionally in a mixture with normal and branched paraffins and/or aromatics in the carbon range C4 to C10.Typically, the olefin-containing stream has a boiling point of from around −15 to around 180° C. In particularly preferred embodiments of the present invention, the hydrocarbon feedstocks comprise C4 mixtures from refineries and steam cracking units.", "Such steam cracking units crack a wide variety of feedstocks, including ethane, propane, butane, naphtha, gas oil, fuel oil, etc.", "Most particularly, the hydrocarbon feedstock may comprises a C4 cut from a fluidised-bed catalytic cracking (FCC) unit in a crude oil refinery which is employed for converting heavy oil into gasoline and lighter products.", "Typically, such a C4 cut from an FCC unit comprises around 50 wt % olefin.", "Alternatively, the hydrocarbon feedstock may comprise a C4 cut from a unit within a crude oil refinery for producing methyl tert-butyl ether (MTBE) which is prepared from methanol and isobutene.", "Again, such a C4 cut from the MTBE unit typically comprises around 50 wt % olefin.", "These C4 cuts are fractionated at the outlet of the respective FCC or MTBE unit.", "The hydrocarbon feedstock may yet further comprise a C4 cut from a naphtha steam-cracking unit of a petrochemical plant in which naphtha, comprising C5 to C9 species having a boiling point range of from about 15 to 180° C., is steam cracked to produce, inter alia, a C4 cut.", "Such a C4 cut typically comprises, by weight, 40 to 50% 1,3-butadiene, around 25% isobutylene, around 15% butene (in the form of but-1-ene and/or but-2-ene) and around 10% n-butane and/or isobutane.", "The olefin-containing hydrocarbon feedstock may also comprise a C4 cut from a steam cracking unit after butadiene extraction (Raffinate 1), or after butadiene hydrogenation.", "In accordance with the present invention, the catalyst for the cracking of the olefins comprises a crystalline silicate of the MFI family which may be a zeolite, a silicalite or any other silicate in that family or the MEL family which may be a zeolite or any other silicate in that family.", "Examples of MFI silicates are ZSM-5 and silicalite.", "An example of an MEL zeolite is ZSM-11 which is known in the art.", "Other examples are Boralite D, and silicalite-2 as described by the International Zeolite Association (Atlas of zeolite structure types, 1987, Butterworths).", "The preferred crystalline silicates have pores or channels defined by ten oxygen rings and a high silicon/aluminium atomic ratio.", "Crystalline silicates are microporous crystalline inorganic polymers based on a framework of XO4 tetrahydra linked to each other by sharing of oxygen ions, where X may be trivalent (e.g.", "Al, B, .", ".", ". )", "or tetravalent (e.g.", "Ge, Si, .", ".", ".", ").", "The crystal structure of a crystalline silicate is defined by the specific order in which a network of tetrahedral units are linked together.", "The size of the crystalline silicate pore openings is determined by the number of tetrahedral units, or, alternatively, oxygen atoms, required to form the pores and the nature of the cations that are present in the pores.", "They possess a unique combination of the following properties: high internal surface area; uniform pores with one or more discrete sizes; ion exchangeability; good thermal stability; and ability to adsorb organic compounds.", "Since the pores of these crystalline silicates are similar in size to many organic molecules of practical interest, they control the ingress and egress of reactants and products, resulting in particular selectivity in catalytic reactions.", "Crystalline silicates with the MFI structure possess a bi-directional intersecting pore system with the following pore diameters: a straight channel along [10]: 0.53-0.56 nm and a sinusoidal channel along [100]: 0.51-0.55nm.", "Crystalline silicates with the MEL structure possess a bi-directional intersecting straight pore system with straight channels along [100] having pore diameters of 0.53-0.54 nm.", "The crystalline silicate catalyst has structural and chemical properties and is employed under particular reaction conditions whereby the catalytic cracking readily proceeds.", "Different reaction pathways can occur on the catalyst.", "Under the process conditions, having an inlet temperature of around 500 to 600° C., preferably from 520 to 600° C., yet more preferably 540 to 580° C., and an olefin partial pressure of from 0.1 to 2 bars, most preferably around atmospheric pressure, the shift of the double bond of an olefin in the feedstock is readily achieved, leading to double bond isomerisation.", "Furthermore, such isomerisation tends to reach a thermodynamic equilibrium.", "Propylene can be, for example, directly produced by the catalytic cracking of hexene or a heavier olefinic feedstock.", "Olefinic catalytic cracking may be understood to comprise a process yielding shorter molecules via bond breakage.", "With such high silicon/aluminum ratio in the crystalline silicate catalyst, a stable olefin conversion can be achieved with a high propylene yield on an olefin basis of from 30 to 50% whatever the origin and composition of the olefinic feedstock.", "Such high ratios reduce the acidity of the catalyst, thereby increasing the stability of the catalyst.", "The MFI catalyst having a high silicon/aluminum atomic ratio for use in the catalytic cracking process of the present invention may be manufactured by removing aluminum from a commercially available crystalline silicate.", "A typical commercially available silicalite has a silicon/aluminum atomic ratio of around 120.The commercially available MFI crystalline silicate may be modified by a steaming process which reduces the tetrahedral aluminum in the crystalline silicate framework and converts the aluminum atoms into octahedral aluminum in the form of amorphous alumina.", "Although in the steaming step aluminum atoms are chemically removed from the crystalline silicate framework structure to form alumina particles, those particles cause partial obstruction of the pores or channels in the framework.", "This inhibits the olefinic cracking processes of the present invention.", "Accordingly, following the steaming step, the crystalline silicate is subjected to an extraction step wherein amorphous alumina is removed from the pores and the micropore volume is, at least partially, recovered.", "The physical removal, by a leaching step, of the amorphous alumina from the pores by the formation of a water-soluble aluminum complex yields the overall effect of de-alumination of the MFI crystalline silicate.", "In this way by removing aluminum from the MFI crystalline silicate framework and then removing alumina formed therefrom from the pores, the process aims at achieving a substantially homogeneous de-alumination throughout the whole pore surfaces of the catalyst.", "This reduces the acidity of the catalyst, and thereby reduces the occurrence of hydrogen transfer reactions in the cracking process.", "The reduction of acidity ideally occurs substantially homogeneously throughout the pores defined in the crystalline silicate framework.", "This is because in the olefin-cracking process hydrocarbon species can enter deeply into the pores.", "Accordingly, the reduction of acidity and thus the reduction in hydrogen transfer reactions which would reduce the stability of the MFI catalyst are pursued throughout the whole pore structure in the framework.", "The framework silicon/aluminum ratio may be increased by this process to a value of at least about 180, preferably from about 180 to 1000, more preferably at least 200, yet more preferably at least 300, and most preferably around 480.The MEL or MFI crystalline silicate catalyst may be mixed with a binder, preferably an inorganic binder, and shaped to a desired shape, e.g.", "extruded pellets.", "The binder is selected so as to be resistant to the temperature and other conditions employed in the catalyst manufacturing process and in the subsequent catalytic cracking process for the olefins.", "The binder is an inorganic material selected from clays, silica, metal oxides such as ZrO2 and/or metals, or gels including mixtures of silica and metal oxides.", "The binder is preferably alumina-free.", "Although aluminium in certain chemical compounds as in AlPO4's may be used as the latter are quite inert and not acidic in nature.", "If the binder which is used in conjunction with the crystalline silicate is itself catalytically active, this may alter the conversion and/or the selectivity of the catalyst.", "Inactive materials for the binder may suitably serve as diluents to control the amount of conversion so that products can be obtained economically and orderly without employing other means for controlling the reaction rate.", "It is desirable to provide a catalyst having a good crush strength.", "This is because in commercial use, it is desirable to prevent the catalyst from breaking down into powder-like materials.", "Such clay or oxide binders have been employed normally only for the purpose of improving the crush strength of the catalyst.", "A particularly preferred binder for the catalyst of the present invention comprises silica.", "The relative proportions of the finely divided crystalline silicate material and the inorganic oxide matrix of the binder can vary widely.", "Typically, the binder content ranges from 5 to 95% by weight, more typically from 20 to 50% by weight, based on the weight of the composite catalyst.", "Such a mixture of crystalline silicate and an inorganic oxide binder is referred to as a formulated crystalline silicate.", "In mixing the catalyst with a binder, the catalyst may be formulated into pellets, spheres, extruded into other shapes, or formed into a spray-dried powder.", "For practising the present invention it is preferred that the formulated catalyst has a very symmetrical shape like in spheres and pellets or extrudates having equal height and wideness.", "It is important that the settling velocity of the catalyst particles in a gas stream is the same for all orientations relative to the gas stream direction.", "In the catalytic cracking process, the process conditions are selected in order to provide high selectivity towards propylene, a stable olefin conversion over time, and a stable olefinic product distribution in the effluent.", "Such objectives are favoured by the use of a low acid density in the catalyst (i.e.", "a high Si/Al atomic ratio) in conjunction with a low pressure, a high inlet temperature and a short contact time, all of which process parameters are interrelated and provide an overall cumulative effect (e.g.", "a higher pressure may be offset or compensated by a yet higher inlet temperature).", "The process conditions are selected to disfavour hydrogen transfer reactions leading to the formation of paraffins, aromatics and coke precursors.", "The process operating conditions thus employ a high space velocity, a low pressure and a high reaction temperature.", "The LHSV ranges from 5 to 30 h−1, preferably from 10 to 30 h−1.The olefin partial pressure ranges from 0.1 to 2 bars, preferably from 0.5 to 1.5 bars.", "A particularly preferred olefin partial pressure is atmospheric pressure (i.e.", "1 bar).", "The hydrocarbon feedstocks are preferably fed at a total inlet pressure sufficient to convey the feedstocks through the reactor.", "The hydrocarbon feedstocks may be fed undiluted or diluted in an inert gas, e.g.", "nitrogen.", "Preferably, the total absolute pressure in the reactor ranges from 0.5 to 10 bars.", "The use of a low olefin partial pressure, for example atmospheric pressure, tends to lower the incidence of hydrogen transfer reactions in the cracking process, which in turn reduces the potential for coke formation which tends to reduce catalyst stability.", "The cracking of the olefins is preferably performed at an inlet temperature of the feedstock of from 500 to 600° C., more preferably from 520 to 600° C., yet more preferably from 540 to 590° C., typically around 560° C. to 585° C. Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: FIG.", "1 is a schematic process scheme in accordance with one embodiment of the present invention for processing refinery and/or petrochemical feedstocks, the process scheme incorporating a process for selectively catalytically cracking olefins into lighter olefins over a crystalline silicate catalyst, and incorporating catalyst regeneration; FIG.", "2 shows a schematic process scheme in accordance with a second embodiment of the present invention for processing refinery and/or petrochemical feedstocks, the process scheme incorporating a process for selectively catalytic cracking olefins into lighter olefins over a crystalline silicate catalyst and catalyst regeneration; FIG.", "3 shows a schematic process scheme in accordance with a third embodiment of the present invention for processing refinery and/or petrochemical feedstocks, the process scheme incorporating a process for selectively catalytically cracking olefins into lighter olefins over a crystalline silicate catalyst and catalyst regeneration; FIG.", "4 shows the relationship between the olefin content of an effluent and time for one example of a catalytic cracking process; and FIG.", "5 shows the relationship between olefin content and time for a second example of a catalytic cracking process.", "FIG.", "1 provides a schematic illustration of a configuration for practising the process of the present invention.", "The description is not intended to exclude certain modifications and in order to simplify the drawing shut-off valves, solid flow controlling valves, pumps, piping and other conventional equipment readily known by the person skilled in the art are not shown.", "The fresh olefin-containing feed to be catalytically cracked and preferably combined with recycle feed, and optionally a diluting gas like hydrogen, steam or any other inert gas, are sent through line 1 to a feed-effluent heat exchanger 2 and further through line 3 to a heater 4 to raise the temperature of the mixture to the desired reaction temperature.", "Through line 5 the hot mixture is sent into a radial-flow reactor 10.The reactor 10 contains an annulus of dense phase catalyst.", "The feed mixture may be injected in the centre of the annulus and may leave the catalyst external to the catalyst bed annulus.", "Optionally, the feed mixture may be injected in the catalyst bed external to the bed annulus and may leave the catalyst bed annulus in the centre of the annulus.", "The reaction products leave the reaction section through line 19 via the feed-effluent heat exchanger 2 to the fractionation section (not shown).", "In the fractionation section the different reaction products are concentrated.", "Unconverted feed or a produced butene-rich C4 fraction may be recycled together with fresh feed to the reaction section through line 1.In accordance with the catalyst regeneration in the moving bed reactor in the present invention, the catalyst travels down under gravity through the catalyst bed annulus and is continuously or intermittently withdrawn through line 20 into a lock hopper 21 where the catalyst is purged with nitrogen in order to remove hydrocarbon vapours from the catalyst.", "In the lock hopper the pressure is equalised to that of a lift engager 22.The catalyst is lifted from the lift engager 22 by means of a lift gas coming through line 23 to a lift dis-engager 30 through a catalyst lift line 24.The gaseous lift gas may be hydrogen, nitrogen, methane, steam or even diluted oxygen in nitrogen.", "The flow rate of the lift gas is sufficient to surpass the settling velocity of the catalyst particles in order to transfer the catalyst through the lift line 24 to a lift dis-engager 30.In the lift dis-engager 30, the catalyst is separated from the lift gases through line 31 and the pressure is equalised to the pressure of a catalyst regeneration vessel 40.The lift gases may be recycled or sent to other purposes.", "The catalyst is fed from the lift dis-engager 30 through line 32 to the regeneration vessel 40.In the regeneration vessel 40 the carbonaceous material laid down on the catalyst is burned off by means of oxygen, to form carbon dioxide.", "The regeneration vessel 40 may consist of a cylindrical moving bed of catalyst travelling down by gravity.", "Alternatively, it may also consist of a radial-flow type catalyst bed.", "The oxidising gases are injected in the centre of the catalyst bed annulus or from the exterior of the annulus.", "Fresh air is provided through line 41, mixed with recycle gas coming through line 48 and compressed by means of a compressor 42 into line 43.The oxygen containing mixture goes from line 43 into the regeneration vessel 40.The combustion gases leave the regeneration vessel through line 44 and goes to a vessel 45.The combustion gases are cooled down or heat exchanged and eventually dried.", "Water is drained off through line 46.Uncondensed gases are partially purged out through line 47, and the remaining may be recycled and mixed with fresh air through line 41.To control the combustion of the carbonaceous material on the catalyst the oxygen should be present at relatively low concentrations.", "The ratio of recycle gas to fresh air is generally high.", "The volume percent of oxygen in the oxidising gas is typically from 0.2 to 2, preferably about 0.6.Other compounds may be present in the oxidising gas, such as carbon dioxide, nitrogen and optionally carbon monoxide.", "During the regeneration the catalyst travels down under gravity and the carbonaceous material is progressively burned off.", "It may be desirable to use higher concentrations of oxygen towards the end of the regeneration vessel 40.A second inlet of oxygen containing gas may be injected into the regeneration vessel 40 more to the lower parts of the catalyst bed where carbonaceous material is already burned off to a great extent.", "As is known, regeneration with oxygen is exothermic and care should be taken not to exceed the temperature at which the catalyst is damaged.", "It is preferred not to surpass 600° C. in the catalyst bed.", "The regeneration is generally started at about 450° C. Therefore the oxygen containing gas may be heated up before entering the regeneration vessel 40.The second oxygen containing stream which may be injected into the regeneration vessel may be heated up to a higher temperature to finish better the burn off of carbonaceous materials laid down on the catalyst.", "The value percent of oxygen in the second oxygen-containing stream is typically from 2 to 100, preferably from 5 to 21.Other compounds may be present in the oxidising gas, such as carbon dioxide, nitrogen and optionally carbon monoxide.", "The catalyst flows through line 50 to a lock hopper 51.Optionally, the regeneration may be finished here by purging first the hopper 51 with pure air at the highest allowable temperature for the catalyst, followed by a nitrogen purge in order to remove any remaining oxygen.", "The catalyst further flows through line 52 to a lift engager 53.By means of a lift gas, coming through line 54, the catalyst is sent to a catalyst collector hopper 61 located above the reactor 10 through a catalyst transfer line 60.The catalyst is separated from the lift gases through line 62.These lift gases may be sent to other purposes or may be recycled and used again as lift gas.", "The pressure in the catalyst collector hopper 61 is equalised to the reactor pressure.", "The regenerated catalyst in the collector hopper 61 flows through line 63 into the reactor vessel 10.New fresh catalyst may be added into the catalyst collector hopper 61 through line 64, while used catalyst can be withdrawn from the regeneration system through line 65.FIG.", "2 shows an alternative embodiment for practising the present invention.", "As the cracking of long-chain olefins into lighter olefins is an endothermic reaction, it may be desired to reheat the reaction mixture.", "FIG.", "2 shows the alternative embodiment with two moving bed reactors 10, 15 in series for the olefin-cracking process.", "The reactor effluent of the first radial-flow reactor 10 leaves the reactor through line 11 and is sent to a reheater 12.The mixture is sent through line 13 into the second reactor 15.The second reactor 15 can be located below the first rector 10 as illustrated or optionally the second reactor 15 is parallel to the first reactor 10.In the latter case, there is provided a catalyst lift transfer line (not shown) between the first and the second reactors 10, 15.The rest of the process scheme is as explained above for FIG.", "1.As the catalyst becomes less active when moving down through the moving bed, it may be desired to increase the contact time of the reaction mixture with the catalyst in the second reactor.", "This can easily be done by increasing the thickness of the catalyst bed annulus.", "A still other embodiment for practising the present invention is shown in FIG.", "3.As the reactors are not very large, it can be advantageous to place the regeneration vessel 40 on top of the first reactor 10 (or the single reactor 10 as shown in FIG.", "1).", "This implies one fewer catalyst transfer line which will reduce the attrition of the catalyst due to the transport step.", "The present invention will now be described with reference to the following non-limiting examples.", "EXAMPLE 1 A feedstock having the feed composition shown in Table 1, consisting of a 50/50 wt % mixture of C4s and LCCS produced on an FCC unit was subjected to olefin catalytic cracking in a fixed bed reactor (not in accordance with the invention) comprising a crystalline silicate catalyst of the MFI-type (as generally disclosed in EP-A-0921179) having a silicon/aluminium atomic ratio of at least 270 at an inlet temperature of 585° C., a liquid hourly space velocity (LHSV) of 20 h−1 and an outlet pressure of 0.5 bara.", "The composition of the effluent over time was measured to determine the propylene (C3—) content, the ethylene (C2—) content, the isobutene (i-C4-) content and the propylene purity and the results are shown in FIG.", "4.The reactor is loaded with 5 litres of catalyst and the reactor operates in an adiabatical mode.", "From FIG.", "4 it may be seen that the propylene content, i.e.", "the yield on an olefin basis towards propylene of the olefin-cracking process, is initially slightly greater than or about 35 wt % up to a period of around 35 hours, after which the propylene content rapidly decreases to a value of as low as about 18 wt % after a period of about 75 hours.", "This shows that the activity of the catalyst towards the production of propylene in the olefin-cracking process reduces over time, specifically for runs greater than around 35 hours.", "In addition, for shorter reaction times on stream, there are problems in that the ethylene content on an olefin basis of the effluent is initially high, starting from greater than 10 wt % and being greater than 5 wt % up to 40 hours on stream, and also the propylene purity (i.e.", "the ratio of propylene to total C3 content) is initially low and increases to a value greater than 94 wt % only after a period of around 10 hours on stream.", "Table 2 shows values of the propylene content, ethylene content, isobutene content and propylene purity after 4 specific times on stream, up to about 35 hours on stream during which the propylene yield is quite constant.", "In accordance with the process of the present invention, by providing a moving bed reactor with continuous catalyst regeneration, the four discrete yields in the effluent are substantially averaged to yield the average values also specified in Table 2.It may thus be seen that by using a moving bed reactor in conjunction with continuous catalyst regeneration, the composition of the effluent may be made more constant, in particular the propylene content and purity.", "Moreover, the formation of less desired products in the effluent, such as ethylene, which requires a relatively difficult fractionation process to be separated from the desired propylene, reduced continuously to an average acceptable level as compared to the initial level in the case of a fixed bed.", "EXAMPLE 2 In accordance with this Example, the same feed having a typical composition illustrated in Table 1 was fed over the same catalyst as in Example 1 and at the same inlet temperature and outlet pressure, but at a lower LHSV of 10 h−1.The relationship between the olefin content and time on stream is illustrated in FIG.", "5.Table 3 shows the variation between the propylene, ethylene and isobutene contents with time, together with the propylene purity variation with time.", "As for Example 1, for Example 2 it may be seen that the use of a moving bed reactor together with catalyst regeneration provides a substantially average value for the composition of the effluent which tends to provide an improved average value for the ethylene content and an improved average value for the propylene purity.", "TABLE 1 FEED COMPOSITION [WT %] C nr F O N A Total Cumulative 1 0.00 2 0.00 0.00 3 0.02 0.28 0.31 0.31 4 17.40 25.74 43.14 43.45 5 6.03 6.90 0.09 13.03 56.48 6 8.84 5.54 1.58 0.52 16.48 72.96 7 3.98 3.13 2.39 3.00 12.49 85.45 8 3.23 1.12 2.06 3.88 10.28 95.74 9 1.49 0.26 0.11 1.86 3.72 99.46 10 0.11 0.00 0.00 0.40 0.51 99.97 11 0.03 0.03 100.00 Total 41.10 42.97 6.23 9.70 100.00 TABLE 2 EXAMPLE 1 TOS [h] 4.17 19.48 34.57 AVERAGE C3-[wt %] 36.27 34.95 35.14 35.45 C2-[wt %] 10.03 7.26 6.35 7.88 i-C4-[wt %] 13.56 18.48 22.66 18.23 c3-purity [wt %] 93.44 94.94 95.45 94.61 TABLE 3 EXAMPLE 2 TOS[h] 2.47 4.92 10.18 17.83 28.37 41.10 AVERAGE C3-[wt %] 32.93 33.71 33.61 33.49 35.53 34.09 33.89 C2-[wt %] 10.59 10.71 9.93 9.11 9.03 7.54 9.48 i-C4-[wt %] 11.51 11.96 12.31 12.93 14.34 15.47 13.09 c3-pur- 88.38 90.42 91.79 93.08 93.81 94.97 92.08 ity[wt %]" ] ]

[ [ "Techniques for hiding network element names and addresses", "A technique for hiding network element names and addresses in communications between first and second networks includes providing a message generated by a network entity in the first network to be delivered to a target network entity in the second network, the messaging including first and second parts.", "The message generated by the network entity in the first network is routed to a contact point disposed between the first and second networks in accordance with the first part of the message and the message generated by the network entity in the first network is routed from the contact point to the target network entity in the second network in accordance with the second part of the message." ], [ "1.A method of hiding at least one of network element names and addresses in communications between first and second networks, the method comprising: providing a message generated by a network entity in the first network to be delivered to a target network entity in the second network, the message comprising first and second parts; routing the message generated by the network entity in the first network to a contact point disposed between the first and second networks in accordance with the first part of the message; and routing the message generated by the network entity in the first network from the contact point to the target network entity in the second network in accordance with the second part of the message.", "2.The method of claim 1, wherein the first part of the message comprises a name resolvable external to the second network to a first address of the contact point and wherein the second part of the message comprises the name resolvable only within the second network to a second address of the target network entity.", "3.The method of claim 2, further comprising providing a DNS (Domain Name System) for resolving a name to an address.", "4.The method of claim 2, further comprising providing a public DNS infrastructure for resolving a name to an address of the contact point.", "5.The method of claim 2, further comprising providing a one of a dedicated or internal DNS infrastructure for resolving a name to an address of a network entity in the second network.", "6.The method of claim 1, wherein the first part of the message comprises a first name usable for routing external to the second network and wherein the second part of the message comprises a second name usable for routing only within the second network.", "7.The method of claim 6, wherein the second name is encrypted.", "8.The method of claim 1, wherein the first part of the message comprises a name usable for routing unmodified external to the second network and wherein the second part of the message comprises a modified version of the name usable for routing only within the second network.", "9.The method of claim 8, wherein the name is encrypted.", "10.The method of claim 1, wherein the first part of the message comprises a modified version of a name usable for routing external to the second network and wherein the second part of the message comprises the name usable for routing unmodified only within the second network.", "11.The method of claim 10, wherein the name is encrypted.", "12.The method of claim 1, wherein the first part of the message comprises a pair of one of either names or addresses, a first member of the pair being usable for routing external to the second network and wherein the second part of the message comprises the pair of one of either names or addresses with a second a member of the pair being encrypted and usable for routing only within the second network.", "13.The method of claim 1, wherein the first part of the message comprises a name that is encrypted and resolvable external to the second network to a first address of the contact point and wherein the second part of the message comprises the encrypted name resolvable only within the second network to a second address of the target network entity.", "14.The method of claim 13, wherein the name is decoded before usage in the second network.", "15.The method of claim 1, wherein the contact point comprises one of an I-CSCF (interrogating Call State Control Function) or a P-CSCF (Proxy-Call State Control Function) or a BGCF (Breakout Gateway Control Function).", "16.The method of any one of the previous claims, wherein the name comprises a logical name and the address comprises an IP address.", "17.The method of any one of the previous claims, wherein the name comprises a hostname and/or FQDN (Fully Qualified Domain Name).", "18.The method of claim 1, further comprising providing IMS (IP Multimedia Core Network Subsystem) networks as the first and second networks.", "19.A communication system comprising: a first network including a network entity disposed therein; a second network including a target network entity disposed therein; and a contact point disposed between said first and second networks; wherein, upon said network entity generating a message comprising first and second parts to be delivered to said target network entity, said message is routed to said contact point in accordance with said first part of said message and then routed from said contact point to said target network entity in accordance with said second part of said message.", "20.The system of claim 19, wherein said first part of said message comprises a name resolvable external to the second network to an address of said contact point and wherein said second part of said message comprises said name resolvable only within said second network to an address of said target network entity.", "21.A contact point apparatus in a system including a first network having a network entity disposed therein and a second network including a target network entity disposed therein, the network entity in the first network generating a message having first and second parts to the target network entity in the second network, the contact point being disposed between said first and second networks and comprising: a means for receiving the message generated by the network entity in the first network the message being routed to the contact point in accordance with the first part of the message; and a means for routing the message generated by the network entity in the first network to the target network entity in the second network in accordance with the second part of the message.", "22.The contact point apparatus of claim 21, wherein the first part of the message comprises a name resolvable external to the second network to an address of the contact point and wherein the second part of the message comprises the name resolvable only within the second network to an address of the target network entity.", "23.The contact point apparatus of claim 21, wherein the contact point comprises one of an I-CSCF (Interrogating Call State Control Function) or a P-CSCF (Proxy-all State Control Function) or a BGCF (Breakout Gateway Control Function)." ], [ "<SOH> BACKGROUND ART <EOH>When a subscriber is registered at a foreign network, such as an IP (Internet Protocol) multimedia network, the home HSS (Home Subscriber Server) normally knows the address of the S-CSCF (Serving Call State Control Function) where the subscriber is registered Since it is desirable to hide the networks, except for the contact points which are usually I-CSCFs (Interrogating Call State Control Functions), the foreign network cannot give the name and/or address of the S-CSCF to the home HSS.", "One proposed solution is that the HSS query should also be used in the visited network to locate the S-CSCF while another proposed solution is that the association between the subscriber and the name and/or address of the S-CSCF should be found from the I-CSCF.", "Locating the associations in the I-CSCF would require a new functionality in the I-CSCF.", "If the HSS contains the associations, it would result in the records of foreign subscribers being stored in the HSS.", "This would disturb the structure of the HSS in that it was designed to only store the records of its own subscribers.", "In addition, the HSS must also include the address of the APSE (Application Server).", "If the home operator does not want the address of the APSE available to other operators, a mechanism is needed to refer to the APSE.", "Furthermore, in the visited network model, as noted above, the name and/or IP address of the S-CSCF are revealed during the registration to the home HSS when the S-CSCF requests the profile of the roaming subscriber from the home HSS of the subscriber.", "In addition, the IP address of the S-CSCF is also revealed in the originating and terminating call cases to/from new operators because the name, i.e.", "the FQDN (Fully Qualified Domain Name) of the S-CSCF has to be publicly resolvable.", "In the home network model, the name and the IP address of the P-CSCF (Proxy CSCF, that is, the initial proxy in the home model) are revealed to the I-CSCF of the home operator during the registration when the P-CSCF sends a REGISTER message to the home I-CSCF of the roaming subscriber.", "The name and the IP address of the S-CSCF in the home network are revealed in originating and, depending on routing, possibly also in terminating call cases to new operators.", "Lastly, in 3GPP IP multimedia network, there is an actual requirement to hide the internal structure of the network with respect to other networks.", "This implies that the names and the IP addresses of network elements, such as the S-CSCF shall not be made known to other networks.", "It has been proposed that the HSS in a visited network behave as a VLS (Visited Location Server) to handle (that is, to select at registration and store for MT call routing) the identity of the S-CSCF in the visited network in order to mask the identity of the S-CSCFv (Serving CSCF in the visited network) to the home network.", "The VLS will be interrogated by the I-CSCFv (Interrogating CSCF in the visited network) when an MT (mobile terminating) call is routed to it by the-home network.", "However, in such a solution, the visited network must maintain a relationship through some mechanism between the identity of the roaming subscriber and the HSSv (HSS in the visited network) that stores the identity of the S-CSCF.", "In addition, the relationship must be available to all I-CSCFv's since the I-CSCFv that receives the MT call routed from the home network cannot be decided nor predicted in advance." ], [ "<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>The foregoing and a better understanding of the present invention will become apparent from the following detailed description of example embodiments and the claims when read in connection with the accompanying drawings, all forming a part of the disclosure of this invention.", "While the foregoing and following written and illustrated disclosure focuses on disclosing example embodiments of the invention, it should be clearly understood that the same as by way of illustration and example only and that the invention is not limited thereto.", "This spirit and scope of the present invention are limited only by the terms of the appended claims.", "The following represents brief descriptions of the drawings, wherein: FIG.", "1 illustrates a salient feature of the present invention.", "FIG.", "2 illustrates a mobile terminal call setup example using the visited network model where the visited network is hidden utilizing the two names for routing with encryption mechanism.", "FIG.", "3 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the two names for routing with encryption mechanism.", "FIG.", "4 illustrates the registration to a home network by a mobile terminal when the S-CSCFh is in the home network, i.e.", "the home network model is used, and the home network is hidden utilizing the double semantics mechanism.", "FIG.", "5 illustrates a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "FIG.", "6 illustrates messages following the first Invite in a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "FIG.", "7 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "FIG.", "8 illustrates a mobile terminated call setup example using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "FIG.", "9 illustrates the main characteristics of address hiding alternatives in accordance with embodiments of the present invention.", "FIG.", "10 illustrates the main characteristics of name hiding alternatives in accordance with embodiments of the present invention.", "detailed-description description=\"Detailed Description\" end=\"lead\"?" ], [ "TECHNICAL FIELD The present invention relates to hiding the configuration of a network with hiding names and/or addresses of network elements in communications between networks.", "More particularly, the present invention relates to hiding names and IP addresses in 3GPP (Third Generation Partnership Program) IP multimedia networks and UMTS (Universal Mobile Telecommunications Systems) networks.", "The terminology in this application is changed compared to the Provisional Application to be more consistent with the 3GPP standardization without changing the scope of the invention.", "The main differences in the words used of the same concepts in this application and in the Provisional Application are presented in the following three column table: Word(s) used Word used of of the same the concept concept in the in this Provisional application: Application: Definition: “name” “address” or input of the name to “logical address” address resolution “address” “IP address” result of the name to address resolution DNS (Domain Name System) may be used as name to address resolution mechanism.", "Also the names of the alternative solutions or embodiments of the invention are changed in this application compared to the Provisional Application according to the following two column table without changing the scope of the invention.", "In the Provisional Application the solutions were also enumerated as alternatives.", "The alternative numbers are marked in the table with abbreviation “ALT”.", "Name in the slide set of Name in this application: Provisional Application: Double semantics Double semantics (ALT2) (not changed) Two names for routing Two addresses for routing (ALT3) One name for two routings One address—two routings (ALT6) Partly encrypted name address pair Partly encrypted address pair (ALT7) Double semantics with encryption Double semantics with encryption (ALT8) (not changed) Two names for routing with encryption Two addresses for routing with encryption (ALT9) One name for two routings with encryption One address—two routings with encryption (ALT10) BACKGROUND ART When a subscriber is registered at a foreign network, such as an IP (Internet Protocol) multimedia network, the home HSS (Home Subscriber Server) normally knows the address of the S-CSCF (Serving Call State Control Function) where the subscriber is registered Since it is desirable to hide the networks, except for the contact points which are usually I-CSCFs (Interrogating Call State Control Functions), the foreign network cannot give the name and/or address of the S-CSCF to the home HSS.", "One proposed solution is that the HSS query should also be used in the visited network to locate the S-CSCF while another proposed solution is that the association between the subscriber and the name and/or address of the S-CSCF should be found from the I-CSCF.", "Locating the associations in the I-CSCF would require a new functionality in the I-CSCF.", "If the HSS contains the associations, it would result in the records of foreign subscribers being stored in the HSS.", "This would disturb the structure of the HSS in that it was designed to only store the records of its own subscribers.", "In addition, the HSS must also include the address of the APSE (Application Server).", "If the home operator does not want the address of the APSE available to other operators, a mechanism is needed to refer to the APSE.", "Furthermore, in the visited network model, as noted above, the name and/or IP address of the S-CSCF are revealed during the registration to the home HSS when the S-CSCF requests the profile of the roaming subscriber from the home HSS of the subscriber.", "In addition, the IP address of the S-CSCF is also revealed in the originating and terminating call cases to/from new operators because the name, i.e.", "the FQDN (Fully Qualified Domain Name) of the S-CSCF has to be publicly resolvable.", "In the home network model, the name and the IP address of the P-CSCF (Proxy CSCF, that is, the initial proxy in the home model) are revealed to the I-CSCF of the home operator during the registration when the P-CSCF sends a REGISTER message to the home I-CSCF of the roaming subscriber.", "The name and the IP address of the S-CSCF in the home network are revealed in originating and, depending on routing, possibly also in terminating call cases to new operators.", "Lastly, in 3GPP IP multimedia network, there is an actual requirement to hide the internal structure of the network with respect to other networks.", "This implies that the names and the IP addresses of network elements, such as the S-CSCF shall not be made known to other networks.", "It has been proposed that the HSS in a visited network behave as a VLS (Visited Location Server) to handle (that is, to select at registration and store for MT call routing) the identity of the S-CSCF in the visited network in order to mask the identity of the S-CSCFv (Serving CSCF in the visited network) to the home network.", "The VLS will be interrogated by the I-CSCFv (Interrogating CSCF in the visited network) when an MT (mobile terminating) call is routed to it by the-home network.", "However, in such a solution, the visited network must maintain a relationship through some mechanism between the identity of the roaming subscriber and the HSSv (HSS in the visited network) that stores the identity of the S-CSCF.", "In addition, the relationship must be available to all I-CSCFv's since the I-CSCFv that receives the MT call routed from the home network cannot be decided nor predicted in advance.", "DISCLOSURE OF INVENTION In the present invention, an indirect reference addressing mechanism is used to hide the names and the IP addresses in both the visited network model and in the home network model both with the S-CSCF in the home network and with the S-CSCF in the visited network.", "In the present invention, the contact points between networks, e.g.", "I-CSCFs, are not hidden.", "The IP addresses as well as the names e.g.", "FQDNs of all of the other network elements remain hidden.", "The present invention provides a solution to the problem of how to find the S-CSCF so that the address of the S-CSCF is not revealed to other network operators.", "Furthermore, the present invention allows the hiding of the names and addresses of the HSSs and P-CSCFs.", "One solution in accordance with the present invention is to identify the S-CSCF with an address pair in which the first part is the address of the I-CSCF and the second part is the encrypted address of the S-CSCF itself.", "In accordance with the present invention, the address of the S-CSCF is hidden from other networks by the adoption of a name assigned to the S-CSCF and translatable by the DNS (Domain Name Service) to the address of the S-CSCF only when the DNS query is performed by a network element belonging to the same network as S-CSCF.", "Accordingly, an object of the present invention is to provide a method of hiding at least one of network element names and addresses in communications between fist and second networks, the method including: providing a message generated by a network entity in the first network to be delivered to a target network entity in the second network, the message comprising first and second parts; routing the message generated by the network entity in the first network to a contact point disposed between the first and second networks in accordance with the first part of the message; and routing the message generated by the network entity in the first network from the contact point to the target network entity in the second network in accordance with the second part of the message.", "Another object of the present invention is to provide a communication system including: a first network including a network entity disposed therein; a second network including a target network entity disposed therein; and a contact point disposed between said first and second networks; wherein, upon said network entity generating a message comprising first and second parts to be delivered to said target network entity, said message is routed to said contact point in accordance with said first part of said message and then routed from said contact point to said target network entity in accordance with said second part of said message.", "Furthermore, still another object of the invention is to provide a contact point apparatus in a system including a first network having a network entity disposed therein and a second network including a target network entity disposed therein, the network entity in the first network generating a message having first and second parts to the target network entity in the second network, the contact point being disposed between said first and second networks and including: a means for receiving the message generated by the network entity in the first network, the message being routed to the contact point in accordance with the first part of the message; and a means for routing the message generated by the network entity in the first network to the target network entity in the second network in accordance with the second part of the message.", "A contact point of a network is a specialized network entity or an ordinary network entity with the functionality needed to route further the incoming messages from outside the network to the target network entities, and to route further the outgoing messages from the network entities keeping hidden the names and/or addresses of the network entities.", "Thus the contact point does not need to be a complete network element.", "The contact point may comprise e.g.", "one of an I-CSCF (Interrogating Call State Control Function) or a P-CSCF (Proxy-Call State Control Function) or a BGCF (Breakout Gateway Control Function) or be a functionality in these network elements.", "A hidden network is a network where names and/or addresses are not available and/or not usable, e.g.", "for routing, outside the network with exception of the names and/or addresses of the so called contact point(s) that is/are the only access point(s) to the network from outside of the network BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and a better understanding of the present invention will become apparent from the following detailed description of example embodiments and the claims when read in connection with the accompanying drawings, all forming a part of the disclosure of this invention.", "While the foregoing and following written and illustrated disclosure focuses on disclosing example embodiments of the invention, it should be clearly understood that the same as by way of illustration and example only and that the invention is not limited thereto.", "This spirit and scope of the present invention are limited only by the terms of the appended claims.", "The following represents brief descriptions of the drawings, wherein: FIG.", "1 illustrates a salient feature of the present invention.", "FIG.", "2 illustrates a mobile terminal call setup example using the visited network model where the visited network is hidden utilizing the two names for routing with encryption mechanism.", "FIG.", "3 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the two names for routing with encryption mechanism.", "FIG.", "4 illustrates the registration to a home network by a mobile terminal when the S-CSCFh is in the home network, i.e.", "the home network model is used, and the home network is hidden utilizing the double semantics mechanism.", "FIG.", "5 illustrates a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "FIG.", "6 illustrates messages following the first Invite in a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "FIG.", "7 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "FIG.", "8 illustrates a mobile terminated call setup example using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "FIG.", "9 illustrates the main characteristics of address hiding alternatives in accordance with embodiments of the present invention.", "FIG.", "10 illustrates the main characteristics of name hiding alternatives in accordance with embodiments of the present invention.", "BEST MODE FOR CARRYING OUT THE INVENTION As noted above, in the present invention, an indirect reference addressing mechanism is used to hide the names and addresses in both the visited network model and in the home network model with the S-CSCF in the visited network and with the S-CSCF in the home network respectively.", "While the most of the examples discussed below described the visited network model with the S-CSCF in the visited network, it is of course understood that the solutions can also be applied to the home network model where the S-CSCF is in the home network.", "In the indirect reference addressing mechanism, the message is routed to a contact point with the first part of the indirect reference.", "The contact point resolves the second part of the indirect reference and routes the message to the target network element.", "The network between the contact point and the target network element remains hidden.", "There are two basic mechanisms which can be used to hide the network, namely, an indirect reference to the hidden network to hide the address of the target network element or an indirect reference to the hidden network is partially encrypted to hide the name of the target network element.", "FIG.", "1 illustrates the salient feature of the present invention, namely, using the first part of the indirect reference, the message to be sent from a network entity in a first network to a target network entity in a second, hidden, network is routed from the network entity in the first network to the contact point disposed between the first network and the second, hidden, network.", "The contact point then resolves the second part of the indirect message and routes the message to the target network entity.", "For simplicity in the example in FIG.", "1, a message is sent to a hidden network The invention also applies to all combinations, whether or not the source or target network is hidden.", "More particularly, various mechanisms may be used to hide the address of the target element, namely, double semantics, two names for routing, and one name for two routings.", "With regard to double semantics, all incoming and outgoing traffic is routed via contact points e.g.", "I-CSCFs.", "All network elements inside the home network can use only an internal DNS (Domain Name Service).", "All network elements outside of the home network can use only a public DNS.", "A contact point can use both the internal and the public DNS, namely, the contact point uses the internal DNS when a name of its own network has to be resolved and uses the public DNS when a name of a foreign network has to be resolved The name of a particular network element will be resolved to the IP address of a contact point, e.g.", "I-CSCF, when resolved in a foreign network and will be resolved to the IP address of the network element itself when resolved inside the network.", "In the double semantics mechanism, the contact point, e.g.", "I-CSCF, has the capability to consult both the internal DNS and the public DNS depending on the name to be resolved and does not have to change incoming and outgoing messages.", "Furthermore, the S-CSCF and HSS and SPD require no extra functionality nor does any network element in the foreign networks.", "Still furthermore, a double DNS database is needed for names of the hidden network The first database is used in the internal DNS servers which are available only from inside the home network where as the second database is used in the public DNS servers available only from the foreign networks.", "Since the contact points are located on the border between the hidden network and the other networks, they can use both databases.", "In the two names for routing mechanism, two names are used for routing, namely, a first name used to route the message to the network (that is, to the contact point) and a second name used to route the message inside the target network to the S-CSCF.", "In the foreign networks, only the first name can be resolved and not the second name where as in the home network, the second name can be resolved.", "In the two names for routing mechanism, it will be necessary for 3GPP to define a new functionality for usage of two names e.g.", "in the SIP (Session Initiation Protocol) protocol.", "Furthermore in the visited network model, the HSS must store two names rather than one.", "In the one name for two routings mechanism, all incoming and outgoing traffic is routed via contact points, e.g.", "I-CSCFs, as in the case of double semantics.", "One name is used for routing both in the hidden and other networks.", "In this mechanism, the single name is used for routing as is as well as being used in a modified format In this regard, there are two alternatives, namely, the name is modified in the hidden network before being used for routing (i.e.", "the name is home modified) or the name is modified in the other network before being-used for routing (i.e.", "the name is foreign modified).", "In the first alternative, there are two possible solutions, namely, the contact point e.g.", "an I-CSCF replaces the original name of the incoming message with a modified name or alternatively, the contact point e.g.", "the I-CSCF doesn't touch the original name of the incoming message and every network element modifies the original name for routing when needed.", "With regard to the DNS service, the name of a certain network element will be resolved to the IP address of the contact point e.g.", "I-CSCF when resolved outside the hidden network and resolved to the IP address of the network element itself when resolved inside the hidden network In the one name for two routings mechanism, no changes to the SIP or HSS are needed.", "In addition, the IP addresses of all of the elements with the exception of the contact point, e.g.", "I-CSCF, can be hidden using the same technique.", "In the first alternative (i.e.", "home modified) no standardization is needed while m the second alternative (i.e.", "foreign modified), the address modification must be standardized.", "In a similar fashion, various mechanisms may be employed to hide the name of the target network element utilizing encryption, namely, a partially encrypted name address pair, double semantics with encryption, two names for routing with encryption, and one name for two routings with encryption.", "These mechanisms can be divided into non-absolute and absolute total hiding solutions depending on whether the encrypted name is, or is not, used as an argument in a DNS query outside of the hidden network.", "In the case of the partially encrypted name address pair mechanism and the two names for routing with encryption mechanism, total hiding solutions are possible.", "The encrypted name is not used for a DNS query in foreign networks.", "The encrypted name is decoded before it is used in the hidden network.", "Thus there is no need to include the encrypted names in either the public DNS database or in the internal DNS database.", "In the case of the double semantics with encryption mechanism and the one name for two routings with encryption mechanism, the encrypted name is used for a DNS query outside the hidden networks and accordingly, the encrypted name must be included in the public DNS database.", "If the encrypted name is also used for a DNS query in the hidden network, then the encrypted name has to be included in the internal DNS database.", "On the other hand, if the encrypted name is not used for a DNS query in the hidden network, then the encrypted name is decoded at the contact point, for example, an I-CSCF, prior to being used and accordingly, there is no need to include the encrypted names in the internal DNS database.", "As to the scope of the encryption key used, the encryption key can be call leg specific, call specific, contact point specific, contact point type specific, or network wide.", "In the case of a call specific encryption key, the key may be generated from the call identity.", "In the case of a contact point specific encryption key, the incoming and outgoing traffic must pass via the same contact point that knows the encryption key.", "In the case of a contact point type specific encryption key, similar contact points use the same encryption key and in the case of a network wide encryption key, all of the contact points in the network utilize the same encryption key.", "As to the scope of the encryption, a portion of the name or the entire name may be encrypted, for example, in the following ways: the hostname may be encrypted, the hostname and the domain name part may be encrypted while leaving the operator domain unencrypted, the hostname and the entire domain name may be encrypted, the entire name may be encrypted, the entire name may be encrypted except for the @ sign, or the entire address including the @ sign may be encrypted.", "Note that the characters of the encryption result are important if the result has to have the format of a valid name.", "In the case of a partially encrypted name address pair, the total hiding is implemented using the name address pair.", "The name address pair is an indirect reference to a network element located inside a totally hidden network.", "The first part of the pair is the name or address of a contact point, for example, an I-CSCF, in the target network and the second part of the pair is the name or address of the target network element The second part of the pair is always encrypted outside of the home network.", "The name address pair is stored as a single address in the HSS and in the SPD.", "The via headers are encrypted.", "With regard to the needed functionality in the case of a partially encrypted name address pair, the originator of the message in a foreign network may build the name or address from the name address pair by using the first part of the name address pair as a maddr parameter and using the second part of the name address pair as a Request-URI, that is, the Request-URI is an encrypted character string.", "As to the network elements outside the hidden network, the message may be routed with the maddr parameter consistent with the SIP specification.", "With respect to name/address checking, either the validity of the Request-URI is not checked or the validity of the Request-URI is checked if the result of the encryption has a format of a valid name or address (to be used as the Request-URI).", "With regard to the contact points, for example, I-CSCFs, in the target network, the maddr parameter of each incoming message is removed and the encrypted Request-URI is replaced by a decoded Request-URI.", "With respect to outgoing messages, the contact points ensure that all necessary names and addresses of the hidden network are in the format of a name address pair where the second part is encrypted.", "If needed the contact point, e.g.", "I-CSCF, encrypts the name or address, inserts the own name and builds the name address pair.", "With regard to ordinary network elements in the target network, no extra functionality is needed with regard to routing since the messages are routed as usual with the Request-URI.", "As to name/address building, if the receiver is outside the hidden network, the home name/address in the address pair format (with the second part encrypted) is used.", "Alternatively, this address building functionality can be replaced by the above-noted functionality of the contact points.", "As to name address pair building, if performed by the contact point, and ordinary network element of the hidden network does not have to build a name address pair when it wants to provide a name or an address that refers to itself.", "In addition, the scope of the encryption key can be smaller than network wide.", "If the name address pair building is performed by an ordinary network element, the contact point does not have to do anything to the outgoing message and the scope of the encryption key has to be network wide in the absence of an encryption key delivery system.", "With regard to the double semantics with encryption mechanism, the concept corresponds to that of the double semantics mechanism where the name is encrypted and since the same encrypted name is used both outside an inside the network, it must be included in both the public and internal DNS databases.", "Outside the hidden network, the encrypted name is resolved to the IP address of the contact point while in the hidden network, the encrypted name is resolved to the IP address of the target network element.", "A suitable scope of encryption for the double semantics with encryption mechanism is that the host name and the domain name part are encrypted while the operator_domain is unencrypted.", "Furthermore, other scopes of the encryption can be used and the encryption can also be a simple character string modification.", "The encryption interval can either be dynamic, static, extreme dynamic, extreme static, or a combination of dynamic and static encryption intervals.", "With a dynamic encryption interval, the names of the network elements are encrypted all the time with new encryption keys and new encrypted names are added to DNS databases.", "The network elements and contact points always utilize a newly encrypted name when available and old encrypted names are not used and they are removed after a predetermined time period from the DNS databases.", "The lifetime of an encrypted name compared with the birth rate of new encrypted names defines the size of an encrypted name space and hiding is based on the encrypted name space which is changing all the time.", "With a static encryption interval, a part or all of the names are always encrypted with a new key after a predetermined time interval and a part or all of the old names are removed.", "The amount of encrypted names can easily be defined and only a part or all of the encrypted names are used randomly or according to a specific algorithm Hiding is based on a sufficiently large encrypted name space.", "With an extreme dynamic encryption interval, the name is encrypted individually every time it is needed with a new key and the encrypted name is inserted in both the public and internal DNS databases prior to use.", "With an extreme static encryption interval, the names are encrypted and stored in both the public and internal DNS databases only once and are not changed after that.", "Hiding is based on a sufficiently large encrypted name space.", "The functionality needed with the double semantics with encryption mechanism is the same as that needed with the double semantics mechanism with the added functionality of an encryption mechanism as well as dynamic or static DNS database handling.", "The two names for routing with encryption mechanism corresponds to the two names for routing mechanism.", "The names of the network elements, for example the S-CSCF located inside the network, are normally encrypted at a contact point for outgoing messages and are normally decrypted at a contact point for incoming messages.", "The encrypted name is not utilized outside the hidden network nor is utilized within the hidden network.", "That is, the encrypted name is not included in either the public or internal DNS databases.", "The scope of the encryption can be chosen freely, e.g.", "only the part of the name may be encrypted or the entire name may be encrypted.", "The functionality needed with the two names for routing with encryption mechanism is the same as that needed with the two names for routing mechanism with the added functionality of an encryption mechanism.", "The one name with two routings with encryption mechanism corresponds to the one name with two routings mechanism where the name is encrypted.", "Everything that has been discussed with regard to the double semantics with encryption mechanism is also applicable to this mechanism with the exception that in the alternative in which the names modified in the home network prior to being used for routing, the encrypted name can be decoded or not decoded at the contact point.", "If it is decoded, it is not included in the internal DNS database while if it is not decoded, it is included in the internal DNS database after modification.", "In the alternative in which the names are modified in the foreign network prior to be used for routing, both the encrypted name and the encrypted name after modification are needed.", "The encrypted name as is included in the internal DNS database and the encrypted name after modification is included in the public DNS database.", "The functionality needed for the one name with two routings with encryption mechanism is the same as that needed with the one name with two routings mechanism with the added functionality of an encryption mechanism and dynamic or static DNS database handling.", "The above-noted examples are merely for exemplary purposes and the present invention is not limited thereto.", "For example, the SIP protocol is merely used as an example and the solutions are valid and can be applied to other call control protocols.", "The solutions can be applied also to other protocols and all types of networks where hiding of the names or addresses on the logical and/or lower-level is needed.", "The maddr parameter is simply a name or address used as a destination address for routing instead of the Request-URI if the maddr parameter exists while the Request-URI is simply a destination name or address used for routing if the maddr parameter does not exist The HSS is simply a location where the name or address of a network element in the hidden network is stored in the visited network model and the I-CSCF is merely a contact point connecting to the hidden network.", "Furthermore, the solutions can be applied to any situation in which a host, located inside a hidden network, must be addressed from outside the network and the network itself is inaccessible except through a contact point or contact points.", "The contact point can be any applicable host or suitable network element in the target network having a connection to another network.", "Still furthermore, as to the use of two separate DNS databases, the application can explicitly choose the resolver that utilizes specific DNS servers or the resolver can make a decision based on a given parameter, argument, etc., as to which DNS server it will use for the query in question That is, the resolver may utilize an internal DNS server for a name in its own network and utilize a public DNS server for foreign names.", "Lastly, in the discussion above, an indication that a name is resolved to an IP address is equally applicable to a name being resolved to more than one IP address.", "In addition, a single contact point can have more than one IP address.", "These concepts and abbreviations are used in the following figures: UE_MSISDN is an identity of a subscriber e.g.", "E.164.LN i.e.", "logical name is an identity of a subscriber e.g.", "[email protected].", "APSE is an abbreviation for Application Server.", "FIG.", "2 illustrates a mobile terminal call setup example using the visited network model where the visited network is hidden utilizing the two names for routing with encryption mechanism.", "As illustrated in FIG.", "2, in step 1, an I-CSCFh receives an Invite and in response thereto, sends a Query to the HSSh in step 2 which in turn, in step 3, sends a Response (FQDN of I-CSCFv and encrypted FQDN of S-CSCFv) back to the I-CSCFh.", "In step 4, the I-CSCFh sends a DNS query in order to resolve FQDN of I-CSCFv to the Public DNS Infrastructure which in turn, in step 5, sends a DNS answer (IP address of I-CSCFv) back to the I-CSCFh.", "In step 6, the I-CSCFh sends using the IP address an Invite to an I-CSCFv of the visited network which, in step 7, decodes the encrypted FQDN of S-CSCFv for routing.", "In step 8, the I-CSCFv sends a DNS query in order to resolve FQDN of S-CSCFv to that portion of the Public DNS Infrastructure under control of the visited network which in turn sends a DNS answer (IP address(es) of S-CSCFv) back to the I-CSCFv in step 9.In step 10, the I-CSCFv sends using the IP address an Invite to a S-CSCFv which in turn, in step 11, sends the Invite to a P-CSCFv of the visited network which in turn sends the Invite to the target mobile terminal UE (User Equipment).", "FIG.", "3 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the two names for routing with encryption mechanist.", "FIG.", "4 illustrates the registration to a home network by a mobile terminal when the S-CSCFh is in the home network, i.e.", "the home network model is used, and the home network is hidden utilizing the double semantics mechanism.", "FIG.", "5 illustrates a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "FIG.", "6 illustrates messages following the first Invite in a mobile originated call when the S-CSCFh is in the home network, i.e.", "the home network model is used, and both the home as well as the other network are hidden utilizing the double semantics mechanism.", "The steps illustrated in the FIGS.", "2-5 are self-explanatory and accordingly, a detailed description thereof has been omitted for the sake of brevity.", "FIG.", "7 illustrates registration of a mobile terminal to a visited network using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "FIG.", "8 illustrates a mobile terminated call setup example using the visited network model where the visited as well as the home network are hidden utilizing the one name for two routings (home modified) mechanism.", "As with FIGS.", "3-6, the steps illustrated in the FIGS.", "7-8 are self-explanatory and accordingly, a detailed description thereof has been omitted for the sake of brevity.", "FIG.", "9 illustrates the main characteristics of the address hiding alternatives in accordance with the present invention.", "Note that alternative ALT01 is not part of the invention but rather is the situation in which the operators of the first and second networks trust each other and therefore, neither network need be hidden.", "Where there is no trust, an indirect reference with one name may be used, that is, alternative ALT2, the double semantics embodiment of the present invention.", "Alternatively, the indirect reference with one name with the help of modification may be used, that is, alternative ALT6, the one name with two routings embodiment of the present invention.", "Furthermore, the indirect reference with two separate names may be used, that is, alternative ALT3, the two names for routing embodiment of the present invention.", "Similarly, FIG.", "10 illustrates the main characteristics of the name hiding alternatives in accordance with the present invention.", "For example, an address pair which becomes one name that has two routings may be used, that is, alternative ALT8, the double semantics with encryption embodiment of the present invention.", "One name having two routings with a help of modification may be used, that is, alternative ALT10, the one name-two routings with encryption embodiment of the present invention.", "Alternatively, when the two separate routings become two separate names, alternative ALT9, the two names for routing with encryption embodiment of the present invention may be used and when two names are merged into the name address pair, alternative ALT7, that is, the partly encrypted name address pair embodiment of the present invention may be used.", "This concludes the description of the example embodiments.", "Although the present invention has been described with reference to a number of illustrated embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within this spirit and scope of the principles of this invention.", "More particularly, reasonable variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the invention.", "In addition to variations and modifications in the component parts and/or arrangements, alternative uses will also be apparent to those skilled in the art.", "Furthermore, the various terms used throughout the specification and drawing figures are well-defined in the art and are publicly available at the WebSite of 3GPP at www.3gpp.org and the definitions of such various terms contained within this WebSite are incorporated by reference herein in their entirety.", "Furthermore, the SIP protocol are defined in RFC 2543 which is publicly available at numerous WebSites including www.faqs.org/rfc/rfc2543.html and this reference is also incorporated by reference herein in its entirety." ] ]

[ [ "Personal message delivery system", "The present invention provides for a personal message system comprised of a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats.", "A group services module is provided configured to maintain communications among groups of the subscribers.", "A platform conversion module is also provided and is coupled to the plurality of interfaces and the group services modules configured to connect each of the plurality of subscribers within a group, regardless of the communication protocols used by the subscribers." ], [ "1.A personal message system comprising: a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats; a group services module configured to maintain communications among groups of said subscribers; and a platform conversion module coupled to said plurality of interfaces and said group services modules configured to connect each of Said plurality of subscribers within a group, regardless of the communication protocols used by said subscribers.", "2.A personal message system as claimed in claim 1, wherein one of said plurality of interfaces is an Hypertext Transfer Protocol/Wireless Application Protocol interface.", "3.A personal message system as claimed in claim 2, wherein one of said plurality of interfaces is a Simple Mail Transfer Protocol interface.", "4.A personal message system as claimed in claim 3, wherein one of said plurality of interfaces is a Short Message Service/2-way paging protocol interface.", "5.A personal message system as claimed in claim 4, wherein one of said plurality of interfaces is an Interactive Voice Response protocol interface.", "6.A personal message system as claimed in claim 1, further comprising a personal home page service module configured to provide personal mobile home pages for subscribers on said messaging system.", "7.A personal message system as claimed in claim 1, wherein said group service module further comprises an alert module configured to provide alerts to said plurality of subscribers so as to alert them to incoming messages.", "8.A personal message system as claimed in claim 1, wherein said group service module further comprises a send message module such that said plurality of subscribers can formulate messages to be sent through said messaging system to other said subscribers.", "9.A personal message system as claimed in claim 1, wherein said group service module further comprises a group module configured to allow said subscribers to create, and send messages to and receive messages from subscribers of said groups.", "10.A personal message system as claimed in claim 9, further comprising a group main page utility configured to display of a list of said groups.", "11.A personal message deliver system as claimed in claim 9, further comprising a group details utility configured to list the details of said groups including number of members, number of messages per day.", "12.A personal message system as claimed in claim 9, further comprising group administration utility configured to provide administration function for said group including allowing in new subscribers, removing existing subscribers and setting parameters for group actions.", "13.A personal message system as claimed in claim 9, further comprising a group history page configured to store the history of messages sent by said subscribers to said group.", "14.A personal message system as claimed in claim 10, further comprising a group search results utility configured to provide search results for searches conducted from said group main page utility.", "15.A personal message system as claimed in claim 9, further comprising a create group utility configured to allow said subscribers to create a new group for said messaging system.", "16.A personal message system as claimed in claim 1, wherein said group service module further comprising a group membership module configured to allow said subscribers with the necessary utilities-to facilitate the membership functions of said groups.", "17.A personal message system as claimed in claim 16, further comprising a request to join utility configured to allow a subscriber to request to join one of said groups.", "18.A personal message system as claimed in claim 16, further comprising an invite to group utility configured to allow a subscriber of one of said groups to invite another non-member subscriber to said group.", "19.A personal message system as claimed in claim 16, further comprising an accept invite utility configured to allow a subscriber who receives an invite to one of said groups to accept or reject the invitation.", "20.A personal message system as claimed in claim 16, further comprising a member directory utility configured to provide a list of the member subscribers to said groups.", "21.A personal message system as claimed in claim 1, further comprising a user database configured to store information relating to the accounts of said plurality of subscribers.", "22.A personal message system as claimed in claim 1, further comprising a multimedia database configured to store information relating to multimedia data for delivery to said plurality of subscribers.", "23.A personal message system comprising: a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats; a channel services module configured to maintain communications from a content provider to said subscribers; and a platform conversion module coupled to said plurality of interfaces and said channel services modules configured to connect each of said plurality of subscribers with said content providers, regardless of the communication protocols used by said subscribers.", "24.A personal message system as claimed in claim 23, wherein one of said plurality of interfaces is an Hypertext Transfer Protocol/Wireless Application Protocol interface.", "25.A personal message system as claimed in claim 24, wherein one of said plurality of interfaces is a Simple Mail Transfer Protocol interface.", "26.A personal message system as claimed in claim 25, wherein one of said plurality of interfaces is a Short Message Service/2-way paging protocol interface.", "27.A personal message system as claimed in claim 26, wherein one of said plurality of interfaces is an Interactive Voice Response protocol interface.", "28.A personal message system as claimed in claim 23, further comprising a personal home page service module configured to provide personal mobile home pages for subscribers on said messaging system.", "29.A personal message system as claimed in claim 23, wherein said channel service module further comprises an alert module configured to provide alerts to said plurality of subscribers so as to alert them to incoming messages.", "30.A personal message system as claimed in claim 23, wherein said channel service module further comprises a send message module such that said content provider can formulate messages to be sent through said messaging system to said subscribers.", "31.A personal message system as claimed in claim 23, wherein said channel service module further comprises a poll module configured to provide said content provider with the ability to send polls to said channel subscribers.", "32.A personal message system as claimed in claim 23, wherein said channel service module further comprises a channel module configured to allow said content providers create and send messages to said subscribers of said channel.", "33.A personal message system as claimed in claim 32, further comprising a channel main page utility configured to display of a list of said channels.", "34.A personal message system as claimed in claim 32, further comprising a channel details utility configured to list the details of said channels including the number of messages per day and a description of the content of those channels.", "35.A personal message system as claimed in claim 32, further comprising a channel categories utility configured to list said channels into categories based on their content.", "36.A personal message system as claimed in claim 32, further comprising a channel history utility configured to store the history of messages sent by said content provider to said channel.", "37.A personal message system as claimed in claim 32, further comprising a channel configuration utility configured to allow said channel subscribers to control the content that they receive from said channel.", "38.A personal message system as claimed in claim 23, wherein said channel service module further comprising a channel administration module configured to allow said content providers administrate said channels.", "39.A personal message system as claimed in claim 38, further comprising a channel text message utility configured to allow said content provider to generate a text message to be sent to said member subscribers of said channel.", "40.A personal message system as claimed in claim 38, further comprising a channel voice message utility configured to allow said content provider to generate a voice message to be sent to said member subscribers of said channel.", "41.A personal message system as claimed in claim 38, further comprising a channel toolset utility configured to allow said content provider to administrate the content of said channel.", "42.A personal message system as claimed in claim 38, further comprising a channel message scheduling utility configured to allow a content provider to pre-schedule the deliver of messages to said member subscribers of said channel in advance.", "43.A personal message system as claimed in claim 38, further comprising a channel poll utility configured to allow said channel provider to enter the information necessary to conduct a poll.", "44.A personal message system as claimed in claim 23, further comprising a user database configured to store information relating to the accounts of said plurality of subscribers.", "44.A personal message system as claimed in claim 23, further comprising a multimedia database configured to store information relating to multimedia data of said content providers for delivery to said plurality of subscribers.", "45 A personal message system comprising: a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats; a group services module configured to maintain communications among groups of said subscribers; a platform conversion module coupled to said plurality of interfaces and said group services modules configured to connect each of said plurality of subscribers within a group, regardless of the communication protocols used by said subscribers; and a message delivery subsystem coupled to one of said plurality of interfaces configured to create a virtual number based on a the devices of both said receiving and sending said subscriber and a randomly generated virtual number which are stored in a call completion data table.", "46.A personal message system as claimed in claim 45, wherein said call completion data table maintains a receiving subscriber device field configured to store the wireless number associated with the device of the call receiving subscriber.", "47.A personal message system as claimed in claim 45, wherein said call completion data table maintains a virtual number field configured to store the virtual number associated with the call between said devices of the call receiving and call sender subscribers.", "48.A personal message system as claimed in claim 45, wherein said call completion data table maintains a sending subscriber device field configured to store the wireless number associated with the device of the call sending subscriber 49.A personal message system comprising: a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats; a group services module configured to maintain communications among groups of said subscribers; a platform conversion module coupled to said plurality of interfaces and said group services modules configured to connect each of said plurality of subscribers within a group, regardless of the communication protocols used by said subscribers; and a wireless application protocol—short message service/interactive voice response handler module coupled to said platform conversion module configured to provide interactive voice response links into a wireless application protocol sessions such that a subscriber in said wireless application protocol sessions can directly link to an interactive voice response session so as to directly download a multimedia file.", "50.A personal message system as claimed in claim 49, further comprises an interactive voice response subsystem handler module, coupled to said wireless application protocol—short message service/interactive voice response handler module configured to provide an external interactive voice response session from an independent multimedia content provider.", "51.A personal message system as claimed in claim 50, further comprises an interactive voice response multimedia database, coupled to said interactive voice response subsystem handler module configured to store multimedia content to be delivered by said interactive voice response subsystem handler module and said wireless application protocol—short message service/interactive voice response handler module.", "52.A personal message system comprising: a plurality of interfaces configured to interface with a plurality of subscribers communication devices using a plurality of formats; a group services module configured to maintain communications among groups of said subscribers; a platform conversion module coupled to said plurality of interfaces and said group services modules configured to connect each of said plurality of subscribers within a group, regardless of the communication protocols used by said subscribers; and a subscriber information table configured to store the information necessary for said platform conversion module to send messages to said subscribers in the proper format.", "53.A personal message system as claimed in claim 52, further comprising a username field configured to store information pertaining to said subscribers username, used to identify said subscriber to said other subscribers on said message system.", "54.A personal message system as claimed in claim 52, further comprising a personal information/e-mail field configured to store information pertaining to said subscribers personal information such as real name, mailing address, phone number and e-mail address for use by said message system in contacting said subscriber.", "55.A personal message system as claimed in claim 52, further comprising a Personal Identification Number (PIN) field configured to store information pertaining to said subscribers personal identification number used by said message system to restrict access to said subscribers account.", "56.A personal message system as claimed in claim 52, further comprising a validation list field configured to store information pertaining to said subscribers personal communication devices which are recognized by said message system.", "57.A personal message system as claimed in claim 52, further comprising a preferred voice field configured to store information pertaining to said subscribers preferred device for receiving voice messages from said message system.", "58.A personal message system as claimed in claim 52, further comprising a preferred text field configured to store information pertaining to said subscribers preferred device for receiving text messages from said message system.", "59.A personal message system as claimed in claim 52, further comprising a groups field configured to store information pertaining to said subscribers group membership record.", "60.A personal message system as claimed in claim 52, further comprising a channel field configured to store information pertaining to said subscribers channel membership record.", "61.A personal message system as claimed in claim 52, flier comprising a addresses field configured to store information pertaining to said subscribers address book of all other subscribers on said message system that said subscriber has previously contacted.", "62.A personal message system as claimed in claim 52, further comprising a history field configured to store information pertaining to said subscribers recently received messages from said message system 63.A method for sending a message on a personal messaging system having a plurality of interfaces, a group services module and a platform conversion module, said method comprising the steps of: sending a message to said message system via any one of said plurality of interfaces; sending said message to said group services module for sending said message to said subscriber members of said group; reformatting said message in said platform conversion module into various formats corresponding to said group member subscriber preferences; and delivering said message through said plurality of interfaces to said members of said group.", "64.A method for delivering a message on a personal messaging system having a plurality of interfaces, a channel services module and a platform conversion module, said method comprising the steps of: creating a channel message on said message system; sending said message to said channel services module for sending said message to said subscribers members of said channel; reformatting said message in said platform conversion module into various formats corresponding to said channel member subscriber preferences; and delivering said message through said plurality of interfaces to said members of said channel." ], [ "<SOH> BACKGROUND OF THE INVENTION <EOH>Many systems are currently in place which interconnect people via electronic messaging services.", "These services include phones (mobile and fixed-line), pager services and text paging (SMS), wireless palm communicators, wireless internet and e-mail, all of which are designed -to provide mobile communication services to users.", "However, each particular system both wireless and standard (e-mail via PC and fixed-line phone) require different protocols to deliver the messages.", "The drawback to these systems is that they are not well integrated As such, communications within a group are limited to the subscribers of a particular service provider.", "Subscribers of various service providers do not have the ability to interact For example, there is currently no ability if for people desire to form a group where, each communicates via different modes of operation; such as, one by cell phone, one by e-mail, one by paging, and one by paging with SMS.", "Therefore, there exists a need for developing an enhanced personal message delivery system that allows people with various communication devices supported by various service providers to communicate in groups of their own design." ], [ "<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with one embodiment of the present invention, a system is provided that fully integrates all personal communication forms including but not limited to mobile and fixed-line voice, SMS (mobile-terminated and mobile-originated), text paging (SMS), standard paging, web, e-mail, WAP & (Phone).com and instant messaging, such that a single message intended to reach a group of users is entered only once in any available format whereby it is automatically converted into all of the other necessary formats and delivered to all members of a group.", "Furthermore, the system comprises integrated components and navigation architecture to allow smooth cross platform transitions during system sessions." ], [ "RELATED APPLICATIONS This application is related to and claims priority to both U.S.", "Provisional Patent Application No.", "60/238,940 entitled “A Personal Message Delivery System”, filed on Oct. 10, 2000, and U.S.", "Provisional Application No.", "60/312,899 entitled “A Personal Message delivery system”, filed on Aug. 15, 2001, the entirety of which are incorporated herein by reference.", "FIELD OF THE INVENTION The present invention relates to a personal message delivery system.", "More specifically, the present invention relates a cross platform personal message delivery system connecting member communities through the operation of a mobile community platform, a mobile content and commerce network and a mobile publishing toolset BACKGROUND OF THE INVENTION Many systems are currently in place which interconnect people via electronic messaging services.", "These services include phones (mobile and fixed-line), pager services and text paging (SMS), wireless palm communicators, wireless internet and e-mail, all of which are designed -to provide mobile communication services to users.", "However, each particular system both wireless and standard (e-mail via PC and fixed-line phone) require different protocols to deliver the messages.", "The drawback to these systems is that they are not well integrated As such, communications within a group are limited to the subscribers of a particular service provider.", "Subscribers of various service providers do not have the ability to interact For example, there is currently no ability if for people desire to form a group where, each communicates via different modes of operation; such as, one by cell phone, one by e-mail, one by paging, and one by paging with SMS.", "Therefore, there exists a need for developing an enhanced personal message delivery system that allows people with various communication devices supported by various service providers to communicate in groups of their own design.", "SUMMARY OF THE INVENTION In accordance with one embodiment of the present invention, a system is provided that fully integrates all personal communication forms including but not limited to mobile and fixed-line voice, SMS (mobile-terminated and mobile-originated), text paging (SMS), standard paging, web, e-mail, WAP & (Phone).com and instant messaging, such that a single message intended to reach a group of users is entered only once in any available format whereby it is automatically converted into all of the other necessary formats and delivered to all members of a group.", "Furthermore, the system comprises integrated components and navigation architecture to allow smooth cross platform transitions during system sessions.", "BRIEF DESCRIPTION OF THE FIGURES FIG.", "1 illustrates a personal message delivery system, in accordance with one embodiment of the present invention.", "FIG.", "2 illustrates a group services module, in accordance with one embodiment of the present invention.", "FIG.", "2A illustrates a message window, in accordance with one embodiment of the present invention.", "FIG.", "2B illustrates an administration web page, in accordance with one embodiment of the present invention.", "FIG.", "2C illustrates a create group page, in accordance with one embodiment of the present invention.", "FIG.", "3 illustrates a channel services module, in accordance with one embodiment of the present invention.", "FIG.", "3A illustrates a channel message history/channel configuration page, in accordance with one embodiment of the present invention.", "FIG.", "4 illustrates a subscriber information table, in accordance with one embodiment of the present invention.", "FIG.", "5 illustrates a message delivery subsystem call completion data table, in accordance with one embodiment of the present invention.", "FIG.", "6 illustrates a flow chart for a login and create account procedure, in accordance with one embodiment of the present invention.", "FIG.", "7 illustrates a flow chart for a subscriber joining a group, in accordance with one embodiment of the present invention.", "FIG.", "8 illustrates a flow chart for a subscriber joining a channel, in accordance with one embodiment of the present invention.", "FIG.", "9 illustrates a flow chart for a subscriber forming a new group, in accordance with one embodiment of the present invention.", "FIG.", "10 illustrates a flow chart for a subscriber sending an invite, in accordance with one embodiment of the present invention.", "FIG.", "11 illustrates a flow chart for a subscriber generating a text message, in accordance with one embodiment of the present invention.", "FIG.", "12 illustrates a flow chart for a subscriber generating a voice message, in accordance with one embodiment of the present invention.", "FIG.", "13 illustrates a flow chart for a subscriber receiving multimedia content via a WAP-SMS/IVR handler module, in accordance with one embodiment of the present invention.", "FIG.", "14 illustrates a flow chart for a subscriber communication via a virtual addressing scheme, in accordance with one embodiment of the present invention.", "FIG.", "15 illustrates a flow chart for the creation and operation of a poll in accordance with one embodiment of the present invention.", "DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one embodiment of the present invention, a cross-platform personal message delivery system 2 is provided, configured to provide a mobile community platform, a mobile content and commerce network and other independent services such as mobile home pages provided through a mobile publishing toolset.", "In one embodiment of the present invention, as illustrated in FIG.", "1, personal message delivery system 2 is comprised of a number of internal modules and components configured to provide the subscribers 4 with a means to communicate with one another through various groups, channels and other means.", "Subscribers 4 is used throughout to refer to individuals with accounts in system 2.Subscribers 4 can refer to non-mobile device users as well as mobile device users, however, for the purposes of illustration, subscribers 4 will generally refer to mobile users of system 2.It should be noted that system 2 is available remotely to subscribers 4 of any mobile carrier and is not limited by any particular cellular provider or device manufacturer.", "As illustrated in FIG.", "1, subscribers 4 contact system 2 via the internet or the public switched telephone network, based on the protocol preferred by subscriber 4.In one embodiment of the present invention, as illustrated in FIG.", "1, a Hyper Text Transfer Protocol (HTTP)/Wireless Application Protocol (WAP) interface 8 is provided.", "HTTP/WAP interface 8 is coupled to a platform conversion module 9, described below, and is configured to interface system 2 with subscribers 4 that connect to system 2 via the internet or wireless internet methods.", "In addition, HTTP/WAP interface 8 is configured to provide the principle interface for subscribers 4 to connect with system 2 both initially and when updating their account information.", "HTTP/WAP interface 8 is preferably a single server or a load balanced cluster of servers Any one of the commercially available HTTP/WAP servers such as Apache, IM, and Tomcat servers or any other servers capable of interfacing between HTTP/WAP devices and system 2 are within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a Simple Mail Transfer Protocol (SMTP) e-mail interface 10 is provide& SMTP interface 10 is coupled to platform conversion module 9 and is configured to interface system 2 with subscribers 4 that connect with system 2 via e-mail and/or 2-way paging methods.", "SMTP interface 10 can be either a single-server or a load balanced cluster of servers.", "SMTP interface 10 can be any commercially available servers capable of interfacing with e-mail and 2-way paging devices.", "In one embodiment of the present invention, as illustrated in FIG.", "1, an SMS and paging messaging interface 12 is provided.", "Messaging interface 12 is coupled to platform conversion module 9 and is configured to interface system 2 with subscribers 4 that connect with system 2 via Short Messaging Service (SMS) and paging methods.", "Messaging interface 12 can be either a single server or an asymmetric cluster of servers.", "Messaging interface 12 can be any commercially available servers capable of interfacing with SMS and paging devices.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a voice telephone and network IVR interface 14 is provided.", "IVR interface 14 is coupled to platform conversion module 9 and is configured to interface system 2 with subscribers 4 that connect with system 2 via voice telephone network methods.", "IVR interfaces 14 can be either a single server or a load balanced cluster of servers.", "IVR interface 14 can be any commercially available servers capable of interfacing with voice operating devices.", "It should be noted that a single interface module could be used to maintain the functions of all the four interface modules for use in interfacing system 2 with subscribers 4 using various mediums including voice communications, SMS, paging, e-mail HTTP and WAP as well as other formats.", "However, for the purposes of illustration, separate modules for the IVR, HTTP/WAP, SMS and e-mail interfaces are used throughout as illustrative of the modules by which system 2 interfaces with their respective types of communications to and from subscribers 4.Any similarly operating cross-platform messaging system 2 which interfaces with subscribers 4 is within the contemplation of the present invention.", "While only four major interface have be described this is in no way intended to limit the scope of the present invention.", "System 2 maintains a software design such that it is easily ungradable and expandable to cover any new technologies which subscribers 4 may be communicating with.", "For example, additional interfaces may include interfaces for platforms such as wireless PALM® devices, 3G mobile networks and other newly developing technologies.", "Any similar system which interfaces with subscribers 4 in order to provide a cross-platform communications network using a server interface, regardless of the platform serviced is within the contemplation of the present invention.", "It should also be noted that the configuration and distribution of interface functions among these modules is intended as only one example of the configuration of interface module sin system 2.Various permutations of the distribution of interface functions between the interface modules is within the contemplation of the present invention.", "In accordance with one embodiment of the present invention, subscribers 4 communicate to system 2 and to each other via virtual assigned phone numbers provided by a message delivery subsystem 17, described in more detail below.", "As such, since the number of subscribers 4 using system is almost always more that the number of trunk lines provided to system 2, in accordance with one embodiment a virtual address scheme is provided as described latter.", "In one embodiment of the present invention, a platform conversion module 9 is provided, configured to connect each of the protocol interfaces 8, 10, 12 and 14 to the functional components of system 2.Platform conversion module 9 converts outgoing messages from system 2 into the appropriate format for each subscriber 4 as set by their preferences.", "Also, platform conversion module 9 converts incoming messages from subscribers 4 to system 2 into the appropriate format for all of the target subscribers 4 such that the sending subscriber 4 does not have to resend the message in different formats.", "In addition to maintaining the functions of cross-platform conversion for incoming and outgoing messages to and from subscribers 4, platform conversion module 9 maintains LAN (Local Area Network)-Connections between interface modules 8, 10, 12 and 14.During ceratin functions of system 2 described below, some of the interfaces need to directly connect with one another to coordinate certain functions.", "For example, during a multimedia file transfer to a WAP user connected to system 2 through HTTP/WAP interface 8, IVR interface 14 and WAP interface 8 communicate with one another in order to coordinate the file transfer.", "It should be noted that the functions listed above for platform conversion module 9 are intended only as certain examples and is in no way intended to limit the scope of the present invention.", "Additional uses for conversion module 9 may become necessary if additional modules are added to system 2.Any platform conversion module which works to translate message formats in a similar cross-platform message system 2 is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a group services module 11 is provided, configured to supply system 2 with the means to manage and control a cross-platform mobile community of subscribers 4.Group services module 11 maintains several internal components described below which provide system 2 with a group feature allowing subscribers 4 to communicate with one another regardless of the devices used by the individual subscriber 4.In one embodiment of the present invention, as illustrated in FIG.", "1 a channel services module 13 is provided, configured to supply system 2 with the means to manage mobile content and commerce between subscribers 4 and advertisers on system 2.Channel services module 13 maintains several internal components discussed below which provide system 2 with a channel feature allowing advertisers and other interests to communicate sales, promotions information and more in a mobile format to interested subscribers 4, regardless of the mobile platform used.", "In one embodiment of the present invention, as illustrated in FIG.", "1 a personal home page module 15 is provided, configured to supply system 2 with the means to manage a personal home page and other similar services for subscribers 4 on system 2.personal home page module 15 provides system 2 with a personal mobile home page feature.", "In one embodiment of the present invention, as illustrated in FIG.", "1, an operations/user database 16 is provided.", "User database 16 is coupled to group services module 11, channel service module 13, personal home page module 15 as well as multi media database 18, described below.", "User database 16 is configured to store the web application information for system 2 as well as the subscribers 4 account information.", "User database 16 can be any commercial database, such as those produced by Oracle®, capable of handling the required information as well as being able to interface with the software and other components of system 2.User database 16 can exist either a single unit or as an asymmetric cluster of servers.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a multimedia storage server 18 is provided.", "Multimedia storage database 18 is connected to group services module 11, channel service module 13, personal mobile web page module 15 and user database 16.Multimedia database 18 is configured to store multimedia content such as WAV (Windows Audio Volume) files associated with IVR interface 14 functions.", "Also, multimedia content from channel advertisers and other channel providers for use in delivery to subscribers 4 can be stored in multimedia storage database 18.Multimedia storage database 18 can be any commercial database capable of handling the required information as well as being able to interface with the components and software of system 2.Multimedia storage database 18 can exist either a single unit or as an asymmetric cluster of servers.", "It should be noted that although user database 16 and multimedia storage database 18 are pictured as separate devices, this is in no way intended to limit the scope of the present invention.", "For example, both operations database 16 and multimedia storage database 18 can be a single database unit or, alternatively, a single asymmetric cluster, provided such single unit or single cluster can handle both the operations data storage and the multimedia storage for system 2.For the purposes of illustration, operations database 16 and multimedia storage database 18 will be depicted as separate databases, however any database configuration capable of supporting the requirements of a similar system 2 is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a message delivery system 17 is provided, coupled to IVR interface 14 configured to create and store the number directories for subscribers 4.In the event a service provider offers system 2 less trunk lines than there are subscribers 4 on system 2 with that provider, message delivery system 17 operates to create a virtual numbering system, so that subscribers 4 can communicate on mobile phone devices between one another using a virtual number system, described in more detail below.", "In one embodiment of the present invention, as illustrated in FIG.", "1, a WAP-SMS/IVR handler module 19 is provided, coupled to platform conversion module 9, group services module 11 and channel services module 13, configured to maintain the logical command flow for the IVR and WAP (a well as passive SMS) functions of system 2.The logical command flow refers to the numeric command structure of the IVR interface (ie.", "Press 1 to hear new messages, press 2 to hear old messages etc.)", "WAP-SMS/IVR handler module 19 of system 2 provides the function of cross platforming the logical command flow such that when a subscriber enters system 2 via WAP protocol instead of using the IVR interface, handler module 19 automatically converts the logical command flow into WAP protocol such that the logical command flow for IVR, WAP and passive SMS (if available) are interchangeable.", "Selections made in either protocol will be treated the same by system 2.In one embodiment of the present invention, as illustrated in FIG.", "1, an IVR subscriber handler 21 is provided, coupled to WAP-SMS/IVR handler module 19 and to an IVR database 23, configured to provide system 2 with outside multimedia content coupled with an IVR response, such that the content functions are managed by TV interface 14R For example, if IVR subscriber handler 21 wished to provide subscribers 4 with some media content, coupled with the ability to listen (option 1), order (option 2), hear different content (option 3), etc., IVR subscriber handler module 21 would provide the IVR logical command flow to WAP-SMS/IVR handler module 19 such that system 2 could administrate and deliver their content to the appropriate subscribers 4.In one embodiment of the present invention, as illustrated in FIG.", "1, an IVR/WAP database 23 is provided, coupled to WAP-SMS/IVR handler module 19 and IVR subscriber handler module 21, configured to store the media content for use in the transfer of media to subscribers 4 of system 2 as dictated by their selections entered in response to the options presented by IVR subscriber handler module 21.A more complete description of the function and operation of the delivery of media content through IVR subscriber handler module 21 and IVR media database 23 is discussed below in more detail.", "In one embodiment of the present invention, system 2 utilizes redundant components for each of the individual modules utilized such that up-time for system 2 is not compromised during updating, high-volume time, and/or in the event of any component failures.", "In one embodiment of the present invention, as illustrated in FIG.", "1, all of the components of system 2 are located in a single location such that all of the physical modules used to run system 2 are located locally.", "However, this is in no way intended to limit the scope of the present invention.", "For example, all of the components of system 2 such as HTTP/WAP interface 8, SMTP interface 10, messaging interface 12, and IVR interface 14 can be located remotely.", "In fact, it is possible that some or all of the components of system 2 be maintained off site by a third party such that system 2 headquarters is solely for the purpose of monitoring system 2 activity.", "As such, any similar system 2 which operates to provide a cross platform messaging service to connect subscribers 4 which utilizes similar modules and software during operation is within the contemplation of the present invention.", "As the physical location is not relevant to the functional aspects of any components, for the purposes of illustration, system 2 will be discussed through out as simply a collection of the appropriate functional modules and their respective connectivity,, regardless of their physical location.", "The software which operates on the modules of system 2 is preferably written in JAVA® programming language.", "The programming language used by system 2 is preferably designed in layers such that all of the programming for the modules of system 2 are insulated against the inner workings of all of the other modules in system 2.Thus, in operation, the programing used by the various modules of system 2 operates on independent layers for each of their respective different functions such that software modifications to any one particular area or module of system 2 will not require knowledge of or alteration of software for any other part or module of system 2.It should be noted that the general software architecture discussed above is intended only as an example of one possible software configuration for system 2, and is in no way intended to limit the scope of the present invention.", "For example, any software architecture either commercially available or independently designed that operates a similar system 2 so as to provide cross-platform communications to a plurality of subscribers 4 is within the contemplation of the present invention.", "The principle means by which subscribers 4 enter and use system 2 is maintained by the web application.", "The software which runs on system 2, HTTP/WAP server 8 and operations database 16 provide a web architecture which supplies subscriber 4 with a means to open an account, select preferences, register mobile and non-mobile devices and generally operate their account The following components provide the bulk of the interface utility architecture used by system 2 to maintain its accounts.", "In one embodiment of the present invention, as illustrated in FIG.", "2, group services module 11 maintains many internal component modules which provide group services module 11 with the ability to carry out its functions in system 2.The internal components of group services module 11 include but are not limited to alert module 20, send message module 22, group module 28, group membership module 40 and group interface module 25.This list of internal components for group services module 11 is intended only as an example of one possible configuration and in is in no way intended to limit the scope of the present invention.", "Additional modules may be added increasing the functional capabilities of group services module 11.Any similar cross-platform group service module operating in a similar cross-platform system is within the contemplation of the present invention.", "Alert module 20, coupled to group interface module 25, is configured to provide alert messages to subscriber 4, displayed on the various group web pages generated by system 2 as an alert button or panel on all of the pages required to provide alert notices functionalities to subscriber 4.In one embodiment of the present invention, alert module 20 provides alert buttons/panels on these web pages to inform subscribers 4 as to certain events and/or issues that need to be addressed.", "For example, such alert messages can include but are not limited to; unheard voice mail alerts, unvalidated device alerts, membership requests, record handle requests, record group name requests, vacation alert/no device in use, invitations to join a group, etc.", "In such instants where an alert button is used for a subscriber 4, the alerts is usually accompanied by a description of the alert and possibly a link to the appropriate web page where that alert can be properly addressed if applicable.", "In addition to alert buttons/panels displayed on web pages, alert module 20 is also configured to send alert messages to subscribers 4 based on their preferences.", "For example, if a subscriber wishes to be notified of a received voice mail, they may wish to have an alert notice sent to their mobile device alerting them to the message.", "Thus, in addition from posting the alert on the subscriber's 4 account in web page format in system 2, alert module also creates and sends SMS/e-mail text message alerts to the same subscriber 4, such that they are notified even before they logon to system 2.Send message module 22, coupled to group interface module 25, is configured to provide system 2 with the ability to allow subscribers 4 that are members of a group to send messages via a send message window 24 displayed on various group web pages which require such message sending functionality.", "Message windows 24, as illustrated in FIG.", "2A, provides subscriber 4 the ability to create a message by filling in the appropriate windows and pressing a send button.", "As illustrated in FIG.", "2A, message window 24 is comprised of address book line 24a, group line 24b, message block 24c and send button 24d.", "In operation, group line 24b is defaulted to the group that subscriber 4 is currently in, however, subscriber can change the addressee by simply using the pull down arrows in address line 24a and group line 24b.", "Message block 24c is configured to receive the text message, which is ultimately sent to addressee upon the activation of send button 24d.", "A more complete description of the function of message window 24 and how it is used in the overall operation of system 2 is discussed in more detail below.", "Group membership module 40, coupled to group module interface 25, is configured to allow subscribers 4 to utilize the group functions of system 2 such as inviting others to join and requesting to join particular groups.", "In operation group membership module 40, provides group members subscribers 4 with a number of window interfaces in which to conduct the operations of inviting members and requesting to join group, as is discussed later.", "Some of the functions of group membership module 40 may include but are not limited to a request to joint utility 42, an invite to group utility 44, an accept invite utility 46, and a member directory utility 48.Request to join utility 42 allow subscribers 4 to request to join an invite-only status group.", "Invite to group utility 44 allows a founder and/or member subscribers 4 of group to invite a non-member subscribers 4 to that group.", "Accept invite utility 46 allow the invited subscriber 4 to accept or reject the offer to join the group.", "Directory utility page 48 allows a subscriber 4 to review all of the member subscribers 4 of a particular group based on the members' user ID so as to check their active/inactive status or any other information that subscriber 4 has chosen to make public.", "Group module 28, coupled to group services module interface 25, is configured to support the basic multiuser cross-platform group functions for system 2.Group module 28 allows subscribers 4 to search, join and/or create, manage and review messaging groups for sending and receiving messages.", "In order to support the group functions of system 2 group module 28 maintains several utilities which facilitate this process.", "Some of the utilities supported by group module 28 may include but are not limited to main page utility 30, group details utility 32, group administration utilities 34, group history utility 36, group search results utility 38 and create group utility 39.Group main page utility 30 provides web pages which give a brief description of what the groups do and/or what the messages are related to.", "Group detail utility 32 provides web pages which explain the details of the group such as frequency of messages, status of group (open, invite only or secret), and other information.", "Group administration utility 34 provides web pages, as illustrated in FIG.", "2B which allow subscribers 4 authorized to manage a group (usually only the creator) a means for doing so.", "The group owner can make administrative decisions for the group including but not limited to approving and denying new member subscribers 4, removing members (presumably for bad behavior or parameter violations) and entering/editing the group description.", "In order to provide these capabilities, the group administration utility provides a web page, illustrated in FIG.", "2B which maintains a list of pending join requests with links to approve or decline, a list of current member subscribers 4 with a link to remove them, a link to toggle public/private/secret group designation and a form for editing club description text and the ability to operate other administrative functions.", "A group history utility 36 provides web pages which allow subscribers 4 to view of the recent history of the messages that have been sent in a particular group so as to evaluate whether or not to join and/or to catch up on missed messages.", "Group search result utility 38 provides web pages which list the results of a search conducted by subscriber 4 so as to list all of the groups which meet the search criteria (except for secret groups).", "Start group utility 39 provides web pages, as illustrated in FIG.", "2C which allows a subscriber 4 to create their own group.", "In order to create the group, create group utility 39 requests a name and description for the group, type of group (public, private or secret), who can send messages to the group, what category the group is in if any (for category searches), what geographic region the group is in and the names/handles of the other subscribers 4 the creator wants to invite to their group.", "A more complete description of the process involved in creating a group is described in more detail below.", "It should be noted that the above description of the utilities provided by group module 28 for performing the various processes available in system 2 regarding group functions are intended only as an example of one possible format for the utilities and is in no way intended to limit the scope of the present invention.", "Any similar utility architecture in a group module which can support member-group functions in a similar cross-platform messaging system 2 is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "3, channel services module 13 is comprised of several components including an alert module 20a, a messaging module 22a, channel module 50, a channel administration module 51, a channel services module interface 25a and a polling module 55.This list of the components of channel services module 13 is intended on as an example of some of the operational components of this module and is in no way intended to limit the scope of the present invention.", "Components may be added and deleted as necessary to accommodate consolidation, upgrade, new features and deleted features.. Any similar channel services module which operates in conjunction with a similar cross-platform messaging system is within the contemplation of the present invention.", "Alert module 20a, coupled to channel interface module 25a, is configured to provide alert messages to subscriber 4, displayed on the various channel web pages generated by system 2 as an alert button or panel on all of the pages required to provide alert notice functionalities to subscriber 4.In one embodiment of the present invention, alert module 20a provides alert buttons/panels on these web pages to inform subscribers 4 as to certain events and/or issues that need to be addressed.", "For example, such alert messages can include but are not limited to; unheard voice mail alerts, unvalidated device alerts, membership requests, record handle requests, vacation alert/no device in use, invitations to join a channel, etc.", "In such instants where an alert button is used for a subscriber 4, the alert notice is usually accompanied by a description of the alert and possibly a link to the appropriate web page where that alert can be properly addressed if applicable.", "In addition to alert buttons/panels displayed on web pages, alert module 20a is also configured to send alert messages to subscribers 4 based on their preferences.", "For example, if a subscriber wishes to be notified of a received voice mail, they may wish to have an alert notice sent to their mobile device alerting them to the message.", "Thus, in addition from posting the alert on the subscriber's 4 account in web page format in system 2, alert module also creates and sends SMS/e-mail text message alerts to the same subscriber 4, such that they are notified even before they logon to system 2.Send message module 22a, coupled to channel interface module 25a is configured to provide system 2 with the ability to allow subscribers 4 to send messages via a send message window 24 displayed on various channel web pages which require such message sending functionality.", "Message windows 24, as illustrated in FIG.", "2A, provides subscriber 4 the ability to create a message by filing in the appropriate windows and pressing a send button.", "As illustrated in FIG.", "2A, message window 24 is comprised of address book line 24a, group line 24b, message block 24c and send button 24d.", "Subscriber can set the addressee by simply using the pull down arrows in address line 24a and group line 24b.", "Message block 24c is configured to receive the text message, which is ultimately sent to addressee upon the activation of send button 24d.", "A more complete description of the function of message window 24 and how it is used in the overall operation of system 2 is discussed in more detail below.", "Channel module 50, coupled to channel service module interface 25a, is configured to support the channel functions of system 2 from a subscriber perspective channel module 50, maintains several utilities which support these functions which may include but are not limited to channel main page utilities 52, channel category utilities 54, channel details utilities 56, channel history utilities 58, and channel configuration utilities 59.Channel main page utilities 52 provides a web page which displays the all of the available channels, channel options as well as feature channels.", "Channel category utilities 54 list the existing channels by category.", "Channel details utilities 56 allow subscriber 4 to review the details of channel before signing up, such as how frequently messages are sent, and what sort of content they provide.", "Channel history utilities 58 provides web material, as illustrated in FIG.", "3A which allows subscriber 4 to review past messages on the channel in order to determine whether or not to join and/or to catch up on missed messages.", "In one embodiment of the present invention, channel configuration utility 59 allow a member subscriber 4 to specify their preferences regarding channel messages, such as how often they wish to receive information, and which particular information they want.", "Channel configuration utility 59 provides a web page, as illustrated in FIG.", "3A, which provides subscriber 4 with a number of choices as to which content they wish to receive and how often they would like to receive it.", "For example, web pages, as illustrated in FIG.", "3 provided by channel configuration pages include various types of messages supplied by the content provider which can be ordered or skipped by subscriber 4 when selecting their preferences.", "It should be noted that the above description of the utilities regarding the various processes available in system 2 regarding channel functions was intended only as an example of one possible format for the pages and is in no way intended to limit the scope of the present invention.", "Any similar web page architecture which can support member-channel functions in a similar cross-platform messaging system 2 is within the contemplation of the present invention.", "Channel provider module 51, coupled to channel service module interface 25a is configured to support the channel functions of system 2 from a channel content provider's perspective.", "Channel provider module 51, maintains several utilities which support these functions which may include but are not limited to send text message to channel utilities 70, send voice message to channel utilities 71, channel tools utilities 72, schedule channel messages utilities 73 and channel polling utilities 74.In one embodiment of the present invention, channel send text message utilities 70 and channel send voice message utilities 71 provide web functions which allow a channel provider to create and send messages to member subscribers 4.In addition to sending basic messages, channel providers may include direct or indirect links to their products via IVR subscriber module 21 and IVR database 23 such that content can be advertised delivered and sold to member subscribers 4 while they are logged in through WAP and/or IVR protocol sessions.", "A complete description of this process is described below.", "In another embodiment of the present invention, the channel operator can also utilize the channel scheduled message utilities 73 which provider greater versatility in creating messages in advance to be sent periodically based on the channel provider's instructions.", "The channel scheduled message utilities 72 also allow the channel provider the opportunity to re-arrange, delete, re-schedule, edit the message before they are sent.", "In one embodiment of the present invention, the channel tools utilities 72 are utilized by the channel providers to create and manage the content of their channel.", "These tools allow the channel provider to set their description, their logo, and their available configurations to send messages to these subscriber 4 (as it appears in the subscriber 4 side configuration utilities 59, described above).", "In another embodiment of the present message, channel poll utilities 74 provide web applications configured to allow the channel provider to create and manage an on-the-fly poll to be delivered to the member-subscribers 4 of that channel.", "The poll questions can range from movie reviews, to sports event outcomes or any other events relevant to that channel's content.", "The poll's interactive distribution and responses are handled by channel poll module 53 discussed below.", "It should be noted that the above description of the utilities provided by channel module 50 and channel provider module 51 for performing the various processes available in system 2 regarding channel functions was intended only as an example of one possible format for the utilities and is in no way intended to limit the scope of the present invention.", "Any similar utilities architecture in a channel module which can support member-channel functions in a similar cross-platform messaging system 2 is within the contemplation of the present invention.", "In one embodiment of the present invention, polling module 53, coupled to channel services module interface 25a and channel provider module 51, is configured to manage the dissemination and responses to polls conducted by the channel provider.", "Poll module 53 works in conjunction with other system 2 components such as WAP-SMS/IVR handler module 19, platform conversion module 9 and the various interface modules of system 2 to disseminate the polls in such a way that the responses can easily be handled by subscribers 4, regardless of the communication protocol that subscribers 4 is using.", "It should be noted that the above list of components in channel services module 13 is intended only as an example and is in now ay intended to limit the scope of the present invention.", "Any similar channel service module operating in conjunction with a similar cross-platform message system is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "4, subscriber information is stored in a subscriber information table 60 as stored in user database 16.Subscriber table 60 maintains all of the pertinent information about subscriber 4 that system 2 requires to properly handle the accounts of and send messages to its users.", "The information contained therein is supplied by the user either initially upon entering system 2 or it is supplied/edited at a later date.", "The information is processed through a web interface, where subscriber 4 can enter their information.", "Additional means for entering the information are available such as through WAP sessions or other protocols that are capable of adequately transferring the necessary information.", "In one embodiment of the present invention as illustrated in FIG.", "4, subscriber table 60 maintains multiple fields so as to organize the subscriber 4 information.", "Fields contained within subscriber table 60 may include but are not limited to: username field 61, personal information field 62 (including e-mail), PIN number field 63, device list field 64 (with validation flags), device/alert message preference fields 65a (text) and 65b (voice), group memberships field 66, channel memberships field 67, address book field 68, and message history fields 69.It should be noted that the examples listed above for fields in subscriber table 60 are intended only as examples, and are in no way intended to limit the scope of the present invention.", "Additional information fields can be added or redundant fields can be deleted provided the aggregate result provides sufficient information to support the functions of system 2.In one embodiment of the present invention, as illustrated in FIG.", "4, subscriber table 60 maintains a username field 61 is configured to store information regarding subscriber's 4 screen name also referred to as handle.", "This username is used through out system 2 to identify a subscriber 4 to other subscribers 4 without revealing their true identity.", "Personal information field 62 is provided, configured to store information regarding the real name, address, telephone number(s), e-mail addresses and other information of subscriber 4.This information is used for accounting purposes and to identify subscriber's 4 e-mail address if that is chosen as a preference for receiving messages.", "PIN (Personal Identification Number) field 63 is provided to store information regarding subscriber's 4 PIN used by subscriber 4 to gain access to their accounts in system 2.In one embodiment of the present invention, as illustrated in FIG.", "4, subscriber table 60 maintains device list field 64 configured to store information regarding all of subscriber's 4 devices that have been validated by system 2.Validated devices are S devices acknowledged by system 2 as compatible with at least some of the functions of system 2.Each listing in the validated devices field, maintains the contact number for that device the maker of the device and the service provider which operates the device.", "Device alerts/messages preferences field 65a (text) and 65b (voice) are configured to store information regarding which validated device subscriber 4 prefers to receive their messages on.", "For example, the device preferences field 65a(text) notes the device subscriber 4 wishes to receive their text messages and device preferences filed 65b(voice) notes which device subscriber 4 wishes to receive their voice messages.", "In one embodiment of the present invention, as illustrated in FIG.", "4, subscriber table 60 maintains group memberships field 66 configured to store information regarding the groups subscriber 4 is a member.", "Channel memberships field 67 is provided to store information regarding the channels subscriber 4 is a member.", "Address book field 68 configured to store information regarding all of the other subscribers 4, that a subscriber has contacted in the past.", "The information in address book filed 68 is stored based on the other subscriber's username information.", "Message history field 69 configured to store information regarding a number of the most recently received messages from groups and/or subscribers from system 2.Subscribers 4 enter and edit all of the information in subscriber table 60 through the use of interface web pages supplied by system 2.It should be noted that the above fields for subscriber table 60 are intended only as an example of one possible format for subscriber table 60 and is in no way intended to limit the scope of the present invention.", "Any similar subscriber table which can support subscriber account information on a similar cross-platform messaging system 2 is within the contemplation of the present invention.", "In one embodiment of the present invention as illustrated in FIG.", "5, a call completion data table 80 is provided configured to store a unique calling code for communications between any two subscribers 4.Call completion table 80 can be stored in several places including but not limited to user database 16, subscriber information table 60, message delivery subsystem 17 and/or any other data storage structure in system 2.Call completion data table 80 stores the necessary information to maintain a virtual number system such that message delivery subsystem 17 can complete a call between any two subscribers 4 using that virtual system.", "In one embodiment of the present invention, as illustrated in FIG.", "5, call completion data table 80 maintains several fields which may include but is not limited to, sending terminal field 81, virtual number field 82, and receiving terminal field 83.Sending terminal field 81 is configured to store information regrading a unique identifier or reference representing a originating terminal.", "Virtual number field 82 is configured to store information regarding a virtual numeric address as generated by system 2 from the existing trunk lines from the various service providers.", "Receiving terminal field 83 is configured to store information regarding a unique identifier or reference representing the addressee of the call A more detailed description of the operation of the virtual number system operated by message deliver module 17 using call completion data table 80 will be described in more detail below.", "It should be noted that the above description of the components of system 2 is intended only as an example of one design for system 2.However, any similar cross platform message system which utilizes similar components to perform similar group and channel functions is within the contemplation of the present invention.", "In Operation, Utilizing the structure and software architecture described above, subscribers 4 utilize system 2 to engage in mobile cross-platform personal message delivery community between subscribers 4, and in a mobile content and commerce network between subscribers 4 and channel providers.", "The various process used in performing these functions is describe below.", "In one embodiment of the present invention, as illustrated in FIG.", "6, subscriber 4 sets up an account with system 2.In this process at step 100, subscriber 4 enters system 2 and elects to create a new account.", "Next, at step 102, subscriber 4 enters the necessary information required to open an account, such that system 2 can create a subscriber information table 60 for that subscriber 4.This information includes but is not limited to username, personal information, and PIN.", "Subscriber 4 then proceeds to logging into system 2.", "(When subscriber 4 signs up with system 2 initially, after entering the required information at step 102, they are already considered logged in and would proceed with operations as if they were already at step 110.)", "At step 104, system 2 attempts to recognize subscriber 4 based on the protocol subscriber 4 is using to contact system 2.For example, if subscriber 4 is calling system to via IVR interface 14 then system 2 attempts to recognize the callers ANI (Automatic Number Identifier) such that it can retrieve the username from username field 61 of subscriber table 60 for that subscriber 4.Alternatively, if subscriber 4 is contacting system 2 via HTTP/WAP interface 8, system 2 may attempt to identify subscriber 4 using a coolie or other web based identification and memory device so as to automatically call up the username of subscriber 4.If the user name is, recovered automatically subscriber 4 proceeds directly to step 108.However if system 2 is unable to identify subscriber 4 automatically, at step 106, subscriber 4 enters their username as it is stored in username field 61.If the username is valid (ie.", "in the system), subscriber 4 continues to step 108.If the username is incorrect, subscriber 4 is returned to step 106 to re-enter their username.", "At step 108, subscriber 4 enters their PIN as it is stored in PIN field 63.If the information is correct then they proceed to step 110, However, if the PIN is incorrect, subscriber 4 is returned to step 108 to re-enter their PIN.", "After a valid PIN is entered, at step 110, subscriber 4 enters into their account at system 2.It should be noted that the-above described login procedure is intended only as an example of one login procedure that could be used by system 2 and is in no way intended to limit the scope of the present invention.", "Additional features can be added at this stage or features may be deleted provided the overall procedure still functions as a login procedure.", "Any similar login feature used in conjunction with a similar cross-platform messaging system is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "7, subscriber 4 joins a group, in system 2.In this process, at step 200, subscriber 4 enters group main page utility 30 and selects a desired group.", "This selection can be made by browsing the web pages provided by group main page utility 30 or subscriber 4 may search for a group based on some criteria and select a group based on the results produced in group search results utility 38.After selecting a group, at step 202, subscriber 4 enters group details utility 32 where subscriber 4 peruses the group information and decides in they selected the right group for themselves.", "Next, at step 204, subscriber 4 can either automatically join a public group or they can request to join a restricted group.", "Secrets groups will not be listed and can only be joined by the invite process which is discussed in more detail below.", "If subscriber 4 selects to join a open group then at step 206, subscriber 4 activates a join button and they are admitted to that group.", "System 2 subsequently distributes this information to all relevant places where it is stored such as on that group's group administration utility 34 and subscriber table 60, group field 66.Subscriber 4 can then proceed to step 216 and begin activity in the group.", "However, if subscriber 4 elects to join a restricted group, at step 208 subscriber 4 activates a request to join button which forwards their request to the group administration utility 34.At step 210, the group administrator/founder, determines if this subscriber is allowed to join their group.", "If subscriber 4 is allowed to join, then, at step 212, system 2 sends an alert via alert module 20 notifying subscriber 4 that they are now active and may proceed to step 216 to conduct group activity.", "If however, at step 210 the group administrator decides not to allow subscriber 4 to join their group, then at step 214, system 2 sends an alert to subscriber 4 via alert module 20 notifying them that they have been denied entry into the group.", "This terminates the process for subscriber 4 unless they attempt again from step 208.Assuming subscriber 4 gets into the group, at step 216, subscriber 4 is free to perform any allowable group function as determine by the group administrator.", "In one embodiment of the present invention, as illustrated in FIG.", "8, subscriber 4 joins a channel in system 2.In this process, at step 300, subscriber 4 enters channel main page utility 52 and selects an interesting channel.", "This selection can be made by browsing the web pages provided by channel main page utility 52 or subscriber 4 may search for a channel based on some criteria and select channel based on the results produced in channel category utility 54.After selecting a channel, at step 302, subscriber 4 enters channel details utility 56 where subscriber 4 peruses the information on the channel and decides in they selected the right group for themselves.", "Next, at step 304, subscriber 4 can automatically join the channel as all channels are public.", "Assuming subscriber 4 joins, system 2 subsequently distributes this information to all relevant places where it is stored such as channel tool utilities 72 and channel field 67 of subscriber table 60.After joining a channel, at step 306, subscriber 4 enters channel configuration utility 59, as illustrated in FIG.", "3A, and sets the parameters for which information they wish to receive.", "As channels often provide a large sum of information to chose from, subscribers 4 often limit their information received to relevant or interesting material.", "Next at step 308, after subscriber 4 enters their preferences, subscriber 4 closes the utility and is officially a member of the channel and will receive messages according to the parameters they selected.", "In one embodiment of the present invention, as illustrated in FIG.", "9, subscriber 4 enters system 2 and sets up a new group.", "In this process, at step 400, subscriber 4 enters group main page utility 30 and selects to be sent to create group utility 39, as illustrated in FIG.", "2C.", "Next, at step 402, subscriber 4 enters all of the necessary information on the pages provided by create group utility 39, as illustrated in FIG.", "2C, which may include but is not limited to: naming the group, picking an abbreviation, entering a brief description, selecting whether the group is public, private or secret, selecting who can send messages to the group (open or restricted to creator) and selecting categories (if applicable).", "It should be noted that, public groups are open to everyone and found on all public search results, private groups are open to public search but can only be joined with approval of the administrator of the group, secret groups are not open to public searches and can only be joined with approval from the creator/administrator.", "Next, after the parameters of the group are set, at step 404, subscriber may choose to send invites out to friends or at random in order to get members to join.", "The entire invite procedure is discussed below in more detail below in routine 500.After the group creator sends invites, at step 406, subscriber 4 selects the create group button and the group is now effectively open for operation (ie.", "Listed on group main page utility 30.At this point, and at any point in the future, at step 408 the creator subscriber 4 of the group may now enter group administration utility 34.Group administration utility 34, as illustrated in FIG.", "2B, provides web interfaces for creator subscriber 4 to edit and control the functions of their group.", "Thus, at step 410, subscriber 4 can perform any of the actions as detailed in FIG.", "2B which may include but are not limited to change group name, record group handle, change group message, boot unruly members, accept or decline requests to join, change the group from/to private/public/secret and/or disband the group.", "It should be noted that upon acceptance or denial to a group by group administrator, alert module 20, at step 411 sends and alert to that subscriber 4 notifying them of the results.", "After group creator subscriber 4 completes the editing/managing functions on administration utility 34, at step 412, subscriber 4 exits administration utility 34 and re-enters the general functions of system 2.In one embodiment of the present invention, as illustrated in FIG.", "10, subscriber 4 enters system 2 and invites a-non-member subscriber into a group.", "It should be noted that the invite procedure from step 404 above substantially runs this subroutine.", "In this process, at step 500, a group member subscriber 4 enters group main page utility 30 and selects the invite option.", "It should be noted that entering the invite process, can be initiated from several other locations within the software of system 2, however, this method has been chosen as an example, the steps to follow are identical, regardless of how the invite process was initiated Next, at step 502, a user selects a group that they wish to invite a non-member subscriber into.", "After selecting a group to invite the non-member to, at step 504, member subscriber 4 selects the non-member they wish to invite to that group.", "This selection can be any member of the system 2 community (ie.", "a subscriber 4).", "This member is selected based on non-member subscriber's 4 username as stored in their user name field 61.It should be noted that mass invitations can be implemented by simply adding additional usernames to the invite, however for the purposes of illustration one non-member subscriber 4 is invited.", "After the invitation is complete, at step 506, member subscriber 4, selects a send Is option which informs group alert module 20 to send a text invite.", "At this point two possible processes can occur.", "If the group is open to the public, then at step 508 alert message is sent directly to the non-member subscriber however if the group is private or secret then, at step 510, alert module 20 send the invite to the group administrator.", "If the group is public, system 2 proceeds to step 518, however if the group is private or secret then at step 512, group administrator enters group administration utility 34 and makes the determination to accept or decline the new non-member.", "If they are denied then, at step 514, the process ends.", "However, if the non-member subscriber is accepted, then, at step 516, alert module 20 sends a text alert to non-member subscriber 4 based the information contained in preferred format field 65a(text) in non-member's subscriber information table 60.Thus, even if member subscriber enters the invite via HTTP/WAP interface 8, the invite will be sent to non-member subscriber 4 in either e-mail, WAP/HTTP or SMS, based on the non-member's preference.", "At step 518, after receiving the invite alert from system 2, non-member subscriber 4 is afforded two option, either accept or decline the invite.", "If the non-member subscriber 4 declines the invite then at step 520 the process is terminated.", "However, if non-member subscriber 4 accepts the invite then the non-member is added to the group and the pertinent information is disseminated through system 2, such as information pertaining to group member field 66 and group administration utility 34.It should be noted that the above described invitation procedure is intended only as an example of one invite procedure that could be used by system 2 and is in no way intended to limit the scope of the present invention.", "Additional features can be added at this stage such as mass invites or invites to multiple groups, provided the overall procedure still functions as an invite to group procedure.", "Any similar invite feature used in conjunction with a similar cross-platform messaging system is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "11, subscriber 4 enters system 2 and sends a text message to another subscriber 4.In this process at step 600 subscriber 4 enters send message module 22 and, as such, enters message window 24, as illustrated in FIG.", "2A.", "It should be noted that there are a number of ways to reach send message module 22 because it appears as a sidebar on most utilities in system 2.Irrespective of the where subscriber 4 reaches sends message module 22, the operation of sending message is substantially the same.", "If, for example, subscriber 4 enters message window 24 from group details utility 32 it is possible that that group will be defaulted to as the addressee, but subscriber 4 can change that addressee as they choose.", "Next at step, 602, sender subscriber 4 selects which subscriber 4 to send a message to.", "This selection can be made from the address window 24a which is populated by the information in subscriber address field 68 of subscriber table 60, from group window 24b, which is populated by group membership field 66 of subscriber table 60 or the address can be selected by simply entering the desired username into address window 24a.", "Next, at step 604, subscriber 4 enters the text of the message into text window 24c of the message window 24.A maximum character length may be imposed at this stage.", "After the message is complete, at step 606, subscriber sends the message by activating the send message button 24d in window 24.It should be noted at this point that because of the cross platform nature of system 2, several means are available to send a text message via system 2.Subscriber 4 can enter the message in any format accepted by system 2 which is capable of receiving a text message.", "For example, text messages can be entered and sent via system 2 by way of WAP/HTTP (as described above), SMS, 2 way paging, and IVR (with text to speech capabilities).", "Thus, the above example of creating a text message is intended only as an example of one possible method of creating text message and is in no way intended to limit the scope of the present invention Any format accepted by system 2 which can facilitate the creation of text message is within the contemplation of the present invention.", "Next at step 608, system 2 reads the addressee information and determines to who and how to the message is to be delivered.", "If the message is to an individual, system 2 refers to that subscriber's device preference field 65a(text).", "If the message is for a group, system 2 determines all of the members of the group from group administration utility 34 and determines each one of their preferred device fields 65a(text) formats.", "After all of the recipient subscribers 4 are determined and their formats are acknowledged, at step 610, the message is forwarded by system 2 to platform conversion module 9 where the message is converted into all of the necessary formats required to complete delivery of the messages in each of the desired formats.", "Upon completion of the format conversion, at step 612 platform conversion module 9, delivers the message to all of the necessary interfaces 8, 10, 12 and 14 as required, and the message is delivered to each addressed subscriber 4.In one embodiment of the present invention, as illustrated in FIG.", "12, subscriber 4 sends a voice message through system 2.In this process, at step 700, subscriber 4 enters send message module 22.For voice messages send message module 22 accepts voice messages from IVR interface 14, or from a WAV file sent via HTTP/WAP interface 8.However, it should be noted that there are a number of ways to reach send a message module 22 for voice messages.", "For example, using speech-to-text conversions, system 2 may accept a voice message via message window 24 as described above.", "Also, in addition to IVR, and HTTP, message module 22 may also accept text to speech message input through other means than window 24 such WAP and SMS protocols.", "Irrespective of the how subscriber 4 reaches message module 22 for recording a voice message, the operation of sending message is substantially the same.", "However for the purposes of illustration a typical voice message will be created by a subscriber 4 interacting with system 2 via IVR interface 14, and, as such, this will be used as the example.", "Next at step, 702, sender subscriber 4 selects which subscriber 4 to send a message to.", "This selection can be made by following an IVR logic flow (ie.", "Press 1 for group XYZ, press 2 for group ABC, press the first four letters of the username etc.).", "If the voice message is being entered as text, subscriber 4 would just follow the procedures outlined above in routine 600.Next, at step 704, subscriber 4 enters the message by speaking it, as it as recorded by IVR interface 14 and stored in multimedia database 18.A maximum length may be imposed at this stage.", "After the message is complete, at step 706, subscriber sends the message by activating send message module 22 as indicated by the IVR.", "Next, at step 708, system 2 reads the addressee information and determines to who and how to the message is to be delivered.", "If the message is to an individual, system 2 refers to that subscriber's device preference field 65b(voice).", "If the message is for a group, system 2 determines all of the members of the group from the group administration utility 34 and determines each one of their preferred device fields 65b(voice) formats.", "After all of the recipient subscribers 4 are determined and their formats are acknowledged, at step 710, alert module 20 sends a text alert notifying recipient subscriber 4 of an incoming voice message.", "This alert is sent by alert module 20 to platform conversion module 9 where the message is converted into all of the necessary formats required to complete delivery of the messages in each of the desired formats.", "The alert is sent in text format and as such the delivery method is dictated by device preference field 65a(text).", "Upon completion of the format conversion, at step 712, platform conversion module 9, delivers the alert to all of the necessary interfaces 8, 10, 12 and 14 as required and the message is delivered to each addressed subscriber 4.Upon receipt of the alert, at step 714, subscriber 4 accesses system 2 in some method such as IVR or HTTP, and elects to either listen to the message or delete it.", "It should be noted that it is possible that subscriber 4 may prefer to receive voice messages via a speech to text message, which system 2 would determine from device preference 65b(voice) field of subscriber table 60.As such, it is in fact possible for a voice message to be originally created in text and eventually received in text.", "The distinguishing factor is that the sending subscriber 4 designated it as a voice message and the message is alerted rather than sent directly.", "It should be noted that the above described message sending procedures are intended only as an examples of message sending procedures that could be used by system 2 and are in no way intended to limit the scope of the present invention Additional features can be added at this stage or features may be deleted provided the overall procedures still function as a message sending procedure.", "Any similar message sending features used in conjunction with a similar cross-platform messaging system is within the contemplation of the present invention.", "In one embodiment of the present invention, as illustrated in FIG.", "13, subscriber 4 enters system 2 receives a multimedia content from a channel provider via WAP/SMS and IVR handler module 19.In this procedure IVR, WAP and SMS interfaces 14, 8 and 12 respectively, work in combination to provide an interactive streaming media function to subscriber 4 of system 2.As such, at step 800, a subscriber logs into system 2 via a WAP session (referring generally to any user currently logged into system 2 via WAP protocol).", "Next, at step 802, while subscriber 4 is navigating through the various menus of system 2, system 2 at the request of any number of channel providers sends an alert via alert module 22a, to “push” an alert to subscriber 4, notifying them of a multimedia file they may wish to receive.", "This multimedia file may be contained on multimedia database 18 if the “pushed” alert is from a channel or it may be contained m IVR database 23 if the message is arriving through system 2 via an outside source.", "Without disconnecting the WAP session, at step 804, WAP interface 8 and IVR interface 14 communicate via LAN connection platform conversion module 9 so as to to seamlessly deliver streaming multimedia files.", "At step 806, subscriber 4 is prompted to activate a soft key in order to receive the multimedia clip.", "This soft key is associated with a WA command to send some form of media.", "The logical control flow used for this message is identical between WAP interface 8 and IVR interface 14.This is made possible by the coordination of the two interfaces by WAP-SMS IVR handler module 19.It should be noted that additional choices could be provided which could allow for other options such as purchase of the multimedia, which would be handled in the same fashion.", "However for the purposes of illustration, the multimedia delivery is used.", "Assuming subscriber 4 decides to accept the invitation and activates the appropriate soft key, at step 808, WAP interface 8 automatically informs IVR interface 14 of an incoming call that is about to be placed from subscriber 4 mobile device.", "At step 810, subscriber's 4 mobile device automatically calls IVR interface 14 on the link provided by WAP interface 8.A similar method of operation for requesting steaming multimedia files is available for subscribers 4 who do not have WAP capabilities by using SMS in “passive mode.” In this mode, subscriber 4 is sent an invitation to receive a multimedia clip via an SMS text message with a special embedded IVR number.", "Simultaneously, SMS interface 12 notifies IVR interface 14 in advance of a potential multimedia request Assuming subscriber 4 accepts the invitation, subscribers mobile device calls the IVR special embedded IVR number provided in the SMS messages and using the initial information provided by SMS interface 12, IVR identifies subscriber 4 and sends the multimedia clip.", "At step 812, IVR interface uses a special routing number, locates subscriber 4, and delivers the multimedia file directly from database 18 or 23 to subscriber 4.These types of deliveries from IVR interface 14 can be based on messages generated in a WAP sessions or the can be based on a flag set in database 18 or 23 associated with subscriber 4 checked when IVR interface 14 background logs-in subscriber 4.Upon completion, at step 814, subscriber 4 is prompted by IVR interface 14 with several options regarding the multimedia files such as: purchase the CD; replay the clip; hear another clip; check out other features; or return to wireless internet (WAP) session.", "Subscriber 4 can either return to the WAP session in progress disconnecting them from the 1R or they can choose from the other options.", "In one embodiment of the present invention, as-illustrated in FIG.", "14, subscriber 4 places a call to another subscriber 4 and message delivery subsystem 17 creates a virtual number, completes the call and stores the virtual number for future use.", "This method utilizes a block of telephone numbers (addresses) in such a way as to uniquely identify each subscriber 4 by a single address on a service provider's network, using a total number of addresses from the service provider which is less than the total number of subscribers 4 on system 2.This feature is employed with a asynchronous text messages, however, that is in no way intended to limit the scope of the present invention.", "Any telephony communications utilizing this virtual addressing system such as with synchronous voice telephone calls is also within the scope of the present invention.", "Typically a provider allocates 10,000 address blocks, however, there are more than 10,000 subscriber 4 on system 2.The virtual addressing method provided by message deliver subsystem 17 on system 2 allows an arbitrarily large number of subscribers 4 to be addressed with a limited range of addresses.", "Thus, for each mobile device in system 2 that receives a message, there are 10,000 (based on this example) numbers which can be assigned.", "If a single user exceeds 10,000 incoming message sources, a least recently used (LRU) system can be implemented.", "As such, message delivery subsystem 17 of system 2, provides a means for each subscriber's 4 mobile device to use the same 10,000 number base to communicate even if more than 10,000 users exist.", "In operation, at step 900, subscriber 4(s) begins by placing a call to subscriber 4(r).", "The designations (s) and (r) are added for this feature to alleviate any confusion as to who the sender and receiver of the call are.", "The calls are managed through system 2 to maintain subscriber 4 anonymity via the use of their username, which necessitates the virtual numbering system.", "If subscribers 4 freely exchange their numbers then they are free to contact one another independent of system 2 in which case there is no need for this numbering system.", "The virtual numbering system described herein is only for subscriber 4 to subscriber 4 communication via usernames through system 2.Next at step 902, after system 2 has identified the sender and receiver, a three-part data structure call completion data table 80 is generated including the fields of: a unique identifier field or reference 81 representing the terminal (sender) on the affected network; a virtual numeric address 82; and a unique identifier field or reference 83 representing the individual who is addressed (receiver).", "This information is used to complete the initial call.", "At step 904, system 2 records call completion data table 80 such that it maintains a database of virtual addresses in either database 16 or in message delivery subsystem 16.Call completion data table contains references not only to the target mobile device of subscriber 4(r) (field 83) but also the mobile device of subscriber 4(s) (field 81) where the message originated.", "Only one address structure is stored in system 2 for any given combination of a numeric address, an originating device and a target device (ie.", "call completion data table 80).", "At step 906, the combined virtual number as contained in call completion data table 80 is stored in subscriber's 4(r) mobile device as a virtual number.", "Next, at step 908, subscriber 4(r) attempts a return call to subscriber 4(s), and system 2 identifies the incoming call as originating with subscriber 4(r) by way of a recognition function housed in IVR interface 14.Thus, at step 910, message deliver subsystem 17, attached to IVR interface 14, recalls call completion data table 80.Upon retrieval, message deliver subsystem 17, at step 912 initiates the return call using the virtual number stored in the directory in call completion data table 80.This virtual number, created when subscriber 4(s) initially called subscriber 4(r), is now used by system 2 to complete all future calls that originate from subscriber 4(r) back to subscriber 4(s).", "This process is repeated for all calls made to subscriber's 4 device until 10,000 incoming calls are reached.", "In one embodiment of the present invention, as illustrated in FIG.", "15, a content provider creates a poll and subscriber 4 responds.", "In this process, at step 1000, a content provider or channel operator enters channel toolset utility 72, where they enter create/manage poll utility 74.Next, at step, 1002, the channel or content provider enters the appropriate information into the required locations, providing the questions and the set of possible answers.", "It should be noted that although this features is generally referred to as a poll, the types of questions that can be responded to include any number of questions related to a particular channel provider's channel.", "For example, the proprietor of movie theater channel may wish to generate a poll which requests movie review results.", "Likewise, a sports related channel may wish to create a poll relating to a featured game, such as “which game would you like for us to feature on our show” Any type of question with a limited (multiple choice) response is within the contemplation of the present invention.", "Upon completion of the poll information, at step 1004, the channel or content provider activates the poll function and the information is sent to poll module 55 which administrates the poll.", "Next, at step 1006, system 2 distributes the poll questions and accompanying response options to subscribers 4 specified by the channel or content provider.", "In the event the content provider maintains a channel, the distribution list is most likely the member subscribers 4 to their channel.", "It should be noted that the existence of polls as type of information to be received is listed on subscriber 4 side channel configuration utility 59 such that subscriber 4 channel members can elect whether or not to receive poll questions.", "During distribution, poll module 55 works in conjunction with the information device preferences field 65a(text) of subscriber table 60 and with platform conversion module 9 to ensure the distribution occurs in the desired formats.", "Next, at step 1008, after subscriber 4 receives the poll question, they can respond to it using any one of the interfaces 8, 10, 12 or 14 provided that the format supports a means to communicate the multiple choice response (most formats).", "At step 1010, after system 2 receives the response, the information is forwarded to poll module 55 for compilation of the results.", "Upon completing the compilation of the results, at step 1012, poll module 55 sends the results back to the content or channel provider where they are stored for viewing in poll utility 74.In another embodiment of the present invention, poll module 55 and poll utility 74 can work in conjunction with scheduling channel utility 73 such that a new poll can be disseminated automatically on a regular basis provided the content is set in advance.", "This would allow a content or channel provider to create polls well in advance and send them on a schedule or possibly even send the same poll repeatedly, assuming the content for the poll is not stale.", "It should be noted that the above described polling procedure is intended only as an example of one polling procedure that could be used by system 2 and is in no way intended to limit the scope of the present invention.", "Additional features can be added at this stage or features may be deleted provided the overall procedure still functions as a polling procedure.", "Any similar polling feature used in conjunction with a similar cross-platform messaging system is within the contemplation of the present invention.", "While only certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes or equivalents will now occur to those skilled in the art It is therefore, to be understood that this application is intended to cover all such modifications and changes that fall within the true spirit of the invention." ] ]

[ [ "Intravascular ultrasonic catheter arrangements", "Apparatus for intravascular ultrasonic imaging comprises a catheter having an ultrasonic transducer array fabricated at least in part from an electrostrictive material, wherein the electrostrictive material is non-polymeric." ], [ "1.An apparatus for intravascular ultrasonic imaging, the apparatus comprising: a catheter having an ultrasonic transducer array fabricated at least in part from non-polymeric electrostrictive material; and a high permitivity ceramic member or layer that couples a common radiofrequency source to elements of the array.", "2.The apparatus of claim 1 further comprising controls for energizing the array and processing signals derived therefrom, the controls being adapted to apply a bias voltage to elements of the array to render those elements substantially piezoelectric.", "3.The apparatus of claim 1 wherein the electrostrictive material is a relaxor ferroelectric material.", "4.The apparatus of claim 3, wherein the electrostrictive material comprises lead magnesium niobate.", "5.The apparatus of claim 2, wherein the controls are adapted to transmit and receive signals only while the bias voltage is applied.", "6.The apparatus of claim 1 further comprising a high permitivity ceramic member or layer that couples a common radiofrequency source to the elements of the array.", "7.The apparatus of claim 1 further comprising a multiplexer arrangement through which the transducer array is energized and through which echo signals received by the transducer array are passed to the proximal end of the catheter, when in use.", "8.The apparatus of claim 7 wherein the multiplexer arrangement comprises a plurality of integrated circuits.", "9.The apparatus as claimed in claim 8 wherein the integrated circuits are arranged in substantially cylindrical configuration.", "10.The apparatus of claim 8 wherein adjacent integrated circuits are spaced from one another by a slot.", "11.The apparatus of claim 8, wherein the integrated circuits are flip-chip bonded to an electrical circuit.", "12.The apparatus of claim 11 wherein the electrical circuit is a printed circuit.", "13.A method of manufacturing a catheter for intravascular ultrasonic imaging having an ultrasonic transducer array fabricated at least in part from a non-polymeric, electrostrictive material, the method comprising: fabricating the transducer in-the-flat; the transducer array comprising a high permitivity ceramic member or layer that couples a common radiofrequency source to elements of the array; and reconfiguring the array into a substantially cylindrical configuration.", "14.The method of claim 13 further comprising electrically coupling controls to the array for energizing the array and processing signals derived therefrom, the controls being adapted to apply a bias voltage to elements of the array to render those elements substantially piezoelectric.", "15.The method of claim 13, wherein fabricating the transducer array in-the-flat further comprises: providing a substrate; forming electrically conductive tracks on the substrate; bonding a piezoelectric block to the conductive tracks; and forming slots in the piezoelectric block to form a plurality of discrete transducer elements.", "16.The method of claim 15, further comprising coupling at least one multiplexer circuit to the conductive tracks.", "17.The method of claim 14, wherein the electrostrictive material comprises a relaxor ferroelectric material.", "18.The method of claims 16, wherein the relaxor ferroelectric material comprises lead magnesium niobate.", "19.The method of claim 15, further comprising forming slot bottoms in the substrate adjacent the electrically conductive tracks prior to bonding the piezoelectric block to the conductive tracks.", "20.The method of claim 19, wherein forming slot bottoms comprises forming slot bottoms with a laser.", "21.The method of claim 19, further comprising performing a photoresist technique within the slot bottoms." ], [ "The present invention relates to intravascular ultrasonic catheter arrangements and more particularly to the construction and manufacture of an ultrasonic transducer array for mounting at or near the distal end of a catheter arrangement.", "Examples of the types of intravascular ultrasonic catheter arrangements to which the present invention may be applied are disclosed in our earlier United Kingdom Patent Nos.", "2,221,267; 2,233,094 and our U.S. Pat.", "Nos.", "5,081,993; 5,257,629 and 5,456,259.With the kinds of ultrasonic transducer array to which the present invention relates, its very small size (typically one millimetre in diameter) means that there are considerable technological problems to overcome in order to firstly make it possible to manufacture the array at an acceptable yield level and secondly to provide the array and its associated control circuitry/software with an acceptable performance particularly as far as the definition of images obtained is concerned.", "Ultrasound arrays to which the present invention is applicable have typically employed piezoelectric materials such as modified PZT (lead zirconate titanate) for the transduction of a radio frequency (rf) electrical signal into an ultrasonic signal.", "In very high frequency applications, such as are relevant to the present invention, the performance of such piezoelectric arrays is limited by the grain size of the piezoelectric ceramic material.", "This is because, due to the very small size of the array, the grain size of the material begins to become comparable with the dimensions of the array elements.", "Furthermore, because of the crystalline nature of the material from which the elements of the array are manufactured, the manufacturing operations such as lapping, polishing, dicing and electroding (ie securing electrodes to the elements of the array) introduce defects which are associated both with the bulk body of the transducer element material and its surface.", "In particular microcracks are generated which significantly reduce the macroscopic fracture resistance of the material.", "One object of the present invention therefore is to overcome or reduce the above mentioned manufacturing problems whilst at the same time not prejudicing the operational performance of the ultrasonic transducer arrangement.", "In order to try and overcome the above discussed problems the inventors have therefore researched alternative materials for the manufacture of ultrasonic transducer elements and have concluded that a class of materials that would be suitable are those known as electrostrictive materials such as “relaxor ferroelectrics”.", "It has been found that these can exhibit a large pseudo-piezoelectric response if a suitable d.c. electric bias field is applied to them.", "These materials have a finer microstructure than the known PZT material discussed earlier together with enhanced fracture toughness.", "An example of a relaxor ferroelectric material is modified PMN (lead magnesium niobate).", "As indicated these relaxor ferroelectrics can exhibit a very large pseudo piezoelectric response, typically d33 −3,000 pC/N.", "In addition such materials are available with high values of permitivity (eg er-12000), with small grain size (eg 1-2 μm) and with improved fracture toughness.", "High values of permittivity will, in general, allow improved ultra-small array elements from the interrogating electronics by reducing the element impedance.", "Fine grain microstructures will reduce surface microcrack dimensions and thus the overall tendency to fracture.", "A further advantageous feature of an electrostrictive material is that it is unpolarised in the absence of the bias field.", "This means that processing steps involving heat and high local stresses will not result in the polarising degradation which can occur with piezoelectric materials such as PZT.", "Thus according to a first aspect of the present invention, in an ultrasonic transducer array arrangement suitable for mounting on a catheter, the elements of the transducer array are manufactured from a non-polymeric electrostrictive material.", "According to a second aspect of the present invention the control arrangement for energising such an array and processing signals derived therefrom comprises means for applying a bias voltage to the elements of the array, to render those elements significantly piezoelectric, and means for transmitting and receiving signals in relation to those transducer elements only when the bias voltage is being applied.", "With this arrangement it is thus possible to apply the rf signals simultaneously (but not continuously) to all elements of the array because only those transducer elements which have the bias field applied to them will transform the rf signal into the correct ultrasonic signal.", "The drive and interrogation of the array is thus by means of the multiplexed bias voltage which opens “windows” during which the rf signal can be effective.", "The rf signal is turned off during the receive interval for a given channel in the usual manner.", "The advantage of coupling the rf signal in this way are (i) the signal losses in the multiplexer are now only seen during the receive mode and (ii) the rf signal size is not limited by the multiplexer.", "So far the present invention has been discussed in relation to the material from which the transducer elements are manufactured and this aspect of the invention is applicable irrespective of other steps in the manufacture of the ultrasonic transducer arrangement.", "There will now be discussed a further aspect of the present invention which relates to the manufacturing process employed for producing an ultrasonic transducer array of the kind previously outlined, this further aspect being independent of the material employed for the manufacture of the transducer array elements.", "One method of manufacturing an annular transducer array of the kind to which the present invention relates is disclosed in our European Patent No 0 671 221 in which the transducer array, and its associated multiplexer/control circuitry, is manufactured in-the-flat initially and then transformed into a cylindrical configuration.", "In such a manufacturing method the functionally independent elements of the eventual annular array are produced by the following method.", "A single block of PZT is mounted on a substrate in-the-flat.", "A plurality of saw cuts are then made in the PZT block to define the individual transducer elements of which there are typically sixty-four.", "The arrangement thus produced is then folded into the final cylindrical configuration to thus produce an annular ultrasonic transducer array made up of the plurality of transducer elements.", "In order to facilitate folding step, the aforementioned saw cuts are continued down to some extent into the polyimide substrate on which the transducer material is mounted.", "This extent is critical because it has to be sufficient to allow the assembly to be folded easily whilst at the same time not weakening the substrate to the point where it will fracture.", "Because of the extremely small size of the arrangement just described the sawing step in the manufacturing process has to be carried out to extremely small tolerances.", "In addition, for the best operating characteristics, the saw cuts should have flat-bottoms but this is difficult to achieve in practice.", "The bottom of the saw cut is hereinafter referred to as the “slot bottom”.", "Therefore, according to a further aspect of the present invention in a manufacturing process for producing an ultrasonic transducer array in which a block of transducer material is divided into a plurality of discrete transducer elements and is mounted on a flexible substrate, flexing slots are formed in the substrate between adjacent transducer elements by means of a laser beam, This enables the depth of the cut to be more easily controlled, when compared with using a saw, and it also makes it possible to more easily approach the ideal rectangular shape for the bottom of the slot, or “slot bottom”.", "How the invention may be carried out will now be described by way of example only with reference to the accompanying drawings in which: FIG.", "1 is a perspective view of the ultrasonic transducer/multiplexer assembly in-the-flat as shown in FIG.", "4 of the applicant's European Patent No 0 671 221; FIG.", "2 is a perspective view of the assembly of FIG.", "1 in its final cylindrical configuration as shown in FIG.", "8 of the applicant's European Patent No 0 671 221; FIG.", "3 is a fragmentary perspective view to an enlarged scale of one embodiment of the present invention showing part of a transducer array and associated multiplexer arrangement in the flat condition prior to its wrapping into the its cylindrical configuration; FIG.", "4 illustrates the basic electronic address circuit employed with the present invention; and FIGS.", "5 to 10 illustrate the various stages, according to the present invention, for forming the slot bottoms in the ultrasonic transducer.", "FIGS.", "1 and 2 A transducer array 110 and multiplexer 111 arrangement is first manufactured in-the-flat as shown in FIG.", "1.It is then wrapped or rolled into the cylindrical configuration shown in FIG.", "2.The transducer array 110 comprises sixty four transducer elements 112 which are electrically connected to four 16-channels multiplexer chips 111a, 111b, 111c and 111d (111b being omitted for clarity and 111c being only partially shown) each chip being in the form of an integrated circuit.", "The advantage of initially manufacturing the assembly shown in FIG.", "1 in-the-flat is that it is easier to manufacture because firstly forming the various components in-the-flat rather than on a cylindrical surface is inherently easier and secondly it is possible to use standard production equipment.", "More particularly standard printed circuit and integrated circuit production methods can be employed.", "A further advantage, is that the thickness of flat material is easier to control to high accuracy than the wall thickness of cylindrical components.", "The transducer array 110 consist of functionally discrete ceramic elements mounted on a flexible substrate 113.Each multiplexer 111a, 111b, 111c and 111d, is in the form of an integrated circuit and this integrated circuit can itself be flip-chip bonded to a circuit comprising electrical connections 114 which are formed on the substrate 113 by means of known printed circuit techniques.", "The transducer array 110 which consists of functionally discrete ceramic elements, is manufactured using the following steps.", "The polyimide substrate material 113 is plated on both sides, with a 1-2 micron thickness of copper, typically by a two stage process in which vacuum deposition or sputtering is used to give a thin base coat of good allocation, and chemical plating techniques to increase the copper thickness to the desired value.", "The conductive tracks 114 are then formed in the layer of copper on one side of the substrate by a standard photolithography technique followed by chemical etching or ion-beam milling to form the circuit pattern.", "A block of piezo-electric material 112 having the desired radial thickness of the final transducer elements and coated on both sides by a metallisation layer, is bonded in one piece to an area of the copper layer which is shaped to define a connection pad on the substrate.", "The bonding is effected by a suitable adhesive which could comprise a low viscosity epoxy resin.", "The polyimide substrate 113 has a copper layer on its bottom surface.", "The piezo-electric transducer array, in use, would be energised through the copper layer, the upper metalised layer on the top of the piezoelectric ceramic transducer block 112 forming an earth return path and being electrically connected to the other copper layer to thus form a common return path.", "The substrate 113 is provided with slots 115 to facilitate the folding or wrapping of the substrate into a cylindrical configuration as shown in FIG.", "2.In FIG.", "2 the same reference numerals have been used as in FIG.", "1 in order to designate the same items.", "The cylindrical transducer array and multiplexer arrangement is mounted on a flexible plastic tubular body member 201 which itself is mounted on the main flexible plastic tubular body of the catheter (not shown).", "The usual guide wire is shown at 202.FIGS.", "3 and 4 The transducer array and associated multiplexers are substantially the same in the present invention as those disclosed in our published European Patent No 0 671 221 and illustrated in FIGS.", "1 and 2, except in the following respects.", "The transducer array 310 itself is fabricated from a PMN (lead magnesium niobate) and the polyimide substrate 313 carries a block 316 of high permitivity low loss ceramic associated with each of the four multiplexers 311 (only 311b is shown).", "Each block 316 functions as a common rf connection between the associated multiplexer 311a, b, c and d and the associated group of sixteen transducer elements for coupling the rf signal to the transducer array input tracks 314.In a modification each ceramic block could be divided into sixteen physically articulated sections, one for each channel associated with each of the sixteen transducer elements.", "The block 316 capacitatively couples the mulitplexer 311b to the individual channels of the array 310 by means of a high permitivity ceramic layer.", "The transducer array 310 may be made from an electrostrictive or equivalent ferroelectric relaxor material.", "The multiplexer arrangement 311 is configured to transmit a DC bias voltage to the array 310.FIG.", "3 illustrates a single quadrant of the IVUS catheter and one method of coupling the rf signal to the array.", "This method is directly applicable to the existing configuration of the “wrap”.", "The principle of operation is as follows.", "PMN requires a bias field to become significantly piezoelectric and thus the PMN elements only transmit and receive when the bias voltage is applied through the multiplexer 311.The rf signals can therefore be applied simultaneously (but not continuously) to all elements of the array 310; because only those transducer elements which have the bias field applied to them will transform the rf signal into an ultrasonic signal.", "The drive and interrogation of the array 310 is thus by means of the multiplexed bias voltage which opens windows during which the rf signal can be effective.", "Clearly the rf signal but not the dc bias is turned off during the receive interval for a given channel.", "Advantages of coupling the rf signal in this way are (i) the signal losses in the multiplexer 311 are now only seen in receive and (ii) the rf signal size is not limited by the multiplexer 311.FIG.", "4 shows the essentials of the electronic addressing of two channels of the array.", "FIG.", "4 illustrates part of the control circuit for two of the transducer elements 410a and 410b.", "The multiplexer 411 is configured to transmit a DC bias voltage 417 to the elements of the array 410.The high permitivity ceramic block 416 functions as a common rf connection between the multiplexer 411 and the array 410 and capacitatively couples the latter to the former.", "U PZT can act as such a component.", "FIGS.", "5 to 10 Existing array-dicing processes have problems with height-control and the shape of the slot bottom.", "The height-control problem is exacerbated by: (i) the non-uniformity of the polyimide substrate; (ii) the difficulties of vacuum-mounting an undulating, flexible substrate containing rigid components with stressed adhesive interfaces; and (iii) dicing-blade wear.", "The slot bottom problem relates primarily to the rounded shape of the cutting edge of the dicing blade which is reproduced approximately in the array slot-bottom.", "Flat-bottomed blades are difficult to achieve in practice because both the blade-dressing procedure prior to dicing and the blade-wear of the dicing process itself tend to yield a rounded blade-edged profile.", "The rounded profile, leading to a similar shape in the polyimide slot bottom, is undesirable from the point of view of the wrap-mechanics and acoustic performance of the final array.", "Furthermore, any subsequent processing of a saw-cut polyimide slot bottom (e.g.", "by laser ablation) would necessarily begin from the “parabolic” shape left by the sawing process, and may not result in the desired rectangular shape.", "The process issues to be addressed are therefore: (i) how can shape-controlled slots be cut into a thin, flexible and variable carrier film?", "(ii) can a pre-cut flex-circuit slot bottom, be ablated accurately using the flex-circuit itself as a mask?", "The present invention relates to the fabrication of rectangular slot bottoms in the polyimide substrate by a laser ablation process prior to array dicing.", "The flex-circuit slot bottom is ablated using the flex-circuit itself as a mask, which automatically aligns the slot bottoms with the flex-circuit.", "The following process achieves a rectangular slot bottom in the polyimide.", "The flex-circuit is utilised as a mask for laser ablation.", "The transducer area of the flex-circuit contains copper tracks 51 of width equal to the element width, namely 30 μm formed in a polyimide matching layer 50.These tracks 51 in conjunction with a rectangular aperture step 52, defining an overall exposure window, are used as a mask for laser ablation of rectangular trenches 53 of width ˜17 μm in the polyamide flex-circuit.", "The intensity-time exposure parameters for the laser yield reproducible slot bottoms 54.It may be advantageous to thicken the tracks 51 to 1-2 μm by use of nickel plating in the transducer area, as on the remainder of the flex-circuit.", "The whole flex-circuit 51 is coated with a layer of photoresist 55 ˜5 μm thick using a spinning technique, giving the result illustrated in FIG.", "7.It is only necessary that the trenches 53 be partly filled with the photoresist material 55.The photoresist layer 55 is ablated and the ˜5 μm thick layer 55a covering the metal tracks 51 is removed.", "This will leave a photoresist layer 55b in the trench-bottoms, as illustrated in FIG.", "8.The same result can also be achieved by “wicking” photoresist (or an alternative slot bottom fill substance) along the trenches (i.e.", "relying on capillary action to cause the resist to move along the trenches to substantially fill them) from one end, or by selective ultra violet curing of the photoresist layer means of an auxiliary mask.", "Having accurately formed the slot bottom 54 using a laser (FIGS.", "5 to 8), the PZT transducer array is then fabricated on top of the arrangement shown in FIG.", "8.This fabrication is illustrated in FIGS.", "9 and 10.A PZT block 91 is bonded by adhesive 92 to the layer 50 using known adhesive techniques.", "This is illustrated in FIG.", "9.The adhesive 92 will fill the trenches 54 above the photoresist layer 55b and will not penetrate to the bottom of the slot bottom 91a, 91b, 91c, 91d etc.", "There is an upper metallisation layer 93 topped by a polyimide ground plane 94.The individual PZT elements of the transducer array are diced in the normal way using a diamond saw to create the slots, ensuring the blade penetrates the polyimide by approximately two thirds of its kerf depth (e.g.", "10 μm if the polyimide kerf is 15 μm deep).", "This is illustrated in FIG.", "10, and shows the saw cuts penetrating into the temporary key-fill material (photoresist) 55b, the saw being indicated at 101 in broken lines.", "The polyimide slot bottoms are cleaned with a suitable solvent to remove the residual photoresist 55b and create an empty flat bottomed slot bottom.", "The advantages of the modified process are the following: (i) A reproducible slot bottom can be created in the polyimide without melting or tearing of the plastic and without the risk of depth overshoot inherent in the existing sawing process.", "That is, the laser will ablate a certain thickness of polyimide irrespective of whether or not the flex-circuit is flat, whereas the sawing process depends critically on mounting-fixture flatness, flex circuit planarity and flex-circuit thickness variability.", "(ii) A rectangular slot bottom is the optimum for strain relief on wrapping and leads to significantly reduced risk of PZT fracture of wrapping stresses.", "(iii) A rectangular slot bottom is in many cases the optimum for reduction of acoustic cross-talk.", "(iv) The modified process removes the need for stringent height-control in the dicing process, and thus removes a demanding process parameter involving significant cost.", "As a further aspect of the present invention there will now be described a modification of the transducer fabrication technique which could address both the shape and depth of the slot bottom.", "This modified method is as follows: (i) Double-metalize the ground-plane polyimide and wax both this and the ceramic spacer material to a ceramic carrier block.", "The ceramic carrier will be a low-density, low-cost PZT material, or some other machineable ceramic such as Macor or Shapal.", "The wax will be of a type soluble in a safe organic solvent, and capable of being pressed under controlled, elevated-temperature, conditions into a thin, uniform layer.", "(ii) Bond the pre-poled, metalized, transducer block, of dimensions 3.2×0.8×0.05, to the ground-plane electrode and, the ground-plane electrode to the spacer using Hysol epoxy in a purpose-built assembly press.", "(iii) Dice the transducer block whilst on the sacrificial ceramic carrier.", "The slot bottom depth will be such as to penetrate the ceramic carrier by a few tens of microns.", "By this means parallel-sided cuts in the PZT and ground-plane are achieved and each full array width is defined.", "(iv) Invert the ceramic carrier block, ground-planes and transducer plates over the polyimide matching layer (flex-circuit) and epoxy bond in a temperature and pressure controlled press.", "This step involves accurate alignment of the transducer array and the flex-circuit.", "The alignment is achieved by dicing through the carrier ceramic block at, or close to, the perimeter of the transducer array, in order to allow the individual positioning of these arrays on the flex-circuit diaphragm.", "Alternative bonding processes are flip-chip solder and indium-gold.", "Epoxy-bonding would involve excess adhesive which may interfere with the laser ablation process.", "(v) Remove the ceramic carrier by immersion of the flex-circuit in the chosen solvent.", "Remaining on the flex-circuit are the diced PZT arrays with their diced ground-planes, each array bonded to the polyimide flex circuit.", "Since the exposed surface of the ground-plane is metalized it acts as a reflector of the laser beam, whereas the polyimide does not.", "(vi) Use the diced PZT-array and ground-plane assembly as a mask for ablation of the polyimide flex-circuit.", "Since the exposed surface of the ground-plane is metalized it acts as a reflector of the laser beam, whereas the polyimide in the slot bottom is exposed to the full-power of the laser and is ablated.", "Clearly, since the slot bottom shape is rectangular before ablation, there is a much greater chance of controlling the thinning to give, say, a 0.005 uniform layer of polyimide remaining after ablation.", "An alternative to this is to ablate the flex circuit before assembly using the tracks in the transducer area as a mask." ] ]

[ [ "Vehicle signaling device", "A signalling aapparatus for a motor vehicle is provided with communication means for receiving information coming from another motor vehicle and for broadcasting information intended for a nearby motor vehicle, and with sensor means for providing inforation about the status or status change of thr respective motor vehicle with regard to at least the safety of this vehicle.", "A computer is present for determining, on the ground of the information coming from the sensor means and/or on the ground of the received information, information to be broadcast and further action signals.", "In addition, means are present to enable, on the ground of the action signals, realization of a status change of the respective motor vehicle, which means are formed by a signalling element for making observable a signalling knowable for the driver of the vehicle or of a nearby vehicle." ], [ "1.A signalling apparatus for a motor vehicle, provided with communication means for receiving information coming from another motor vehicle and for broadcasting information with regard to at least the safety of the first-mentioned motor vehicle to a further nearby vehicle, characterized in that sensor means are present for providing information about the status or a status change of the respective motor vehicle with regard to at least the safety of this vehicle, as well as a computer which is arranged for generating action signals and information to be broadcast, on the ground of a combination of the information provided by the sensor means and information received from a nearby vehicle, and -means to enable, on the ground of these action signals, initiation of a status change of the respective vehicle or of a nearby vehicle, which means comprise a signalling element for making observable a signalling knowable for the driver of the respective vehicle or the nearby vehicle.", "2.A signalling apparatus according to claim 1, characterized in that the computer is arranged to generate combined information for the information to be broadcast by weakened transfer of an extent of exceptionality of the information received from the nearby vehicle and/or enhancement of the extent of exceptionality of the combined information depending on the information provided by the sensor means.", "3.A signalling apparatus according to claim 1, characterized in that the communication means are arranged on the front side and rear side of the vehicle.", "4.A signalling apparatus according to claim 1, characterized in that the communication means comprise at least one infrared transmitter and infrared receiver.", "5.A signalling apparatus according to claim 1, characterized in that the communication means are formed by connecting means which enable a line-of-sight connection between two vehicles driving within view of each other, as well as radio connecting means, wherein, when according to the line-of-sight a connection between two vehicles driving within view of each other has been established, a data transmission by the broadcasting vehicle to the receiving vehicle is realized with the aid of the radio connection.", "6.A signalling apparatus according to claim 1, characterized in that the information to be broadcast is formed by a single safety status signal.", "7.A signalling apparatus according to claim 6, characterized in that in the vehicle a display is present for displaying, on the ground of a received safety status signal, a warning signal for the driver of this vehicle.", "8.A signalling apparatus according to claim 6, characterized in that at the back of the vehicle, a display is present for displaying, on the ground of a received signal and on the ground of signals produced by the sensor means, a warning signal for the driver of a following vehicle.", "9.A signalling apparatus according to claim 6, characterized in that the vehicle is provided with brake lights for displaying, on the ground of a received safety status signal, a warning signal for the driver of a following vehicle.", "10.A signalling apparatus according to claim 7, characterized in that the warning signal is in the form of a warning triangle which lights up to an increasing extent as to its dimensions according as the safety status signal represents a greater danger.", "11.A signalling apparatus according to claim 6, characterized in that the means for enabling a status change of the vehicle or of a nearby vehicle are formed by brake energization means controlled by the safety status signal.", "12.A signalling apparatus according to claim 1, characterized in that the sensor means comprise means indicating the position of the brake pedal.", "13.A signalling apparatus according to claim 1, characterized in that the sensor means comprise means determining the speed and/or the acceleration of the vehicle.", "14.A signalling apparatus according to claim 1, characterized in that the sensor means comprise a global positioning system, which functions as sensor for measuring at least the position of the respective vehicle." ], [ "The present invention relates to a signalling apparatus for a motor vehicle, provided with communication means for receiving information coming from another motor vehicle and for broadcasting information with regard to at least the safety of the first-mentioned motor vehicle to a further nearby vehicle.", "A well-known phenomenon on highways where traffic density is high and vehicles are driving close to each other, is that at a system level a wave motion is involved.", "The ‘nodes’ here represent a locally high traffic density and the ‘troughs’ represent a relatively empty road section.", "The individual motorist experiences this phenomenon as disturbances in the form of sudden speed reductions of vehicles in front of him.", "When a driver is not sufficiently alert and does not react sufficiently fast, this leads to dangerous situations with an accident as a possible result.", "Partly prompted by society's wish to that effect, much attention is paid to safety in the design of vehicles.", "This especially concerns passive safety, that is, safety aimed at minimizing the consequences of an accident.", "Improving passive safety is inherently realized at the level of a single vehicle.", "A well-known example is the crushable zone.", "Efforts in the field of active safety, that is, safety aimed at preventing accidents, take place at different levels.", "First, at a global level, that is, with regard to a whole road section.", "This has resulted in particular in infrastructure adaptations.", "Thus, at present, via electronic motorway signals, a lane can be closed off in case obstacles are located there.", "A second level at which active safety is realized is the vehicle level; safety is then focused on an autonomous vehicle.", "Thus, for instance, in the '90s, the third brake light was introduced.", "This makes it possible for drivers to observe a braking action not only of the vehicle immediately downstream, but also of vehicles further downstream.", "Thus, drivers can anticipate dangerous situations as mentioned above.", "An important disadvantage of the third brake light is that very often it is not possible to observe the brake light of several vehicles.", "The view is obstructed, for instance, by truck traffic or other large vehicles.", "Moreover, weather conditions may lead to strongly reduced visibility.", "However, experience shows that, also in situations where these physical impediments do not occur, the number of observable brake lights is always small.", "In addition, the information content is not high: the brake light only indicates that the vehicle is being braked, but not to what extent.", "As a consequence, by way of the third brake light of other vehicles, a driver only gets a very limited picture of the traffic situation in this system.", "This makes it difficult to realize a cooperative driving behavior, which is necessary to realize active safety.", "In the above, it has been assumed that the driver is alert.", "However, under normal circumstances too, there are always shorter or longer periods of inattention, for instance due to distraction or fatigue.", "A development addressing this problem has yielded so-called advanced driver assistance (ADA) systems.", "This includes inter alia autonomous cruise control (ACC); this is a system which, in addition to the normal ‘cruise control’ function, is also capable of automatically maintaining a desired distance to the vehicle in front.", "ADA systems are focused primarily on increasing comfort.", "The vehicle autonomously tries to form a picture of its immediate environment This information is obtained with the aid of on-board sensors, while additional information can be obtained via infrastructure-bound communication systems (radio, GSM, and the like).", "From this abundance of signals, a status, that is, the condition of the vehicle, must be obtained, which leads to a possible intervention in the control of the vehicle, such as braking, steering, and the like.", "The intervention performed is only in the interest of the ADA vehicle.", "It is therefore highly questionable if ADA systems can enhance the safety of a group of vehicles on the road.", "For the above reason, it has been sought to make active safety possible at a third level, between the global level and the vehicle level, that is, at a safety level pertaining to a group of vehicles, that is, a vehicle system, with an associated driving behavior.", "Thus, a signalling apparatus as described in the opening paragraph hereof, operative at this third safety level, is known from the French patent specification FRA-2,624,454.This known apparatus is intended, on the ground of received information broadcast by a vehicle in front, to adjust the lighting intensity of the lights of the respective vehicle, in other words, to realize one specific status change of the respective vehicle, and further to transmit adapted information to a following vehicle.", "The respective information is therefore very limited here, while automatically one specific status change of the respective vehicle is realized.", "In addition, a warning signal is transmitted from one vehicle to the next.", "The object of the invention is to expand such a known system and, with the aid of supplementary measures, to farther enhance active safety, allowing status changes to be realized on a larger scale.", "According to the invention, to that end, the signalling apparatus is characterized in that sensor means are present for providing information about the status or a status change of the respective motor vehicle with regard to at least the safety of this vehicle, as well as a computer which is arranged for generating action signals and information to be broadcast, on the ground of a combination of the information provided by the sensor means and information received from a nearby vehicle, and means to enable, on the ground of these action signals, initiation of a status change of the respective vehicle or of a vehicle present in the immediate vicinity, which means comprise a signalling element for making observable a signalling knowable for the driver of the respective vehicle or the vehicle present in the immediate vicinity.", "The information about the status or a status change comprises, for instance, deceleration information, speed information and/or position information.", "The means further comprise, for instance, actuators for the execution of autonomous actions by the respective vehicle on the basis of available information.", "When a vehicle is the first vehicle in a group of vehicles driving in mutual proximity, then, in the computer, with the aid of the information about the status or a status change of the respective motor vehicle with regard to at least the safety of this vehicle, the above-mentioned action signals and the abovementioned information to be broadcast are determined.", "However, the computer will also be arranged to determine the action signals and information to be broadcast partly on the ground of information received from a nearby vehicle, typically a downstream vehicle.", "A further nearby vehicle can be both an upstream vehicle (a vehicle following immediately or after a number of intervening vehicles), and a vehicle driving past laterally.", "In a simpler embodiment, the communication means are arranged on the front and rear side of the vehicle, allowing communication only with a following vehicle.", "In a more advanced embodiment, it is also possible to communicate with fixed infrastructure.", "Naturally, it remains possible to provide communication means on opposite sides of the respective vehicle, thereby enabling communication with a vehicle driving on the side, which may be of importance when a vehicle is joining traffic or is overtaking.", "With the aid of such a signalling apparatus, it is rendered possible to gather time-critical information about the actions of other road users and to combine this information with the vehicle's own status.", "As a result, for several road users, an instantaneous and reliable picture of the traffic situation is obtained and a cooperative driving behavior is enabled.", "The signalling apparatus is able, on the basis of the combined information, and through the signalling element, to offer a driving advice to a respective driver, so that the latter obtains instantaneous and reliable support in driving or otherwise manipulating the vehicle.", "The information can also be used for initiating automatic actions by the vehicle with no or only limited driver intervention.", "In an embodiment of the signalling apparatus according to the invention, the computer is arranged to generate combined information for the information to be broadcast, by weakened transfer of an extent of exceptionality of the information received from the nearby vehicle and/or enhancement of the extent of exceptionality of the combined information depending on the information provided by the sensor means.", "The idea behind this is that in addition to the internal status of a particular vehicle, also the status of downstream vehicles plays a role in generating the information to be broadcast to an upstream vehicle.", "The idea is further that the upstream vehicles need to be influenced to a lesser extent by information from downstream vehicles according as the downstream vehicles are further ahead.", "The extent of exceptionality is, for instance, a warning signal which can take different values of warning intensity or, more particularly, status information about braking actions which can take different values of increasing warning intensity.", "The combined signal is, for instance, determined by broadcasting information about a force of a braking action of a particular vehicle to a following vehicle, unless it appears from information of a vehicle in front that a weakened version of the force of braking action of this vehicle in front (or several such vehicles) is greater than the force of the braking action of the particular vehicle, in which case this weakened version is broadcast.", "Alternatively, combining can also lead to the joint broadcast by the vehicle involved of the information from the sensor means of the vehicle involved and received information from other vehicles, preferably with an indication of which information comes from the sensor means in the vehicle involved and which information comes from the other vehicles.", "Thus, a further receiving signalling apparatus receiving this combined information can itself combine this combined information.", "The signalling apparatus has the following general advantages: Drivers can anticipate possibly dangerous situations because they obtain information about a traffic situation along a particular proximal road section.", "In other words, the safety of the whole vehicle system is enhanced.", "Much time is gained because drivers are no longer dependent on the propagation of conventional brake lights in a stream of vehicles.", "In the communication chain, the reaction time of other drivers required, for instance, to depress the brake pedal, is therefore no longer critical.", "The information exchange is not dependent on the field of vision of the driver.", "In principle, much more information can be exchanged than just information about whether or not downstream vehicles are braking or not.", "As a result, drivers can better react to the incident circumstances.", "To determine with which vehicles communication is to take place, it is important to know each other's relative positions.", "This can be realized when the information to be passes comprises position and direction data, whereafter in a respective vehicle, on the basis of position and direction data coming from other vehicles, it is calculated which vehicles are relevant for the safety of the respective vehicle The disadvantage of this is that each vehicle must be equipped with a device to determine it own position and direction, for instance a global positioning system (GPS).", "Moreover, it requires much arithmetic capacity to determine from all received information which vehicle is eligible for communication from a safety point of view.", "To circumvent this disadvantage, the communication means can he formed by an infrared communication system, more particularly, by an infrared transmitter and an infrared receiver on or in the vehicle.", "As an infrared transmitter involves a so-called transmission cone, the broadcast signal has a main direction which coincides with the axis of the cone.", "In case of a tailback, therefore, this allows well directed broadcast in rearward direction.", "When a next upstream car is equipped with an infrared receiver, that car will only receive signals coming from the front.", "This is referred to as line-of-sight communication: only connections between two immediately consecutive vehicles are effected.", "Thus the relative position of the two communicating vehicles is known.", "The infrared signal cannot penetrate through a car.", "The infrared system at the same time ensures that the information exchange is also maintained in the event of fog and heavy rain or snow.", "In a preferred embodiment, the communication means are formed by connecting means which enable a line-of-sight connection between two vehicles driving behind each other, in combination with radio connecting means, wherein, when according to the line-of-sight, a connection between two vehicles driving behind each other has been effected, a data transmission by the vehicle in front to the following vehicle is realized with the aid of the radio connection.", "After the vehicle driving in front has broadcast a connecting signal, the following vehicle can broadcast an address signal, whereafter, with the aid of the radio connection, data can be transmitted to the vehicle defined by the address.", "The advantage of a line-of-sight communication system is that it enables communication with a single vehicle relevant for the road user, without complicated analysis of the received information.", "Naturally, it remains possible to communicate with several vehicles, for instance both with a following vehicle and with a vehicle driving past on the side.", "Because in bends the infrared connection may be briefly broken, however, combination with a radio connection provides a solution for that as well.", "This implementation of inter-vehicle communication therefore combines the advantage of line-of-sight communication with that of a radio connection.", "The status of a vehicle is determined by a large number of parameters, or signals.", "The transmission of all this raw information to other vehicles is consequently susceptible to error and requires powerful transmitter and receiver equipment.", "Also, a large arithmetic capacity of the board computers is required for analysis of the data.", "It is therefore favorable, instead of transmitting the raw information, to process this information already in the respective vehicle and to compress is to one safety status signal, which indicates the condition of the vehicle with regard to safety aspects.", "As a consequence, in principle, no conversion step is necessary to present this safety status signal in a manner comprehensible to the driver of the receiving vehicle.", "A simple possibility here is, for instance, a number on a scale from 0 to 5, with 5 indicating a serious situation which requires extreme attention on the part of the drivers in the receiving vehicles.", "As the information transfer is now less susceptible to error, less and hence cheaper equipment will suffice.", "Moreover, such a safety status signal can be made visible to the driver in a simple manner in the respective vehicle with the aid of a human machine interface and/or be used for generating an automatic reaction by the vehicle.", "The received safety status signal can be linked to the information from the sensor means of the vehicle.", "Through such linking, unnecessary control of the human machine interface can be prevented.", "The fact is, when the driver has already initiated the correct intervention, he no longer needs to be alerted.", "These sensor means can comprise means that indicate the position of the brake pedal.", "The sensor means can also comprise means determining the speed and/or acceleration of the vehicle.", "An efficient and effective communication of the vehicle status received by a vehicle can be achieved through a brief, automatic depression of the brake pedal, based on the received information, while the extent of depression is related to the seriousness of the situation such as it appears from the received information.", "The reaction time of a driver is determined by the time required to process the received information and to actually initiate braking.", "This time is determined by the voluntary motor movements of the driver.", "The reaction time of the driver can now be effectively utilized in that during this time the means to enable a status change of the vehicle provide for an automatic braking.", "Moreover, the mechanical response time, due to a lost motion when a braking action is being performed, can be eliminated through this intervention.", "An efficient and effective communication of the vehicle status received by a vehicle can further be achieved by means of a display, for instance in the form of a warning triangle, this triangle becoming larger or smaller in accordance with the seriousness of the situation.", "In particular the triangular form is completely in line with the conventional method (through road signs) of alerting road users to danger.", "It shortens the time the driver requires for interpretation, and he can react faster to critical situations in which surrounding road users are involved.", "Moreover, the interpretation requires little attention, which is beneficial to a safe conduct of the driver himself.", "The display can be built into a respective vehicle, naturally within the field of vision of the driver, or be arranged at the back of a vehicle and hence within the field of vision of the following vehicle.", "Instead of a display, brake lights can be used.", "If, for instance, the first vehicle in a row of three vehicles brakes, the brake lights of the second vehicle will go on, also if this second vehicle does not brake (yet).", "The third vehicle can then react to this, for instance by applying the brakes in anticipation.", "The advantage of this solution is that no displays need to be fitted on or in the vehicles.", "Moreover, the brake lights are always in the immediate field of vision of the driver.", "A disadvantage, however, is that the information provided by the brake lights is limited: on or off.", "The signalling apparatus described here can obtain an advanced functionality, such that complex sensors, such as radar equipment, are required, with which the position and optionally the speed of the vehicle relative to other, neighboring vehicles is measured.", "In the vehicle status which is made knowable for the driver, the distance to a vehicle in front could then be taken into account as well: if the vehicle is very far away, the display is never fully controlled, even if this downstream vehicle makes an emergency stop.", "For this purpose, it is necessary to measure this distance, which, as mentioned, requires complex sensors.", "This raises the price and reduces the reliability of the system.", "If the functionality relates not only to the situation where vehicles are driving behind each other, but also to the situation where they are driving next to each other, it is even necessary to arrange these complex sensors all around the vehicles.", "Starting from the assumption that the vehicles are provided with means to communicate with each other, the above problem can be solved by providing each vehicle with a global positioning system based on, for instance GPS, GPRS (General Packet Radio Service) or UMTS (universal Mobile Telecommunications System), which functions as sensor for measuring at least the position of the respective vehicle.", "If presently, by way of the inter-vehicle communication, at least information about each other's position is transferred, it is possible in a simple manner to calculate the relative distance for the purpose of, for instance, a more accurate determination of the vehicle status and/or the status to be represented on the display.", "Because such systems can typically determine the speed of the vehicle as well, it is possible, when the speed is also transferred, to calculate the relative differential speed and use it for the purpose of status determination and status representation.", "Global positioning systems are more and more often included in road vehicles, typically to generate information for the purpose of an on-board navigation system.", "For this reason, these positioning systems are relatively inexpensive and reliable.", "Complex sensors for generating position and speed information are then redundant.", "It should be noted that the positioning system is not used here to determine which road vehicles are relevant to each other from the viewpoint of safety.", "For this is the task of the line-of-sight communication system.", "The invention will now be further elucidated with reference to the accompanying drawing.", "In the drawing: FIG.", "1 shows a block diagram of a first embodiment of the signalling apparatus according to the invention; and FIG.", "2 shows a schematic representation of a second embodiment of the signalling apparatus according to the invention, shown for a group of vehicles.", "In the figures, corresponding parts are indicated by the same reference numerals.", "The signalling apparatus represented in FIG.", "1, which relates to an embodiment in which the vehicles are assumed to drive wholly behind each other, comprises communication means 1, a computer 2, sensor means 3, means 4 to enable realization of a status change of the vehicle, a display 5 and an automatic brake operation 6.The communication means 1 are formed by an infrared transmitter 7 which can be fitted on or in the back of a vehicle, an infrared receiver 8 which can be fitted on or in the front of the vehicle, and a radio transmitter and receiver installation 9.When a following vehicle has approached the vehicle in front to a sufficient extent, the following vehicle receives an infrared signal broadcast by the vehicle in front.", "With the aid of the infrared transmitter 7, an address is passed to the vehicle in front, which, with the aid of the radio transmitter and receiver installation 9, thereupon broadcasts a safety status signal intended exclusively for the following vehicle.", "It is also possible that the vehicle in front passes on a connecting signal with address to the following vehicle, which in turn transmits a confirmation signal to the vehicle in front.", "The latter, with the aid of the radio transmitter and receiver installation 9, can then broadcast a safety status signal intended exclusively for the following vehicle again.", "However, other solutions with regard to the communication also remain possible.", "By way of the radio transceiver 9 of the following vehicle, the safety status signal is received, detected and processed and then fed to the computer 2.Together with the information of the sensor means 3 in the following vehicle, a new safety status signal is determined, which can be broadcast again.", "Also, on the basis of the received safety status signal in combination with the information of the sensor means 3, in this case a speedometer and an accelerometer, action signals can be determined which are fed to the means 4.These means enable a change in the condition of the vehicle and to that end comprise, for instance, a display arranged in the vehicle, on which a warning triangle varying in size can be displayed, so that the driver can see immediately when an action, such as braking or corrective steering, is to be taken.", "According as the triangle increases in size, the danger is greater and action by the driver more urgent.", "On the basis of the action signals mentioned, with the aid of the means 4, at the same time a signal can be generated for the unit 6 to enable an automatic braking operation (and/or accelerator pedal operation) to be performed.", "In order that, apart from the transfer of a safety status signal, the driver of a following vehicle—when, for instance, it is not provided with a signalling apparatus as described here—be warned anyhow, a display 5 may be provided on the back of the vehicle.", "On this display, too, a warning triangle varying in size may be represented.", "However, this information is not necessarily identical to that on the display in the vehicle.", "In the embodiment of FIG.", "2, the communication means are limited to an infrared transmitter and infrared receiver 7 and 8, respectively.", "The information transmitted consists of a safety status signal which indicates the extent of deceleration of the vehicle or of vehicles in front of it.", "For this purpose, use is made of the information from the sensor means 3 in the vehicle, which sensor means in this example measure the position of the brake pedal and/or the acceleration in forward direction.", "This status signal is calculated as follows: Let is be assumed that ak is the deceleration in [m/s2] of vehicle k. ak therefore indicates that the vehicle is being braked.", "This braking action is compressed by providing a limited number of levels therein.", "If this compression is represented by the function f, then f(ak) is a number giving an indication of the extent of braking.", "Starting from the “resolution” of a driver, f(ak) is chosen to have six values: 0-5.The determination of the status xm,k of vehicle k is now done with the aid of the braking action f(ak) and the status of the vehicle in front xm,k-1.For this purpose, the following recursive function is used: xm,k=max {f(ak), xm,k-1-q} wherein xm,k-1 is the status of the vehicle in front k−1.The constant q is a “weakening term”.", "The status xm,k-1, just like f(ak), has a value varying from 0 to 5.By reducing this value by q, a weakening is obtained; if it is chosen that q=1, then, after five vehicles, the status will have become equal to 0.The idea behind this is that upstream vehicles are less dependent on, or are less influenced by, the front vehicle according as they are further away from the front vehicle.", "In addition to the status of a vehicle in front, also the internal condition plays a role.", "This is taken into account by taking the maximum of the incoming weakened status (xm,k-1-q) and the vehicle's own braking action f(ak).", "The status xm,k-1 is presented to the driver of vehicle k, viz.", "on a display on which a warning triangle varying in size is displayed.", "He reacts to it, which yields a value for f(ak).", "With the aid of the above relation, next, the status xm,k is calculated, which, finally, is forwarded to the following vehicle k+1.Also when the driver does not react, this algorithm results in a useful status to be transmitted.", "The invention is not limited to the exemplary embodiments described here with reference to the figures, but comprises all kinds of modifications thereon, naturally insofar as they fall within the scope of protection of the following claims." ] ]